The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR–Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR–Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.

中文翻译：

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APOBEC3在CRISPR–Cas9产生的DNA断裂修复过程中诱导突变

胞苷脱氨酶的APOBEC-AID家族更倾向于将单链核酸用于胞嘧啶-尿苷脱氨。DNA修复系统通常涉及单链核酸，以解决CRISPR–Cas9产生的断裂。在这里，我们在人细胞中显示，APOBEC3可以触发单链寡聚脱氧核苷酸的胞苷脱氨，这最终通过Cas9产生的双链断裂的同源性定向修复（HDR）导致基因组DNA中的碱基取代突变。另外，在人细胞中Cas9切口酶产生的单链断裂的修复过程中形成的基因组单链DNA中，APOBEC3催化的脱氨作用可以进一步处理以产生主要涉及插入或缺失（indels）的突变。APOBEC3介导的脱氨和DNA修复蛋白在这些indel的产生中都起着重要作用。因此，优化条件以修复CRISPR–Cas9产生的DNA断裂，例如在HDR中使用双链供体或暂时抑制内源性APOBEC3，可以抑制基因组DNA中这些不需要的突变。