DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA–DNA base-pairing to target foreign DNA in bacteria. Cas9–guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9–RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9–RNA. Competition assays provide evidence that DNA strand separation and RNA–DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

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Figure 1: DNA curtains assay for target binding by Cas9–RNA.
Figure 2: Cas9–RNA remains bound to cleaved products and localizes to PAM-rich regions during the target search.
Figure 3: Cas9–RNA searches for PAMs and unwinds dsDNA in a directional manner.
Figure 4: PAM recognition regulates Cas9 nuclease activity.

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Acknowledgements

We thank P. Bhat, A. Smith and K. Zhou for technical assistance, and members of the Doudna and Greene laboratories and J. Cate for discussions and critical reading of the manuscript. S.H.S. acknowledges support from the National Science Foundation and National Defense Science & Engineering Graduate Research Fellowship programs. Funding was provided by the National Institutes of Health (GM074739 to E.C.G.) and the National Science Foundation (MCB-1154511 to E.C.G. and MCB-1244557 to J.A.D.). M.J. was a Research Specialist, E.C.G. is an Early Career Scientist, and J.A.D. is an Investigator of the Howard Hughes Medical Institute.

Author information

S.H.S. generated RNAs, conducted biochemical and single-molecule experiments, and assisted with single-molecule data analysis. S.R. conducted single-molecule experiments and data analysis, and assisted with the design and analysis of biochemical assays. M.J. cloned and purified Cas9, and assisted with the design and interpretation of initial single-molecule experiments. S.H.S., S.R., M.J., E.C.G. and J.A.D. discussed the data and wrote the manuscript.

Correspondence to Eric C. Greene or Jennifer A. Doudna.

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Competing interests

J.A.D. is an inventor on a related patent.

Extended data figures and tables

Extended Data Figure 1 Activity assays of reagents used in single-molecule experiments.

a, Cleavage assays were conducted using radiolabelled 55-bp DNA substrates that contained the six λ-DNA sequences targeted in Fig. 1d. Each DNA substrate (1 nM) was incubated with 100 nM Cas9–RNA complex reconstituted using the corresponding guide RNA, and reaction products were resolved by 10% denaturing polyacrylamide gel electrophoresis (PAGE). Reactions contained 3×-Flag-tagged Cas9 (where indicated) or untagged, wild-type Cas9. The asterisk denotes further trimming of the non-target strand. b, Cleavage assay of λ-DNA under conditions identical to those used in single-molecule experiments. Full-length λ-DNA (25 ng μl−1) was incubated with 10 nM Cas9–RNA reconstituted using the λ6 guide RNA, and reaction products were resolved by agarose gel electrophoresis. Successful cleavage is expected to generate DNA products that are 42,051 and 6,451 bp in length. When present, imaging components included anti-Flag antibody-coated quantum dots, YOYO1, BSA, glucose, and glucose oxidase/catalase.

Extended Data Figure 2 Binding histograms and Gaussian fits for λ-DNA target binding, and analysis of off-target binding.

a, Binding distributions for dCas9 programmed with λ1–λ6 guide RNAs were measured as described in Methods, and the data from each individual experiment were then bootstrapped and fit with a Gaussian curve. Error bars represent 95% confidence intervals; n = 366, 378, 373, 420, 397 and 363 for experiments with λ1–λ6 guide RNAs, respectively. Shown in number of base pairs is the mean, μ, and standard deviation, σ, obtained from each Gaussian fit, as well as the expected location of each target site in λ-DNA. b, Distribution of Cas9–RNA binding events for λ2 crRNA (n = 2,330, top) and spacer 2 crRNA (n = 2,190, bottom); error bars represent 95% confidence intervals. The density of PAM sites throughout the λ-DNA substrate is shown in red. c, Survival probabilities for non-target binding events with λ2 (n = 632) and spacer 2 (n = 607) crRNA; error bars represent 70% confidence intervals. Data were collected at 25 mM KCl.

Extended Data Figure 3 DNA binding by apo-dCas9 and dCas9–RNA.

a, Electrophoretic mobility gel shift assay (left) with radiolabelled 55-bp target DNA and increasing concentrations of dCas9–RNA, using a 10× excess of crRNA–tracrRNA over dCas9. The quantified data (right) were fit with a standard binding isotherm (solid line), and data from three such experiments yielded an equilibrium dissociation constant (Kd) of 0.49 ± 0.21 nM. b, Results for apo-dCas9 shown as in panel a. Data from three independent experiments yielded a Kd of 26 ± 15 nM. c, crRNA–tracrRNA duplex and heparin dissociate apo-dCas9 bound to nonspecific DNA, but not dCas9–RNA complexes bound to target DNA. 55-bp DNA substrates were pre-incubated with the indicated reagent for 15 min at 37 °C, at which point non-targeting crRNA–tracrRNA duplex (10–1,000 nM) or heparin (0.01–100 µg ml−1) was added. Reactions were incubated an additional 15 min at 37 °C and then resolved by 5% native PAGE. Reactions at the far right show that apo-dCas9 pre-bound to target DNA can be dissociated by complementary crRNA–tracrRNA and re-bind the same DNA in complex with RNA. Note the distinct mobilities of DNA in complex with apo-dCas9 versus DNA in complex with dCas9–RNA.

