Extensible combinatorial CRISPR screening in mammalian cells

• Published in: STAR protocols Vol. 2; no. 1; p. 100255
• Main Authors: Zhou, Peng, Wan, Yuk Kei, Chan, Becky K.C., Choi, Gigi C.G., Wong, Alan S.L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19-03-2021; Elsevier
• Subjects: Animals; Cell Line; CRISPR; CRISPR-Cas Systems; Genetic Testing; High Throughput Screening; Humans; Protocol; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; High Throughput Screening; CRISPR
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100255

The CRISPR-Cas system coupled with Combinatorial Genetics En Masse (CombiGEM) enables systematic analysis of high-order genetic perturbations that are important for understanding biological processes and discovering therapeutic target combinations. Here, we present detailed steps and technical considerations for building multiplexed guide RNA libraries and carrying out a combinatorial CRISPR screen in mammalian cells. We also present an analytical pipeline, CombiPIPE, for mapping two- and three-way genetic interactions.
For complete details on the use and execution of this protocol, please refer to Zhou et al. (2020).
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•Engineered promoter and scaffold sequences for multiplexed guide RNA expression•Extensible assembly and screening of barcoded multiplexed guide RNA libraries•CombiPIPE established for analyzing CombiGEM-CRISPR screen results
The CRISPR-Cas system coupled with Combinatorial Genetics En Masse (CombiGEM) enables systematic analysis of high-order genetic perturbations that are important for understanding biological processes and discovering therapeutic target combinations. Here, we present detailed steps and technical considerations for building multiplexed guide RNA libraries and carrying out a combinatorial CRISPR screen in mammalian cells. We also present an analytical pipeline, CombiPIPE, for mapping two- and three-way genetic interactions.