Extended Data Fig. 2: The direction of EvolvR-mediated mutagenesis relative to the gRNA is dependent on which strand is nicked.

Our previous fluctuation analysis in Fig. 1e demonstrated that nCas9(D10A)–PolI3M mutates a window 3′ of the nick site. Here we directly tested whether mutations are generated 5′ of the nick site using a different gRNA. Because DNA polymerases synthesize in the 5′-to-3′ direction, we anticipated that nCas9(D10A)–PolI3M would not provide an elevated mutation rate 5′ of the nick site. We indeed found that expressing a guide RNA which targeted nCas9(D10A)–PolI3M to nick 16 nucleotides 3′ from the nonsense mutation (indicated by a red cross) did not show targeted mutagenesis. We hypothesized that we could induce targeted mutagenesis using the same gRNA by using a Cas9 variant harbouring the H840A mutation, which nicks the DNA strand non-complementary to the gRNA, rather than the D10A mutation, which nicks the strand complementary to the gRNA. nCas9(H840A)–PolI3M increased the mutation rate 16 nucleotides 3′ from the nick by 52-fold compared to the global mutation rate of cells expressing an off-target gRNA. We used the D10A nCas9 variant for all subsequent experiments. Data are mean ± 95% confidence intervals from ten biologically independent samples. *P < 0.0001; two-sided Student’s t-test.