Creating knockin alleles in mouse embryonic stem cells by CRISPR/Cas9-mediated homologous recombination without drug selection

Abstract

The rapidly evolving CRISPR/Cas9-mediated genome editing provides the convenience of genome manipulation directly in mouse zygotes for a number of genomic manipulations; but knockins of large insertions prove to be relatively inefficient at least with double-strand DNA as targeting constructs. Here, we describe an alternative approach to the direct genome editing in mouse zygotes by generating knockin alleles in mouse embryonic stem cells first with CRIPSR-mediated homologous recombination. Our results show this approach is efficient and requires no drug selection in mouse embryonic stem cells as in classic gene targeting. As the result, knockin alleles across many target loci are created in mouse embryonic stem cells and readily transmitted through germline. The knockin alleles created in ES cells can also serve as valuable tools for in vitro stem cell differentiation.