A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome

ABSTRACT To interrogate genes essential for cell growth, proliferation and survival in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribosome biogenesis and stress responses. Essential genes exhibited a bimodal distribution across 10 different cell lines, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively spliced exons in essential genes were dispensable and were enriched for disordered regions. Fitness defects were observed for 44 newly evolved hypothetical reading frames. These results illuminate the contextual nature and evolution of essential gene functions in human cells.

Download Full-text

Contextual diversity of the human cell-essential proteome

10.1101/107797 ◽

2017 ◽

Author(s):

Thierry Bertomeu ◽

Jasmin Coulombe-Huntington ◽

Andrew Chatr-Aryamontri ◽

Karine Bourdages ◽

Yu Xia ◽

...

Keyword(s):

Human Cell ◽

Essential Gene ◽

B Cell Lymphoma ◽

Cell Types ◽

Bimodal Distribution ◽

Essential Genes ◽

Analysis Tool ◽

Contextual Diversity ◽

Alternatively Spliced ◽

Alternatively Spliced Exons

AbstractEssential genes define central biological functions required for cell growth, proliferation and survival, but the nature of gene essentiality across human cell types is not well understood. We assessed essential gene function in a Cas9-inducible human B-cell lymphoma cell line using an extended knockout (EKO) library of 278,754 sgRNAs that targeted 19,084 RefSeq genes, 20,852 alternatively-spliced exons and 3,872 hypothetical genes. A new statistical analysis tool called RANKS identified 2,280 essential genes, 234 of which had not been reported previously. Essential genes exhibited a bimodal distribution across 10 cell lines screened in different studies, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines were associated with more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes. Genes essential in fewer lines tended to form context-specific modules and encode subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively-spliced exons in essential genes were dispensable and tended to encode disordered regions. We also detected a significant fitness defect for 44 newly evolved hypothetical reading frames. These results illuminate the nature and evolution of essential gene functions in human cells.

Download Full-text

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

Nucleic Acids Research ◽

10.1093/nar/gku576 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9838-9853 ◽

Cited By ~ 6

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.

Download Full-text

Comprehensive Genome-Wide Analysis of The Catalase Enzyme Toolbox In Potato (Solanum Tuberosum L.)

10.21203/rs.3.rs-1020759/v1 ◽

2021 ◽

Author(s):

Rania Jbir Koubaa ◽

Mariem Ayadi ◽

Mohamed Najib Saidi ◽

Safa Charfeddine ◽

Radhia Gargouri Bouzid ◽

...

Keyword(s):

Stress Responses ◽

Expression Profiling ◽

Phylogenetic Trees ◽

Solanum Tuberosum L ◽

Potato Cultivar ◽

Promoter Sequence ◽

3D Protein Structure ◽

Genome Wide Analysis ◽

Genome Wide ◽

A Genome

Abstract As antioxidant enzymes, catalase (CAT) protects organisms from oxidative stress via the production of reactive oxygen species (ROS). These enzymes play important roles in diverse biological processes. However, little is known about the CAT genes in potato plants despite its important economical rank of this crop in the world. Yet, abiotic and biotic stresses severely hinder growth and development of the plants which affects the production and quality of the crop. To define the possible roles of CAT genes under various stresses, a genome-wide analysis of CAT gene family has been performed in potato plant.In this study, the StCAT gene’s structure, secondary and 3D protein structure, physicochemical properties, synteny analysis, phylogenetic tree and also expression profiling under various developmental and environmental cues were predicted using bioinformatics tools. The expression analysis by RT-PCR was performed using commercial potato cultivar. Three genes encoding StCAT that code for three proteins each of size 492 aa, interrupted by seven introns have been identified in potatoes. StCAT proteins were found to be localized in the peroxisome which is judged as the main H2O2 cell production site during different processes. Many regulating cis-elements related to stress responses and plant hormones signaling were found in the promoter sequence of each gene. The analysis of motifs and phylogenetic trees showed that StCAT are closer to their homologous in S. lycopersicum and share a 41% – 95% identity with other plants’ CATs. Expression profiling revealed that StCAT1 is the constitutively expressive member; while StCAT2 and StCAT3 are the stress-responsive members.

