Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.

TitleGenome-Scale CRISPR-Mediated Control of Gene Repression and Activation.
Publication TypeJournal Article
Year of Publication2014
AuthorsGilbert, LA, Horlbeck, MA, Adamson, B, Villalta, JE, Chen, Y, Whitehead, EH, Guimaraes, C, Panning, B, Ploegh, HL, Bassik, MC, Qi, LS, Kampmann, M, Weissman, JS
JournalCell
Volume159
Issue3
Pagination647-61
Date Published2014 Oct 23
ISSN1097-4172
KeywordsCell Line, Cholera Toxin, CRISPR-Cas Systems, Diphtheria Toxin, Genetic Techniques, Genome, Human, Humans, Transcription, Genetic
Abstract

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

DOI10.1016/j.cell.2014.09.029
Alternate JournalCell
PubMed ID25307932
PubMed Central IDPMC4253859
Grant ListU01 CA168370 / CA / NCI NIH HHS / United States
K99 CA181494 / CA / NCI NIH HHS / United States
DP2 HD084069 / HD / NICHD NIH HHS / United States
T32 GM008284 / GM / NIGMS NIH HHS / United States
P01 AG010770 / AG / NIA NIH HHS / United States
P50 GM081879 / GM / NIGMS NIH HHS / United States
T32 EB009383 / EB / NIBIB NIH HHS / United States
DP5 OD017887 / OD / NIH HHS / United States
P50 GM102706 / GM / NIGMS NIH HHS / United States
R01 DA036858 / DA / NIDA NIH HHS / United States
U01CA168370 / CA / NCI NIH HHS / United States
K99CA181494 / CA / NCI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 GM100518 / GM / NIGMS NIH HHS / United States
P50GM102706 / GM / NIGMS NIH HHS / United States