2016
DOI: 10.1186/s12864-016-3331-9
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Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

Abstract: 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-dir… Show more

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Cited by 70 publications
(76 citation statements)
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“…In fact, we observed higher positive colony-forming efficiency using PITCh knock-in than using HR-mediated knock-in in HeLa cells 5 . In addition, a recent study showed that the overexpression of MMEJ-related genes such as exonuclease 1 can further enhance the knock-in efficiency 8 . Second, the lengths of the microhomologies on the PITCh donor vector are extremely short; thus, they can easily be added by a single PCR without any amplification from genomic DNA.…”
Section: Introductionmentioning
confidence: 66%
“…In fact, we observed higher positive colony-forming efficiency using PITCh knock-in than using HR-mediated knock-in in HeLa cells 5 . In addition, a recent study showed that the overexpression of MMEJ-related genes such as exonuclease 1 can further enhance the knock-in efficiency 8 . Second, the lengths of the microhomologies on the PITCh donor vector are extremely short; thus, they can easily be added by a single PCR without any amplification from genomic DNA.…”
Section: Introductionmentioning
confidence: 66%
“…In this case, the PITCh experiment used Exo1 nuclease that is an enhancer of targeted insertion [Aida et al 2016]. A drawback of ssODN is that lengths over 2 kb are not commercially available [Paix et al 2016; Quadros et al 2017].…”
Section: Recent Advances For Site-specific Insertion Of Relatively Lamentioning
confidence: 99%
“…In addition, the detailed protocol, freely available materials, and online design tool for the CRISPR-mediated PITCh knock-in were provided by the authors Nakamae et al 2017). Along with these user-friendly resources, the application examples of CRISPR-mediated PITCh have become more diversified, e.g., high-throughput epitope tagging in cultured cells for chromatin immunoprecipitation (ChIP)-seq (Xiong et al 2017), the application in mammalian zygotes (Aida et al 2016;Nakagawa et al 2017), and in vivo somatic knock-in in mice (Yao et al 2017a) were reported one after another. Note that recent design of CRISPR-mediated PITCh has employed longer microhomologies (~40 bp); thus, it might be based on SSA rather than MMEJ.…”
Section: Mmej/ssa-mediated Gene Knock-inmentioning
confidence: 99%