Josh Cofsky
Josh Stamp


CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

Joshua C Cofsky, Deepti Karandur, Carolyn J Huang, Isaac P Witte, John Kuriyan and Jennifer A Doudna

ELife 2020;9:e55143     (local copy)

Abstract

Type V CRISPR-Cas interference proteins use a single RuvC active site to make RNAguided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one strand of a double-helical DNA substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the doublestrand break. Here, we detect intrinsic instability in DNA flanking the RNA-3' side of R-loops, which Cas12a can exploit to expose second-strand DNA for cutting. Interestingly, DNA flanking the RNA-5' side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

Figures from the paper

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Figure 1 from paper

Figure 1 - Structure of Cas12a and comparison of its DNA cleavage pathway to that of Cas9.


(A) Crystal structure of the DNA-bound Cas12a interference complex from Francisella novicida (FnCas12a, PDB 61IK) (Swarts and Jinek, 2019). While the protein ortholog used for most experiments in this manuscript is from Acidaminoccus species (AsCas12a,~40% identity to FnCas12a), the FnCas12a crystal structure shown here represents the most complete structure of such a complex to date, most notably with respect to the DNA at the target-strand cleavage sites. We did not perform any experiments with the particular DNA sequence used by Swarts and Jinek in crystallization, so the scissile phosphodiesters indicated were determined for a different sequence (see Appendix 2—figure 1, Appendix 2—figure 1—figure supplement 6) and superimposed onto the structural model according to their distance from the PAM (in terms of number of nucleotides). The discontinuity modeled into the non-target strand corresponds to positions of weak electron density in the crystal structure, which could have been due to some combination of disorder of the (intact) intervening tract and/or in crystallo hydrolysis and dissociation of the intervening tract.
Figure 1 from paper

Figure 1 continued - Structure of Cas12a and comparison of its DNA cleavage pathway to that of Cas9.


(B) For Cas12a, successful R-loop formation results in activation of the RuvC DNase active site to cleave three classes of DNA substrates (yellow scissors): the non-target strand (in cis), the target strand (in cis), and non-specific ssDNA (in trans). Circled numbers indicate the required order of cis strand cleavage; three conserved active site carboxylates of the RuvC DNase are shown in yellow and red; ‘PAM’ indicates the protospacer-adjacent motif; red arrow indicates the direction in which the R-loop is opened. Cas9 contains two DNase domains: the RuvC domain cleaves the non-target strand, and the HNH domain cleaves the target strand.
Figure 2 from paper

Figure 2 - The target-strand cleavage site becomes distorted upon R-loop formation.


(A) Denaturing PAGE phosphorimages of piperidine-treated permanganate oxidation products, demonstrating the assay’s ability to detect non-B-form DNA conformations within and adjacent to a dCas12agenerated R-loop. Permanganate reactions were quenched after 10 s at 30˚C. Each thymine in the DNA substrate is shown as a circled T.
Figure 2 from paper

Figure 2 continued - The target-strand cleavage site becomes distorted upon R-loop formation.


(B) Permanganate reactivity of a PAM-distal R-loop flank whose sequence was changed (as compared to the native protospacer sequence that was probed in A) to contain more thymines, with an intact or cleaved non-target strand (‘cleaved NTS’ indicates that there is a 5-nt gap in the NTS—see Appendix 2). Permanganate reactions were quenched after 2 min at 30˚C. A raw phosphorimage is shown in Figure 2—figure supplement 3. The permanganate reactivity index (PRI) is an estimate of the rate of oxidation at each thymine, normalized such that PRI = 1 for a fully single-stranded thymine (see Materials and methods). Columns and associated error bars indicate the mean and standard deviation of three replicates. The phosphodiester bonds normally cleaved by WT Cas12a are indicated with arrows on the substrate schematic for reference, but note that the complexes being probed with permanganate were formed with dCas12a.

Figure 3 from paper

Figure 3 - DNA distortion in the R-loop flank facilitates target-strand cleavage.


