Abstract
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.
Highlights
CRISPR/Cas9 RNA-guided endonucleases are a transformative tool for eukaryotic genome engineering [1,2] due to their unique ability to bind and cleave specific DNA sequences that are complementary to a Cas9-bound singleguide RNA
While the guide RNAs were constitutively expressed, expression of dead’ mutant of Cas9 (dCas9) was under the control of the pGAL promoter [32]. dCas9 expression was induced by growth on galactose-containing plates for ∼6 days prior to assaying for CAN1 mutations on glucose medium with canavanine
Targeting dCas9 to the CAN1 transcribed strand (TS) significantly increased CanR frequency (P = 0.026). The magnitude of this increase (∼10-fold relative to the ‘No singleguide RNA (sgRNA)’ control in Figure 1D) was significantly lower than when the non-transcribed strand (NTS) was targeted (P = 0.0022). These results indicate that dCas9 targeting significantly stimulates mutagenesis, when the dCas9/guide RNA complex is targeted to the CAN1 NTS
Summary
CRISPR/Cas RNA-guided endonucleases are a transformative tool for eukaryotic genome engineering [1,2] due to their unique ability to bind and cleave specific DNA sequences that are complementary to a Cas9-bound singleguide RNA (sgRNA). Specific targeting of Cas (or dCas9) relies on the formation of an ∼20-nucleotide-long R-loop, consisting of an RNA:DNA hybrid between the target DNA strand and the Cas9-bound sgRNA [15]. Following PAM recognition, Cas promotes the progressive unwinding of the DNA target and sgRNA invasion, beginning with the ∼10 nucleotide ‘seed’ region of the sgRNA that is immediately proximal to the PAM [17]. This results in the formation of an R-loop between the target DNA strand and the sgRNA, which induces a conformational change in the Cas protein that triggers DNA cleavage [18,19]
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