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  • Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.
    Author: Aida T, Nakade S, Sakuma T, Izu Y, Oishi A, Mochida K, Ishikubo H, Usami T, Aizawa H, Yamamoto T, Tanaka K.
    Journal: BMC Genomics; 2016 Nov 28; 17(1):979. PubMed ID: 27894274.
    Abstract:
    BACKGROUND: Although 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. RESULTS: Here, 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. CONCLUSIONS: Our results provide a technical platform for high-throughput knock-in.
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