Select recent publications
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2023
Liu, Bingxu; Carlson, Rebecca J; Pires, Ivan S; Gentili, Matteo; Feng, Ellie; Hellier, Quentin; Schwartz, Marc A; Blainey, Paul C; Irvine, Darrell J; Hacohen, Nir
Human STING is a proton channel Journal Article
In: Science, vol. 381, no. 6657, pp. 508–514, 2023, ISSN: 1095-9203.
@article{pmid37535724,
title = {Human STING is a proton channel},
author = {Bingxu Liu and Rebecca J Carlson and Ivan S Pires and Matteo Gentili and Ellie Feng and Quentin Hellier and Marc A Schwartz and Paul C Blainey and Darrell J Irvine and Nir Hacohen},
doi = {10.1126/science.adf8974},
issn = {1095-9203},
year = {2023},
date = {2023-08-01},
journal = {Science},
volume = {381},
number = {6657},
pages = {508--514},
abstract = {Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING's channel activity is critical for these two processes. Thus, STING's interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Proton leakage from organelles is a common signal for noncanonical light chain 3B (LC3B) lipidation and inflammasome activation, processes induced upon stimulator of interferon genes (STING) activation. On the basis of structural analysis, we hypothesized that human STING is a proton channel. Indeed, we found that STING activation induced a pH increase in the Golgi and that STING reconstituted in liposomes enabled transmembrane proton transport. Compound 53 (C53), a STING agonist that binds the putative channel interface, blocked STING-induced proton flux in the Golgi and in liposomes. STING-induced LC3B lipidation and inflammasome activation were also inhibited by C53, suggesting that STING's channel activity is critical for these two processes. Thus, STING's interferon-induction function can be decoupled from its roles in LC3B lipidation and inflammasome activation.
Borrajo, Jacob; Javanmardi, Kamyab; Griffin, James; Martin, Susan J St; Yao, David; Hill, Kaisle; Blainey, Paul C; Al-Shayeb, Basem
Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing
2023.
@{pmid37645763,
title = {Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing},
author = {Jacob Borrajo and Kamyab Javanmardi and James Griffin and Susan J St Martin and David Yao and Kaisle Hill and Paul C Blainey and Basem Al-Shayeb},
doi = {10.1101/2023.08.18.553620},
year = {2023},
date = {2023-08-01},
journal = {bioRxiv},
abstract = {Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.
ONE-SENTENCE SUMMARY: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.},
keywords = {},
pubstate = {published},
tppubtype = {}
}
Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.
ONE-SENTENCE SUMMARY: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.
ONE-SENTENCE SUMMARY: CRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.
Ramezani, Meraj; Bauman, Julia; Singh, Avtar; Weisbart, Erin; Yong, John; Lozada, Maria; Way, Gregory P; Kavari, Sanam L; Diaz, Celeste; Haghighi, Marzieh; Batista, Thiago M; Pérez-Schindler, Joaquín; Claussnitzer, Melina; Singh, Shantanu; Cimini, Beth A; Blainey, Paul C; Carpenter, Anne E; Jan, Calvin H; Neal, James T
A genome-wide atlas of human cell morphology
2023.
@{pmid37609130,
title = {A genome-wide atlas of human cell morphology},
author = {Meraj Ramezani and Julia Bauman and Avtar Singh and Erin Weisbart and John Yong and Maria Lozada and Gregory P Way and Sanam L Kavari and Celeste Diaz and Marzieh Haghighi and Thiago M Batista and Joaquín Pérez-Schindler and Melina Claussnitzer and Shantanu Singh and Beth A Cimini and Paul C Blainey and Anne E Carpenter and Calvin H Jan and James T Neal},
doi = {10.1101/2023.08.06.552164},
year = {2023},
date = {2023-08-01},
journal = {bioRxiv},
abstract = {A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting ) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale.},
keywords = {},
pubstate = {published},
tppubtype = {}
}
A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting ) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale.
Al'Khafaji, Aziz M; Smith, Jonathan T; Garimella, Kiran V; Babadi, Mehrtash; Popic, Victoria; Sade-Feldman, Moshe; Gatzen, Michael; Sarkizova, Siranush; Schwartz, Marc A; Blaum, Emily M; Day, Allyson; Costello, Maura; Bowers, Tera; Gabriel, Stacey; Banks, Eric; Philippakis, Anthony A; Boland, Genevieve M; Blainey, Paul C; Hacohen, Nir
High-throughput RNA isoform sequencing using programmed cDNA concatenation Journal Article
In: Nat Biotechnol, 2023, ISSN: 1546-1696.
@article{pmid37291427,
title = {High-throughput RNA isoform sequencing using programmed cDNA concatenation},
author = {Aziz M Al'Khafaji and Jonathan T Smith and Kiran V Garimella and Mehrtash Babadi and Victoria Popic and Moshe Sade-Feldman and Michael Gatzen and Siranush Sarkizova and Marc A Schwartz and Emily M Blaum and Allyson Day and Maura Costello and Tera Bowers and Stacey Gabriel and Eric Banks and Anthony A Philippakis and Genevieve M Boland and Paul C Blainey and Nir Hacohen},
doi = {10.1038/s41587-023-01815-7},
issn = {1546-1696},
year = {2023},
date = {2023-06-01},
journal = {Nat Biotechnol},
abstract = {Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.
Carlson, Rebecca J; Leiken, Michael D; Guna, Alina; Hacohen, Nir; Blainey, Paul C
A genome-wide optical pooled screen reveals regulators of cellular antiviral responses Journal Article
In: Proc Natl Acad Sci U S A, vol. 120, no. 16, pp. e2210623120, 2023, ISSN: 1091-6490.
@article{pmid37043539,
title = {A genome-wide optical pooled screen reveals regulators of cellular antiviral responses},
author = {Rebecca J Carlson and Michael D Leiken and Alina Guna and Nir Hacohen and Paul C Blainey},
doi = {10.1073/pnas.2210623120},
issn = {1091-6490},
year = {2023},
date = {2023-04-01},
journal = {Proc Natl Acad Sci U S A},
volume = {120},
number = {16},
pages = {e2210623120},
abstract = {The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches.
Thakku, Sri Gowtham; Lirette, Jackson; Murugesan, Kanagavel; Chen, Julie; Theron, Grant; Banaei, Niaz; Blainey, Paul C; Gomez, James; Wong, Sharon Y; Hung, Deborah T
Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13 Journal Article
In: Nat Commun, vol. 14, no. 1, pp. 1803, 2023, ISSN: 2041-1723.
@article{pmid37002219,
title = {Genome-wide tiled detection of circulating Mycobacterium tuberculosis cell-free DNA using Cas13},
author = {Sri Gowtham Thakku and Jackson Lirette and Kanagavel Murugesan and Julie Chen and Grant Theron and Niaz Banaei and Paul C Blainey and James Gomez and Sharon Y Wong and Deborah T Hung},
doi = {10.1038/s41467-023-37183-8},
issn = {2041-1723},
year = {2023},
date = {2023-03-01},
journal = {Nat Commun},
volume = {14},
number = {1},
pages = {1803},
abstract = {Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Detection of microbial cell-free DNA (cfDNA) circulating in the bloodstream has emerged as a promising new approach for diagnosing infection. Microbial diagnostics based on cfDNA require assays that can detect rare and highly fragmented pathogen nucleic acids. We now report WATSON (Whole-genome Assay using Tiled Surveillance Of Nucleic acids), a method to detect low amounts of pathogen cfDNA that couples pooled amplification of genomic targets tiled across the genome with pooled CRISPR/Cas13-based detection of these targets. We demonstrate that this strategy of tiling improves cfDNA detection compared to amplification and detection of a single targeted locus. WATSON can detect cfDNA from Mycobacterium tuberculosis in plasma of patients with active pulmonary tuberculosis, a disease that urgently needs accurate, minimally-invasive, field-deployable diagnostics. We thus demonstrate the potential for translating WATSON to a lateral flow platform. WATSON demonstrates the ability to capitalize on the strengths of targeting microbial cfDNA to address the need for point-of-care diagnostic tests for infectious diseases.
Reyes, Miguel; Leff, Samantha M; Gentili, Matteo; Hacohen, Nir; Blainey, Paul C
Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands Journal Article
In: Sci Adv, vol. 9, no. 8, pp. eade5090, 2023, ISSN: 2375-2548.
@article{pmid36827376,
title = {Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands},
author = {Miguel Reyes and Samantha M Leff and Matteo Gentili and Nir Hacohen and Paul C Blainey},
doi = {10.1126/sciadv.ade5090},
issn = {2375-2548},
year = {2023},
date = {2023-02-01},
journal = {Sci Adv},
volume = {9},
number = {8},
pages = {eade5090},
abstract = {Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells' potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space. Single-cell genomics coupled with multiplexing techniques provide a useful tool for characterizing cell states across several experimental conditions. However, current multiplexing strategies require programmatic handling of many samples in macroscale arrayed formats, precluding their application in large-scale combinatorial analysis. Here, we introduce StimDrop, a method that combines antibody-based cell barcoding with parallel droplet processing to automatically formulate cell population × stimulus combinations in a microfluidic device. We applied StimDrop to profile the effects of 512 sequential stimulation conditions on human dendritic cells. Our results demonstrate that priming with viral ligands potentiates hyperinflammatory responses to a second stimulus, and show transcriptional signatures consistent with this phenomenon in myeloid cells of patients with severe COVID-19.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells' potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space. Single-cell genomics coupled with multiplexing techniques provide a useful tool for characterizing cell states across several experimental conditions. However, current multiplexing strategies require programmatic handling of many samples in macroscale arrayed formats, precluding their application in large-scale combinatorial analysis. Here, we introduce StimDrop, a method that combines antibody-based cell barcoding with parallel droplet processing to automatically formulate cell population × stimulus combinations in a microfluidic device. We applied StimDrop to profile the effects of 512 sequential stimulation conditions on human dendritic cells. Our results demonstrate that priming with viral ligands potentiates hyperinflammatory responses to a second stimulus, and show transcriptional signatures consistent with this phenomenon in myeloid cells of patients with severe COVID-19.
Mead, Benjamin E; Kummerlowe, Conner; Liu, Nuo; Kattan, Walaa E; Cheng, Thomas; Cheah, Jaime H; Soule, Christian K; Peters, Josh; Lowder, Kristen E; Blainey, Paul C; Hahn, William C; Cleary, Brian; Bryson, Bryan; Winter, Peter S; Raghavan, Srivatsan; Shalek, Alex K
Compressed phenotypic screens for complex multicellular models and high-content assays
2023.
@{pmid36747859,
title = {Compressed phenotypic screens for complex multicellular models and high-content assays},
author = {Benjamin E Mead and Conner Kummerlowe and Nuo Liu and Walaa E Kattan and Thomas Cheng and Jaime H Cheah and Christian K Soule and Josh Peters and Kristen E Lowder and Paul C Blainey and William C Hahn and Brian Cleary and Bryan Bryson and Peter S Winter and Srivatsan Raghavan and Alex K Shalek},
doi = {10.1101/2023.01.23.525189},
year = {2023},
date = {2023-01-01},
journal = {bioRxiv},
abstract = {High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.},
keywords = {},
pubstate = {published},
tppubtype = {}
}
High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.
2022
Funk, Luke; Su, Kuan-Chung; Ly, Jimmy; Feldman, David; Singh, Avtar; Moodie, Brittania; Blainey, Paul C; Cheeseman, Iain M
The phenotypic landscape of essential human genes Journal Article
In: Cell, vol. 185, no. 24, pp. 4634–4653.e22, 2022, ISSN: 1097-4172.
