Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice.

ABSTRACT: Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Mor

SUBMITTER: Ozawa M 

PROVIDER: S-EPMC9748034 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature