Journal
BMC GENOMICS
Volume 17, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s12864-016-3331-9
Keywords
MMEJ; CRISPR/Cas; Gene cassette; Reporter; Flox; Knock-in; Mouse; Exo1; High throughput; Cloning-free
Funding
- Japan Society for the Promotion of Science [26830131, 16 K07085, 15 K19988, 16 K18478]
- Ministry of Education, Culture, Sports, Science and Technology of Japan [26112010]
- Nakatani Foundation
- Takeda Science Foundation
- SENSHIN Medical Research Foundation
- Mochida Memorial Foundation for Medical and Pharmaceutical Research
- MRI of TMDU
- TMDU
- Brain Sciences Project of the Center for Novel Science Initiatives (CNSI), National Institutes of Natural Sciences (NINS) [BS281001]
- Nakatomi Foundation
- Ichiro, Kanehara Foundation
- Sasakawa Foundation
- Uehara Memorial Foundation
- Ministry of Health, Labor, and Welfare of Japan
- joint Usage/Research Program from MRI of TMDU
- Grants-in-Aid for Scientific Research [26290070, 16H01383, 26290021, 16K18478, 15K19988, 16J03164, 26112010, 26430008, 16K07085, 26830131] Funding Source: KAKEN
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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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