Molecular Mechanism of the Cytosine CRISPR Base Editing Process and the Roles of Translesion DNA Polymerases
Molecular Mechanism of the Cytosine CRISPR Base Editing Process and the Roles of Translesion DNA Polymerases
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DOI：
10.1021/acssynbio.1c00293
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发表时间：
2021-12-17
影响因子：
4.7
通讯作者：
Zhang, Xueli
中科院分区：
文献类型：
--
作者：
Jiang, Guo;Wang, Jie;Zhang, Xueli
CRISPR介导的碱基编辑会对DNA造成损伤，主要是尿嘧啶、脱嘌呤/脱嘧啶（AP）位点和缺口，这些损伤需要各种DNA修复机制来完成碱基转换过程。目前，只有假说解释碱基编辑过程，但修复系统在该过程中的分子机制和作用相对未知。为了探索碱基编辑修复的机制，将碱基编辑器nCas 9-PmCDA 1应用于模型真核生物酿酒酵母（Saccharomyces cerevisiae）中，其中野生型或其衍生物具有编码跨损伤DNA合成（TLS）聚合酶的基因敲除。我们发现，C到G和C到A的转换主要是由Ung创建的AP位点的修复引起的，并且需要Pol zeta作为扩展剂。Rev 1是主要的TLS聚合酶，用于在AP位点的相对位置上特异性地掺入Cs以引起显性的C-to-G转换，而Pol delta在AP位点的相对位置上掺入Ts或As，导致C-to-A和C-to-T转换。Pol eta不参与由碱基编辑器引起的AP位点的修复。此外，我们的数据表明，碱基编辑的插入缺失主要是由AP位点的断裂引起的。与目前碱基编辑机制的假设模型不同，本工作首先阐明了TLS聚合酶在胞嘧啶碱基编辑过程中的关键作用。这项工作还通过采用、操纵和工程化TLS聚合酶为基因组和碱基编辑技术的发展提出了一个新的方向。
CRISPR-mediated base editing causes damage to DNA, mainly uracil, apurinic/apyrimidinic (AP) sites, and nicks, which require various DNA repair mechanisms to complete the base conversion process. Currently, there are only hypotheses explaining the base editing process, but the molecular mechanism and roles of the repair systems in the process are relatively unknown. To explore the mechanism of base editing repair, a base editor, nCas9-PmCDA1, was applied in the model eukaryote, Saccharomyces cerevisiae, either with the wild type or its derivatives with genes encoding translesion DNA synthesis (TLS) polymerases knocked out. We found that C-to-G and C-to-A conversions resulted mainly from the repair of AP sites created by Ung and required Pol zeta as an extender. Rev1 is the main TLS polymerase for specifically incorporating Cs on the opposite position of AP sites to cause the dominant C-to-G conversion, while Pol delta incorporates Ts or As on the opposite of AP sites, resulting in C-to-A and C-to-T conversions. Pol eta is not involved in the repair of AP sites caused by the base editor. Furthermore, our data suggested that the indels of base editing are mainly caused by the breakage of AP sites. Different from the current hypothesis model of the base editing mechanism, this work first elucidates the key roles of TLS polymerases in the cytosine base editing process. This work also suggests a new direction for the development of genomic and base editing techniques by employing, manipulating, and engineering TLS polymerases.