TY - JOUR
T1 - Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing
AU - Replogle, Joseph M.
AU - Norman, Thomas M.
AU - Xu, Albert
AU - Hussmann, Jeffrey A.
AU - Chen, Jin
AU - Cogan, J. Zachery
AU - Meer, Elliott J.
AU - Terry, Jessica M.
AU - Riordan, Daniel P.
AU - Srinivas, Niranjan
AU - Fiddes, Ian T.
AU - Arthur, Joseph G.
AU - Alvarado, Luigi J.
AU - Pfeiffer, Katherine A.
AU - Mikkelsen, Tarjei S.
AU - Weissman, Jonathan S.
AU - Adamson, Britt
N1 - Funding Information:
We thank S.E. Vazquez, A. Guna, M. Jost, D. Yang, R. Saunders, X. Qiu, E. Chow, R. Sit and all members of the Weissman and Adamson laboratories and 10x Genomics for helpful discussions. This work was funded by National Institutes of Health grant nos. P50 GM102706, U01 CA168370, R01 DA036858 and RM1HG009490 (all to J.S.W.), the Defense Advanced Research Projects Agency (DARPA) (grant no. HR0011-19-2-0007), the Chan Zuckerberg Initiative and Princeton University. J.S.W. is a Howard Hughes Medical Institute Investigator. J.M.R. is an NIH/NINDS Ruth L. Kirschstein National Research Service Award fellow (no. F31 NS115380). T.M.N. is a fellow of the Damon Runyon Cancer Research Foundation (no. DRG-(2211-15)). J.A.H. is the Rebecca Ridley Kry Fellow of the Damon Runyon Cancer Research Foundation (no. DRG-2262-16). J.C. is funded by the Jane Coffin Childs Memorial Fund for Medical Research and the NIH K99/R00 Pathway to Independence Award (no. GM134154).
Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2020/8/1
Y1 - 2020/8/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41587-020-0470-y
DO - 10.1038/s41587-020-0470-y
M3 - Article
C2 - 32231336
AN - SCOPUS:85083057725
SN - 1087-0156
VL - 38
SP - 954
EP - 961
JO - Nature Biotechnology
JF - Nature Biotechnology
IS - 8
ER -