Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

[摘要] BackgroundAlthough CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.ResultsHere, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.ConclusionsOur results provide a technical platform for high-throughput knock-in.

[发布日期] 2016-11-28

[关键词] MMEJ;CRISPR/Cas;Gene cassette;Reporter;Flox;Knock-in;Mouse;Exo1;High throughput;Cloning-free