@article{pmid36347254,
title = {The phenotypic landscape of essential human genes},
author = {Luke Funk and Kuan-Chung Su and Jimmy Ly and David Feldman and Avtar Singh and Brittania Moodie and Paul C Blainey and Iain M Cheeseman},
url = {https://vesuvius.wi.mit.edu/},
doi = {10.1016/j.cell.2022.10.017},
issn = {1097-4172},
year = {2022},
date = {2022-11-01},
urldate = {2022-11-01},
journal = {Cell},
volume = {185},
number = {24},
pages = {4634--4653.e22},
abstract = {Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.
Walton, Russell T; Singh, Avtar; Blainey, Paul C
Pooled genetic screens with image-based profiling Journal Article
In: Mol Syst Biol, vol. 18, no. 11, pp. e10768, 2022, ISSN: 1744-4292.
@article{pmid36366905,
title = {Pooled genetic screens with image-based profiling},
author = {Russell T Walton and Avtar Singh and Paul C Blainey},
doi = {10.15252/msb.202110768},
issn = {1744-4292},
year = {2022},
date = {2022-11-01},
journal = {Mol Syst Biol},
volume = {18},
number = {11},
pages = {e10768},
abstract = {Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.
Metsky, Hayden C; Welch, Nicole L; Pillai, Priya P; Haradhvala, Nicholas J; Rumker, Laurie; Mantena, Sreekar; Zhang, Yibin B; Yang, David K; Ackerman, Cheri M; Weller, Juliane; Blainey, Paul C; Myhrvold, Cameron; Mitzenmacher, Michael; Sabeti, Pardis C
Designing sensitive viral diagnostics with machine learning Journal Article
In: Nat Biotechnol, vol. 40, no. 7, pp. 1123–1131, 2022, ISSN: 1546-1696.
@article{pmid35241837,
title = {Designing sensitive viral diagnostics with machine learning},
author = {Hayden C Metsky and Nicole L Welch and Priya P Pillai and Nicholas J Haradhvala and Laurie Rumker and Sreekar Mantena and Yibin B Zhang and David K Yang and Cheri M Ackerman and Juliane Weller and Paul C Blainey and Cameron Myhrvold and Michael Mitzenmacher and Pardis C Sabeti},
doi = {10.1038/s41587-022-01213-5},
issn = {1546-1696},
year = {2022},
date = {2022-07-01},
journal = {Nat Biotechnol},
volume = {40},
number = {7},
pages = {1123--1131},
abstract = {Design of nucleic acid-based viral diagnostics typically follows heuristic rules and, to contend with viral variation, focuses on a genome's conserved regions. A design process could, instead, directly optimize diagnostic effectiveness using a learned model of sensitivity for targets and their variants. Toward that goal, we screen 19,209 diagnostic-target pairs, concentrated on CRISPR-based diagnostics, and train a deep neural network to accurately predict diagnostic readout. We join this model with combinatorial optimization to maximize sensitivity over the full spectrum of a virus's genomic variation. We introduce Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT), a system for automated design, and use it to design diagnostics for 1,933 vertebrate-infecting viral species within 2 hours for most species and within 24 hours for all but three. We experimentally show that ADAPT's designs are sensitive and specific to the lineage level and permit lower limits of detection, across a virus's variation, than the outputs of standard design techniques. Our strategy could facilitate a proactive resource of assays for detecting pathogens.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Design of nucleic acid-based viral diagnostics typically follows heuristic rules and, to contend with viral variation, focuses on a genome's conserved regions. A design process could, instead, directly optimize diagnostic effectiveness using a learned model of sensitivity for targets and their variants. Toward that goal, we screen 19,209 diagnostic-target pairs, concentrated on CRISPR-based diagnostics, and train a deep neural network to accurately predict diagnostic readout. We join this model with combinatorial optimization to maximize sensitivity over the full spectrum of a virus's genomic variation. We introduce Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT), a system for automated design, and use it to design diagnostics for 1,933 vertebrate-infecting viral species within 2 hours for most species and within 24 hours for all but three. We experimentally show that ADAPT's designs are sensitive and specific to the lineage level and permit lower limits of detection, across a virus's variation, than the outputs of standard design techniques. Our strategy could facilitate a proactive resource of assays for detecting pathogens.
Quan, Kathleen A; Sater, Mohamad R A; Uy, Cherry; Clifton-Koeppel, Robin; Dickey, Linda L; Wilson, William; Patton, Pat; Chang, Wayne; Samuelson, Pamela; Lagoudas, Georgia K; Allen, Teri; Merchant, Lenny; Gannotta, Rick; Bittencourt, Cassiana E; Soto, J C; Evans, Kaye D; Blainey, Paul C; Murray, John; Shelton, Dawn; Lee, Helen S; Zahn, Matthew; Wolfe, Julia; Madey, Keith; Yim, Jennifer; Gohil, Shruti K; Grad, Yonatan H; Huang, Susan S
In: Infect Control Hosp Epidemiol, pp. 1–8, 2022, ISSN: 1559-6834.
@article{pmid35706396,
title = {Epidemiology and genomics of a slow outbreak of methicillin-resistant (MRSA) in a neonatal intensive care unit: Successful chronic decolonization of MRSA-positive healthcare personnel},
author = {Kathleen A Quan and Mohamad R A Sater and Cherry Uy and Robin Clifton-Koeppel and Linda L Dickey and William Wilson and Pat Patton and Wayne Chang and Pamela Samuelson and Georgia K Lagoudas and Teri Allen and Lenny Merchant and Rick Gannotta and Cassiana E Bittencourt and J C Soto and Kaye D Evans and Paul C Blainey and John Murray and Dawn Shelton and Helen S Lee and Matthew Zahn and Julia Wolfe and Keith Madey and Jennifer Yim and Shruti K Gohil and Yonatan H Grad and Susan S Huang},
doi = {10.1017/ice.2022.133},
issn = {1559-6834},
year = {2022},
date = {2022-06-01},
journal = {Infect Control Hosp Epidemiol},
pages = {1--8},
abstract = {OBJECTIVE: To describe the genomic analysis and epidemiologic response related to a slow and prolonged methicillin-resistant (MRSA) outbreak.
DESIGN: Prospective observational study.
SETTING: Neonatal intensive care unit (NICU).
METHODS: We conducted an epidemiologic investigation of a NICU MRSA outbreak involving serial baby and staff screening to identify opportunities for decolonization. Whole-genome sequencing was performed on MRSA isolates.
RESULTS: A NICU with excellent hand hygiene compliance and longstanding minimal healthcare-associated infections experienced an MRSA outbreak involving 15 babies and 6 healthcare personnel (HCP). In total, 12 cases occurred slowly over a 1-year period (mean, 30.7 days apart) followed by 3 additional cases 7 months later. Multiple progressive infection prevention interventions were implemented, including contact precautions and cohorting of MRSA-positive babies, hand hygiene observers, enhanced environmental cleaning, screening of babies and staff, and decolonization of carriers. Only decolonization of HCP found to be persistent carriers of MRSA was successful in stopping transmission and ending the outbreak. Genomic analyses identified bidirectional transmission between babies and HCP during the outbreak.
CONCLUSIONS: In comparison to fast outbreaks, outbreaks that are "slow and sustained" may be more common to units with strong existing infection prevention practices such that a series of breaches have to align to result in a case. We identified a slow outbreak that persisted among staff and babies and was only stopped by identifying and decolonizing persistent MRSA carriage among staff. A repeated decolonization regimen was successful in allowing previously persistent carriers to safely continue work duties.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVE: To describe the genomic analysis and epidemiologic response related to a slow and prolonged methicillin-resistant (MRSA) outbreak.
DESIGN: Prospective observational study.
SETTING: Neonatal intensive care unit (NICU).
METHODS: We conducted an epidemiologic investigation of a NICU MRSA outbreak involving serial baby and staff screening to identify opportunities for decolonization. Whole-genome sequencing was performed on MRSA isolates.
RESULTS: A NICU with excellent hand hygiene compliance and longstanding minimal healthcare-associated infections experienced an MRSA outbreak involving 15 babies and 6 healthcare personnel (HCP). In total, 12 cases occurred slowly over a 1-year period (mean, 30.7 days apart) followed by 3 additional cases 7 months later. Multiple progressive infection prevention interventions were implemented, including contact precautions and cohorting of MRSA-positive babies, hand hygiene observers, enhanced environmental cleaning, screening of babies and staff, and decolonization of carriers. Only decolonization of HCP found to be persistent carriers of MRSA was successful in stopping transmission and ending the outbreak. Genomic analyses identified bidirectional transmission between babies and HCP during the outbreak.
CONCLUSIONS: In comparison to fast outbreaks, outbreaks that are "slow and sustained" may be more common to units with strong existing infection prevention practices such that a series of breaches have to align to result in a case. We identified a slow outbreak that persisted among staff and babies and was only stopped by identifying and decolonizing persistent MRSA carriage among staff. A repeated decolonization regimen was successful in allowing previously persistent carriers to safely continue work duties.
DESIGN: Prospective observational study.
SETTING: Neonatal intensive care unit (NICU).
METHODS: We conducted an epidemiologic investigation of a NICU MRSA outbreak involving serial baby and staff screening to identify opportunities for decolonization. Whole-genome sequencing was performed on MRSA isolates.
RESULTS: A NICU with excellent hand hygiene compliance and longstanding minimal healthcare-associated infections experienced an MRSA outbreak involving 15 babies and 6 healthcare personnel (HCP). In total, 12 cases occurred slowly over a 1-year period (mean, 30.7 days apart) followed by 3 additional cases 7 months later. Multiple progressive infection prevention interventions were implemented, including contact precautions and cohorting of MRSA-positive babies, hand hygiene observers, enhanced environmental cleaning, screening of babies and staff, and decolonization of carriers. Only decolonization of HCP found to be persistent carriers of MRSA was successful in stopping transmission and ending the outbreak. Genomic analyses identified bidirectional transmission between babies and HCP during the outbreak.
CONCLUSIONS: In comparison to fast outbreaks, outbreaks that are "slow and sustained" may be more common to units with strong existing infection prevention practices such that a series of breaches have to align to result in a case. We identified a slow outbreak that persisted among staff and babies and was only stopped by identifying and decolonizing persistent MRSA carriage among staff. A repeated decolonization regimen was successful in allowing previously persistent carriers to safely continue work duties.
Welch, Nicole L; Zhu, Meilin; Hua, Catherine; Weller, Juliane; Mirhashemi, Marzieh Ezzaty; Nguyen, Tien G; Mantena, Sreekar; Bauer, Matthew R; Shaw, Bennett M; Ackerman, Cheri M; Thakku, Sri Gowtham; Tse, Megan W; Kehe, Jared; Uwera, Marie-Martine; Eversley, Jacqueline S; Bielwaski, Derek A; McGrath, Graham; Braidt, Joseph; Johnson, Jeremy; Cerrato, Felecia; Moreno, Gage K; Krasilnikova, Lydia A; Petros, Brittany A; Gionet, Gabrielle L; King, Ewa; Huard, Richard C; Jalbert, Samantha K; Cleary, Michael L; Fitzgerald, Nicholas A; Gabriel, Stacey B; Gallagher, Glen R; Smole, Sandra C; Madoff, Lawrence C; Brown, Catherine M; Keller, Matthew W; Wilson, Malania M; Kirby, Marie K; Barnes, John R; Park, Daniel J; Siddle, Katherine J; Happi, Christian T; Hung, Deborah T; Springer, Michael; MacInnis, Bronwyn L; Lemieux, Jacob E; Rosenberg, Eric; Branda, John A; Blainey, Paul C; Sabeti, Pardis C; Myhrvold, Cameron
Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants Journal Article
In: Nat Med, vol. 28, no. 5, pp. 1083–1094, 2022, ISSN: 1546-170X.
