Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.

TitleCombinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.
Publication TypeJournal Article
Year of Publication2020
AuthorsReplogle, JM, Norman, TM, Xu, A, Hussmann, JA, Chen, J, J Cogan, Z, Meer, EJ, Terry, JM, Riordan, DP, Srinivas, N, Fiddes, IT, Arthur, JG, Alvarado, LJ, Pfeiffer, KA, Mikkelsen, TS, Weissman, JS, Adamson, B
JournalNat Biotechnol
Date Published2020 Mar 30
ISSN1546-1696
Abstract

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

DOI10.1038/s41587-020-0470-y
Alternate JournalNat. Biotechnol.
PubMed ID32231336
Grant ListU01 CA168370 / CA / NCI NIH HHS / United States
GM134154 / / U.S. Department of Health & Human Services | National Institutes of Health (NIH) /
DRG-2262-16 / / Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation) /
F31 NS115380 / NS / NINDS NIH HHS / United States
R01 DA036858 / DA / NIDA NIH HHS / United States
DRG-[2211-15] / / Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation) /
K99 GM134154 / GM / NIGMS NIH HHS / United States
P50 GM102706 / GM / NIGMS NIH HHS / United States
RM1HG009490 / / U.S. Department of Health & Human Services | National Institutes of Health (NIH) /
HR0011-19-2-0007 / / United States Department of Defense | Defense Advanced Research Projects Agency (DARPA) /