Supplementary Figure 6: Bulk cleavage assays with various bubble sizes and location, and bona fide cellular EMX1-1 off-targets.

a Cleavage assay for OT2 with different PAM sequences. Efficient cleavage is only observed in the presence of canonical (NGG) or non-canonical (NAG) PAM sequences with equal efficiency. b Cleavage assay for OT3 with λ2-crRNA:tracrRNA:wtCas9, and bubbles from 0–20 nt. Cleavage is only observed with ≥14 nt bubbles. c Cleavage assay for ON and OT1–3 with small (6 nt) PAM-proximal, middle and -distal bubbles. Cleavage is only observed for ON. d Cleavage assay for ON and OT1–3 with double-stranded (ds), single-stranded (ss), double-stranded PAM (dsPAM) and full bubble constructs (bub). Most efficient OT cleavage is only observed in the presence both dsPAM and non-target strand. All with 100 nM wtCas9 complex (see methods). e Genomic locations and sequences of EMX1-1 target (ON) and four off-targets (OT) identified with CIRCLE-seq and validated in cells. Seed sequence in bold, mismatches (4 each) highlighted in red and underlined. f Denaturing PAGE cleavage assay (left) and quantification (right, n = 3) shows that CRISPR-Cas9 cuts bubbled (bub) OT substrates with four mismatches and not double-stranded ones (ds). Error bars = s.d.