scholarly journals Genome-wide CRISPR-dCas9 screens inE. coliidentify essential genes and phage host factors

2018 ◽  
Author(s):  
François Rousset ◽  
Lun Cui ◽  
Elise Siouve ◽  
Florence Depardieu ◽  
David Bikard
Keyword(s):  
High Throughput ◽  
Essential Genes ◽  
Host Factors ◽  
Null Mutant ◽  
Genetic Screens ◽  
E Coli ◽  
Colanic Acid ◽  
Genome Wide ◽  
A Genome

AbstractHigh-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a pool of ~ 92,000 sgRNAs which target random positions in the chromosome ofE. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome ofE. coli. We then apply the screen to discoverE. coligenes required by phages λ, T4 and 186 to kill their host. In particular, we show that colanic acid capsule is a barrier to all three phages. Finally, cloning the library on a plasmid that can be packaged by λ enables to identify genes required for the formation of functional λ capsids. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria in order to identify genes linked to a given phenotype.

scholarly journals Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors

PLoS Genetics ◽  
2018 ◽  
Vol 14 (11) ◽  
pp. e1007749 ◽  
Author(s):  
François Rousset ◽  
Lun Cui ◽  
Elise Siouve ◽  
Christophe Becavin ◽  
Florence Depardieu ◽  
...  
Keyword(s):  
Essential Genes ◽  
Host Factors ◽  
E Coli ◽  
Genome Wide

scholarly journals A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽  
2021 ◽  
Vol 34 (11) ◽  
pp. 108859
Author(s):  
Jessie Kulsuptrakul ◽  
Ruofan Wang ◽  
Nathan L. Meyers ◽  
Melanie Ott ◽  
Andreas S. Puschnik
Keyword(s):  
Virus Infection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals Fast and efficient Rmap assembly using the Bi-labelled de Bruijn graph

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Kingshuk Mukherjee ◽  
Massimiliano Rossi ◽  
Leena Salmela ◽  
Christina Boucher
Keyword(s):  
Single Molecule ◽  
De Bruijn Graph ◽  
Anabas Testudineus ◽  
E Coli ◽  
Genome Wide ◽  
A Genome ◽  
De Bruijn ◽  
Optical Maps ◽  
Definition Of ◽  
Numeric Representation

AbstractGenome wide optical maps are high resolution restriction maps that give a unique numeric representation to a genome. They are produced by assembling hundreds of thousands of single molecule optical maps, which are called Rmaps. Unfortunately, there are very few choices for assembling Rmap data. There exists only one publicly-available non-proprietary method for assembly and one proprietary software that is available via an executable. Furthermore, the publicly-available method, by Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006), follows the overlap-layout-consensus (OLC) paradigm, and therefore, is unable to scale for relatively large genomes. The algorithm behind the proprietary method, Bionano Genomics’ Solve, is largely unknown. In this paper, we extend the definition of bi-labels in the paired de Bruijn graph to the context of optical mapping data, and present the first de Bruijn graph based method for Rmap assembly. We implement our approach, which we refer to as rmapper, and compare its performance against the assembler of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) and Solve by Bionano Genomics on data from three genomes: E. coli, human, and climbing perch fish (Anabas Testudineus). Our method was able to successfully run on all three genomes. The method of Valouev et al. (Proc Natl Acad Sci USA 103(43):15770–15775, 2006) only successfully ran on E. coli. Moreover, on the human genome rmapper was at least 130 times faster than Bionano Solve, used five times less memory and produced the highest genome fraction with zero mis-assemblies. Our software, rmapper is written in C++ and is publicly available under GNU General Public License at https://github.com/kingufl/Rmapper.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

Elucidating acetate tolerance in E. coli using a genome-wide approach

2011 ◽  
Vol 13 (2) ◽  
pp. 214-224 ◽  
Author(s):  
Nicholas R. Sandoval ◽  
Tirzah Y. Mills ◽  
Min Zhang ◽  
Ryan T. Gill
Keyword(s):  
E Coli ◽  
Genome Wide ◽  
A Genome

scholarly journals A genome-wide screen in the mouse liver reveals sex-specific and cell non-autonomous regulation of cell fitness

2021 ◽  
Author(s):  
Heather R. Keys ◽  
Kristin A. Knouse
Keyword(s):  
Functional Genomics ◽  
High Throughput ◽  
Mouse Liver ◽  
Ex Vivo ◽  
Screening Method ◽  
Living Organism ◽  
Cellular Processes ◽  
Autonomous Regulation ◽  
Genome Wide ◽  
A Genome

ABSTRACTOur ability to understand and modulate mammalian physiology and disease requires knowing how all genes contribute to any given phenotype in the organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for the genetic dissection of cellular processes1,2, but the need to stably deliver single guide RNAs to millions of cells has restricted its implementation to ex vivo systems. These ex vivo systems cannot reproduce all of the cellular phenotypes observed in vivo nor can they recapitulate all of the factors that influence these phenotypes. There thus remains a pressing need for high-throughput functional genomics in a living organism. Here, we establish accessible genome-wide screening in the mouse liver and use this approach to uncover the complete regulation of cellular fitness in a living organism. We discover novel sex-specific and cell non-autonomous regulation of cell growth and viability. In particular, we find that the class I major histocompatibility complex is essential for preventing immune-mediated clearance of hepatocytes. Our approach provides the first comprehensive picture of cell fitness in a living organism and highlights the importance of investigating cellular phenomena in their native context. Our screening method is robust, scalable, and easily adapted to examine diverse cellular processes using any CRISPR application. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into mammalian physiology and disease.

