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

这项研究旨在发现一种新的策略，用于基于单倍型（一组倾向于一起遗传的SNP）特异性方法与CRISPR/Cas9系统校正显性突变。我们检查了三种常规方法：通过引入indel（插入和缺失）敲除突变等位基因，使用同基因供体载体校正突变（MhAX; Kim et al，2018），以及通过两个等位基因特异性DSB（双链断裂）去除周围序列的突变。我们证明了基于生物信息学设计的每种策略都导致了基因型的预期变化，但效率不同。这表明利用单倍型进行等位基因特异性靶向是一种实用的基因编辑策略，可以精确调控突变周围的单倍型。为此，我们在人诱导多能干细胞（hiPSC）中产生了一种新的基于荧光的报告系统（由Maurissen和Woltjen，2020修改），并证实当引入DSB时，有丝分裂IHR在hiPSC中发生的频率较低。此外，我们发现在靶DSB位点的下游和上游同时切口导致比单独DSB更高的IHR频率。此外，已知电穿孔后的细胞周期调节和冷休克可以提高HDR（同源定向修复）介导的基因编辑效率，也证明可以有效提高IHR频率。

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.