@article{pmid35130561,
title = {Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants},
author = {Nicole L Welch and Meilin Zhu and Catherine Hua and Juliane Weller and Marzieh Ezzaty Mirhashemi and Tien G Nguyen and Sreekar Mantena and Matthew R Bauer and Bennett M Shaw and Cheri M Ackerman and Sri Gowtham Thakku and Megan W Tse and Jared Kehe and Marie-Martine Uwera and Jacqueline S Eversley and Derek A Bielwaski and Graham McGrath and Joseph Braidt and Jeremy Johnson and Felecia Cerrato and Gage K Moreno and Lydia A Krasilnikova and Brittany A Petros and Gabrielle L Gionet and Ewa King and Richard C Huard and Samantha K Jalbert and Michael L Cleary and Nicholas A Fitzgerald and Stacey B Gabriel and Glen R Gallagher and Sandra C Smole and Lawrence C Madoff and Catherine M Brown and Matthew W Keller and Malania M Wilson and Marie K Kirby and John R Barnes and Daniel J Park and Katherine J Siddle and Christian T Happi and Deborah T Hung and Michael Springer and Bronwyn L MacInnis and Jacob E Lemieux and Eric Rosenberg and John A Branda and Paul C Blainey and Pardis C Sabeti and Cameron Myhrvold},
doi = {10.1038/s41591-022-01734-1},
issn = {1546-170X},
year = {2022},
date = {2022-05-01},
journal = {Nat Med},
volume = {28},
number = {5},
pages = {1083--1094},
abstract = {The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.
Thakku, Sri Gowtham; Ackerman, Cheri M; Myhrvold, Cameron; Bhattacharyya, Roby P; Livny, Jonathan; Ma, Peijun; Gomez, Giselle Isabella; Sabeti, Pardis C; Blainey, Paul C; Hung, Deborah T
Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays Journal Article
In: PNAS Nexus, vol. 1, no. 1, pp. pgac021, 2022, ISSN: 2752-6542.
@article{pmid35450424,
title = {Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays},
author = {Sri Gowtham Thakku and Cheri M Ackerman and Cameron Myhrvold and Roby P Bhattacharyya and Jonathan Livny and Peijun Ma and Giselle Isabella Gomez and Pardis C Sabeti and Paul C Blainey and Deborah T Hung},
doi = {10.1093/pnasnexus/pgac021},
issn = {2752-6542},
year = {2022},
date = {2022-03-01},
journal = {PNAS Nexus},
volume = {1},
number = {1},
pages = {pgac021},
abstract = {Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.
Feldman, David; Funk, Luke; Le, Anna; Carlson, Rebecca J; Leiken, Michael D; Tsai, FuNien; Soong, Brian; Singh, Avtar; Blainey, Paul C
Pooled genetic perturbation screens with image-based phenotypes Journal Article
In: Nat Protoc, vol. 17, no. 2, pp. 476–512, 2022, ISSN: 1750-2799.
@article{pmid35022620,
title = {Pooled genetic perturbation screens with image-based phenotypes},
author = {David Feldman and Luke Funk and Anna Le and Rebecca J Carlson and Michael D Leiken and FuNien Tsai and Brian Soong and Avtar Singh and Paul C Blainey},
doi = {10.1038/s41596-021-00653-8},
issn = {1750-2799},
year = {2022},
date = {2022-02-01},
journal = {Nat Protoc},
volume = {17},
number = {2},
pages = {476--512},
abstract = {Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >10 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >10 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.
2021
Kehe, Jared; Ortiz, Anthony; Kulesa, Anthony; Gore, Jeff; Blainey, Paul C; Friedman, Jonathan
Positive interactions are common among culturable bacteria Journal Article
In: Sci Adv, vol. 7, no. 45, pp. eabi7159, 2021, ISSN: 2375-2548.
@article{pmid34739314,
title = {Positive interactions are common among culturable bacteria},
author = {Jared Kehe and Anthony Ortiz and Anthony Kulesa and Jeff Gore and Paul C Blainey and Jonathan Friedman},
doi = {10.1126/sciadv.abi7159},
issn = {2375-2548},
year = {2021},
date = {2021-11-01},
journal = {Sci Adv},
volume = {7},
number = {45},
pages = {eabi7159},
abstract = {Interspecies interactions shape the structure and function of microbial communities. In particular, positive, growth-promoting interactions can substantially affect the diversity and productivity of natural and engineered communities. However, the prevalence of positive interactions and the conditions in which they occur are not well understood. To address this knowledge gap, we used kChip, an ultrahigh-throughput coculture platform, to measure 180,408 interactions among 20 soil bacteria across 40 carbon environments. We find that positive interactions, often described to be rare, occur commonly and primarily as parasitisms between strains that differ in their carbon consumption profiles. Notably, nongrowing strains are almost always promoted by strongly growing strains (85%), suggesting a simple positive interaction–mediated approach for cultivation, microbiome engineering, and microbial consortium design.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Interspecies interactions shape the structure and function of microbial communities. In particular, positive, growth-promoting interactions can substantially affect the diversity and productivity of natural and engineered communities. However, the prevalence of positive interactions and the conditions in which they occur are not well understood. To address this knowledge gap, we used kChip, an ultrahigh-throughput coculture platform, to measure 180,408 interactions among 20 soil bacteria across 40 carbon environments. We find that positive interactions, often described to be rare, occur commonly and primarily as parasitisms between strains that differ in their carbon consumption profiles. Notably, nongrowing strains are almost always promoted by strongly growing strains (85%), suggesting a simple positive interaction–mediated approach for cultivation, microbiome engineering, and microbial consortium design.
Banal, James L; Shepherd, Tyson R; Berleant, Joseph; Huang, Hellen; Reyes, Miguel; Ackerman, Cheri M; Blainey, Paul C; Bathe, Mark
Random access DNA memory using Boolean search in an archival file storage system Journal Article
In: Nat Mater, vol. 20, no. 9, pp. 1272–1280, 2021, ISSN: 1476-4660.
@article{pmid34112975,
title = {Random access DNA memory using Boolean search in an archival file storage system},
author = {James L Banal and Tyson R Shepherd and Joseph Berleant and Hellen Huang and Miguel Reyes and Cheri M Ackerman and Paul C Blainey and Mark Bathe},
doi = {10.1038/s41563-021-01021-3},
issn = {1476-4660},
year = {2021},
date = {2021-09-01},
journal = {Nat Mater},
volume = {20},
number = {9},
pages = {1272--1280},
abstract = {DNA is an ultrahigh-density storage medium that could meet exponentially growing worldwide demand for archival data storage if DNA synthesis costs declined sufficiently and if random access of files within exabyte-to-yottabyte-scale DNA data pools were feasible. Here, we demonstrate a path to overcome the second barrier by encapsulating data-encoding DNA file sequences within impervious silica capsules that are surface labelled with single-stranded DNA barcodes. Barcodes are chosen to represent file metadata, enabling selection of sets of files with Boolean logic directly, without use of amplification. We demonstrate random access of image files from a prototypical 2-kilobyte image database using fluorescence sorting with selection sensitivity of one in 10 files, which thereby enables one in 10 selection capability using N optical channels. Our strategy thereby offers a scalable concept for random access of archival files in large-scale molecular datasets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
DNA is an ultrahigh-density storage medium that could meet exponentially growing worldwide demand for archival data storage if DNA synthesis costs declined sufficiently and if random access of files within exabyte-to-yottabyte-scale DNA data pools were feasible. Here, we demonstrate a path to overcome the second barrier by encapsulating data-encoding DNA file sequences within impervious silica capsules that are surface labelled with single-stranded DNA barcodes. Barcodes are chosen to represent file metadata, enabling selection of sets of files with Boolean logic directly, without use of amplification. We demonstrate random access of image files from a prototypical 2-kilobyte image database using fluorescence sorting with selection sensitivity of one in 10 files, which thereby enables one in 10 selection capability using N optical channels. Our strategy thereby offers a scalable concept for random access of archival files in large-scale molecular datasets.
Zhu, Meilin; Tse, Megan W; Weller, Juliane; Chen, Julie; Blainey, Paul C
The future of antibiotics begins with discovering new combinations Journal Article
In: Ann N Y Acad Sci, vol. 1496, no. 1, pp. 82–96, 2021, ISSN: 1749-6632.
@article{pmid34212403,
title = {The future of antibiotics begins with discovering new combinations},
author = {Meilin Zhu and Megan W Tse and Juliane Weller and Julie Chen and Paul C Blainey},
doi = {10.1111/nyas.14649},
issn = {1749-6632},
year = {2021},
date = {2021-07-01},
journal = {Ann N Y Acad Sci},
volume = {1496},
number = {1},
pages = {82--96},
abstract = {Antibiotic resistance is a worldwide and growing clinical problem. With limited drug development in the antibacterial space, combination therapy has emerged as a promising strategy to combat multidrug-resistant bacteria. Antibacterial combinations can improve antibiotic efficacy and suppress antibacterial resistance through independent, synergistic, or even antagonistic activities. Combination therapies are famously used to treat viral and mycobacterial infections and cancer. However, antibacterial combinations are only now emerging as a common treatment strategy for other bacterial infections owing to challenges in their discovery, development, regulatory approval, and commercial/clinical deployment. Here, we focus on discovery-where the sheer scale of combinatorial chemical spaces represents a significant challenge-and discuss how combination therapy can impact the treatment of bacterial infections. Despite these challenges, recent advancements, including new in silico methods, theoretical frameworks, and microfluidic platforms, are poised to identify the new and efficacious antibacterial combinations needed to revitalize the antibacterial drug pipeline.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Antibiotic resistance is a worldwide and growing clinical problem. With limited drug development in the antibacterial space, combination therapy has emerged as a promising strategy to combat multidrug-resistant bacteria. Antibacterial combinations can improve antibiotic efficacy and suppress antibacterial resistance through independent, synergistic, or even antagonistic activities. Combination therapies are famously used to treat viral and mycobacterial infections and cancer. However, antibacterial combinations are only now emerging as a common treatment strategy for other bacterial infections owing to challenges in their discovery, development, regulatory approval, and commercial/clinical deployment. Here, we focus on discovery-where the sheer scale of combinatorial chemical spaces represents a significant challenge-and discuss how combination therapy can impact the treatment of bacterial infections. Despite these challenges, recent advancements, including new in silico methods, theoretical frameworks, and microfluidic platforms, are poised to identify the new and efficacious antibacterial combinations needed to revitalize the antibacterial drug pipeline.
Reyes, Miguel; Filbin, Michael R; Bhattacharyya, Roby P; Sonny, Abraham; Mehta, Arnav; Billman, Kianna; Kays, Kyle R; Pinilla-Vera, Mayra; Benson, Maura E; Cosimi, Lisa A; Hung, Deborah T; Levy, Bruce D; Villani, Alexandra-Chloe; Sade-Feldman, Moshe; Baron, Rebecca M; Goldberg, Marcia B; Blainey, Paul C; Hacohen, Nir
In: Sci Transl Med, vol. 13, no. 598, 2021, ISSN: 1946-6242.
@article{pmid34103408,
title = {Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro},
author = {Miguel Reyes and Michael R Filbin and Roby P Bhattacharyya and Abraham Sonny and Arnav Mehta and Kianna Billman and Kyle R Kays and Mayra Pinilla-Vera and Maura E Benson and Lisa A Cosimi and Deborah T Hung and Bruce D Levy and Alexandra-Chloe Villani and Moshe Sade-Feldman and Rebecca M Baron and Marcia B Goldberg and Paul C Blainey and Nir Hacohen},
doi = {10.1126/scitranslmed.abe9599},
issn = {1946-6242},
year = {2021},
date = {2021-06-01},
journal = {Sci Transl Med},
volume = {13},
number = {598},
abstract = {Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14 monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14 MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14 monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14 MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection.
Elacqua, Joshua J; Ranu, Navpreet; DiIorio, Sarah E; Blainey, Paul C
DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution Journal Article
In: Genome Res, vol. 31, no. 1, pp. 75–87, 2021, ISSN: 1549-5469.
