Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

• Published in: eLife Vol. 5
• Main Authors: Horlbeck, Max A, Gilbert, Luke A, Villalta, Jacqueline E, Adamson, Britt, Pak, Ryan A, Chen, Yuwen, Fields, Alexander P, Park, Chong Yon, Corn, Jacob E, Kampmann, Martin, Weissman, Jonathan S
• Format: Journal Article
• Language: English
• Published: England eLife Sciences Publications Ltd 23-09-2016; eLife Sciences Publications, Ltd
• Subjects: Animals; Bacterial Proteins - metabolism; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Computational and Systems Biology; CRISPR; CRISPR-Associated Protein 9; Endonucleases - metabolism; Gene Targeting - methods; Genes and Chromosomes; genetic screening; Humans; Mice; nucleosomes; Nucleosomes - metabolism; Research Advance; RNA, Guide, CRISPR-Cas Systems - metabolism; computational biology; systems biology; CRISPR; genetic screening; genes; nucleosomes; chromosomes; human
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/elife.19760

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.