School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
Center for Reproductive Biology, Washington State University, Pullman, WA 99164, USA.
Nucleic Acids Res. 2019 Mar 18;47(5):2389-2401. doi: 10.1093/nar/gky1278.
Cas9 binds and cleaves specific DNA sequences by inducing the formation of an R-loop between the guide RNA and its genomic target. While targeting of active Cas9 to a genomic locus is highly mutagenic because Cas9 creates DNA double strand breaks, targeting of dead Cas9 (dCas9) is presumed not to be mutagenic, as dCas9 lacks DNA endonuclease activity. Here, we show that dCas9 targeting induces mutations in yeast, particularly when targeted to the non-transcribed strand of a gene. dCas9-induced mutations cluster near the guide RNA target region and are comprised of single nucleotide substitutions, small insertions and deletions, and even complex mutations, depending upon the particular guide RNA target. We show that many of these mutations are a consequence of cytosine deamination events occurring on the non-target strand of the dCas9-induced R-loop, while others are associated with homopolymer instability or translesion DNA synthesis. Targeting of dCas9 by a mismatch-containing guide RNA also increases CAN1 mutation frequency, particularly in an ung1Δ mutant strain, suggesting that dCas9 induces mutations through similar mechanisms at off-target sites. These findings indicate that DNA binding by dCas9 is mutagenic in yeast, likely because dCas9 induces the formation of an R-loop at its target site.
Cas9 通过在向导 RNA 与其基因组靶标之间诱导 R 环的形成来结合并切割特定的 DNA 序列。虽然将活性 Cas9 靶向基因组位置具有高度突变性，因为 Cas9 会造成 DNA 双链断裂，但假定靶向失活 Cas9 (dCas9) 不会产生突变性，因为 dCas9 缺乏 DNA 内切酶活性。在这里，我们表明 dCas9 靶向会在酵母中诱导突变，特别是当靶向基因的非转录链时。dCas9 诱导的突变聚集在向导 RNA 靶标区域附近，并且由单核苷酸取代、小插入和缺失、甚至复杂突变组成，具体取决于特定的向导 RNA 靶标。我们表明，这些突变中的许多是 dCas9 诱导的 R 环中非靶标链上胞嘧啶脱氨酶事件的结果，而其他突变与同聚物不稳定性或跨损伤 DNA 合成有关。含有错配的向导 RNA 靶向 dCas9 也会增加 CAN1 突变频率，特别是在 ung1Δ 突变株中，这表明 dCas9 通过在非靶位点的类似机制诱导突变。这些发现表明 dCas9 在酵母中通过 DNA 结合具有致突变性，可能是因为 dCas9 在其靶位点诱导 R 环的形成。