@article{pmid33355294,
title = {DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution},
author = {Joshua J Elacqua and Navpreet Ranu and Sarah E DiIorio and Paul C Blainey},
doi = {10.1101/gr.265223.120},
issn = {1549-5469},
year = {2021},
date = {2021-01-01},
journal = {Genome Res},
volume = {31},
number = {1},
pages = {75--87},
abstract = {DNA single-strand breaks (SSBs), or "nicks," are the most common form of DNA damage. Oxidative stress, endogenous enzyme activities, and other processes cause tens of thousands of nicks per cell per day. Accumulation of nicks, caused by high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to characterize the mechanisms of these processes and learn how the location and number of nicks affect cells, disease progression, and health outcomes. In addition to natural processes, including DNA repair, leading genome editing technologies rely on nuclease activity, including nick generation, at specific target sites. There is currently a pressing need for methods to study off-target nicking activity genome-wide to evaluate the side effects of emerging genome editing tools on cells and organisms. Here, we developed a new method, DENT-seq, for efficient strand-specific profiling of nicks in complex DNA samples with single-nucleotide resolution and low false-positive rates. DENT-seq produces a single deep sequence data set enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand with single-base resolution at penetrance as low as one strand per thousand. We apply DENT-seq to profile the off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. DENT-seq will be useful in exploring the activity of engineered nucleases in genome editing and other biotechnological applications as well as spontaneous and therapeutic-associated strand breaks.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
DNA single-strand breaks (SSBs), or "nicks," are the most common form of DNA damage. Oxidative stress, endogenous enzyme activities, and other processes cause tens of thousands of nicks per cell per day. Accumulation of nicks, caused by high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to characterize the mechanisms of these processes and learn how the location and number of nicks affect cells, disease progression, and health outcomes. In addition to natural processes, including DNA repair, leading genome editing technologies rely on nuclease activity, including nick generation, at specific target sites. There is currently a pressing need for methods to study off-target nicking activity genome-wide to evaluate the side effects of emerging genome editing tools on cells and organisms. Here, we developed a new method, DENT-seq, for efficient strand-specific profiling of nicks in complex DNA samples with single-nucleotide resolution and low false-positive rates. DENT-seq produces a single deep sequence data set enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand with single-base resolution at penetrance as low as one strand per thousand. We apply DENT-seq to profile the off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. DENT-seq will be useful in exploring the activity of engineered nucleases in genome editing and other biotechnological applications as well as spontaneous and therapeutic-associated strand breaks.
Al’Khafaji, Aziz M.; Smith, Jonathan T.; Garimella, Kiran V; Babadi, Mehrtash; Sade-Feldman, Moshe; Gatzen, Michael; Sarkizova, Siranush; Schwartz, Marc A.; Popic, Victoria; Blaum, Emily M.; Day, Allyson; Costello, Maura; Bowers, Tera; Gabriel, Stacey; Banks, Eric; Philippakis, Anthony A.; Boland, Genevieve M.; Blainey, Paul C.; Hacohen, Nir
High-throughput RNA isoform sequencing using programmable cDNA concatenation Unpublished
2021.
@unpublished{Al’Khafaji2021.10.01.462818,
title = {High-throughput RNA isoform sequencing using programmable cDNA concatenation},
author = {Aziz M. Al’Khafaji and Jonathan T. Smith and Kiran V Garimella and Mehrtash Babadi and Moshe Sade-Feldman and Michael Gatzen and Siranush Sarkizova and Marc A. Schwartz and Victoria Popic and Emily M. Blaum and Allyson Day and Maura Costello and Tera Bowers and Stacey Gabriel and Eric Banks and Anthony A. Philippakis and Genevieve M. Boland and Paul C. Blainey and Nir Hacohen},
url = {https://www.biorxiv.org/content/early/2021/10/01/2021.10.01.462818},
doi = {10.1101/2021.10.01.462818},
year = {2021},
date = {2021-01-01},
urldate = {2021-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Alternative splicing is a core biological process that enables profound and essential diversification of gene function. Short-read RNA sequencing approaches fail to resolve RNA isoforms and therefore primarily enable gene expression measurements - an isoform unaware representation of the transcriptome. Conversely, full-length RNA sequencing using long-read technologies are able to capture complete transcript isoforms, but their utility is deeply constrained due to throughput limitations. Here, we introduce MAS-ISO-seq, a technique for programmably concatenating cDNAs into single molecules optimal for long-read sequencing, boosting the throughput >15 fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. We validated unambiguous isoform assignment with MAS-ISO-seq using a synthetic RNA isoform library and applied this approach to single-cell RNA sequencing of tumor-infiltrating T cells. Results demonstrated a >30 fold boosted discovery of differentially spliced genes and robust cell clustering, as well as canonical PTPRC splicing patterns across T cell subpopulations and the concerted expression of the associated hnRNPLL splicing factor. Methods such as MAS-ISO-seq will drive discovery of novel isoforms and the transition from gene expression to transcript isoform expression analyses.Competing Interest StatementA.M.A., K.V.G., J.S., M.B., P.B., and N.H. have filed a patent on the MAS-seq method. A.A.P. is a Venture Partner and Employee of GV. He has received funding from Verily, Microsoft, Illumina, Bayer, Pfizer, Biogen, Abbvie, Intel, and IBM. M.S.F. receives funding from Bristol-Myers Squibb. G.M.B. has served on SAB and on the steering committee for Nektar Therapeutics. She has SRAs with Olink proteomics and Palleon Pharmaceuticals. She served on SAB and as a speaker for Novartis N.H. holds equity in BioNTech and is a founder and equity holder of Danger Bio. P.C.B. is a consultant to and/or holds equity in companies that develop or apply genomic or genome editing technologies: 10X Genomics, General Automation Lab Technologies, Celsius Therapeutics, Next Gen Diagnostics LLC, Cache DNA, and Concerto Biosciences. P.C.B. receives funding from industry for unrelated work.},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Alternative splicing is a core biological process that enables profound and essential diversification of gene function. Short-read RNA sequencing approaches fail to resolve RNA isoforms and therefore primarily enable gene expression measurements - an isoform unaware representation of the transcriptome. Conversely, full-length RNA sequencing using long-read technologies are able to capture complete transcript isoforms, but their utility is deeply constrained due to throughput limitations. Here, we introduce MAS-ISO-seq, a technique for programmably concatenating cDNAs into single molecules optimal for long-read sequencing, boosting the throughput >15 fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. We validated unambiguous isoform assignment with MAS-ISO-seq using a synthetic RNA isoform library and applied this approach to single-cell RNA sequencing of tumor-infiltrating T cells. Results demonstrated a >30 fold boosted discovery of differentially spliced genes and robust cell clustering, as well as canonical PTPRC splicing patterns across T cell subpopulations and the concerted expression of the associated hnRNPLL splicing factor. Methods such as MAS-ISO-seq will drive discovery of novel isoforms and the transition from gene expression to transcript isoform expression analyses.Competing Interest StatementA.M.A., K.V.G., J.S., M.B., P.B., and N.H. have filed a patent on the MAS-seq method. A.A.P. is a Venture Partner and Employee of GV. He has received funding from Verily, Microsoft, Illumina, Bayer, Pfizer, Biogen, Abbvie, Intel, and IBM. M.S.F. receives funding from Bristol-Myers Squibb. G.M.B. has served on SAB and on the steering committee for Nektar Therapeutics. She has SRAs with Olink proteomics and Palleon Pharmaceuticals. She served on SAB and as a speaker for Novartis N.H. holds equity in BioNTech and is a founder and equity holder of Danger Bio. P.C.B. is a consultant to and/or holds equity in companies that develop or apply genomic or genome editing technologies: 10X Genomics, General Automation Lab Technologies, Celsius Therapeutics, Next Gen Diagnostics LLC, Cache DNA, and Concerto Biosciences. P.C.B. receives funding from industry for unrelated work.
2020
Shand, Megan; Soto, Jose; Lichtenstein, Lee; Benjamin, David; Farjoun, Yossi; Brody, Yehuda; Maruvka, Yosef; Blainey, Paul C; Banks, Eric
A validated lineage-derived somatic truth data set enables benchmarking in cancer genome analysis Journal Article
In: Commun Biol, vol. 3, no. 1, pp. 744, 2020, ISSN: 2399-3642.
@article{pmid33293579,
title = {A validated lineage-derived somatic truth data set enables benchmarking in cancer genome analysis},
author = {Megan Shand and Jose Soto and Lee Lichtenstein and David Benjamin and Yossi Farjoun and Yehuda Brody and Yosef Maruvka and Paul C Blainey and Eric Banks},
doi = {10.1038/s42003-020-01460-9},
issn = {2399-3642},
year = {2020},
date = {2020-12-01},
journal = {Commun Biol},
volume = {3},
number = {1},
pages = {744},
abstract = {Existing cancer benchmark data sets for human sequencing data use germline variants, synthetic methods, or expensive validations, none of which are satisfactory for providing a large collection of true somatic variation across a whole genome. Here we propose a data set, Lineage derived Somatic Truth (LinST), of short somatic mutations in the HT115 colon cancer cell-line, that are validated using a known cell lineage that includes thousands of mutations and a high confidence region covering 2.7 gigabases per sample.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Existing cancer benchmark data sets for human sequencing data use germline variants, synthetic methods, or expensive validations, none of which are satisfactory for providing a large collection of true somatic variation across a whole genome. Here we propose a data set, Lineage derived Somatic Truth (LinST), of short somatic mutations in the HT115 colon cancer cell-line, that are validated using a known cell lineage that includes thousands of mutations and a high confidence region covering 2.7 gigabases per sample.
Feldman, David; Tsai, FuNien; Garrity, Anthony J; O'Rourke, Ryan; Brenan, Lisa; Ho, Patricia; Gonzalez, Elizabeth; Konermann, Silvana; Johannessen, Cory M; Beroukhim, Rameen; Bandopadhayay, Pratiti; Blainey, Paul C
CloneSifter: enrichment of rare clones from heterogeneous cell populations Journal Article
In: BMC Biol, vol. 18, no. 1, pp. 177, 2020, ISSN: 1741-7007.
@article{pmid33234154,
title = {CloneSifter: enrichment of rare clones from heterogeneous cell populations},
author = {David Feldman and FuNien Tsai and Anthony J Garrity and Ryan O'Rourke and Lisa Brenan and Patricia Ho and Elizabeth Gonzalez and Silvana Konermann and Cory M Johannessen and Rameen Beroukhim and Pratiti Bandopadhayay and Paul C Blainey},
doi = {10.1186/s12915-020-00911-3},
issn = {1741-7007},
year = {2020},
date = {2020-11-01},
journal = {BMC Biol},
volume = {18},
number = {1},
pages = {177},
abstract = {BACKGROUND: Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones.
RESULTS: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population.
CONCLUSIONS: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones.
RESULTS: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population.
CONCLUSIONS: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.
RESULTS: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population.
CONCLUSIONS: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.
Ackerman, Cheri M; Myhrvold, Cameron; Thakku, Sri Gowtham; Freije, Catherine A; Metsky, Hayden C; Yang, David K; Ye, Simon H; Boehm, Chloe K; Kosoko-Thoroddsen, Tinna-Sólveig F; Kehe, Jared; Nguyen, Tien G; Carter, Amber; Kulesa, Anthony; Barnes, John R; Dugan, Vivien G; Hung, Deborah T; Blainey, Paul C; Sabeti, Pardis C
Massively multiplexed nucleic acid detection with Cas13 Journal Article
In: Nature, vol. 582, no. 7811, pp. 277–282, 2020, ISSN: 1476-4687.
