Abstract
AbstractCRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.
Funder
Japan Agency for Medical Research and Development
MEXT | Japan Society for the Promotion of Science
Uehara Memorial Foundation
Takeda Medical Research Foundation
Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care
Princess Takamatsu Cancer Research Fund
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary