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

Autor: Replogle JM; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.; Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, USA.; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA., Norman TM; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA.; Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA., Xu A; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA., Hussmann JA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA.; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA., Chen J; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA., Cogan JZ; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA., Meer EJ; 10x Genomics Inc., Pleasanton, CA, USA., Terry JM; 10x Genomics Inc., Pleasanton, CA, USA., Riordan DP; 10x Genomics Inc., Pleasanton, CA, USA., Srinivas N; 10x Genomics Inc., Pleasanton, CA, USA., Fiddes IT; 10x Genomics Inc., Pleasanton, CA, USA., Arthur JG; 10x Genomics Inc., Pleasanton, CA, USA., Alvarado LJ; 10x Genomics Inc., Pleasanton, CA, USA., Pfeiffer KA; 10x Genomics Inc., Pleasanton, CA, USA., Mikkelsen TS; 10x Genomics Inc., Pleasanton, CA, USA., Weissman JS; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. jonathan.weissman@ucsf.edu.; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA. jonathan.weissman@ucsf.edu.; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA. jonathan.weissman@ucsf.edu., Adamson B; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. badamson@princeton.edu.; Department of Molecular Biology, Princeton University, Princeton, NJ, USA. badamson@princeton.edu.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2020 Aug; Vol. 38 (8), pp. 954-961. Date of Electronic Publication: 2020 Mar 30.
DOI: 10.1038/s41587-020-0470-y
Abstrakt: 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.
Databáze: MEDLINE
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