@article{pmid32349121,
title = {Massively multiplexed nucleic acid detection with Cas13},
author = {Cheri M Ackerman and Cameron Myhrvold and Sri Gowtham Thakku and Catherine A Freije and Hayden C Metsky and David K Yang and Simon H Ye and Chloe K Boehm and Tinna-Sólveig F Kosoko-Thoroddsen and Jared Kehe and Tien G Nguyen and Amber Carter and Anthony Kulesa and John R Barnes and Vivien G Dugan and Deborah T Hung and Paul C Blainey and Pardis C Sabeti},
doi = {10.1038/s41586-020-2279-8},
issn = {1476-4687},
year = {2020},
date = {2020-06-01},
journal = {Nature},
volume = {582},
number = {7811},
pages = {277--282},
abstract = {The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples while simultaneously testing for many pathogens. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples while simultaneously testing for many pathogens. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health.
Reyes, Miguel; Filbin, Michael R; Bhattacharyya, Roby P; Billman, Kianna; Eisenhaure, Thomas; Hung, Deborah T; Levy, Bruce D; Baron, Rebecca M; Blainey, Paul C; Goldberg, Marcia B; Hacohen, Nir
An immune-cell signature of bacterial sepsis Journal Article
In: Nat Med, vol. 26, no. 3, pp. 333–340, 2020, ISSN: 1546-170X.
@article{pmid32066974,
title = {An immune-cell signature of bacterial sepsis},
author = {Miguel Reyes and Michael R Filbin and Roby P Bhattacharyya and Kianna Billman and Thomas Eisenhaure and Deborah T Hung and Bruce D Levy and Rebecca M Baron and Paul C Blainey and Marcia B Goldberg and Nir Hacohen},
doi = {10.1038/s41591-020-0752-4},
issn = {1546-170X},
year = {2020},
date = {2020-03-01},
journal = {Nat Med},
volume = {26},
number = {3},
pages = {333--340},
abstract = {Dysregulation of the immune response to bacterial infection can lead to sepsis, a condition with high mortality. Multiple whole-blood gene-expression studies have defined sepsis-associated molecular signatures, but have not resolved changes in transcriptional states of specific cell types. Here, we used single-cell RNA-sequencing to profile the blood of people with sepsis (n = 29) across three clinical cohorts with corresponding controls (n = 36). We profiled total peripheral blood mononuclear cells (PBMCs, 106,545 cells) and dendritic cells (19,806 cells) across all subjects and, on the basis of clustering of their gene-expression profiles, defined 16 immune-cell states. We identified a unique CD14 monocyte state that is expanded in people with sepsis and validated its power in distinguishing these individuals from controls using public transcriptomic data from subjects with different disease etiologies and from multiple geographic locations (18 cohorts, n = 1,467 subjects). We identified a panel of surface markers for isolation and quantification of the monocyte state and characterized its epigenomic and functional phenotypes, and propose a model for its induction from human bone marrow. This study demonstrates the utility of single-cell genomics in discovering disease-associated cytologic signatures and provides insight into the cellular basis of immune dysregulation in bacterial sepsis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Dysregulation of the immune response to bacterial infection can lead to sepsis, a condition with high mortality. Multiple whole-blood gene-expression studies have defined sepsis-associated molecular signatures, but have not resolved changes in transcriptional states of specific cell types. Here, we used single-cell RNA-sequencing to profile the blood of people with sepsis (n = 29) across three clinical cohorts with corresponding controls (n = 36). We profiled total peripheral blood mononuclear cells (PBMCs, 106,545 cells) and dendritic cells (19,806 cells) across all subjects and, on the basis of clustering of their gene-expression profiles, defined 16 immune-cell states. We identified a unique CD14 monocyte state that is expanded in people with sepsis and validated its power in distinguishing these individuals from controls using public transcriptomic data from subjects with different disease etiologies and from multiple geographic locations (18 cohorts, n = 1,467 subjects). We identified a panel of surface markers for isolation and quantification of the monocyte state and characterized its epigenomic and functional phenotypes, and propose a model for its induction from human bone marrow. This study demonstrates the utility of single-cell genomics in discovering disease-associated cytologic signatures and provides insight into the cellular basis of immune dysregulation in bacterial sepsis.
Ysasi, Alexandra B; Bennett, Robert D; Wagner, Willi; Valenzuela, Cristian D; Servais, Andrew B; Tsuda, Akira; Pyne, Saumyadipta; Li, Shuqiang; Grimsby, Jonna; Pokharel, Prapti; Livak, Kenneth J; Ackermann, Maximilian; Blainey, Paul C; Mentzer, Steven J
Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts Journal Article
In: Front Med (Lausanne), vol. 7, pp. 112, 2020, ISSN: 2296-858X.
@article{pmid32373614,
title = {Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts},
author = {Alexandra B Ysasi and Robert D Bennett and Willi Wagner and Cristian D Valenzuela and Andrew B Servais and Akira Tsuda and Saumyadipta Pyne and Shuqiang Li and Jonna Grimsby and Prapti Pokharel and Kenneth J Livak and Maximilian Ackermann and Paul C Blainey and Steven J Mentzer},
doi = {10.3389/fmed.2020.00112},
issn = {2296-858X},
year = {2020},
date = {2020-01-01},
journal = {Front Med (Lausanne)},
volume = {7},
pages = {112},
abstract = {Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative "hotspots" in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis of the murine alveolar duct cells was performed using the C1 integrated fluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi-dimensional data set was analyzed using visualization software based on the tSNE algorithm. The analysis identified 6 cell clusters; 1 cell cluster was present only after pneumonectomy. This post-pneumonectomy cluster was significantly less transcriptionally active than 3 other clusters and may represent a transitional cell population. A provisional cluster identity for 4 of the 6 cell clusters was obtained by embedding bulk transcriptional data into the tSNE analysis. The transcriptional pattern of the 6 clusters was further analyzed for genes associated with lung repair, matrix production, and angiogenesis. The data demonstrated that multiple cell-types (clusters) transcribed genes linked to these basic functions. We conclude that the coordinated gene expression across multiple cell clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative "hotspots" in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis of the murine alveolar duct cells was performed using the C1 integrated fluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi-dimensional data set was analyzed using visualization software based on the tSNE algorithm. The analysis identified 6 cell clusters; 1 cell cluster was present only after pneumonectomy. This post-pneumonectomy cluster was significantly less transcriptionally active than 3 other clusters and may represent a transitional cell population. A provisional cluster identity for 4 of the 6 cell clusters was obtained by embedding bulk transcriptional data into the tSNE analysis. The transcriptional pattern of the 6 clusters was further analyzed for genes associated with lung repair, matrix production, and angiogenesis. The data demonstrated that multiple cell-types (clusters) transcribed genes linked to these basic functions. We conclude that the coordinated gene expression across multiple cell clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts.
Peters, Joshua M.; Blainey, Paul C.; Bryson, Bryan D.
2020.
@unpublished{Peters2020.08.06.240424,
title = {Consensus transcriptional states describe human mononuclear phagocyte diversity in the lung across health and disease},
author = {Joshua M. Peters and Paul C. Blainey and Bryan D. Bryson},
url = {https://www.biorxiv.org/content/early/2020/08/07/2020.08.06.240424},
doi = {10.1101/2020.08.06.240424},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Monocytes, dendritic cells, and macrophages, commonly referred to as mononuclear phagocytes (MNPs), are innate immune cells capable of adopting diverse homeostatic and pathogenic phenotypes. Recent single-cell RNA-sequencing studies across many diseases in the lung have profiled this diversity transcriptionally, defining new cellular states and their association with disease. Despite these massive cellular profiling efforts, many studies have focused on defining myeloid dysfunction in specific diseases without identifying common pan-disease trends in the mononuclear phagocyte compartment within the lung. To address these gaps in our knowledge, we collate, process, and analyze 561,390 cellular transcriptomes from 12 studies of the human lung across multiple human diseases. We develop a computational framework to identify and compare dominant gene markers and gene expression programs and characterize MNP diversity in the lung, proposing a conserved dictionary of gene sets. Utilizing this reference, we efficiently identify disease-associated and rare MNP populations across multiple diseases and cohorts. Furthermore, we demonstrate the utility of this dictionary in characterizing a recently published dataset of bronchoalveolar lavage cells from COVID-19 patients and healthy controls which further reveal novel transcriptional shifts directly relatable to other diseases in the lung. These results underline conserved MNP transcriptional programs in lung disease, provide an immediate reference for characterizing the landscape of lung MNPs and establish a roadmap to dissecting MNP transcriptional complexity across tissues.Competing Interest StatementJ.M.P. and B.D.B. declare no competing interests. P.C.B. is a consultant to and/or holds equity in companies that develop or apply genomic, microfluidic, or single-cell technologies: 10X Genomics, General Automation Lab Technologies, Celsius Therapeutics, Next Gen Diagnostics, LLC, and Cache DNA.},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
Monocytes, dendritic cells, and macrophages, commonly referred to as mononuclear phagocytes (MNPs), are innate immune cells capable of adopting diverse homeostatic and pathogenic phenotypes. Recent single-cell RNA-sequencing studies across many diseases in the lung have profiled this diversity transcriptionally, defining new cellular states and their association with disease. Despite these massive cellular profiling efforts, many studies have focused on defining myeloid dysfunction in specific diseases without identifying common pan-disease trends in the mononuclear phagocyte compartment within the lung. To address these gaps in our knowledge, we collate, process, and analyze 561,390 cellular transcriptomes from 12 studies of the human lung across multiple human diseases. We develop a computational framework to identify and compare dominant gene markers and gene expression programs and characterize MNP diversity in the lung, proposing a conserved dictionary of gene sets. Utilizing this reference, we efficiently identify disease-associated and rare MNP populations across multiple diseases and cohorts. Furthermore, we demonstrate the utility of this dictionary in characterizing a recently published dataset of bronchoalveolar lavage cells from COVID-19 patients and healthy controls which further reveal novel transcriptional shifts directly relatable to other diseases in the lung. These results underline conserved MNP transcriptional programs in lung disease, provide an immediate reference for characterizing the landscape of lung MNPs and establish a roadmap to dissecting MNP transcriptional complexity across tissues.Competing Interest StatementJ.M.P. and B.D.B. declare no competing interests. P.C.B. is a consultant to and/or holds equity in companies that develop or apply genomic, microfluidic, or single-cell technologies: 10X Genomics, General Automation Lab Technologies, Celsius Therapeutics, Next Gen Diagnostics, LLC, and Cache DNA.
2019
Schmidt, Stephanie Tzouanas; Yu, Feiqiao Brian; Blainey, Paul C; May, Andrew P; Quake, Stephen R
Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium Journal Article
In: Proc Natl Acad Sci U S A, vol. 116, no. 46, pp. 23100–23105, 2019, ISSN: 1091-6490.
@article{pmid31659048,
title = {Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium},
author = {Stephanie Tzouanas Schmidt and Feiqiao Brian Yu and Paul C Blainey and Andrew P May and Stephen R Quake},
doi = {10.1073/pnas.1904273116},
issn = {1091-6490},
year = {2019},
date = {2019-11-01},
journal = {Proc Natl Acad Sci U S A},
volume = {116},
number = {46},
pages = {23100--23105},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Cas9 (SpyCas9) and the 70 °C limit of both Cas9 (GeoCas9) and T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Cas9 (SpyCas9) and the 70 °C limit of both Cas9 (GeoCas9) and T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.
Reyes, Miguel; Billman, Kianna; Hacohen, Nir; Blainey, Paul C
Simultaneous profiling of gene expression and chromatin accessibility in single cells Journal Article
In: Adv Biosyst, vol. 3, no. 11, 2019, ISSN: 2366-7478.