scholarly journals sPepFinder expedites genome-wide identification of small proteins in bacteria

2020 ◽  
Author(s):  
Lei Li ◽  
Yanjie Chao
Keyword(s):  
De Novo ◽  
Bacterial Species ◽  
Computational Prediction ◽  
Ribosome Profiling ◽  
Support Vector ◽  
Initiation Rate ◽  
E Coli ◽  
Small Proteins ◽  
Genome Wide ◽  
A Genome

ABSTRACTSmall proteins shorter than 50 amino acids have been long overlooked. A number of small proteins have been identified in several model bacteria using experimental approaches and assigned important functions in diverse cellular processes. The recent development of ribosome profiling technologies has allowed a genome-wide identification of small proteins and small ORFs (smORFs), but our incomplete understanding of small proteins hinders de novo computational prediction of smORFs in non-model bacterial species. Here, we have identified several sequence features for smORFs by a systematic analysis of all the known small proteins in E. coli, among which the translation initiation rate is the strongest determinant. By integrating these features into a support vector machine learning model, we have developed a novel sPepFinder algorithm that can predict conserved smORFs in bacterial genomes with a high accuracy of 92.8%. De novo prediction in E. coli has revealed several novel smORFs with evidence of translation supported by ribosome profiling. Further application of sPepFinder in 549 bacterial species has led to the identification of > 100,000 novel smORFs, many of which are conserved at the amino acid and nucleotide levels under purifying selection. Overall, we have established sPepFinder as a valuable tool to identify novel smORFs in both model and non-model bacterial organisms, and provided a large resource of small proteins for functional characterizations.

scholarly journals A Genome-Wide Arrayed cDNA Screen to Identify Functional Modulators of α7 Nicotinic Acetylcholine Receptors

2016 ◽  
Vol 22 (2) ◽  
pp. 155-165 ◽  
Author(s):  
Elizabeth B. Rex ◽  
Nikhil Shukla ◽  
Shenyan Gu ◽  
David Bredt ◽  
Daniel DiSepio
Keyword(s):  
Cdna Library ◽  
High Throughput ◽  
Protein Interactions ◽  
Nicotinic Acetylcholine Receptors ◽  
Calcium Flux ◽  
Functional Assay ◽  
Α7 Nicotinic Acetylcholine Receptor ◽  
Nicotinic Acetylcholine ◽  
Genome Wide ◽  
A Genome

Cellular signaling is in part regulated by the composition and subcellular localization of a series of protein interactions that collectively form a signaling complex. Using the α7 nicotinic acetylcholine receptor (α7nAChR) as a proof-of-concept target, we developed a platform to identify functional modulators (or auxiliary proteins) of α7nAChR signaling. The Broad cDNA library was transiently cotransfected with α7nAChR cDNA in HEK293T cells in a high-throughput fashion. Using this approach in combination with a functional assay, we identified positive modulators of α7nAChR activity. We identified known positive modulators/auxiliary proteins present in the cDNA library that regulate α7nAChR signaling, in addition to identifying novel modulators of α7nAChR signaling. These included NACHO, SPDYE11, TCF4, and ZC3H12A, all of which increased PNU-120596-mediated nicotine-dependent calcium flux. Importantly, these auxiliary proteins did not modulate GluR1(o)-mediated Ca flux. To elucidate a possible mechanism of action, we employed an α7nAChR-HA surface staining assay. NACHO enhanced α7nAChR surface expression; however, the mechanism responsible for the SPDYE11-, TCF4-, and ZC3H12A-dependent modulation of α7nAChR has yet to be defined. This report describes the development and validation of a high-throughput, genome-wide cDNA screening platform coupled to FLIPR functional assays in order to identify functional modulators of α7nAChR signaling.

scholarly journals A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

2002 ◽  
Vol 43 (6) ◽  
pp. 1387-1400 ◽  
Author(s):  
R. Allyn Forsyth ◽  
Robert J. Haselbeck ◽  
Kari L. Ohlsen ◽  
Robert T. Yamamoto ◽  
Howard Xu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Essential Genes ◽  
Genome Wide ◽  
A Genome

scholarly journals Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael G. Connor ◽  
Amanda R. Pulsifer ◽  
Donghoon Chung ◽  
Eric C. Rouchka ◽  
Brian K. Ceresa ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Host Factors ◽  
Endocytic Recycling ◽  
Recycling Endosome ◽  
Independent Manner ◽  
Genome Wide ◽  
Type Three Secretion System ◽  
A Genome ◽  
Recycling Pathway ◽  
Bacterial Replication

ABSTRACTYersinia pestishas evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis,Y. pestisprevents phagolysosome maturation and establishes a modified compartment termed theYersinia-containing vacuole (YCV).Y. pestisactively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requiresY. pestisinteractions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required forY. pestissurvival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival ofY. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated thatY. pestisactively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV byY. pestisto resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered thatY. pestisdisrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence thatY. pestistargets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestiscan infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellularY. pestissurvival. This approach revealed that the host endocytic recycling pathway is essential forY. pestisto avoid clearance by the macrophage. We further demonstrate thatY. pestisremodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection withY. pestisdisrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence thatY. pestistargets the host endocytic recycling pathway in order to evade killing by macrophages.

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