Download Full-text

Genome-wide identification and analysis of cystatin family genes in Sorghum (Sorghum bicolor (L.) Moench)

PeerJ ◽

10.7717/peerj.10617 ◽

2021 ◽

Vol 9 ◽

pp. e10617

Author(s):

Jie Li ◽

Xinhao Liu ◽

Qingmei Wang ◽

Junyan Sun ◽

Dexian He

Keyword(s):

Gene Family ◽

Seed Development ◽

Stress Responses ◽

Abiotic Stresses ◽

Expression Profiles ◽

Seed Formation ◽

Aphid Infestation ◽

Genome Wide ◽

A Genome ◽

Cystatin Family

To set a systematic study of the Sorghum cystatins (SbCys) gene family, a genome-wide analysis of the SbCys family genes was performed by bioinformatics-based methods. In total, 18 SbCys genes were identified in Sorghum, which were distributed unevenly on chromosomes, and two genes were involved in a tandem duplication event. All SbCys genes had similar exon/intron structure and motifs, indicating their high evolutionary conservation. Transcriptome analysis showed that 16 SbCys genes were expressed in different tissues, and most genes displayed higher expression levels in reproductive tissues than in vegetative tissues, indicating that the SbCys genes participated in the regulation of seed formation. Furthermore, the expression profiles of the SbCys genes revealed that seven cystatin family genes were induced during Bipolaris sorghicola infection and only two genes were responsive to aphid infestation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that 17 SbCys genes were induced by one or two abiotic stresses (dehydration, salt, and ABA stresses). The interaction network indicated that SbCys proteins were associated with several biological processes, including seed development and stress responses. Notably, the expression of SbCys4 was up-regulated under biotic and abiotic stresses, suggesting its potential roles in mediating the responses of Sorghum to adverse environmental impact. Our results provide new insights into the structural and functional characteristics of the SbCys gene family, which lay the foundation for better understanding the roles and regulatory mechanism of Sorghum cystatins in seed development and responses to different stress conditions.

Download Full-text

Timing Polymerase Pausing with TV-PRO-seq

10.1101/461442 ◽

2018 ◽

Author(s):

Jie Zhang ◽

Massimo Cavallaro ◽

Daniel Hebenstreit

Keyword(s):

High Resolution ◽

Human Cells ◽

Rrna Genes ◽

Dna Motifs ◽

Single Base ◽

Chromatin States ◽

Genome Wide ◽

A Genome ◽

Single Base Resolution ◽

Polymerase Pausing

Transcription of many genes in metazoans is subject to polymerase pausing, which corresponds to the transient arrest of transcriptionally engaged polymerase. It occurs mainly at promoter proximal regions and is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking.We present here an extension of PRO-seq, time variant PRO-seq (TV-PRO-seq), that allowed us to estimate genome-wide pausing times at single base resolution. Its application to human cells reveals that promoter proximal pausing is surprisingly short compared to other regions and displays an intricate pattern. We also find precisely conserved pausing profiles at tRNA and rRNA genes and identified DNA motifs associated with pausing time. Finally, we show how chromatin states reflect differences in pausing times.

Download Full-text

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

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.02832.x ◽

2002 ◽

Vol 43 (6) ◽

pp. 1387-1400 ◽

Cited By ~ 328

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

Download Full-text

Genome-wide identification of citrus calmodulin-like genes and their expression in response to arbuscular mycorrhizal fungi colonization and drought

Canadian Journal of Plant Science ◽

10.1139/cjps-2020-0160 ◽

2021 ◽

Author(s):

Bo Shu ◽

YaChao Xie ◽

Fei Zhang ◽

Dejian Zhang ◽

Chunyan Liu ◽

...

Keyword(s):

Abiotic Stress ◽

Drought Stress ◽

Arbuscular Mycorrhizal Fungi ◽

Stress Responses ◽

Mycorrhizal Fungi ◽

Expression Patterns ◽

Arbuscular Mycorrhizal ◽

Biotic And Abiotic Stress ◽

Genome Wide ◽

A Genome

Calmodulin-like (CML) proteins represent a diverse family of protein in plants, and play significant roles in biotic and abiotic stress responses. However, the involvement of citrus CMLs in plant responses to drought stress (abiotic stress) and arbuscular mycorrhizal fungi (AMF) colonization remain relatively unknown. We characterized the citrus CML genes by analyzing the EF-hand domains and a genome-wide search, and identified a total of 38 such genes, distributed across at least nine chromosomes. Six tandem duplication clusters were observed in the CsCMLs, and 12 CsCMLs exhibited syntenic relationships with Arabidopsis thaliana CMLs. Gene expression analysis showed that 29 CsCMLs were expressed in the roots, and exhibited differential expression patterns. The regulation of CsCMLs expression was not consistent with the cis-elements identified in their promoters. CsCML2, 3, and 5 were upregulated in response to drought stress, and AMF colonization repressed the expression of CsCML7, 9, 12, 13,20, 27, 28, and 35,and induced that of CsCML1, 2, 3, 5, 8, 10, 11, 14, 15, 16, 18, 25, 30, 33, and 37. Furthermore, AMF colonization and drought stress exerted a synergistic effect, evident from the enhanced repression of CsCML7, 9, 12, 13, 27, 28, and 35 and enhanced expression of CsCML2, 3, and 5 under AMF colonization and drought stress. The present study provides valuable insights into the CsCML gene family and its responses to AMF colonization and drought stress.