@article{pmid31853478,
title = {Simultaneous profiling of gene expression and chromatin accessibility in single cells},
author = {Miguel Reyes and Kianna Billman and Nir Hacohen and Paul C Blainey},
doi = {10.1002/adbi.201900065},
issn = {2366-7478},
year = {2019},
date = {2019-11-01},
journal = {Adv Biosyst},
volume = {3},
number = {11},
abstract = {Profiling multiple omic layers in a single cell enables the discovery and analysis of biological phenomena that are not apparent from analysis of mono-omic data. While methods for multi-omic profiling have been reported, their adoption has been limited due to high cost and complex workflows. Here, we present a simple method for joint profiling of gene expression and chromatin accessibility in tens to hundreds of single cells. We assess the quality of resulting single cell ATAC- and RNA-seq data across three cell types, examine the link between accessibility and expression at the and loci in human primary T cells and monocytes, and compare the accuracy of clustering solutions for mono-omic and combined data. The new method allows biological laboratories to perform simultaneous profiling of gene expression and chromatin accessibility using standard reagents and instrumentation. This technique, in conjunction with other advances in multi-omic profiling, will enable highly-resolved cell state classification and more specific mechanistic hypothesis generation than is possible with mono-omic analysis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Profiling multiple omic layers in a single cell enables the discovery and analysis of biological phenomena that are not apparent from analysis of mono-omic data. While methods for multi-omic profiling have been reported, their adoption has been limited due to high cost and complex workflows. Here, we present a simple method for joint profiling of gene expression and chromatin accessibility in tens to hundreds of single cells. We assess the quality of resulting single cell ATAC- and RNA-seq data across three cell types, examine the link between accessibility and expression at the and loci in human primary T cells and monocytes, and compare the accuracy of clustering solutions for mono-omic and combined data. The new method allows biological laboratories to perform simultaneous profiling of gene expression and chromatin accessibility using standard reagents and instrumentation. This technique, in conjunction with other advances in multi-omic profiling, will enable highly-resolved cell state classification and more specific mechanistic hypothesis generation than is possible with mono-omic analysis.
Feldman, David; Singh, Avtar; Schmid-Burgk, Jonathan L; Carlson, Rebecca J; Mezger, Anja; Garrity, Anthony J; Zhang, Feng; Blainey, Paul C
Optical Pooled Screens in Human Cells Journal Article
In: Cell, vol. 179, no. 3, pp. 787–799.e17, 2019, ISSN: 1097-4172.
@article{pmid31626775,
title = {Optical Pooled Screens in Human Cells},
author = {David Feldman and Avtar Singh and Jonathan L Schmid-Burgk and Rebecca J Carlson and Anja Mezger and Anthony J Garrity and Feng Zhang and Paul C Blainey},
doi = {10.1016/j.cell.2019.09.016},
issn = {1097-4172},
year = {2019},
date = {2019-10-01},
journal = {Cell},
volume = {179},
number = {3},
pages = {787--799.e17},
abstract = {Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.
Guo, Syuan-Ming; Veneziano, Remi; Gordonov, Simon; Li, Li; Danielson, Eric; de Arce, Karen Perez; Park, Demian; Kulesa, Anthony B; Wamhoff, Eike-Christian; Blainey, Paul C; Boyden, Edward S; Cottrell, Jeffrey R; Bathe, Mark
Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes Journal Article
In: Nat Commun, vol. 10, no. 1, pp. 4377, 2019, ISSN: 2041-1723.
@article{pmid31558769,
title = {Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes},
author = {Syuan-Ming Guo and Remi Veneziano and Simon Gordonov and Li Li and Eric Danielson and Karen Perez de Arce and Demian Park and Anthony B Kulesa and Eike-Christian Wamhoff and Paul C Blainey and Edward S Boyden and Jeffrey R Cottrell and Mark Bathe},
doi = {10.1038/s41467-019-12372-6},
issn = {2041-1723},
year = {2019},
date = {2019-09-01},
journal = {Nat Commun},
volume = {10},
number = {1},
pages = {4377},
abstract = {Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.
Kehe, Jared; Kulesa, Anthony; Ortiz, Anthony; Ackerman, Cheri M; Thakku, Sri Gowtham; Sellers, Daniel; Kuehn, Seppe; Gore, Jeff; Friedman, Jonathan; Blainey, Paul C
Massively parallel screening of synthetic microbial communities Journal Article
In: Proc Natl Acad Sci U S A, vol. 116, no. 26, pp. 12804–12809, 2019, ISSN: 1091-6490.
@article{pmid31186361,
title = {Massively parallel screening of synthetic microbial communities},
author = {Jared Kehe and Anthony Kulesa and Anthony Ortiz and Cheri M Ackerman and Sri Gowtham Thakku and Daniel Sellers and Seppe Kuehn and Jeff Gore and Jonathan Friedman and Paul C Blainey},
doi = {10.1073/pnas.1900102116},
issn = {1091-6490},
year = {2019},
date = {2019-06-01},
journal = {Proc Natl Acad Sci U S A},
volume = {116},
number = {26},
pages = {12804--12809},
abstract = {Microbial communities have numerous potential applications in biotechnology, agriculture, and medicine. Nevertheless, the limited accuracy with which we can predict interspecies interactions and environmental dependencies hinders efforts to rationally engineer beneficial consortia. Empirical screening is a complementary approach wherein synthetic communities are combinatorially constructed and assayed in high throughput. However, assembling many combinations of microbes is logistically complex and difficult to achieve on a timescale commensurate with microbial growth. Here, we introduce the kChip, a droplets-based platform that performs rapid, massively parallel, bottom-up construction and screening of synthetic microbial communities. We first show that the kChip enables phenotypic characterization of microbes across environmental conditions. Next, in a screen of ∼100,000 multispecies communities comprising up to 19 soil isolates, we identified sets that promote the growth of the model plant symbiont in a manner robust to carbon source variation and the presence of additional species. Broadly, kChip screening can identify multispecies consortia possessing any optically assayable function, including facilitation of biocontrol agents, suppression of pathogens, degradation of recalcitrant substrates, and robustness of these functions to perturbation, with many applications across basic and applied microbial ecology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microbial communities have numerous potential applications in biotechnology, agriculture, and medicine. Nevertheless, the limited accuracy with which we can predict interspecies interactions and environmental dependencies hinders efforts to rationally engineer beneficial consortia. Empirical screening is a complementary approach wherein synthetic communities are combinatorially constructed and assayed in high throughput. However, assembling many combinations of microbes is logistically complex and difficult to achieve on a timescale commensurate with microbial growth. Here, we introduce the kChip, a droplets-based platform that performs rapid, massively parallel, bottom-up construction and screening of synthetic microbial communities. We first show that the kChip enables phenotypic characterization of microbes across environmental conditions. Next, in a screen of ∼100,000 multispecies communities comprising up to 19 soil isolates, we identified sets that promote the growth of the model plant symbiont in a manner robust to carbon source variation and the presence of additional species. Broadly, kChip screening can identify multispecies consortia possessing any optically assayable function, including facilitation of biocontrol agents, suppression of pathogens, degradation of recalcitrant substrates, and robustness of these functions to perturbation, with many applications across basic and applied microbial ecology.
Järvenpää, Marko; Sater, Mohamad R Abdul; Lagoudas, Georgia K; Blainey, Paul C; Miller, Loren G; McKinnell, James A; Huang, Susan S; Grad, Yonatan H; Marttinen, Pekka
A Bayesian model of acquisition and clearance of bacterial colonization incorporating within-host variation Journal Article
In: PLoS Comput Biol, vol. 15, no. 4, pp. e1006534, 2019, ISSN: 1553-7358.
@article{pmid31009452,
title = {A Bayesian model of acquisition and clearance of bacterial colonization incorporating within-host variation},
author = {Marko Järvenpää and Mohamad R Abdul Sater and Georgia K Lagoudas and Paul C Blainey and Loren G Miller and James A McKinnell and Susan S Huang and Yonatan H Grad and Pekka Marttinen},
doi = {10.1371/journal.pcbi.1006534},
issn = {1553-7358},
year = {2019},
date = {2019-04-01},
journal = {PLoS Comput Biol},
volume = {15},
number = {4},
pages = {e1006534},
abstract = {Bacterial populations that colonize a host can play important roles in host health, including serving as a reservoir that transmits to other hosts and from which invasive strains emerge, thus emphasizing the importance of understanding rates of acquisition and clearance of colonizing populations. Studies of colonization dynamics have been based on assessment of whether serial samples represent a single population or distinct colonization events. With the use of whole genome sequencing to determine genetic distance between isolates, a common solution to estimate acquisition and clearance rates has been to assume a fixed genetic distance threshold below which isolates are considered to represent the same strain. However, this approach is often inadequate to account for the diversity of the underlying within-host evolving population, the time intervals between consecutive measurements, and the uncertainty in the estimated acquisition and clearance rates. Here, we present a fully Bayesian model that provides probabilities of whether two strains should be considered the same, allowing us to determine bacterial clearance and acquisition from genomes sampled over time. Our method explicitly models the within-host variation using population genetic simulation, and the inference is done using a combination of Approximate Bayesian Computation (ABC) and Markov Chain Monte Carlo (MCMC). We validate the method with multiple carefully conducted simulations and demonstrate its use in practice by analyzing a collection of methicillin resistant Staphylococcus aureus (MRSA) isolates from a large recently completed longitudinal clinical study. An R-code implementation of the method is freely available at: https://github.com/mjarvenpaa/bacterial-colonization-model.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bacterial populations that colonize a host can play important roles in host health, including serving as a reservoir that transmits to other hosts and from which invasive strains emerge, thus emphasizing the importance of understanding rates of acquisition and clearance of colonizing populations. Studies of colonization dynamics have been based on assessment of whether serial samples represent a single population or distinct colonization events. With the use of whole genome sequencing to determine genetic distance between isolates, a common solution to estimate acquisition and clearance rates has been to assume a fixed genetic distance threshold below which isolates are considered to represent the same strain. However, this approach is often inadequate to account for the diversity of the underlying within-host evolving population, the time intervals between consecutive measurements, and the uncertainty in the estimated acquisition and clearance rates. Here, we present a fully Bayesian model that provides probabilities of whether two strains should be considered the same, allowing us to determine bacterial clearance and acquisition from genomes sampled over time. Our method explicitly models the within-host variation using population genetic simulation, and the inference is done using a combination of Approximate Bayesian Computation (ABC) and Markov Chain Monte Carlo (MCMC). We validate the method with multiple carefully conducted simulations and demonstrate its use in practice by analyzing a collection of methicillin resistant Staphylococcus aureus (MRSA) isolates from a large recently completed longitudinal clinical study. An R-code implementation of the method is freely available at: https://github.com/mjarvenpaa/bacterial-colonization-model.
Berghuis, Bojk A; Yu, Feiqiao Brian; Schulz, Frederik; Blainey, Paul C; Woyke, Tanja; Quake, Stephen R
Hydrogenotrophic methanogenesis in archaeal phylum Verstraetearchaeota reveals the shared ancestry of all methanogens Journal Article
In: Proc Natl Acad Sci U S A, vol. 116, no. 11, pp. 5037–5044, 2019, ISSN: 1091-6490.