(A) Permanganate reactivity of A/T tract in a 20-nt R-loop and an 18-nt R-loop. Permanganate experiments were conducted as in Figure 2B (2 minutes, 30˚C). Purple rectangles alongside DNA schematics indicate the location of the tract of DNA whose permanganate reactivity is being quantified. The y-axis denotes the fraction of DNA molecules estimated to have been oxidized on at least one thymine within the A/T tract (see Materials and methods). Columns and associated error bars indicate the mean and standard deviation of three replicates.
(B) Target-strand cut-site distribution with a shrinking R-loop, as resolved by denaturing PAGE and phosphorimaging (n = 3). 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 37˚C for 1 hr, prior to quenching and resolution by denaturing PAGE (kinetics shown in Figure 3—figure supplement 4). Each lane corresponds to a different DNA target, bearing varying numbers of PAM-distal mismatches with respect to the crRNA. Indicated above each lane is the number of base pairs of complementarity between the target strand and the crRNA spacer, starting with the base immediately adjacent to the PAM. For the lane lacking an asterisk, the DNA target was fully duplex. For the lanes that bear asterisks, the DNA target contained a bubble across the region of crRNA:TS complementarity, which stabilized the interaction of the DNA with the Cas12a/crRNA complex. Numbers to the left of the phosphorimage indicate the position (distance from the PAM, as numbered in C) of the dinucleotide whose phosphodiester was cleaved to yield the labeled band. Black arrows are drawn on the substrate diagrams to indicate cleaved phosphodiesters (as determined from the phosphorimage), and relative arrow lengths are roughly reflective of relative band intensities.
Figure 3 from paper

Figure 3 continued - DNA distortion in the R-loop flank facilitates target-strand cleavage.


(C)Target-strand cut-site distribution with various sequences in the R-loop flank (all with a 20-nt R-loop), as resolved by denaturing PAGE and phosphorimaging (n = 3). 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 25˚C for 10 min, prior to quenching and resolution by denaturing PAGE (kinetics shown in Figure 3—figure supplement 7). All DNA targets were 5’-radiolabeled on the target strand. The non-target strand contained a gap from positions 14–18 (see Appendix 2) but was complementary to the target strand at positions 1–13 and 19–20. In each lane, the DNA target was varied to contain different sequences in the R-loop flank, which either formed a perfect duplex (substrates A, C, and E) or contained a 3-bp NTS:TS mismatch (substrates B, D, and F). Black arrows are drawn on the substrate diagrams as in B.
Figure 4 from paper

Figure 4 - DNA distortion is protein-independent and unique to 3’ R-loop flanks.


(A) Permanganate reactivity of the A/T tract in a dCas12a R-loop, a dCas9 R-loop, and their protein-free mimics. The y-axis denotes the fraction of DNA molecules estimated to have been oxidized on at least one thymine within the A/T tract (see Materials and methods). Purple rectangles alongside DNA schematics indicate the location of the tract of DNA whose permanganate reactivity is being quantified. Columns and associated error bars indicate the mean and standard deviation of three replicates.
(B) Model for the relative conformational dynamics of 3’ and 5’ R-loop boundaries, as suggested by permanganate reactivity experiments. The depth of fraying shown (three base pairs) was chosen arbitrarily for the schematic and should not be interpreted as a uniquely stable ‘open’ structure (see Materials and methods).

Figure 5 from paper

Figure 5 - Energetics of base stacking at the R-loop boundary probed by optical measurements and molecular dynamics simulations.


(A) Melting temperatures of nicked-dumbbell constructs that recapitulate each type of R-loop boundary, determined by monitoring absorbance of ultraviolet light while slowly cooling samples from 95˚C to 2˚C. Reported values show mean and standard deviation of three replicates. See Figure 5—figure supplement 1 for refolding curves and control constructs.
(B) Molecular dynamics simulations reveal nucleobase unstacking in 3’ R-loop boundaries but not in 5’ R-loop boundaries. At the top left is a schematized version of the true structural model shown immediately below (this coaxially stacked conformation is the starting structure that was used for simulation); hydrogens were present in the simulated model and analyses but are omitted from representations here for clarity. The simulated model contained only the nucleic acid molecules shown in stick representation; the protein and remainder of the R-loop are drawn in a schematic only to orient the reader as to where the simulated structure would fit into a full DNA-bound CRISPR interference complex; the Cas9-orientation R-loop is drawn with a Cas12a-like NTS gap to reflect the simulated model.

Figure 5 continued- Energetics of base stacking at the R-loop boundary probed by optical measurements and molecular dynamics simulations.


The inset is a closeup of the two nucleotides on the ‘flapped’ side of the junction in the structural model; the 2’-OH is shown as a red sphere. Envelope surface area (ESA) was determined by isolating two nucleobases of the interhelical stack—that on the RNA terminus and that stacked upon it from the NTS—and calculating the surface area of the volume they jointly occupy over the course of each trajectory (envelope shown in cyan). High ESA values reflect unstacking of nucleobases, whereas low ESA values reflect a stacked architecture similar to that of the starting conformation. Pale lines are absolute ESA values, and bold lines are moving averages (1-ns sliding window). Data from ten independent 50-ns trajectories are shown in different colors. Simulations of a second set of sequences are described in Figure 5—figure supplement 2.
Figure 6 from paper

Figure 6 - Model for the double-strand-break formation pathway of Cas12a and that of an analogous (hypothetical) enzyme with inverted R-loop topology.


