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Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis

Nature Structural & Molecular Biologyvolume 25pages347354 (2018) | Download Citation

Abstract

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding–rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.

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Acknowledgements

The project was supported by grants from the National Science Foundation (PHY-1430124 to T.H.) and National Institutes of Health (GM065367; GM112659 to T.H. and GM097330 to S.B.); T.H. is supported by the Howard Hughes Medical Institute. J.M. is supported by the National Institutes of Health Chemical Biology Interface training program (T32GM080189). We thank J. A. Doudna and S. H. Sternberg for useful early discussions about the design of experiments. We also thank S. H. Sternberg and J. S. Chen of the Doudna laboratory (University of California-Berkeley) for generously providing Cas9 stocks and EngCas9 expression plasmids, respectively.

Author information

Author notes

    • Jingyi Fei

    Present address: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA

Affiliations

  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Digvijay Singh
    • , Yanbo Wang
    • , Olivia Yang
    • , Anustup Poddar
    • , Scott Bailey
    •  & Taekjip Ha
  2. Bloomberg School of Public Health, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • John Mallon
    •  & Scott Bailey
  3. Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    • Jingyi Fei
    •  & Taekjip Ha
  4. Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    • Damon Ceylan
    •  & Taekjip Ha
  5. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    • Taekjip Ha
  6. Howard Hughes Medical Institute, Baltimore, MD, USA

    • Taekjip Ha

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Contributions

D.S. and T.H. designed the experiments. D.S. and Y.W. performed smFRET DNA-interrogation experiments. D.S. performed smFRET DNA-unwinding experiments. D.S. and J.M. performed gel-based experiments. D.S. and J.M. expressed and purified Cas9s. D.S., Y.W., O.Y., J.F., A.P., and D.C. performed or helped with the data analysis. O.Y. assisted with some experiments. A.P. assisted with PEG passivation of some slides. D.S., T.H., and S.B. discussed the data. D.S. and T.H. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Taekjip Ha.

Integrated supplementary information

  1. Supplementary Figure 1 FRET probe locations for DNA interrogation by Cas9–RNA and determination of Kd between Cas9–RNA and DNA.

    (a) Schematic of Cas9-RNA-DNA complex. The hybridized crRNA and tracrRNA are referred to as guide-RNA. Sequences in red denote guide sequence of the guide-RNA and the matching sequence of the DNA. The target strand is complementary to the guide-RNA. The non-target strand contains the PAM (5’-NGG-3’). A 22 nt biotin-labeled adaptor strand was used for surface immobilization of DNA and is highlighted in grey. (b) Cy3 and Cy5 labeling locations in Cas9-RNA-DNA complex (PDB ID: 4UN3)(Anders et al., Nature. 513, 569-573, 2014). (c) E histograms for cognate DNA and DNA with nPD=11 for dCas9, deCas9 and dCas9-HF1 vs. [Cas9-RNA] obtained in smFRET DNA interrogation experiments. (d-e) The apparent bound fraction vs. [Cas9-RNA] and fits for K d estimation. The number of PAM-distal mismatches (nPD) and PAM-proximal mismatches (nPP) are shown in cyan and orange respectively.

  2. Supplementary Figure 2 E histograms and Transition density plots for different DNA targets obtained from smFRET DNA-interrogation experiments at 20 nM Cas9–RNA.

    E histograms for (a) deCas9. (b) dCas9-HF1. (c) WT Cas9. (d) Transition density plots pictorialize the relative number of transitions between different FRET states, as identified by hidden Markov modeling. A small fraction of binding events showed rapid fluctuations between high and mid FRET states. The source of these fluctuations, also observed for WT Cas9(Singh et al., Nat Commun. 7, 12778, 2016), is unknown and dwells in these states were not included in the lifetime calculation. The number of PAM-distal mismatches (nPD) and PAM-proximal mismatches (nPP) are shown in cyan and orange, respectively.

  3. Supplementary Figure 3 Ultrastable binding of Cas9–RNA to DNA and parameters of DNA interrogation by Cas9–RNA (20 nM Cas9–RNA).

    (a-b) E histograms before and >60 min after washing away free Cas9-RNA in solution obtained from smFRET DNA interrogation experiments. Like WT Cas9-RNA(Singh et al., Nat Commun. 7, 12778, 2016; Sternberg et al., Nature. 507, 62-67, 2014), EngCas9-RNA remains near-irreversibly bound to the DNA targets if there are >8-9 PAM-proximal matches. (a) deCas9-RNA for cognate DNA and DNA with nPD = 11. (b) dCas9-HF1-RNA for cognate DNA and DNA with nPD = 10. (c-d) Like WT Cas9-RNA(Singh et al., Nat Commun. 7, 12778, 2016; Sternberg et al., Nature. 507, 62-67, 2014), catalytically capable EngCas9s do not release cleaved DNA products. The release of cleaved products would have resulted in disappearance of fluorescent/FRET spots. (c) E histograms before and after >60 minutes after removing free eCas9-RNA and Cas9-HF1-RNA for cognate DNA. (d) Representative images showing no reduction in the number of spots. (e) Normalized fraction of DNA molecules bound with Cas9-RNA. Fractions were normalized relative to the bound fraction of cognate DNA for each Cas9-RNA. (f) Average bound state lifetime obtained from dwell times of E >0.2 states. (g) Average lifetime of high FRET (E>0.6) binding events (τhigh), obtained from the dwell times of E>0.6 states. (h) Average lifetime of mid FRET binding events (τmid) obtained from dwell times of mid FRET (E>0.2 & E<0.6) states. (i) Fraction of binding events with mid FRET. (j) Average unbound state lifetime obtained from the single-exponential fits to the distributions of dwell times of E<0.2 state. The number of PAM-distal mismatches (nPD) and PAM-proximal mismatches (nPP) are shown in cyan and orange, respectively. Error bars represent s.d. for n = 3 or 2. Data for WT Cas9-RNA is taken from our previous study(Singh et al., Nat Commun. 7, 12778, 2016).

  4. Supplementary Figure 4 FRET probe locations for smFRET DNA-unwinding experiments and experiments showing that fluorescent labeling for smFRET DNA-unwinding experiments does not affect cleavage.

    (a) Schematic of Cas9-RNA-DNA complex. The hybridized crRNA and tracrRNA are referred to as guide-RNA. Sequences in red denote guide sequence of the guide-RNA and the matching sequence of the DNA. The target strand is complementary to the guide-RNA. The non-target strand contains the PAM (5’-NGG-3’). A 22 nt biotin-labeled adaptor strand was used for surface immobilization of DNA and is highlighted in grey. The donor is attached to the target strand in the 6th position from PAM and the acceptor is attached to the non-target strand in the 16th position from PAM. Formation of Cas9-RNA-DNA complex leads to the unwinding of DNA target, causing a decrease in E. (b) Similar FRET changes upon Cas9-RNA induced unwinding was observed when the donor and acceptor positions were swapped. (c) Probe locations in the Cas9-RNA-DNA complex (PDB ID: 5F9R(Jiang et al., Science. 351, 867-871, 2016)). (d) Schematic of cognate DNA target used in smFRET DNA unwinding experiments. Protospacer is highlighted in red and PAM in yellow. Also indicated are Cas9-RNA induced cleavage positions. Cleavage of the DNA targets analyzed by denaturing polyacrylamide gel electrophoresis and Cy5 imaging. Multiple cleaved products for the non-target strand likely arises from 3’-5’ additional exonuclease activity of RuvC domain(Jinek et al., Science. 337, 816-821, 2012). [DNA] =5 nM and [Cas9-RNA] =100 nM in 10 μl reaction. DNA was incubated with Cas9-RNA for ~75 minutes before being denatured by formamide loading buffer and heating at 95 °C for 10 minutes. The denatured sample products were then resolved using 15% polyacrylamide denaturing gel electrophoresis and imaged via Cy5 fluorescence. (e) Time courses of cognate DNA cleavage by eCas9-RNA with and without fluorescent probes (Cy3 and Cy5 pair) on the DNA show that labeling does not affect cleavage kinetics. Aliquots from a running reaction were taken at different time points and analyzed by PAGE. DNA targets were radio-labeled at the 5’ end of the target strand with 32-P via a T4 polynucleotide kinase reaction for visualization. (f) Time evolution of multiple cleavage products of non-target strand due to additional 3’-5’ exonuclease activity of RuvC. HNH has no exonuclease activity and consequently does not result in cleaved products of different sizes. The number of PAM-distal mismatches (nPD) is shown in cyan.

