Journal
BIO-PROTOCOL
Volume 7, Issue 7, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.2222
Keywords
CRISPR; Cas9; Multiple gene knockout; Lentivirus; Primary human leukemia; Xenograft; sgRNA design
Categories
Funding
- Stiftung Kinderkrebsforschung Schweiz
- MAM-Fonds of the Children's Research Centre of the University Children's Hospital Zurich
- Empiris foundation
- clinical research focus program 'human hemato-lymphatic diseases' of the University of Zurich
- Swiss Cancer League [KFS 3609-02-2015]
- Novartis Foundation for Biomedical Research
- Swiss national Science Foundation SNF [310030-133108]
- Canadian institutes for health research CIHR
- Forschungskredit of the University of Zurich [FK-14-016]
- Fondation Panacee
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CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.
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