School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.
Nat Struct Mol Biol. 2018 Jan;25(1):45-52. doi: 10.1038/s41594-017-0004-6. Epub 2017 Dec 11.
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.
APOBEC-AID 家族的胞嘧啶脱氨酶偏爱单链核酸进行胞嘧啶到尿嘧啶的脱氨反应。单链核酸通常参与由 CRISPR-Cas9 产生的断裂的 DNA 修复系统。在这里，我们在人类细胞中表明，APOBEC3 可以触发单链寡脱氧核苷酸的胞嘧啶脱氨反应，最终通过 Cas9 产生的双链断裂的同源定向修复 (HDR) 将碱基替换突变引入基因组 DNA。此外，在人类细胞中修复 Cas9 尼克酶产生的单链断裂期间形成的基因组单链 DNA 中的 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 介导的脱氨作用和 DNA 修复蛋白在这些 indels 的产生中都起着重要作用。因此，优化 Cas9 产生的 DNA 断裂修复条件，例如在 HDR 中使用双链供体或暂时抑制内源性 APOBEC3，可以抑制这些基因组 DNA 中的不必要突变。
