title: Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Max A
authors.identifier: 0000-0002-3875-871X
authors.initials: MA
authors.lastname: Horlbeck
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Gilbert
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Villalta
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Britt
authors.initials: B
authors.lastname: Adamson
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Innovative Genomics Initiative, University of California, Berkeley, Berkeley, United States.
authors.forename: Ryan A
authors.identifier: 0000-0003-3507-3122
authors.initials: RA
authors.lastname: Pak
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Fields
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Innovative Genomics Initiative, University of California, Berkeley, Berkeley, United States.
authors.forename: Chong Yon
authors.initials: CY
authors.lastname: Park
authors.affiliation: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, United States.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
authors.forename: Jacob E
authors.identifier: 0000-0002-7798-5309
authors.initials: JE
authors.lastname: Corn
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.|Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, United states.
authors.forename: Martin
authors.identifier: 0000-0002-3819-7019
authors.initials: M
authors.lastname: Kampmann
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States.|Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.|California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, United States.|Center for RNA Systems Biology, University of California, San Francisco, San Francisco, United States.
authors.forename: Jonathan S
authors.identifier: 0000-0003-2445-670X
authors.initials: JS
authors.lastname: Weissman
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D002874:Chromosome Mapping; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D051379:Mice; D009707:Nucleosomes / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009707:Nucleosomes; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR; chromosomes; computational biology; genes; genetic screening; human; nucleosomes; systems biology