Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Abstract Identifying new gene functions and pathways underlying diseases and biological processes are major challenges in genomics research. Particularly, most methods for interpreting the pathways characteristic of an experimental gene list defined by genomic data are limited by their dependence on assessing the overlapping genes or their interactome topology, which cannot account for the variety of functional relations. This is particularly problematic for pathway discovery from single-cell genomics with low gene coverage or interpreting complex pathway changes such as during change of cell states. Here, we exploited the comprehensive sets of molecular concepts that combine ontologies, pathways, interactions and domains to help inform the functional relations. We first developed a universal concept signature (uniConSig) analysis for genome-wide quantification of new gene functions underlying biological or pathological processes based on the signature molecular concepts computed from known functional gene lists. We then further developed a novel concept signature enrichment analysis (CSEA) for deep functional assessment of the pathways enriched in an experimental gene list. This method is grounded on the framework of shared concept signatures between gene sets at multiple functional levels, thus overcoming the limitations of the current methods. Through meta-analysis of transcriptomic data sets of cancer cell line models and single hematopoietic stem cells, we demonstrate the broad applications of CSEA on pathway discovery from gene expression and single-cell transcriptomic data sets for genetic perturbations and change of cell states, which complements the current modalities. The R modules for uniConSig analysis and CSEA are available through https://github.com/wangxlab/uniConSig.

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The worm's turn: Genome-wide RNAi in Caenorhabditis elegans

The Biochemist ◽

10.1042/bio02605025 ◽

2004 ◽

Vol 26 (5) ◽

pp. 25-28

Author(s):

Gino Poulin ◽

David P. Welchman

Keyword(s):

Caenorhabditis Elegans ◽

New Technology ◽

Biological Processes ◽

Systematic Screening ◽

C Elegans ◽

Genome Wide ◽

Powerful Approach ◽

Gene Functions ◽

New Gene ◽

Multiple Species

RNAi-mediated silencing is now being used in multiple species, from plants to humans, to identify gene functions. This new technology is fully exploited in Caenorhabditis elegans, since an RNAi feeding library that has the potential to inactivate 86% of the predicted genes of the C. elegans genome is publicly available. This tool made systematic screening of genes involved in biological processes of interest easier, but there are some pitfalls: specificity, reproducibility and hypomorphic effects. However, as with any other technology, RNAi is getting better as time goes on. Once these caveats are taken into account, RNAi-mediated silencing is still a very powerful approach to identify new gene functions and to dissect complex pathways.

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Integrative analysis and refined design of CRISPR knockout screens

10.1101/106534 ◽

2017 ◽

Cited By ~ 1

Author(s):

Chen-Hao Chen ◽

Wei Li ◽

Tengfei Xiao ◽

Han Xu ◽

Peng Jiang ◽

...

Keyword(s):

Safe Harbor ◽

Signal To Noise ◽

Protein Protein Interaction ◽

Guide Rnas ◽

Maximum Likelihood Approach ◽

Genome Wide ◽

Gene Functions ◽

Crispr Screen ◽

Likelihood Approach ◽

Negative Controls

AbstractGenome-wide CRISPR-Cas9 screen has been widely used to interrogate gene functions. However, the analysis remains challenging and rules to design better libraries beg further refinement. Here we present MAGeCK-NEST, which integrates protein-protein interaction (PPI), improves the inference accuracy when fewer guide-RNAs (sgRNAs) are available, and assesses screen qualities using information on PPI. MAGeCK-NEST also adopts a maximum-likelihood approach to remove sgRNA outliers, which are characterized with higher G-nucleotide counts, especially in regions distal from the PAM motif. Using MAGeCK-NEST, we found that choosing non-targeting sgRNAs as negative controls lead to strong bias, which can be mitigated by sgRNAs targeting the “safe harbor” regions. Custom-designed screens confirmed our findings, and further revealed that 19nt sgRNAs consistently gave the best signal-to-noise separation. Collectively, our method enabled robust calling of CRISPR screen hits and motivated the design of an improved genome-wide CRISPR screen library.

