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MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

ABSTRACT: Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9's homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a "plug and play" module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

SUBMITTER: Ma L

PROVIDER: S-EPMC7695827 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

Ma Linyuan L Ruan Jinxue J Song Jun J Wen Luan L Yang Dongshan D Zhao Jiangyang J Xia Xiaofeng X Chen Y Eugene YE Zhang Jifeng J Xu Jie J

Nature communications 20201127 1

Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9's homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to s ...[more]

PMID: 33247137

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Dataset Information

MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

Publications

MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

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