@article{pmid30814220,
title = {Hydrogenotrophic methanogenesis in archaeal phylum Verstraetearchaeota reveals the shared ancestry of all methanogens},
author = {Bojk A Berghuis and Feiqiao Brian Yu and Frederik Schulz and Paul C Blainey and Tanja Woyke and Stephen R Quake},
doi = {10.1073/pnas.1815631116},
issn = {1091-6490},
year = {2019},
date = {2019-03-01},
journal = {Proc Natl Acad Sci U S A},
volume = {116},
number = {11},
pages = {5037--5044},
abstract = {Methanogenic archaea are major contributors to the global carbon cycle and were long thought to belong exclusively to the euryarchaeal phylum. Discovery of the methanogenesis gene cluster methyl-coenzyme M reductase (Mcr) in the Bathyarchaeota, and thereafter the Verstraetearchaeota, led to a paradigm shift, pushing back the evolutionary origin of methanogenesis to predate that of the Euryarchaeota. The methylotrophic methanogenesis found in the non-Euryarchaota distinguished itself from the predominantly hydrogenotrophic methanogens found in euryarchaeal orders as the former do not couple methanogenesis to carbon fixation through the reductive acetyl-CoA [Wood-Ljungdahl pathway (WLP)], which was interpreted as evidence for independent evolution of the two methanogenesis pathways. Here, we report the discovery of a complete and divergent hydrogenotrophic methanogenesis pathway in a thermophilic order of the Verstraetearchaeota, which we have named Candidatus Methanohydrogenales, as well as the presence of the WLP in the crenarchaeal order Desulfurococcales. Our findings support the ancient origin of hydrogenotrophic methanogenesis, suggest that methylotrophic methanogenesis might be a later adaptation of specific orders, and provide insight into how the transition from hydrogenotrophic to methylotrophic methanogenesis might have occurred.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Methanogenic archaea are major contributors to the global carbon cycle and were long thought to belong exclusively to the euryarchaeal phylum. Discovery of the methanogenesis gene cluster methyl-coenzyme M reductase (Mcr) in the Bathyarchaeota, and thereafter the Verstraetearchaeota, led to a paradigm shift, pushing back the evolutionary origin of methanogenesis to predate that of the Euryarchaeota. The methylotrophic methanogenesis found in the non-Euryarchaota distinguished itself from the predominantly hydrogenotrophic methanogens found in euryarchaeal orders as the former do not couple methanogenesis to carbon fixation through the reductive acetyl-CoA [Wood-Ljungdahl pathway (WLP)], which was interpreted as evidence for independent evolution of the two methanogenesis pathways. Here, we report the discovery of a complete and divergent hydrogenotrophic methanogenesis pathway in a thermophilic order of the Verstraetearchaeota, which we have named Candidatus Methanohydrogenales, as well as the presence of the WLP in the crenarchaeal order Desulfurococcales. Our findings support the ancient origin of hydrogenotrophic methanogenesis, suggest that methylotrophic methanogenesis might be a later adaptation of specific orders, and provide insight into how the transition from hydrogenotrophic to methylotrophic methanogenesis might have occurred.
Jin, Chengcheng; Lagoudas, Georgia K; Zhao, Chen; Bullman, Susan; Bhutkar, Arjun; Hu, Bo; Ameh, Samuel; Sandel, Demi; Liang, Xu Sue; Mazzilli, Sarah; Whary, Mark T; Meyerson, Matthew; Germain, Ronald; Blainey, Paul C; Fox, James G; Jacks, Tyler
Commensal Microbiota Promote Lung Cancer Development via γδ T Cells Journal Article
In: Cell, vol. 176, no. 5, pp. 998–1013.e16, 2019, ISSN: 1097-4172.
@article{pmid30712876,
title = {Commensal Microbiota Promote Lung Cancer Development via γδ T Cells},
author = {Chengcheng Jin and Georgia K Lagoudas and Chen Zhao and Susan Bullman and Arjun Bhutkar and Bo Hu and Samuel Ameh and Demi Sandel and Xu Sue Liang and Sarah Mazzilli and Mark T Whary and Matthew Meyerson and Ronald Germain and Paul C Blainey and James G Fox and Tyler Jacks},
doi = {10.1016/j.cell.2018.12.040},
issn = {1097-4172},
year = {2019},
date = {2019-02-01},
journal = {Cell},
volume = {176},
number = {5},
pages = {998--1013.e16},
abstract = {Lung cancer is closely associated with chronic inflammation, but the causes of inflammation and the specific immune mediators have not been fully elucidated. The lung is a mucosal tissue colonized by a diverse bacterial community, and pulmonary infections commonly present in lung cancer patients are linked to clinical outcomes. Here, we provide evidence that local microbiota provoke inflammation associated with lung adenocarcinoma by activating lung-resident γδ T cells. Germ-free or antibiotic-treated mice were significantly protected from lung cancer development induced by Kras mutation and p53 loss. Mechanistically, commensal bacteria stimulated Myd88-dependent IL-1β and IL-23 production from myeloid cells, inducing proliferation and activation of Vγ6Vδ1 γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. Our findings clearly link local microbiota-immune crosstalk to lung tumor development and thereby define key cellular and molecular mediators that may serve as effective targets in lung cancer intervention.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Lung cancer is closely associated with chronic inflammation, but the causes of inflammation and the specific immune mediators have not been fully elucidated. The lung is a mucosal tissue colonized by a diverse bacterial community, and pulmonary infections commonly present in lung cancer patients are linked to clinical outcomes. Here, we provide evidence that local microbiota provoke inflammation associated with lung adenocarcinoma by activating lung-resident γδ T cells. Germ-free or antibiotic-treated mice were significantly protected from lung cancer development induced by Kras mutation and p53 loss. Mechanistically, commensal bacteria stimulated Myd88-dependent IL-1β and IL-23 production from myeloid cells, inducing proliferation and activation of Vγ6Vδ1 γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. Our findings clearly link local microbiota-immune crosstalk to lung tumor development and thereby define key cellular and molecular mediators that may serve as effective targets in lung cancer intervention.
Reyes, Miguel; Vickers, Dwayne; Billman, Kianna; Eisenhaure, Thomas; Hoover, Paul; Browne, Edward P; Rao, Deepak A; Hacohen, Nir; Blainey, Paul C
Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures Journal Article
In: Sci Adv, vol. 5, no. 1, pp. eaau9223, 2019, ISSN: 2375-2548.
@article{pmid30746468,
title = {Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures},
author = {Miguel Reyes and Dwayne Vickers and Kianna Billman and Thomas Eisenhaure and Paul Hoover and Edward P Browne and Deepak A Rao and Nir Hacohen and Paul C Blainey},
doi = {10.1126/sciadv.aau9223},
issn = {2375-2548},
year = {2019},
date = {2019-01-01},
journal = {Sci Adv},
volume = {5},
number = {1},
pages = {eaau9223},
abstract = {Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.
Ranu, Navpreet; Villani, Alexandra-Chloé; Hacohen, Nir; Blainey, Paul C
Targeting individual cells by barcode in pooled sequence libraries Journal Article
In: Nucleic Acids Res, vol. 47, no. 1, pp. e4, 2019, ISSN: 1362-4962.
@article{pmid30256981,
title = {Targeting individual cells by barcode in pooled sequence libraries},
author = {Navpreet Ranu and Alexandra-Chloé Villani and Nir Hacohen and Paul C Blainey},
doi = {10.1093/nar/gky856},
issn = {1362-4962},
year = {2019},
date = {2019-01-01},
journal = {Nucleic Acids Res},
volume = {47},
number = {1},
pages = {e4},
abstract = {Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.
2018
Brody, Yehuda; Kimmerling, Robert J; Maruvka, Yosef E; Benjamin, David; Elacqua, Joshua J; Haradhvala, Nicholas J; Kim, Jaegil; Mouw, Kent W; Frangaj, Kristjana; Koren, Amnon; Getz, Gad; Manalis, Scott R; Blainey, Paul C
Quantification of somatic mutation flow across individual cell division events by lineage sequencing Journal Article
In: Genome Res, vol. 28, no. 12, pp. 1901–1918, 2018, ISSN: 1549-5469.
@article{pmid30459213,
title = {Quantification of somatic mutation flow across individual cell division events by lineage sequencing},
author = {Yehuda Brody and Robert J Kimmerling and Yosef E Maruvka and David Benjamin and Joshua J Elacqua and Nicholas J Haradhvala and Jaegil Kim and Kent W Mouw and Kristjana Frangaj and Amnon Koren and Gad Getz and Scott R Manalis and Paul C Blainey},
doi = {10.1101/gr.238543.118},
issn = {1549-5469},
year = {2018},
date = {2018-12-01},
journal = {Genome Res},
volume = {28},
number = {12},
pages = {1901--1918},
abstract = {Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon () proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon () proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.
Kulesa, Anthony; Kehe, Jared; Hurtado, Juan E; Tawde, Prianca; Blainey, Paul C
Combinatorial drug discovery in nanoliter droplets Journal Article
In: Proc Natl Acad Sci U S A, vol. 115, no. 26, pp. 6685–6690, 2018, ISSN: 1091-6490.
@article{pmid29899149,
title = {Combinatorial drug discovery in nanoliter droplets},
author = {Anthony Kulesa and Jared Kehe and Juan E Hurtado and Prianca Tawde and Paul C Blainey},
doi = {10.1073/pnas.1802233115},
issn = {1091-6490},
year = {2018},
date = {2018-06-01},
journal = {Proc Natl Acad Sci U S A},
volume = {115},
number = {26},
pages = {6685--6690},
abstract = {Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here, we report a high-throughput system for nanoliter-scale phenotypic screening that formulates a chemical library in nanoliter droplet emulsions and automates the construction of chemical combinations en masse using parallel droplet processing. We applied this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against , a model gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against gram-positive bacteria but are not effective by themselves to resolve gram-negative infections.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here, we report a high-throughput system for nanoliter-scale phenotypic screening that formulates a chemical library in nanoliter droplet emulsions and automates the construction of chemical combinations en masse using parallel droplet processing. We applied this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against , a model gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against gram-positive bacteria but are not effective by themselves to resolve gram-negative infections.
Vestergaard, Christian L; Blainey, Paul C; Flyvbjerg, Henrik
Single-particle trajectories reveal two-state diffusion-kinetics of hOGG1 proteins on DNA Journal Article
In: Nucleic Acids Res, vol. 46, no. 5, pp. 2446–2458, 2018, ISSN: 1362-4962.
@article{pmid29361033,
title = {Single-particle trajectories reveal two-state diffusion-kinetics of hOGG1 proteins on DNA},
author = {Christian L Vestergaard and Paul C Blainey and Henrik Flyvbjerg},
doi = {10.1093/nar/gky004},
issn = {1362-4962},
year = {2018},
date = {2018-03-01},
journal = {Nucleic Acids Res},
volume = {46},
number = {5},
pages = {2446--2458},
abstract = {We reanalyze trajectories of hOGG1 repair proteins diffusing on DNA. A previous analysis of these trajectories with the popular mean-squared-displacement approach revealed only simple diffusion. Here, a new optimal estimator of diffusion coefficients reveals two-state kinetics of the protein. A simple, solvable model, in which the protein randomly switches between a loosely bound, highly mobile state and a tightly bound, less mobile state is the simplest possible dynamic model consistent with the data. It yields accurate estimates of hOGG1's (i) diffusivity in each state, uncorrupted by experimental errors arising from shot noise, motion blur and thermal fluctuations of the DNA; (ii) rates of switching between states and (iii) rate of detachment from the DNA. The protein spends roughly equal time in each state. It detaches only from the loosely bound state, with a rate that depends on pH and the salt concentration in solution, while its rates for switching between states are insensitive to both. The diffusivity in the loosely bound state depends primarily on pH and is three to ten times higher than in the tightly bound state. We propose and discuss some new experiments that take full advantage of the new tools of analysis presented here.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We reanalyze trajectories of hOGG1 repair proteins diffusing on DNA. A previous analysis of these trajectories with the popular mean-squared-displacement approach revealed only simple diffusion. Here, a new optimal estimator of diffusion coefficients reveals two-state kinetics of the protein. A simple, solvable model, in which the protein randomly switches between a loosely bound, highly mobile state and a tightly bound, less mobile state is the simplest possible dynamic model consistent with the data. It yields accurate estimates of hOGG1's (i) diffusivity in each state, uncorrupted by experimental errors arising from shot noise, motion blur and thermal fluctuations of the DNA; (ii) rates of switching between states and (iii) rate of detachment from the DNA. The protein spends roughly equal time in each state. It detaches only from the loosely bound state, with a rate that depends on pH and the salt concentration in solution, while its rates for switching between states are insensitive to both. The diffusivity in the loosely bound state depends primarily on pH and is three to ten times higher than in the tightly bound state. We propose and discuss some new experiments that take full advantage of the new tools of analysis presented here.
