Extended Data Fig. 4: Fusing a highly processive DNA polymerase to enCas9 increases the target window length.

PolI was exchanged for a more processive and higher-fidelity bacteriophage Phi29 DNA polymerase (Phi29). Owing to Phi29 not having a flap endonuclease, residues 1–325 of PolI were inserted between enCas9 and Phi29. Using gRNAs targeting different distances from the nonsense mutation, we found that Phi29 with two previously reported fidelity-reducing mutations (N62D and L384R) elevated the mutation rate 56 nucleotides from the nick compared to the global mutation rate^28,29. When we expressed Phi29’s single-stranded binding protein (ssb), which is known to improve the activity of Phi29, we observed an elevation in the targeted mutation rate³⁰. Finally, because the activity of Phi29 is known to decrease at temperatures above 30 °C and the fluctuation analysis was performed at 37 °C, we added mutations previously reported to improve the thermostability of Phi29 (iPhi29) and observed a targeted mutation rate 347 nucleotides from the nick site that was significantly greater than the global mutation rate³¹. Unfortunately, mutations decreasing Phi29’s fidelity are known to decrease its processivity explaining our inability to identify Phi29 variants that retain high processivity while offering as high of a mutation rate as PolI3M²⁸. Data are mean ± 95% confidence intervals from ten biologically independent samples. *P < 0.0001; two-sided Student’s t-test.