Scissile DNA tracts are shown in yellow. The stable interhelical stack in the hypothetical inverted complex is highlighted in white.


Appendix

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Figure 7 from paper

Appendix 1—figure 1 - Substrate specificity of Cas12a trans-active holoenzyme


Phosphorimage of AsCas12a cleavage products, resolved by denaturing PAGE. Trans-active AsCas12a holoenzyme (115 nM of each component: protein, crRNA, pre-cleaved activator) was incubated with 1 nM of the indicated substrate for 2 hours at 30˚C prior to quenching. Substrate (a) was a single-stranded DNA oligonucleotide with no homology to the crRNA. To generate substrates (b) through (l), substrate (a) was hybridized to a variety of unlabeled complementary DNA oligonucleotides. Substrate (c) contained a nick. Substrates (d), (e), and (f) contained gaps of 1, 4, and 8 nt, respectively. Substrates (g), (h), and (i) contained bubbles of 1, 4, and 8 nt, respectively. Substrates (j), (k), and (l) contained bulges of 1, 4, and 8 nt, respectively.

Figure 8 from paper

Appendix 2—figure 1. Cas12a forms a gap in the non-target strand and cleaves the target strand outside the R-loop.


(A) Target-strand cleavage products over time, as quantified by denaturing PAGE. 100 nM AsCas12a and 120 nM crRNA were incubated with 5 nM radiolabeled DNA target at 37˚C for the indicated timepoints, followed by quenching and resolution by denaturing PAGE. Representative phosphorimages are shown in Appendix 2— figure 1—figure supplement 1. Data shown here are the average of three replicates. Each circle denotes a phosphodiester at which cleavage was observed. The intensity of color in each half-circle (‘cleavage signal’) reflects the fraction (band volume for a given cleavage product) / (total volume in lane). The left half of each circle (red) corresponds to the cleavage product detected with a PAM-proximal radiolabel. The right half of each circle (blue) corresponds to the cleavage product detected with a PAM-distal radiolabel.
(B) Non-target-strand cleavage products over time, as quantified by denaturing PAGE (phosphorimage in Appendix 2— figure 1—figure supplement 1). Data representation as in A.

Figure 9 from paper

Appendix 2—figure 2. Non-target-strand gap formation poses a kinetic barrier to target-strand cleavage for AsCas12a.


(A) Extent of target-strand cleavage by wild type AsCas12a in the presence of various non-target-strand variants, as resolved by denaturing PAGE (phosphorimage in Appendix 2—figure 2—figure supplement 5). Cas12a surveillance complex (100 nM AsCas12a, 120 nM crRNA) was added to 1 nM pre-hybridized target DNA radiolabeled on the 5’ end of the TS and allowed to incubate in cleavage buffer with 5 mM CaCl2 for 1 hr at 37˚C prior to quenching. In the schematic, the red portion of the NTS denotes phosphorothioate (PS) linkages; the gray portion denotes phosphodiester (PO) linkages. In the graph, red bars denote reactions with a PS-containing NTS variant; gray bars denote reactions either with no NTS or with an NTS variant containing only PO-linkages. From left to right (omitting the no-NTS control), the NTS variants used were A, B, D, G, J, N, Q, T, W, Y, Z, as schematized in Appendix 2—figure 2—figure supplement 4. Columns and associated error bars indicate the mean and standard deviation of three replicates.
(B) Cleavage kinetics of NTS, TS, and TS complexed with a pre-gapped NTS (NTS contains a 5-nt gap). 100 nM protein and 120 nM cognate crRNA were incubated with 2 nM DNA target with a 5’ radiolabel on the indicated strand at 37˚C for various timepoints, followed by quenching and resolution by denaturing PAGE. Representative phosphorimages and quantifications are shown in Appendix 2—figure 2—figure supplement 8. Columns and associated error bars indicate the mean and standard deviation of three replicates.


Supplemental figures from the paper

(Click on the small image to get a higher-resolution version.)
Supplemental Figure 1 from paper

Figure 2—figure supplement 1 - Method used to 3′-end radiolabel DNA oligonucleotides.


See Materials and methods for details.
Supplemental Figure 2 from paper

Figure 2—figure supplement 2 - A gap in the non-target strand increases the affinity of dCas12a for its DNA target.


