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. 2016 Sep 14;7:12778.
doi: 10.1038/ncomms12778.

Real-time Observation of DNA Recognition and Rejection by the RNA-guided Endonuclease Cas9

Free PMC article

Real-time Observation of DNA Recognition and Rejection by the RNA-guided Endonuclease Cas9

Digvijay Singh et al. Nat Commun. .
Free PMC article


Binding specificity of Cas9-guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s(-1) to >2 s(-1) upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s(-1)), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

Conflict of interest statement

S.H.S and J.A.D. are inventors on a related patent application. The other authors declare no competing financial interests.


Figure 1
Figure 1. Cas9–RNA binding to a cognate sequence.
(a) Schematic of single-molecule FRET assay. High-FRET signal resulted when Cas9 in complex with an acceptor (Cy5)-labelled guide-RNA (Cas9–RNA) bound a surface-immobilized, donor (Cy3)-labelled target DNA that contains the cognate sequence (red DNA segment) and PAM (yellow segment). (b) A representative smFRET time trajectory of a stably bound Cas9–RNA in the presence of 20 nM Cas9–RNA in solution. (c) FRET histograms obtained with cognate DNA (top) and negative controls with a non-cognate DNA (middle) and with RNA only (without Cas9; bottom). The number of molecules included ranged from 568 to 1,314. Corresponding images of donor and acceptor channels are shown. (d) A representative smFRET time trajectory of real-time binding of Cas9–RNA in a single step after 20 nM Cas9–RNA is added at the time point indicated.
Figure 2
Figure 2. Cas9–RNA binding to DNA with proximal or distal mismatches.
(a) A series of fully duplexed DNA targets with a varying number of mismatches (black segments) relative to the guide RNA. An xymm target has a contiguous mismatch running from position x to y relative to PAM. (b,c) FRET histograms of Cas9–RNA binding to DNA constructs carrying PAM-distal (b) and PAM-proximal (c) mismatches. The number of molecules for each histogram ranged from 568 to 3,053. [Cas9–RNA]=20 nM. (d) The fraction of Cas9–RNA-bound DNA molecules for different DNA targets. All the data shown in the figure are from independent experiments and error bars represent s.d. for n=3 (n=2 for few sets).
Figure 3
Figure 3. Cas9–RNA-bound state lifetimes for different DNA targets.
(a) smFRET time trajectory (donor and acceptor intensities, top, and idealized FRET via hidden Markov modelling (HMM) analysis, bottom) for 9–20mm DNA target in the presence of 20 nM Cas9–RNA. Reversible Cas9–RNA association to high- and mid-FRET states and disassociation to zero-FRET state are shown. (b) Transition density plots show relative transition frequencies between different FRET states for 9–20mm and 5–20mm DNA targets. [Cas9–RNA]=20 nM. (c) The amplitude-weighted lifetime, τavg, of the putative bound state, lifetime of high to zero and mid to zero FRET state transitions and biomolecular rate association constants for different DNA targets. On the basis of our model, the mid- and high-FRET states correspond to sampling and RNA–DNA heteroduplex modes, respectively. (d) Lifetime comparison of DNA targets with the respective DNA targets containing mismatches after the roadblock. All the data shown in the figure are from independent experiments and error bars represent s.d. for n=3 (n=2 for few sets).
Figure 4
Figure 4. The proposed model of bimodal Cas9–RNA binding along with the kinetics of Cas9–RNA DNA targeting as a function of mismatches.
Cas9-RNA targeting occurs in predominantly two steps. The first step, that is, initial Cas9-RNA binding to DNA target is a transient PAM surveillance step, independent of the DNA sequence. In the second step, following the PAM detection, Cas9-RNA proceeds to form RNA-DNA heteroduplex in a unidirectional manner, that is, from PAM-proximal to PAM-distal end. Rate of transition between various Cas9-RNA targeting steps for different DNA targets is indicated by the size of arrows.

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