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

• Published in: Nature communications Vol. 11; no. 1; pp. 6082 - 11
• Main Authors: Ma, Linyuan, Ruan, Jinxue, Song, Jun, Wen, Luan, Yang, Dongshan, Zhao, Jiangyang, Xia, Xiaofeng, Chen, Y. Eugene, Zhang, Jifeng, Xu, Jie
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 27-11-2020; Nature Publishing Group; Nature Portfolio
• Subjects: 13/31; 45/15; 45/41; 49/22; 49/23; 631/1647/1513/1967; 631/337/4041/3196; 631/61/201/2110; 96/109; Amino Acid Sequence; Amino acids; BRCA2 protein; Breast cancer; Cell Line; CRISPR-Associated Protein 9 - chemistry; CRISPR-Associated Protein 9 - metabolism; Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Gene deletion; Gene Editing; Gene Knock-In Techniques; Genetic modification; Genome editing; Green Fluorescent Proteins - metabolism; Homology; Humanities and Social Sciences; Humans; INDEL Mutation - genetics; Insertion; Loci; multidisciplinary; Non-homologous end joining; Nuclease; Oligonucleotides; Plug & play; Proteins; Rad51 Recombinase - metabolism; Repair; Science; Science (multidisciplinary); Vascular Endothelial Growth Factor A - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-19842-2

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.
Cas9 fused to DNA damage repair proteins can improve rates of gene knock-in but the chimeric protein is often large. Here the authors fuse Cas9 to a minimal Brex27 motif from BRCA2 consisting of thirty-six amino acids to enhance the efficacy and safety of gene editing.