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Genome-wide analysis oftrans-splicing in the nematodePristionchus pacificusunravels conserved gene functions for germline and dauer development in divergent operons

RNA ◽

10.1261/rna.041954.113 ◽

2014 ◽

Vol 20 (9) ◽

pp. 1386-1397 ◽

Cited By ~ 8

Author(s):

Amit Sinha ◽

Claudia Langnick ◽

Ralf J. Sommer ◽

Christoph Dieterich

Keyword(s):

Genome Wide Analysis ◽

Genome Wide ◽

Gene Functions ◽

Conserved Gene

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Abstract 4538: UniConSig: A new algorithm for genome wide quantification of gene functions and disease associations

10.1158/1538-7445.am2017-4538 ◽

2017 ◽

Author(s):

Xu Chi ◽

Meenakshi Anurag ◽

Sartor A. Maureen ◽

Xiaosong Wang

Keyword(s):

Genome Wide ◽

Gene Functions ◽

Disease Associations

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Genome-Wide Analysis of the UDP-Glycosyltransferase Family Reveals Its Roles in Coumarin Biosynthesis and Abiotic Stress in Melilotus albus

International Journal of Molecular Sciences ◽

10.3390/ijms221910826 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10826

Author(s):

Zhen Duan ◽

Qi Yan ◽

Fan Wu ◽

Yimeng Wang ◽

Shengsheng Wang ◽

...

Keyword(s):

Abiotic Stress ◽

Developmental Regulation ◽

Differentially Expressed ◽

Future Studies ◽

Genome Wide Analysis ◽

Genome Wide ◽

Gene Functions ◽

Melilotus Albus ◽

The Relationship ◽

Heterologous Expression In Yeast

Coumarins, natural products abundant in Melilotus albus, confer features in response to abiotic stresses, and are mainly present as glycoconjugates. UGTs (UDP-glycosyltransferases) are responsible for glycosylation modification of coumarins. However, information regarding the relationship between coumarin biosynthesis and stress-responsive UGTs remains limited. Here, a total of 189 MaUGT genes were identified from the M. albus genome, which were distributed differentially among its eight chromosomes. According to the phylogenetic relationship, MaUGTs can be classified into 13 major groups. Sixteen MaUGT genes were differentially expressed between genotypes of Ma46 (low coumarin content) and Ma49 (high coumarin content), suggesting that these genes are likely involved in coumarin biosynthesis. About 73.55% and 66.67% of the MaUGT genes were differentially expressed under ABA or abiotic stress in the shoots and roots, respectively. Furthermore, the functions of MaUGT68 and MaUGT186, which were upregulated under stress and potentially involved in coumarin glycosylation, were characterized by heterologous expression in yeast and Escherichia coli. These results extend our knowledge of the UGT gene family along with MaUGT gene functions, and provide valuable findings for future studies on developmental regulation and comprehensive data on UGT genes in M. albus.

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Genome-Wide SNP Genotyping to Infer the Effects on Gene Functions in Tomato

DNA Research ◽

10.1093/dnares/dst005 ◽

2013 ◽

Vol 20 (3) ◽

pp. 221-233 ◽

Cited By ~ 40

Author(s):

H. Hirakawa ◽

K. Shirasawa ◽

A. Ohyama ◽

H. Fukuoka ◽

K. Aoki ◽

...

Keyword(s):

Snp Genotyping ◽

Genome Wide ◽

Gene Functions

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Biological interpretation of genome-wide association studies using predicted gene functions

Nature Communications ◽

10.1038/ncomms6890 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 387

Author(s):

Tune H. Pers ◽

◽

Juha M. Karjalainen ◽

Yingleong Chan ◽

Harm-Jan Westra ◽

...

Keyword(s):

Association Studies ◽

Genome Wide Association ◽

Genome Wide Association Studies ◽

Genome Wide ◽

Gene Functions ◽

Biological Interpretation

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A genome-wide almanac of co-essential modules assigns function to uncharacterized genes

10.1101/827071 ◽

2019 ◽

Cited By ~ 10

Author(s):

Michael Wainberg ◽

Roarke A. Kamber ◽

Akshay Balsubramani ◽

Robin M. Meyers ◽

Nasa Sinnott-Armstrong ◽

...

Keyword(s):

Protein Complexes ◽

Functional Modules ◽

Web Tool ◽

Coated Pits ◽

Robust Identification ◽

Genome Wide ◽

A Genome ◽

Gene Functions ◽

Key Enzyme ◽

Transferrin Uptake

SUMMARYA central remaining question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into ‘co-essential’ pathways, but this approach has been limited by ubiquitous false positives. Here, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This almanac recapitulates diverse pathways and protein complexes and predicts the functions of 102 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, the long-sought key enzyme for plasmalogen synthesis. We also show that C15orf57 binds the AP2 complex, localizes to clathrin-coated pits, and enables efficient transferrin uptake. Finally, we provide an interactive web tool for the community to explore the results (coessentiality.net). Our results establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of novel gene functions.

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