  5. Supplementary Figure 5 Cas9–RNA binding to FRET-labeled DNA and smFRET DNA-unwinding experiments at different Cas9–RNA concentrations and different frame rates of image acquisition.

    (a) Schematic of the DNA unwinding experiment as described in Fig. 2a. (b) E Histograms of DNA with nPD=0 and 1 without dCas9-RNA (red) and with increasing concentration of dCas9-RNA (blue). The concentration of dCas9-RNA is indicated within the inset for each histogram. For these DNA targets, the unwound fraction was taken as the fraction of DNA bound by dCas9-RNA. (c) The unwound fraction with increasing concentration of dCas9-RNA. These titrations experiments underlie the low Kd of dCas9-RNA binding to FRET labeled DNA constructs, as had been observed for unlabeled DNA(Sternberg et al., Nature. 507, 62-67, 2014). (d-e) E histograms (left) of DNA targets and their representative single molecule time traces of donor and acceptor intensities (middle) and E values (right). (d) DNA unwinding smFRET experiments performed at 5 nM of dCas9-RNA produce results similar to those observed with 100 nM Cas9-RNA. We used 100 nM for all unwinding experiments reported elsewhere in this study. (e-f) Frame rate of image acquisition for these experiments (e) 35 ms and (f) 100 ms. Both these experiments show similar E distributions. Although the overall signal was noisier for 35 ms data, fast transitions are better resolved.

  6. Supplementary Figure 6 Transition density plots for smFRET DNA-unwinding experiments and smFRET DNA-unwinding experiments using catalytically active Cas9–RNA.

    (a) Transition density plots from DNA unwinding experiments. These plots pictorialize the relative number of transitions between different FRET states, as identified by hidden Markov modeling(McKinney et al., Biophys J. 91, 1941-1951, 2006). (b) smFRET assay to investigate the Cas9-RNA induced DNA unwinding as described in Fig. 2a. smFRET data of DNA target only is shown. (c-e) E histograms vs nPD (left) and their representative time traces of donor and acceptor intensities (middle) and E values (right) for (c) WT Cas9 (d) eCas9 (e) dCas9-HF1. (f) Possible scenario of altered DNA unwinding-rewinding dynamics with cleaved strands in Cas9-RNA-DNA. The number of PAM-distal mismatches (nPD) is shown in cyan.

  7. Supplementary Figure 7 smFRET DNA-unwinding using surface-tethered Cas9–RNA and DNA-unwinding dynamics upon initial formation of Cas9–RNA–DNA complex.

    (a) Cartoon schematic of smFRET DNA unwinding experiments using surface-tethered Cas9-RNA is shown in Fig. 3. E histograms vs nPD obtained >~10 minutes after addition of labeled DNA (left), and the corresponding representative single molecule time traces of donor and acceptor intensities (middle) and their idealized E values (right) (b) The tracrRNA with the indicated 3’ extension for the biotinylated DNA adaptor, for surface-tethering Cas9-RNA, did not affect Cas9-RNA activity as shown by cleavage products analyzed by 15% denaturing polyacrylamide gel electrophoresis and Cy5 imaging. (c) Representative images of the donor and acceptor imaging channels before and after addition of labeled DNA with nPD = 2. (d) Schematic of experiment to observe the DNA unwinding dynamics during very first moments of Cas9-RNA-DNA complex formation. The Cas9-RNA immobilized flow chamber surface was imaged during the addition of the FRET pair labeled DNA target to the flow chamber. Results with catalytically dead Cas9s are in: (e) dCas9 (f) deCas9 (g) dCas9-HF1. Representative single molecule time traces of donor and acceptor intensities (left) and their idealized E values (middle). E density maps (right) show the real-time evolution of different FRET states synchronized to the moment of DNA binding to Cas9-RNA. Histograms (in border blue bars) show E distribution at the moment of binding (time = 0). (h) Average time it takes for catalytically dead Cas9-RNA to go from initial binding (no unwinding) to its first unwound state configuration (τunwinding) is described by a dark yellow line in the schematic shown in (a). (i) Data obtained using catalytically active WT Cas9. The number of PAM-distal mismatches (nPD) is shown in cyan.

