PT  - JOURNAL ARTICLE
AU  - Toshinori Hyodo
AU  - Md Lutfur Rahman
AU  - Sivasundaram Karnan
AU  - Takuji Ito
AU  - Atsushi Toyoda
AU  - Akinobu Ota
AU  - Md Wahiduzzaman
AU  - Shinobu Tsuzuki
AU  - Yohei Okada
AU  - Yoshitaka Hosokawa
AU  - Hiroyuki Konishi
TI  - Tandem paired nicking promotes precise genome editing with scarce interference by p53
AID  - 10.1101/810333
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 810333
4099  - http://biorxiv.org/content/early/2019/10/18/810333.short
4100  - http://biorxiv.org/content/early/2019/10/18/810333.full
AB  - Targeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Holliday’s model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.