Top panel: The affinity of dAsCas12a/crRNA for a cognate DNA target was assessed by an electrophoretic mobility shift assay (EMSA) and a filter-binding (FB) assay. dAsCas12a was titrated in a solution with fixed [crRNA] (750 nM) and [DNA probe] (100 pM), followed by separation of protein-bound DNA from free DNA. The EMSA indicated that the oligonucleotide annealing protocol yields 100% duplex DNA probe and that the binding conditions yield one major protein-bound species. ‘Fraction bound’ is defined as (background-subtracted volume of upper band)/(total background-subtracted lane volume) for the EMSA and (background-subtracted volume of Protran spot)/(total background-subtracted volume of Protran spot + Hybond N+ spot) for the filter-binding assay. The value of ‘fraction bound’ was 0 at [dAsCas12a]=0 for both assays (not shown in plot due to the logarithmic x-axis). When appropriate, data were fit to the sum of a hyperbola and a line (y = Bmax*x/(KD+x)+NS*x), where NS describes a non-specific binding mode. It is common to see Bmax values below 1 in EMSAs and filter-binding assays, in which the process of physical separation can disrupt bound species. KD for the EMSA was 8.2 nM (n = 1). KD for the filter-binding assay was 8.1 nM ±0.8 (SD) (n = 3).

Supplemental Figure 3 from paper

Figure 2—figure supplement 2 continued


Bottom panel: Using the filter binding assay, we assessed the affinity of dAsCas12a/crRNA for various cognate DNA targets.Protospacer 2 (used in Figure 2B) is the version of protospacer 1 (used in Figure 2A) modified for permanganate probing of the R-loop flank. Differences between protospacer 1 and protospacer 2 are highlighted in red (A/T base pairs substituted into the R-loop flank, G/C base pairs substituted elsewhere to maintain stable association between the two DNA strands). ‘Intact’ protospacers are as shown in the sequence schematic. ‘Pre-gapped’ protospacers are missing nt 14–18 of the NTS (as measured from the PAM, see Appendix 2). The value of ‘fraction bound’ was 0 at [dAsCas12a]=0 for all substrates (not shown due to the logarithmic x-axis). Data were analyzed as described for the top panel. KD for protospacer 1 (intact) was 5 nM ±1 (SD) (n = 3). KD for protospacer 2 (intact) was 54 nM ±12 (SD) (n = 3). KD for protospacer 2 (pre-gapped) was 2.8 nM ±0.5 (SD) (n = 3). Data from the protospacer 1 (pre-gapped) experiment indicated that the KD was near or below [DNA probe], preventing accurate KD determination by hyperbolic fitting. The reason for the low observed affinity of dAsCas12a for protospacer 2 (intact) is unknown.

Supplemental Figure 4 from paper

Figure 2—figure supplement 3 - Translating raw phosphorimages into quantitative permanganate reactivity metrics.


Left panel: Kinetics of permanganate reaction with an unpaired thymine. The depicted substrate was subject to the standard permanganate reaction protocol with quenching at 0, 5, 10, 30, 60, and 120 seconds. Black arrows indicate chemical cleavage fragments that resulted from oxidation of the annotated thymine. ‘Fraction oxidized at thymine X,’ plotted in the graph at the bottom, was determined as described in Materials and methods and is equivalent to the variable pi. The phosphorimage and graph shown are from a single representative replicate (n = 3). Data were fit to an exponential decay (y = (y0-plateau)*exp(-k*x)+plateau), with y0 constrained to 0 and the plateau value constrained to 1. The value of k was determined to be 0.998 min−1 ±0.027 (SD) (n = 3), which, when corrected to the reference thymine, yielded the value of kss,corr=0.79 min−1 that was used for normalization of the permanganate reactivity index in all other permanganate experiments.
Right panel: Raw phosphorimage of quantified data presented in Figure 2B. Black arrows indicate chemical cleavage fragments that resulted from oxidation of the annotated thymine. The method to determine the 'permanganate reactivity index' and 'fraction oxidized' metrics from a raw phosphorimage is described in Materials and methods.

Supplemental Figure 5 from paper

Figure 3—figure supplement 1 - dCas12a ribonucleoprotein binds tightly to pre-gapped/pre-unwound targets despite PAM-distal mismatches.


