%0 Journal Article
%T CRISPR¨CCas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development
%J -
%D 2018
%R https://doi.org/10.1038/s41556-018-0202-4
%X CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein¨Cprotein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function
%U https://www.nature.com/articles/s41556-018-0202-4