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

TitleCompact and highly active next-generation libraries for CRISPR-mediated gene repression and activation.
Publication TypeJournal Article
Year of Publication2016
AuthorsHorlbeck, MA, Gilbert, LA, Villalta, JE, Adamson, B, Pak, RA, Chen, Y, Fields, AP, Park, CYon, Corn, JE, Kampmann, M, Weissman, JS
JournalElife
Volume5
Date Published2016 09 23
ISSN2050-084X
KeywordsAnimals, Bacterial Proteins, Chromosome Mapping, Clustered Regularly Interspaced Short Palindromic Repeats, Endonucleases, Gene Targeting, Humans, Mice, Nucleosomes, RNA, Guide
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.

DOI10.7554/eLife.19760
Alternate JournalElife
PubMed ID27661255
PubMed Central IDPMC5094855
Grant ListU01 CA168370 / CA / NCI NIH HHS / United States
T32 GM008284 / GM / NIGMS NIH HHS / United States
T32 GM007618 / GM / NIGMS NIH HHS / United States
K99 CA204602 / CA / NCI NIH HHS / United States
T32 EB009383 / EB / NIBIB NIH HHS / United States
P50 GM102706 / GM / NIGMS NIH HHS / United States
R01 DA036858 / DA / NIDA NIH HHS / United States
DP2 GM119139 / GM / NIGMS NIH HHS / United States