CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture

• Published in: Methods (San Diego, Calif.) Vol. 164-165; pp. 49 - 58
• Main Authors: Spiegel, Aleksandra, Bachmann, Mandy, Jurado Jiménez, Gabriel, Sarov, Mihail
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.07.2019
• Subjects: Alleles; Animals; Cell Culture Techniques; CRISPR-Cas Systems - genetics; CRISPR/Cas9; DNA End-Joining Repair; Electroporation - instrumentation; Electroporation - methods; Gene Knockout Techniques - instrumentation; Gene Knockout Techniques - methods; Gene targeting; Gene trap; Genetic Loci - genetics; Genetic Vectors - genetics; Genotyping Techniques - instrumentation; Genotyping Techniques - methods; HCT116 Cells; Humans; Plasmids - genetics; Recombinational DNA Repair; Reverse Genetics - instrumentation; Reverse Genetics - methods; RNA, Guide, CRISPR-Cas Systems - genetics; Selectable knockout; Gene trap; Gene targeting; Selectable knockout; CRISPR/Cas9
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2019.04.016

•Gene knockout strategy applicable to most mammalian cell lines.•CRISPR/Cas9-mediated gene trap cassette insertion for targeted gene disruption.•Isolation of targeted clones is simplified by antibiotic selection.•Biallelic gene trap cassette insertion is not necessary for complete loss of function. We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for.