Mutational impact of PolE exonuclease domain mutants

Several PolE exonuclease-domain mutants were introduce in RPE-1 cells by CRISPR-Cas9 gene editing in a homozygous manner. As a control, the wildtype sequence was edited similarly, but in a way to keep the wildtype sequence. Single cell clones were established and cultivated for around 60 cell cycles. Then, single cell clones from these cultures were expanded to purify enough gDNA for whole genome sequencing. This approach let us quantify the amount of mutations acquired in PolE mutant clones compared with the amount of PolE wildtype clones and thus the mutational impact of the knock-in PolE mutation.

本研究通过CRISPR-Cas9基因编辑技术，以纯合方式在RPE-1细胞中引入了数种PolE核酸酶结构域突变体（PolE exonuclease-domain mutants）。作为对照，我们以相同流程编辑野生型序列，通过精准设计使编辑后的序列仍维持野生型状态。随后，我们成功构建单细胞克隆并将其培养约60个细胞周期。进一步将上述培养物中的单细胞克隆扩增至足够体量，以提取足量基因组DNA（genomic DNA，gDNA）用于全基因组测序（whole genome sequencing）。本实验策略可量化PolE突变克隆相较于野生型克隆所积累的突变数量，进而明确该敲入型PolE突变的突变效应。