Fig. 4

PAM preferences and cleavage positions of Brevibacillus laterosporus SSP360D4 (Blat) Cas9 enzyme. Blat Cas9 PAM preferences when 1 μg of library DNA was cleaved with 0.5 nM or 50 nM Cas9-sgRNA complex (a), extended out to position 10 by shifting the protospacer target by 3 bp (b). Frequency of nucleotides at each PAM position was independently calculated using a position frequency matrix (PFM) [21] and plotted as a WebLogo [23]. c Cleavage rates of supercoiled plasmid DNA substrates containing mutations (shown in red) in GTCCCGAA PAM sequence. All data points are mean values from ≥3 independent experiments. Error bars are given as S.D. d Run-off sequencing from both sense and anti-sense directions of plasmid DNA cleaved with Blat Cas9