Extended Data Figure 4 Target DNA cleavage products remain bound to Cas9–RNA.

a, b, DNA substrates 72 nucleotides in length were radiolabelled at either their 5′ or 3′ ends and annealed to an unlabelled complementary strand, where indicated (top). The non-target strand contains the PAM (yellow box), whereas the target strand contains the sequence complementary to crRNA (red). Each DNA substrate (1 nM) was incubated with 100 nM Cas9–RNA complex for 30 min at room temperature, using nuclease-inactive D10A/H840A Cas9 (d), both nickase mutants (D10A, n1; H840A, n2), and wild type (WT). Half the reaction volume was quenched with formamide gel loading buffer containing 50 mM EDTA and analysed by 10% denaturing PAGE to verify the expected cleavage pattern of each sample (a). The other half of each reaction was analysed by 5% native PAGE to determine whether the radiolabelled DNA fragment remained bound to Cas9–RNA (b). Aside from an apparent reduced affinity for the single-stranded target strand after cleavage, wild-type Cas9–RNA shows an affinity for all four possible DNA products that is indistinguishable from the affinity of dCas9–RNA for uncleaved DNA substrates. Note that the order of samples in a and b is identical. The additional band present for double-stranded DNA substrates in panel a results from incomplete denaturation and partial migration of intact duplex into the gel (marked with an asterisk).

Extended Data Figure 5 Cas9–RNA acts as a single-turnover enzyme.

a, Agarose gel electrophoresis (1%, TAE buffer) was used to assess cleavage of plasmid DNA containing a λ2 target sequence as a function of Cas9–RNA concentration. DNA (25 nM) was incubated with the indicated concentration of Cas9–RNA, and aliquots were removed at each time point and quenched with gel loading buffer containing 50 mM EDTA. The gel was stained with ethidium bromide, and the quantified data are presented in Fig. 2b. b, Similar turnover experiments were conducted with 25 nM radiolabelled λ2 oligoduplex substrates and increasing concentrations of Cas9–RNA. Cleavage data were visualized by phosphorimaging; an asterisk denotes further trimming of the non-target strand. c, Turnover experiments with 25 nM Cas9–RNA were repeated at 37 °C and with a 10× excess of crRNA–tracrRNA over Cas9; neither condition significantly stimulates turnover. d, Quantified data from experiments in panels b and c show that each reaction reaches its maximum yield after 1 min and does not increase with further incubation time, demonstrating that Cas9–RNA exhibits single-turnover activity. Note that the observed requirement for a slight stoichiometric excess of Cas9–RNA over DNA to reach reaction completion is probably a result of our enzyme preparations not being 100% active. Although modest turnover (2.5-fold) was observed at a single enzyme–substrate stoichiometry in ref. 6, our results clearly demonstrate that the reaction yield remains proportional to the molar ratio between Cas9–RNA and DNA across a range of concentrations.

Extended Data Figure 6 Analysis of competition cleavage assays.

a, Representative cleavage assays as a function of competitor DNA concentration, using a competitor containing 12 PAM sites. Radiolabelled λ1 target DNA (1 nM) was incubated with 10 nM Cas9–RNA and increasing concentrations of the competitor, and reaction products at each time point were resolved by 10% denaturing PAGE. Cleavage data were visualized by phosphorimaging; an asterisk denotes further trimming of the non-target strand. b, Shown are the conditional survival probabilities for the radiolabelled target DNA at each concentration of 12-PAM competitor. c, Shown is the change in survival probability of the target DNA, ΔPs(t), for each 12-PAM competitor concentration. The area under each curve represents the amount of time that Cas9–RNA spent on the competitor DNA during the reaction. d, Competition data with a panel of substrates that have no complementarity to the guide RNA and variable numbers of PAMs, and a perfect target sequence with single-base-pair mutation in the PAM. The data are presented similarly to Fig. 3c, but the time bound to competitor is shown for all five concentrations of competitor tested.

