An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system

Sci Rep. 2014 Sep 19;4:6420. doi: 10.1038/srep06420.

Abstract

The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems*
  • Cell Line
  • Genome
  • Genotyping Techniques*
  • Humans
  • INDEL Mutation / genetics*
  • Mice