Download Full-text

Genome-Wide Analysis of the TCP Gene Family in Switchgrass (Panicum virgatum L.)

International Journal of Genomics ◽

10.1155/2019/8514928 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 2

Author(s):

Yuzhu Huo ◽

Wangdan Xiong ◽

Kunlong Su ◽

Yu Li ◽

Yawen Yang ◽

...

Keyword(s):

Salt Stress ◽

Plant Growth ◽

Gene Family ◽

Stress Responses ◽

Panicum Virgatum ◽

Expression Profiles ◽

Specific Expression ◽

Genome Wide Analysis ◽

Genome Wide ◽

A Genome

The plant-specific transcription factor TCPs play multiple roles in plant growth, development, and stress responses. However, a genome-wide analysis of TCP proteins and their roles in salt stress has not been declared in switchgrass (Panicum virgatum L.). In this study, 42 PvTCP genes (PvTCPs) were identified from the switchgrass genome and 38 members can be anchored to its chromosomes unevenly. Nine PvTCPs were predicted to be microRNA319 (miR319) targets. Furthermore, PvTCPs can be divided into three clades according to the phylogeny and conserved domains. Members in the same clade have the similar gene structure and motif localization. Although all PvTCPs were expressed in tested tissues, their expression profiles were different under normal condition. The specific expression may indicate their different roles in plant growth and development. In addition, approximately 20 cis-acting elements were detected in the promoters of PvTCPs, and 40% were related to stress response. Moreover, the expression profiles of PvTCPs under salt stress were also analyzed and 29 PvTCPs were regulated after NaCl treatment. Taken together, the PvTCP gene family was analyzed at a genome-wide level and their possible functions in salt stress, which lay the basis for further functional analysis of PvTCPs in switchgrass.

Download Full-text

A genome‐wide screen identifies IRF2 as a key regulator of caspase‐4 in human cells

EMBO Reports ◽

10.15252/embr.201948235 ◽

2019 ◽

Vol 20 (9) ◽

Cited By ~ 14

Author(s):

Sacha Benaoudia ◽

Amandine Martin ◽

Marta Puig Gamez ◽

Gabrielle Gay ◽

Brice Lagrange ◽

...

Keyword(s):

Human Cells ◽

Genome Wide ◽

A Genome

Download Full-text

Identification and expression analysis of chloroplast ribonucleoproteins (cpRNPs) in Arabidopsis and rice

Genome ◽

10.1139/gen-2020-0007 ◽

2020 ◽

Author(s):

Jiawen Wu ◽

Huimin Liu ◽

Shan Lu ◽

Jian Hua ◽

Baohong Zou

Keyword(s):

Expression Analysis ◽

Stress Responses ◽

Expression Patterns ◽

Tissue Expression ◽

Stability Control ◽

Rna Recognition Motif ◽

Chloroplast Targeting ◽

Genome Wide ◽

A Genome ◽

Targeting Signal

Chloroplast ribonucleoproteins (cpRNPs) are implicated in splicing, editing and stability control of chloroplast RNAs as well as in regulating development and stress tolerance. To facilitate a comprehensive understanding of their functions, we carried out a genome-wide identification, curation, and phylogenetic analysis of cpRNP genes in Oryza sativa (rice) and Arabidopsis thaliana (Arabidopsis). Ten cpRNP genes were identified in each of Arabidopsis and rice genomes based on the presence of two RRM (RNA recognition motif) domains and an N-terminal chloroplast targeting signal peptide in the predicted proteins. These proteins are localized to chloroplasts. Gene expression analysis revealed that cpRNPs have differential tissue expression patterns and some cpRNPs are induced by abiotic stresses such as cold, heat and drought. Taken together, our study provides a comprehensive annotation of the cpRNP gene family and their expression patterns in Arabidopsis and rice which will facilitate further studies on their roles in plant growth and stress responses.

Download Full-text