Extended Data Figure 7 PAM sites in non-target DNA are bound specifically by dCas9–RNA.

a, None of the competitors from Fig. 3c can be cleaved, including one that bears full complementarity to the crRNA but contains a single-base-pair mutation in the PAM. Radiolabelled competitor DNAs and target DNA (1 nM) were incubated with 100 nM wild-type Cas9–RNA for the indicated time, and reaction products were assessed by 10% denaturing PAGE. The asterisk denotes further trimming of the non-target strand. b, PAM-rich competitor DNAs interfere with target DNA binding by dCas9–RNA. The same radiolabelled 55-bp target DNA from Fig. 3b, c was pre-mixed with increasing concentrations of the indicated competitor DNA and then incubated with 10 nM dCas9–RNA for 60 min at 37 °C. Binding reactions were resolved by 5% native PAGE. c, dCas9–RNA has increased affinity for non-target DNA containing multiple PAM sequences. The indicated radiolabelled DNA substrates (0.02 nM) were incubated with increasing concentrations of dCas9–RNA for 60 min at 37 °C, and reactions were resolved by 5% native PAGE. The observed well-shifting at high concentrations may result from multiple dCas9–RNA molecules binding the same DNA substrate.

Extended Data Figure 8 Cas9–RNA binds and cleaves bubble-containing DNA substrates with mismatches to the crRNA that are otherwise discriminated against within the context of perfect duplexes.

a, dCas9–RNA has weak affinity for a substrate containing a 2-bp mismatch to the crRNA (middle), whereas a substrate presenting the same mismatches within a small 2-nucleotide bubble (right) is bound with an affinity nearly indistinguishable from a perfect target substrate (left), in agreement with data presented in Fig. 3e. The indicated DNA substrates were incubated with increasing concentrations of dCas9–RNA for 60 min at 37 °C, and reactions were resolved by 5% native PAGE. b, The same bubble-containing substrate in panel a is cleaved with similar kinetics as a perfect substrate (compare right and left time courses), whereas a perfectly base-paired substrate with the same pattern of complementarity to the crRNA is cleaved with substantially reduced kinetics (middle). Radiolabelled DNA substrates (1 nM) were incubated with 100 nM wild-type Cas9–RNA for the indicated time, and reaction products were resolved by 10% denaturing PAGE. The asterisk denotes further trimming of the non-target strand.

Extended Data Figure 9 PAM recognition activates the nuclease activity of Cas9.

a, The indicated DNA substrates were prepared using the λ2 target sequence where the flanking region extending beyond the PAM was 16 bp (cleavage experiments) or 26 bp (binding experiments). b, For cleavage experiments, substrates were prepared by annealing the radiolabelled target strand (that is, substrate 2) to a 5× excess of cold complement, and 1 nM DNA was reacted with 50 nM Cas9–RNA at room temperature. Reaction products were resolved by 10% denaturing PAGE, and the quantified data were fit with single-exponential decays (solid lines). Results from three independent experiments yielded apparent pseudo-first-order cleavage rate constants of 9.0 ± 2.0 min−1 (substrate 1), 0.067 ± 0.027 min−1 (substrate 2), 0.066 ± 0.024 min−1 (substrate 3) and 7.3 ± 3.2 min−1 (substrate 4), and are presented as values relative to substrate 1 in Fig. 4b. Rate constants for substrates 2 and 3 are probably overestimates, as the reactions did not approach completion and the data were best fit with amplitudes well below 1. c, For binding experiments, substrates were gel purified after annealing the radiolabelled target strand to a 10× excess of cold complement. Binding reactions contained 0.1 nM DNA and increasing concentrations of dCas9–RNA, and were incubated at 37 °C for 1 h before being resolved by 5% native PAGE. The quantified data were fit with standard binding isotherms (solid lines). Results from three independent experiments yielded apparent Kd values of 0.27 ± 0.14 nM (substrate 1), 0.28 ± 0.12 nM (substrate 2), 0.59 ± 0.18 nM (substrate 3) and 0.21 ± 0.06 nM (substrate 4), and are presented as values relative to substrate 1 in Fig. 4b.

Extended Data Table 1 RNA and DNA substrates used in this study

Supplementary information

dCas9:RNA binds specific targets on single-tethered DNA curtains

Quantum dot-tagged dCas9:RNA (magenta) is shown bound to the λ2 target site in λ-DNA (green). At the beginning of the video, the DNA is extended via buffer flow. Subsequent interruption of flow results in the retraction of DNA and DNA-bound protein. (MOV 849 kb)

Cas9:RNA locates target sequences directly from solution

A single molecule of Cas9:RNA (white dot) is shown locating and stably binding the λ2 target site in λ-DNA (left). The unlabeled DNA is tethered at both ends, and the reaction occurs in the absence of buffer flow. Shown at right is tracking data of the binding event, which demonstrates the lack of 1D motion along the DNA axis. (MP4 4288 kb)

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Sternberg, S., Redding, S., Jinek, M. et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014) doi:10.1038/nature13011

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