Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair

Kazuhiro Nakajima, Yue Zhou, Akiko Tomita, Yoshihiro Hirade, Channabasavaiah B Gurumurthy, Shinichiro Nakada

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.

Original languageEnglish (US)
Pages (from-to)223-230
Number of pages8
JournalGenome Research
Volume28
Issue number2
DOIs
StatePublished - Feb 2018

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Nucleotides
Plasmids
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Genes
Double-Stranded DNA Breaks
Deoxyribonuclease I
Sequence Homology
Genetic Therapy
DNA Damage
DNA

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair. / Nakajima, Kazuhiro; Zhou, Yue; Tomita, Akiko; Hirade, Yoshihiro; Gurumurthy, Channabasavaiah B; Nakada, Shinichiro.

In: Genome Research, Vol. 28, No. 2, 02.2018, p. 223-230.

Research output: Contribution to journalArticle

Nakajima, Kazuhiro ; Zhou, Yue ; Tomita, Akiko ; Hirade, Yoshihiro ; Gurumurthy, Channabasavaiah B ; Nakada, Shinichiro. / Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair. In: Genome Research. 2018 ; Vol. 28, No. 2. pp. 223-230.
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