  8. Supplementary Figure 8 Appearance of unwound state over time and Cas9–RNA induced unwinding of DNA with roadblocks of single base pair mismatches.

    (a) Schematics of smFRET DNA unwinding experiments as described in Fig. 2a. E Histogram of DNA target only (nPD=1) is shown. (b) E histograms of 1 nPD DNA target before and at different time points after adding 100 nM dCas9-HF1-RNA. Rapid appearance of a peak near E=0.5 indicates that Cas9-RNA induced DNA unwinding occurs as soon as Cas9-RNA-DNA complex forms. The number of PAM-distal mismatches (nPD) is shown in cyan. (c) Unwinding of DNA with internal single base mismatches at positions 16th and 18th. E histograms were obtained at [Cas9-RNA] =100 nM or in its absence. Idealized E traces, accompanied with the time traces of donor and acceptor intensities, show transient unwinding observed in a subset of molecules. It is also possible that a significant fraction of such transitions, with lifetime <0.1s, were not detected due to limited time-resolution of these experiments (0.1s). No FRET transitions were observed for DNA only (without Cas9-RNA).

  9. Supplementary Figure 9 smFRET unwinding experiments using pre-unwound DNA and probe locations for smFRET assay to investigate Cas9–RNA-induced DNA unwinding in PAM-proximal site.

    (a) Schematics of smFRET DNA unwinding experiments utilizing pre-unwound DNA targets as described in Fig. 5c. (b-d) E histograms vs nPD (left) and representative single molecule time traces of donor and acceptor intensities (middle) and idealized E values (right). (e) Locations of EngCas9 mutations in dCas9-RNA-DNA complex (PDB ID: 4UN3)(Anders et al., Nature. 513, 569-573, 2014) (f) Schematics of smFRET DNA unwinding experiments to investigate Cas9-RNA induced DNA unwinding in PAM-proximal site (g) Probe locations for smFRET experiments to investigate Cas9-RNA induced DNA unwinding in PAM-proximal site, mapped to the structure of Cas9-RNA-DNA complex (PDB ID: 5F9R(Jiang et al., Science. 351, 867-871, 2016)).

  10. Supplementary Figure 10 Unwinding in absence of magnesium and rare constant high FRET traces in DNA-unwinding experiments.

    (a) smFRET assay to investigate the Cas9-RNA induced DNA unwinding in dsDNA targets as described in Fig. 2a. (b) Without free divalent cations, the unwound fraction is unchanged for cognate DNA although final E value is slightly higher. But unwound fraction is much lower for DNA with nPD=3 with its smFRET traces showing rare cases of transient excursions to low FRET state (data not shown). These results indicate that divalent cations are not required for DNA unwinding but may help in unwinding. A fraction of high FRET population with nPD=3 in experiments without free divalent cations, could also be due to deleterious binding of Cas9 in absence of free divalent cations. In all unwinding experiments, amidst smFRET time-trajectories that showed FRET transitions, a small fraction of it did not show any and always remain at high FRET. These results indicate that Cas9-RNA remains bound to a small subset of DNA targets without ever trying to unwind them. A recent study found(Gong et al., Cell Rep. 22, 359-371, 2018) that ~15% Cas9-RNA-DNA complexes exist in a cleavage incompetent state and the aforementioned subset likely represents this cleavage incompetent state. (c-e) FRET histograms of DNA targets with increasing PAM-proximal mismatches and their corresponding representative smFRET time trajectories for different Cas9-RNA (dCas9; c), (deCas9; d) and (dCas9-HF1; e). Highlighted in black are rare constant high FRET traces, which indicates, that in small number of cases, Cas9-RNA could stably bind DNA and not unwind the DNA or even try to. Number of PAM-distal mismatches (nPD) is shown in cyan.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Tables 1 and 2

  2. Reporting Summary

  3. Supplementary Dataset 1

    Uncropped gel images.

  4. Supplementary Dataset 2

    Combined source data Excel spreadsheets for all main and supplementary figure files.

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https://doi.org/10.1038/s41594-018-0051-7

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