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
Luke A Gilbert
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
Jacqueline E Villalta
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
Britt Adamson
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
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
Yuwen Chen
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
Alexander P Fields
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
Chong Yon Park
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
Innovative Genomics Initiative, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
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
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
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.
