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

Linyuan Ma, Jinxue Ruan, Jun Song, Luan Wen, Dongshan Yang, Jiangyang Zhao, Xiaofeng Xia, Y. Eugene Chen (), Jifeng Zhang () and Jie Xu ()
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Linyuan Ma: University of Michigan Medical School
Jinxue Ruan: University of Michigan Medical School
Jun Song: University of Michigan Medical School
Luan Wen: University of Michigan Medical School
Dongshan Yang: University of Michigan Medical School
Jiangyang Zhao: ATGC Inc.
Xiaofeng Xia: ATGC Inc.
Y. Eugene Chen: University of Michigan Medical School
Jifeng Zhang: University of Michigan Medical School
Jie Xu: University of Michigan Medical School

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-19842-2

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