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Tandem paired nicking promotes precise genome editing with scarce interference by p53

Toshinori Hyodo, Md Lutfur Rahman, Sivasundaram Karnan, Takuji Ito, Atsushi Toyoda, Akinobu Ota, Md Wahiduzzaman, Shinobu Tsuzuki, Yohei Okada, Yoshitaka Hosokawa, View ORCID ProfileHiroyuki Konishi
doi: https://doi.org/10.1101/810333
Toshinori Hyodo
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Md Lutfur Rahman
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Sivasundaram Karnan
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Takuji Ito
2Department of Neurology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Atsushi Toyoda
3Department of Genomics and Evolutionary Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
4Advanced Genomics Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
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Akinobu Ota
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Md Wahiduzzaman
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Shinobu Tsuzuki
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Yohei Okada
2Department of Neurology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Yoshitaka Hosokawa
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Hiroyuki Konishi
1Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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  • ORCID record for Hiroyuki Konishi
  • For correspondence: hkonishi@aichi-med-u.ac.jp
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Summary

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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 18, 2019.
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Tandem paired nicking promotes precise genome editing with scarce interference by p53
Toshinori Hyodo, Md Lutfur Rahman, Sivasundaram Karnan, Takuji Ito, Atsushi Toyoda, Akinobu Ota, Md Wahiduzzaman, Shinobu Tsuzuki, Yohei Okada, Yoshitaka Hosokawa, Hiroyuki Konishi
bioRxiv 810333; doi: https://doi.org/10.1101/810333
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Tandem paired nicking promotes precise genome editing with scarce interference by p53
Toshinori Hyodo, Md Lutfur Rahman, Sivasundaram Karnan, Takuji Ito, Atsushi Toyoda, Akinobu Ota, Md Wahiduzzaman, Shinobu Tsuzuki, Yohei Okada, Yoshitaka Hosokawa, Hiroyuki Konishi
bioRxiv 810333; doi: https://doi.org/10.1101/810333

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