Title | Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. |
Publication Type | Journal Article |
Year of Publication | 2016 |
Authors | Horlbeck, MA, Gilbert, LA, Villalta, JE, Adamson, B, Pak, RA, Chen, Y, Fields, AP, Park, CYon, Corn, JE, Kampmann, M, Weissman, JS |
Journal | Elife |
Volume | 5 |
Date Published | 2016 Sep 23 |
ISSN | 2050-084X |
Keywords | Animals, Bacterial Proteins, Chromosome Mapping, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Protein 9, Endonucleases, Gene Targeting, Humans, Mice, Nucleosomes, RNA, Guide, Kinetoplastida |
Abstract | <p>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.</p> |
DOI | 10.7554/eLife.19760 |
Alternate Journal | Elife |
PubMed ID | 27661255 |
PubMed Central ID | PMC5094855 |
Grant List | U01 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 |