Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ. (December 2016)

• Record Type: Journal Article
• Authors: Aida, Tomomi; Nakade, Shota; Sakuma, Tetsushi; Izu, Yayoi; Oishi, Ayu; Mochida, Keiji; Ishikubo, Harumi; Usami, Takako; Aizawa, Hidenori; Yamamoto, Takashi; Tanaka, Kohichi
• Is Part Of: BMC genomics. Volume 17:Number 1(2016)
• Page Start: 1
• Page End: 18
• Publication Date: 2016-12
• Subjects: MMEJ -- CRISPR/Cas -- Gene cassette -- Reporter -- Flox -- Knock-in -- Mouse -- Exo1 -- High throughput -- Cloning-free; Genomes -- Periodicals; Gene mapping -- Periodicals; Genomics -- Periodicals; Base Sequence -- Periodicals; Chromosome Mapping -- Periodicals; Genetic Techniques -- Periodicals; Sequence Analysis, DNA -- Periodicals; 572.8605
• DOI: 10.1186/s12864-016-3331-9
• Languages: English
• ISSNs: 1471-2164

Abstract

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 identifiedexonuclease 1 (Exo1 ) as an enhancer for PITCh in human cells. By combining theExo1 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.