Inducing multiple nicks promotes interhomolog homologous recombination to correct heterozygous mutations in somatic cells.
Akiko TomitaHiroyuki SasanumaTomoo OwaYuka NakazawaMayuko ShimadaTakahiro FukuokaTomoo OgiShinichiro NakadaPublished in: Nature communications (2023)
CRISPR/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.
Keyphrases
- copy number
- dna repair
- genome wide
- dna damage
- crispr cas
- induced apoptosis
- early onset
- genome editing
- dna methylation
- genome wide identification
- cell cycle arrest
- circulating tumor
- poor prognosis
- endoplasmic reticulum stress
- cell free
- oxidative stress
- gene expression
- risk assessment
- signaling pathway
- mass spectrometry
- high resolution
- room temperature
- cell proliferation
- pi k akt