Targeted sequencing of the can1 gene and its background control gene ALP1 in Canavanine-resistant mutants.

It is well known that ssDNA is more susceptible to mutagenesis than dsDNA due to the exposure of nucleobases. Previous studies have shown that DSBs induced in the presence of UV, alkylating agents, bisulfite, or APOBEC can lead to mutagenesis in their flanking regions, resembling kataegis or clustered mutations observed in cancer genomes. Our lab has demonstrated that dSpCas9 can stall the progression of the DSB resection machinery. Based on this, we hypothesized that dSpCas9 could be applied to control or restrict the region susceptible to experimentally induced kataegis: the DSB distal side of the dSpCas9 binding site exhibits a lower mutation rate than the DSB proximal side and is thus protected from kataegis. We termed this approach of region restricted random mutagenesis controlled kataegis. We performed a proof of concept experiment for controlled kataegis using the CAN1 gene in a reporter Saccharomyces cerevisiae strain. In the presence of bisulfite, binding dSpCas9 to the region between an upstream DSB and the CAN1 gene resulted in a reduced frequency of Canavanine resistant mutants compared to the control strain, which contained the DSBs but no the dSpCas9 binding. To confirm that the Canavanine resistant mutations arose from ssDNA generated by resection rather than ssDNA from transcription of CAN1, and to assess the distribution of mutations in the mutants, we performed targeted sequencing of the CAN1 region.

众所周知，单链DNA（single-stranded DNA，ssDNA）因核碱基暴露，相较于双链DNA（double-stranded DNA，dsDNA）更易发生诱变。既往研究表明，在紫外线（UV）、烷化剂、亚硫酸氢盐或APOBEC存在的条件下诱导产生的DNA双链断裂（double-strand breaks，DSBs），可在其侧翼区域引发诱变效应，该现象与癌症基因组中观察到的kataegis或簇状突变特征相似。本课题组此前已证实，失活型化脓链球菌Cas9（dSpCas9）可阻滞DNA双链断裂切除加工复合体的行进过程。基于此，我们提出假说：可利用dSpCas9调控或限制实验诱导的kataegis易感区域：dSpCas9结合位点的DNA双链断裂远端侧突变率低于近端侧，因此可免受kataegis诱变效应的影响。我们将这种区域限制性随机诱变的方法命名为可控型kataegis。我们在报告型酿酒酵母（Saccharomyces cerevisiae）菌株中，以CAN1基因为标记，开展了可控型kataegis的概念验证实验。在亚硫酸氢盐存在的条件下，相较于仅存在DNA双链断裂但无dSpCas9结合的对照菌株，将dSpCas9结合至上游DNA双链断裂与CAN1基因之间的区域时，抗刀豆氨酸突变体的出现频率显著降低。为证实抗刀豆氨酸突变源于DNA切除过程产生的单链DNA，而非CAN1基因转录产生的单链DNA，并评估突变体中的突变分布情况，我们对CAN1基因区域开展了靶向测序。