dCas9 binding inhibits the initiation of base excision repair in vitro.
dCas9 binding inhibits the initiation of base excision repair in vitro.
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DOI：
10.1016/j.dnarep.2021.103257
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发表时间：
2022-01
期刊：
影响因子：
3.8
通讯作者：
Hinz JM
中科院分区：
文献类型：
--
作者：
Antony JS;Roberts SA;Wyrick JJ;Hinz JM
Cas9 在基因组编辑过程中通过在 Cas9 结合的引导 RNA 和目标 DNA 链之间形成 RNA:DNA 异源双链体（R 环）来靶向 DNA。我们最近证明，催化失活 Cas9 (dCas9) 形成的 R 环本质上具有诱变性，部分原因是通过促进 dCas9 诱导的 R 环非靶向单链 DNA 内的自发胞嘧啶脱氨基作用。然而，dCas9 结合和 R 环形成在多大程度上影响尿嘧啶损伤或其他受损 DNA 碱基的后续修复尚不清楚。在这里，我们证明 dCas9 与 DNA 结合可抑制体外尿嘧啶损伤的碱基切除修复 (BER) 的启动。我们的数据表明，在 dCas9:sgRNA 结合的靶链 (TS) 或单链非靶链 (NT) 中，尿嘧啶 DNA 糖基化酶 (UDG) 对尿嘧啶损伤的切割通常在 dCas9 结合的 DNA 处受到抑制。然而，NT 链碱基编辑器窗口内尿嘧啶损伤的裂解比其他位置受到的抑制较少，表明该位点更容易发生 UDG 活性。此外，我们的数据表明 dCas9 与 PAM 位点结合可以抑制 UDG 活性。然而，这种非特异性抑制可以通过添加与 DNA 底物缺乏序列互补性的 sgRNA 来缓解。此外，我们发现 dCas9 结合还能抑制人单链选择性单功能尿嘧啶 DNA 糖基化酶 (SMUG1)。随后对 Cas9 结合靶位点的结构分析表明了 BER 抑制的分子机制。综上所述，我们的结果表明 dCas9（或 Cas9）结合可能通过抑制 BER 对 DNA 碱基损伤的去除来促进背景诱变。
Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear. Here, we show that DNA binding by dCas9 inhibits initiation of base excision repair (BER) for uracil lesions in vitro. Our data indicate that cleavage of uracil lesions by Uracil-DNA glycosylase (UDG) is generally inhibited at dCas9-bound DNA, in both the dCas9:sgRNA-bound target strand (TS) or the single-stranded non-target strand (NT). However, cleavage of a uracil lesion within the base editor window of the NT strand was less inhibited than at other locations, indicating that this site is more permissive to UDG activity. Furthermore, our data suggest that dCas9 binding to PAM sites can inhibit UDG activity. However, this non-specific inhibition can be relieved with the addition of an sgRNA lacking sequence complementarity to the DNA substrate. Moreover, we show that dCas9 binding also inhibits human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Structural analysis of a Cas9-bound target site subsequently suggests a molecular mechanism for BER inhibition. Taken together, our results imply that dCas9 (or Cas9) binding may promote background mutagenesis by inhibiting the removal of DNA base lesions by BER.
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