Establishment of an allele-specific genome editing method for the correction of disease-causing mutations

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This research aims to discover a novel strategy for the correction of dominant mutation based on haplotype (a group of SNPs which tend to inherit together)-specific approaches with the CRISPR/Cas9 system. We examined three conventional methods: Knocking out the mutant allele by introducing indels (insertions and deletions), correcting the mutation using an isogenic donor vector (MhAX; Kim et al, 2018), and removing the mutation with surrounding sequences by two allele-specific DSBs (double strand breaks). We demonstrated that each strategy designed based on bioinformatics resulted in desired changes in genotype, yet with different efficiencies. This suggests allele-specific targeting using haplotype is a practical gene editing strategy modulating haplotypes around mutations in a precise manner.As one of the novel haplotype-specific gene editing strategies, interhomolog repair (IHR) was studied in this research. To this end, we generated a new fluorescent-based reporter system (modified from Maurissen and Woltjen, 2020) in human induced pluripotent stem cells (hiPSCs) and confirmed that mitotic IHR happens with low frequencies in hiPSCs when DSB is introduced. Additionally, we found nicking downstream and upstream of the target DSB site at the same time led to higher IHR frequency than DSB alone. Moreover, cell cycle modulation and cold shock after electroporation, which were known to improve HDR (homology-directed repair)-mediated gene editing efficiencies, proved effective in increasing IHR frequencies as well.