Kanjilal, Sanjat; Sater, Mohamad R Abdul; Thayer, Maile; Lagoudas, Georgia K; Kim, Soohong; Blainey, Paul C; Grad, Yonatan H
Trends in Antibiotic Susceptibility in Staphylococcus aureus in Boston, Massachusetts, from 2000 to 2014 Journal Article
In: J Clin Microbiol, vol. 56, no. 1, 2018, ISSN: 1098-660X.
@article{pmid29093105,
title = {Trends in Antibiotic Susceptibility in Staphylococcus aureus in Boston, Massachusetts, from 2000 to 2014},
author = {Sanjat Kanjilal and Mohamad R Abdul Sater and Maile Thayer and Georgia K Lagoudas and Soohong Kim and Paul C Blainey and Yonatan H Grad},
doi = {10.1128/JCM.01160-17},
issn = {1098-660X},
year = {2018},
date = {2018-01-01},
journal = {J Clin Microbiol},
volume = {56},
number = {1},
abstract = {The rate of infection by methicillin-resistant (MRSA) has declined over the past decade, but it is unclear whether this represents a decline in infections overall. To evaluate the trends in the annual rates of infection by subtypes and mean antibiotic resistance, we conducted a 15-year retrospective observational study at two tertiary care institutions in Boston, MA, of 31,753 adult inpatients with isolated from clinical specimens. We inferred the gain and loss of methicillin resistance through genome sequencing of 180 isolates from 2016. The annual rates of infection by declined from 2003 to 2014 by 4.2% (2.7% to 5.6%), attributable to an annual decline in MRSA of 10.9% (9.3% to 12.6%). Penicillin-susceptible (PSSA) increased by 6.1% (4.2% to 8.1%) annually, and rates of methicillin-susceptible penicillin-resistant (MSSA) did not change. Resistance in decreased from 2000 to 2014 by 0.8 antibiotics (0.7 to 0.8). Within common MRSA clonal complexes, 3/14 MSSA and 2/21 PSSA isolates arose from the loss of resistance-conferring genes. Overall, in two tertiary care institutions in Boston, MA, a decline in infections has been accompanied by a shift toward increased antibiotic susceptibility. The rise in PSSA makes penicillin an increasingly viable treatment option.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The rate of infection by methicillin-resistant (MRSA) has declined over the past decade, but it is unclear whether this represents a decline in infections overall. To evaluate the trends in the annual rates of infection by subtypes and mean antibiotic resistance, we conducted a 15-year retrospective observational study at two tertiary care institutions in Boston, MA, of 31,753 adult inpatients with isolated from clinical specimens. We inferred the gain and loss of methicillin resistance through genome sequencing of 180 isolates from 2016. The annual rates of infection by declined from 2003 to 2014 by 4.2% (2.7% to 5.6%), attributable to an annual decline in MRSA of 10.9% (9.3% to 12.6%). Penicillin-susceptible (PSSA) increased by 6.1% (4.2% to 8.1%) annually, and rates of methicillin-susceptible penicillin-resistant (MSSA) did not change. Resistance in decreased from 2000 to 2014 by 0.8 antibiotics (0.7 to 0.8). Within common MRSA clonal complexes, 3/14 MSSA and 2/21 PSSA isolates arose from the loss of resistance-conferring genes. Overall, in two tertiary care institutions in Boston, MA, a decline in infections has been accompanied by a shift toward increased antibiotic susceptibility. The rise in PSSA makes penicillin an increasingly viable treatment option.
2017
Xiong, Kan; Blainey, Paul C
A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA Journal Article
In: J Vis Exp, no. 128, 2017, ISSN: 1940-087X.
@article{pmid28994817,
title = {A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA},
author = {Kan Xiong and Paul C Blainey},
doi = {10.3791/55923},
issn = {1940-087X},
year = {2017},
date = {2017-10-01},
journal = {J Vis Exp},
number = {128},
abstract = {We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-λ-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to >80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-λ-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to >80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.
Yu, Feiqiao Brian; Blainey, Paul C; Schulz, Frederik; Woyke, Tanja; Horowitz, Mark A; Quake, Stephen R
Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples Journal Article
In: Elife, vol. 6, 2017, ISSN: 2050-084X.
@article{pmid28678007,
title = {Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples},
author = {Feiqiao Brian Yu and Paul C Blainey and Frederik Schulz and Tanja Woyke and Mark A Horowitz and Stephen R Quake},
doi = {10.7554/eLife.26580},
issn = {2050-084X},
year = {2017},
date = {2017-07-01},
journal = {Elife},
volume = {6},
abstract = {Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.
Brito, I L; Yilmaz, S; Huang, K; Xu, L; Jupiter, S D; Jenkins, A P; Naisilisili, W; Tamminen, M; Smillie, C S; Wortman, J R; Birren, B W; Xavier, R J; Blainey, P C; Singh, A K; Gevers, D; Alm, E J
Corrigendum: Mobile genes in the human microbiome are structured from global to individual scales Journal Article
In: Nature, vol. 544, no. 7648, pp. 124, 2017, ISSN: 1476-4687.
@article{pmid28329759,
title = {Corrigendum: Mobile genes in the human microbiome are structured from global to individual scales},
author = {I L Brito and S Yilmaz and K Huang and L Xu and S D Jupiter and A P Jenkins and W Naisilisili and M Tamminen and C S Smillie and J R Wortman and B W Birren and R J Xavier and P C Blainey and A K Singh and D Gevers and E J Alm},
doi = {10.1038/nature20774},
issn = {1476-4687},
year = {2017},
date = {2017-04-01},
journal = {Nature},
volume = {544},
number = {7648},
pages = {124},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Kim, Soohong; Jonghe, Joachim De; Kulesa, Anthony B; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C
High-throughput automated microfluidic sample preparation for accurate microbial genomics Journal Article
In: Nat Commun, vol. 8, pp. 13919, 2017, ISSN: 2041-1723.
@article{pmid28128213,
title = {High-throughput automated microfluidic sample preparation for accurate microbial genomics},
author = {Soohong Kim and Joachim De Jonghe and Anthony B Kulesa and David Feldman and Tommi Vatanen and Roby P Bhattacharyya and Brittany Berdy and James Gomez and Jill Nolan and Slava Epstein and Paul C Blainey},
doi = {10.1038/ncomms13919},
issn = {2041-1723},
year = {2017},
date = {2017-01-01},
journal = {Nat Commun},
volume = {8},
pages = {13919},
abstract = {Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.
2016
Xiong, Kan; Erwin, Graham S; Ansari, Aseem Z; Blainey, Paul C
Sliding on DNA: From Peptides to Small Molecules Journal Article
In: Angew Chem Int Ed Engl, vol. 55, no. 48, pp. 15110–15114, 2016, ISSN: 1521-3773.
@article{pmid27813331,
title = {Sliding on DNA: From Peptides to Small Molecules},
author = {Kan Xiong and Graham S Erwin and Aseem Z Ansari and Paul C Blainey},
doi = {10.1002/anie.201606768},
issn = {1521-3773},
year = {2016},
date = {2016-11-01},
journal = {Angew Chem Int Ed Engl},
volume = {55},
number = {48},
pages = {15110--15114},
abstract = {Many DNA binding proteins utilize one-dimensional (1D) diffusion along DNA to accelerate their DNA target recognition. Although 1D diffusion of proteins along DNA has been studied for decades, a quantitative understanding is only beginning to emerge and few chemical tools are available to apply 1D diffusion as a design principle. Recently, we discovered that peptides can bind and slide along DNA-even transporting cargo along DNA. Such molecules are known as molecular sleds. Here, to advance our understanding of structure-function relationships governing sequence nonspecific DNA interaction of natural molecular sleds and to explore the potential for controlling sliding activity, we test the DNA binding and sliding activities of chemically modified peptides and analogs, and show that synthetic small molecules can slide on DNA. We found new ways to control molecular sled activity, novel small-molecule synthetic sleds, and molecular sled activity in N-methylpyrrole/N-methylimidazole polyamides that helps explain how these molecules locate rare target sites.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Many DNA binding proteins utilize one-dimensional (1D) diffusion along DNA to accelerate their DNA target recognition. Although 1D diffusion of proteins along DNA has been studied for decades, a quantitative understanding is only beginning to emerge and few chemical tools are available to apply 1D diffusion as a design principle. Recently, we discovered that peptides can bind and slide along DNA-even transporting cargo along DNA. Such molecules are known as molecular sleds. Here, to advance our understanding of structure-function relationships governing sequence nonspecific DNA interaction of natural molecular sleds and to explore the potential for controlling sliding activity, we test the DNA binding and sliding activities of chemically modified peptides and analogs, and show that synthetic small molecules can slide on DNA. We found new ways to control molecular sled activity, novel small-molecule synthetic sleds, and molecular sled activity in N-methylpyrrole/N-methylimidazole polyamides that helps explain how these molecules locate rare target sites.
Xu, Liyi; Brito, Ilana L; Alm, Eric J; Blainey, Paul C
Virtual microfluidics for digital quantification and single-cell sequencing Journal Article
In: Nat Methods, vol. 13, no. 9, pp. 759–762, 2016, ISSN: 1548-7105.
@article{pmid27479330,
title = {Virtual microfluidics for digital quantification and single-cell sequencing},
author = {Liyi Xu and Ilana L Brito and Eric J Alm and Paul C Blainey},
doi = {10.1038/nmeth.3955},
issn = {1548-7105},
year = {2016},
date = {2016-09-01},
journal = {Nat Methods},
volume = {13},
number = {9},
pages = {759--762},
abstract = {We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions.
Blainey, Paul C
Funding: Interdisciplinary challenges Journal Article
In: Nat Microbiol, vol. 1, no. 8, pp. 16118, 2016, ISSN: 2058-5276.
@article{pmid27573117,
title = {Funding: Interdisciplinary challenges},
author = {Paul C Blainey},
doi = {10.1038/nmicrobiol.2016.118},
issn = {2058-5276},
year = {2016},
date = {2016-07-01},
journal = {Nat Microbiol},
volume = {1},
number = {8},
pages = {16118},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Brito, I L; Yilmaz, S; Huang, K; Xu, L; Jupiter, S D; Jenkins, A P; Naisilisili, W; Tamminen, M; Smillie, C S; Wortman, J R; Birren, B W; Xavier, R J; Blainey, P C; Singh, A K; Gevers, D; Alm, E J
Mobile genes in the human microbiome are structured from global to individual scales Journal Article
In: Nature, vol. 535, no. 7612, pp. 435–439, 2016, ISSN: 1476-4687.
@article{pmid27409808,
title = {Mobile genes in the human microbiome are structured from global to individual scales},
author = {I L Brito and S Yilmaz and K Huang and L Xu and S D Jupiter and A P Jenkins and W Naisilisili and M Tamminen and C S Smillie and J R Wortman and B W Birren and R J Xavier and P C Blainey and A K Singh and D Gevers and E J Alm},
doi = {10.1038/nature18927},
issn = {1476-4687},
year = {2016},
date = {2016-07-01},
journal = {Nature},
volume = {535},
number = {7612},
pages = {435--439},
abstract = {Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.
Xiong, Kan; Blainey, Paul C
Molecular sled sequences are common in mammalian proteins Journal Article
In: Nucleic Acids Res, vol. 44, no. 5, pp. 2266–2273, 2016, ISSN: 1362-4962.
@article{pmid26857546,
title = {Molecular sled sequences are common in mammalian proteins},
author = {Kan Xiong and Paul C Blainey},
doi = {10.1093/nar/gkw035},
issn = {1362-4962},
year = {2016},
date = {2016-03-01},
journal = {Nucleic Acids Res},
volume = {44},
number = {5},
pages = {2266--2273},
abstract = {Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins.