CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
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We thank E. Gordon (AstraZeneca) for preparing the Cas9 purified proteins, M. Modesti (CRCM, Marseille) for providing GFP-hRPA, and A. Candelli and J. Cabanas (LUMICKS B.V.) for assistance with the initial tweezers experiments. The Single Molecule Imaging Group is funded by a core grant of the MRC-London Institute of Medical Sciences (UKRI MC-A658-5TY10), a Wellcome Trust Collaborative Grant (206292/Z/17/Z), a Leverhulme Grant (RPG-2016-214), and a BBSRC CASE-studentship (to M.D.N.). The Computational Regulatory Genomics Group is supported by the Medical Research Council UK (MC UP 1102/1), L.R. is supported by the Wellcome Trust (106954) and N.C. is supported by EMBO LTF (EMBO ALTF 1279-2016).
R.P.C.D. is an employee and shareholder of LUMICKS.
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Integrated supplementary information
Supplementary Figure 1 No binding is observed with crRNA alone, with crRNA:tracrRNA or crRNA:Cas9 at low or high force or with nt-crRNA:tracrRNA:dCas9 at low force.
a Kymograph of force-stretched λ-DNA in the presence of 5′-Cy3-λ2-crRNA without tracrRNA or dCas9. b Kymograph of force-stretched λ-DNA in the presence of annealed 5′-Cy3-λ2-crRNA:tracrRNA. c Kymograph of force-stretched λ-DNA in the presence of dCas9 and 5’-Cy3-λ2-crRNA without tracrRNA. d Kymograph of low force-stretched (10 pN) λ-DNA in the presence of dCas9 and 5′-Cy3-nt-crRNA:tracrRNA. Vertical scale bar = 1 μm. e Bulk cleavage assay of Cy3-labeled λ2 On-Target DNA (20 nM) (Supplementary Table 1) with λ2-crRNA:tracrRNA:wtCas9 (50 nM). Visualized by 18% denaturing PAGE with separate fluorescent detection for Cy3 and Cy5 fluorescence. Lane 1, no Cas9 complex, Lane 2, unlabeled λ2-crRNA:tracrRNA and wtCas9 (Supplementary Table 1), Lane 3 5′-Cy5-λ2-crRNA:tracrRNA and wtCas9 (Supplementary Table 1). Cy5 labeling of the modified crRNA does not affect λ2 target cleavage efficiency. f Image Analysis Pipeline i) Kymographs are time-binned and their average intensity calculated ii) Bead edge is defined as point where intensity hits background intensity of region known to be DNA iii) Bead trimmed DNA is linearly mapped across known λ-DNA sequence, alignments corrected on the basis of on-target, then smoothed by FFT and intensities within each time window normalized iv) Binding events are detected using ridge detection ImageJ plugin v) Lengths of binding events are calculated and used to build dwell-time histograms. Vi) Position of each event is calculated, mapped back to λ-DNA sequence and used to build histograms of binding sites.
a Dwell-time histogram of Cy3-λ2-crRNA:tracrRNA:dCas9 off-target λ-DNA binding events induced at forces from 20–50 pN. Bin size = 2 s. b Integrated cumulative probability density plots with corresponding exponential fits and fitting values. Data at 20 pN was fit with single exponential. Data for 30–50 pN was fit with a double exponential. Fit coefficient ± s.d.
Supplementary Figure 3 Off-target binding locations occur at non-random, guide sequence-specific sites.
a GC content or PAM site distribution across the λ-genome. Distribution of canonical PAM sites (NGG) and alternative PAM sites (NAG) across λ-DNA (Top). Distribution of GC content across λ-DNA (Bottom). Bin size = 200 bp. b Average time-binned intensity histograms for λ2, λ4 and nt guide. Off-targets are induced on force stretching DNA to 40 pN and show recurrent non-random off-target binding locations. Arrows marks on-target sites for λ2 (18.5 kb) and λ4 (30.5 kb). c,d,e Histograms (500 nt bin-width) of mapped off-target binding events at 40 pN for 3 guides (λ2-, λ4- and n.t.-crRNA, respectively). Each guide experiment was repeated in triplicate (λ2-crRNA, n = 278, 645 and 311; λ4-crRNA, n = 437, 210 and 150; n.t.-crRNA, n = 605, 876 and 469). f Pearson’s correlation analysis between count normalized histograms of each binding localization repeat (Rep. 1–3) and guide. A strong correlation (dark red) is observed between experiments with the same crRNA guide but not (light red) between different guides, demonstrating that force-induced off-target binding is DNA and crRNA sequence specific. g Kymograph of λ-DNA in the presence of dCas9 with 5′-Cy3-nt-crRNA:tracrRNA (green) and hRPA-eGFP (blue). Off-target binding is observed with increasing force. hRPA binding is observed on nicked DNA and large ssDNA regions. To maintain constant force, extension increases to compensate for DNA nick formation. Off-target Cas9 binding and hRPA binding are mutually exclusive suggesting that two DNA strands are required for Cas9 binding. h Kymograph of 100 nM wtCas9 in complex with non-target 5′-Cy3-nt-crRNA:tracrRNA. Overstretched nicked DNA generates large ssDNA regions, which Cas9 no longer binds. Source data.
a Example processing of the λ2-crRNA intensity. Raw intensities were normalized to maximum signal, aligned to account for small peak shifts caused by force stretching the λ-DNA, and smoothed using FFT function to reduce the noise. b Heatmap representation of guide-RNA sequence weighted nucleotide matches that fall within a peak, that is Cas9 binding event (Type of match: yes) versus outside the peak, that is Cas9 not bound (Type of match: no). A minimum 60% sequence match (12/20 nucleotides) of crRNA is defined. Identified matches were extracted and its sequence composition analyzed. All matching sequences were ordered by distance to the closest peak maxima and clustered according to in vs outside peak location (in peak is up to 200 bp distance from the peak maxima). Matching to the PAM sequence (NGG) was analyzed separately.
Supplementary Figure 5 Example FRET trajectories for binding to bubble constructs and FRET histograms of crRNA only, double-stranded PAM and ssDNA.
a FRET histograms of the full bubble DNA constructs with 10 nM 5′-Cy5-λ2-crRNA:tracrRNA only (no dCas9). A small population (21%, green, n = 1530) of high FRET molecules bind on-target bubble DNA but not off-targets 1–3 (n = 1610, 2094, and 2120 respectively). b Representative FRET trajectories for λ2-crRNA:tracrRNA:dCas9 binding on-target and off-target bubble constructs. Stable high FRET is observed for on-target and off-targets 1 and 3 followed by single-step photobleaching. For off-target 2 dynamic equilibrium between FRET of ~0.55 and ~0.85 is observed followed by single-step photobleaching. c FRET histograms for ssDNA targets with 10 nM 5′-Cy5-λ2-crRNA:tracrRNA:dCas9. A small population (25%, green, n = 3717) of high FRET molecules bind the on-target constructs but not off-targets 1–3 (n = 2234, n = 1499, n = 1012 for OT1, OT2 and OT3 respectively). d FRET histograms for double-stranded PAM constructs with 10 nM 5′-Cy5-λ2-crRNA:tracrRNA:dCas9 show a small high FRET population (20%, n = 1976; 22%, n = 3062; 23%, n = 2127; 31%, n = 2874; for ON, OT1, OT2 and OT3, respectively). e FRET histograms for a small (6 nt) PAM-proximal bubble. A bound population is observed for on-target (FRET = 0.97 ± 0.02, 36%, n = 3863), OT1 (FRET = 0.23 ± 0.02, 52%, n = 2466) and OT2 (FRET = 0.27 ± 0.02, 75%, n = 3704) but not for OT3 (n = 626).
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.
Supplementary Figures 1–6, Supplementary Tables 1–3 and Supplementary Note 1
DNA stretching induces Cas9 off-target binding Force stretching λ-DNA from 5 pN to 50 pN induces reversible off-target binding at multiple sites. Confocal time-lapse movie initially shows a single λ2-crRNA-dCas9 complex (green dot) bound on-target to λ-DNA held between two beads by optical tweezers at low force (5 pN). As force is slowly increased to 50 pN, multiple off-target binding events appear. Off-targets dissociate while the force is decreased back to 5 pN, and only the on-target complex remains bound. Time stamp in mm:ss.
Gel images for Fig. 4f. Raw images of gels used for quantification for Figure 4f.
Table of dwell times at different forces of all events used for kinetic analysis in Figure 2b,c and Supplementary Figure 2.
Table of locations of detected binding events used to generate binding distribution histograms in Figure 3c and Supplementary Figure 3c–f. Contains three repeats of each of the three guides tested.
Quantifications of cleavage efficiencies of different bubble sizes for the three off-targets tested used to plot Figure 4f.
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Newton, M.D., Taylor, B.J., Driessen, R.P.C. et al. DNA stretching induces Cas9 off-target activity. Nat Struct Mol Biol 26, 185–192 (2019). https://doi.org/10.1038/s41594-019-0188-z
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