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Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design
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Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design
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Journal Article
Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design
2016
Overview
Background
Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in
Saccharomyces cerevisiae
.
Results
We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.
Conclusions
Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.
Publisher
BioMed Central,Springer Nature B.V
Subject
/ Biomedical and Life Sciences
/ Cloning
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ genes
/ Genomes
/ Genomics
/ gRNA
/ Humans
/ loci
/ Microbial Genetics and Genomics
/ Mixed Function Oxygenases - genetics
/ Nuclease
/ plasmids
/ Proteins
/ RNA
/ RNA, Guide, CRISPR-Cas Systems - genetics
/ Saccharomyces cerevisiae - genetics
/ Saccharomyces cerevisiae Proteins - genetics
/ Toxicity
/ Transcription Initiation Site
/ Yeast
/ yeasts
