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

Methods. 2019 Jul 15:164-165:49-58. doi: 10.1016/j.ymeth.2019.04.016. Epub 2019 Apr 30.

Abstract

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.

Keywords: CRISPR/Cas9; Gene targeting; Gene trap; Selectable knockout.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Culture Techniques
  • DNA End-Joining Repair
  • Electroporation / instrumentation
  • Electroporation / methods
  • Gene Knockout Techniques / instrumentation
  • Gene Knockout Techniques / methods*
  • Genetic Loci / genetics
  • Genetic Vectors / genetics
  • Genotyping Techniques / instrumentation
  • Genotyping Techniques / methods
  • HCT116 Cells
  • Humans
  • Plasmids / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Recombinational DNA Repair*
  • Reverse Genetics / instrumentation
  • Reverse Genetics / methods*

Substances

  • RNA, Guide, CRISPR-Cas Systems