R-loop formation by dCas9 is mutagenic in Saccharomyces cerevisiae

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.