The affinity of dAsCas12a/crRNA for various cognate DNA targets was assessed by a filter-binding assay. ‘Pre-gapped’ indicates the presence of a 5-nt gap in the non-target strand (see Appendix 2). ‘Pre-unwound’ indicates the presence of a stretch of NTS:TS mismatches in the DNA substrate. In Figure 3A, protospacer 3 is annotated as ‘DNA substrate 1;’ protospacer 4 is annotated as ‘DNA substrate 2;’ and crRNA 3 is the depicted crRNA. For each combination of crRNA/DNA target, crRNA was titrated in a solution with fixed [dAsCas12a] (400 nM), [DNA probe] (100 pM), and [non-specific DNA competitor] (500 nM). The identities of the titrant/fixed component were inverted in this experiment (as compared to all other binding experiments) because crRNA can form a stable complex with pre-unwound DNA targets in the absence of protein. Keeping [dAsCas12a] at 400 nM favored the formation of (dAsCas12a/crRNA):DNA complexes over crRNA:DNA complexes (which would be indistinguishable from free DNA in the filter binding assay). In the presence of high [apo protein], 500 nM non-specific DNA competitor (a duplex with a short ssDNA overhang) was also included to disfavor non-specific interactions between radiolabeled DNA and apo protein.

Supplemental Figure 5 from paper

Figure 3—figure supplement 1 continued - dCas12a ribonucleoprotein binds tightly to pre-gapped/pre-unwound targets despite PAM-distal mismatches.


The value of ‘fraction bound’ was 0 at [crRNA]=0 for all substrates (not shown due to the logarithmic x-axis).For all pre-unwound DNA targets, the fraction bound was essentially concentration-independent across all nonzero concentrations tested, suggesting that the lowest concentration tested had already saturated the specific binding interaction being probed. The high stability is in line with thermodynamic expectations for an interaction involving hybridization of two complementary 18-nt or 20-nt oligonucleotides (Tm > 40°C) (Kibbe, 2007). The fact that the saturated bound fraction is less than 1 could be due to (1) a common feature of filter-binding assays in which the process of physical separation disrupts bound species or (2) a stable population of protein-free crRNA:DNA complexes. In any case, the important conclusion to be drawn from these data is that each protospacer exhibits the same fraction bound regardless of the presence of mismatches at positions 19 and 20 in the crRNA. Thus, the crRNA-dependent effects seen in Figure 3A and Figure 3—figure supplement 2 must emerge from fundamental differences in conformational dynamics and not from differences in binding occupancy of Cas12a/crRNA on the DNA probe.

Supplemental Figure 6 from paper

Figure 3—figure supplement 2 - Effect of R-loop truncation on permanganate reactivity of the A/T tract.


Permanganate reactivity of the A/T tract in a 20-nt R-loop and an 18-nt R-loop. In Figure 3A, protospacer 3 is annotated as ‘DNA substrate 1;’ protospacer 4 is annotated as ‘DNA substrate 2;’ and crRNA 3 is the depicted crRNA. Permanganate experiments were conducted as in Figure 2B (2 minutes, 30°C). See Materials and methods for description of the parameters plotted on the y-axis. Columns and associated error bars indicate the mean and standard deviation of three replicates. Columns 1, 2, 4, and 6 are equivalent to the data shown in Figure 3A. Columns 3 and 5 use a crRNA with compensatory mutations at positions 19–20, showing that the effect is dependent upon base pairing topology and not a particular sequence.

Supplemental Figure 7 from paper

Figure 3—figure supplement 3- Effect of PAM-distal mismatches on non-target-strand and target-strand cleavage kinetics and position with fully duplex DNA targets.


100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 37°C for 20 s, 1 min, 5 min, and 30 min, prior to quenching and resolution by denaturing PAGE. Each group of four lanes corresponds to a different DNA target, with varying numbers of PAM-distal mismatches with respect to the crRNA. Indicated above each group of four lanes is the number of base pairs of complementarity between the TS and the crRNA spacer, starting with the base immediately adjacent to the PAM. All DNA targets in this gel were fully duplex (not pre-unwound/bubbled), resulting in enhanced discrimination against PAM-distal mismatches as compared to the bubbled DNA targets in Figure 3B and Figure 3—figure supplement 4.

Supplemental Figure 8 from paper

Figure 3—figure supplement 4 - Effect of PAM-distal mismatches on non-target-strand and target-strand cleavage kinetics and position with bubbled DNA targets.


100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 37°C for 0 s, 15 s, 2 min, 10 min, and 1 hr, prior to quenching and resolution by denaturing PAGE. Each time series corresponds to a different DNA target, bearing varying numbers of PAM-distal mismatches with respect to the crRNA. Indicated above each time series is the number of base pairs of complementarity between the TS and the crRNA spacer, starting with the base immediately adjacent to the PAM. For the time series lacking an asterisk, the DNA target was fully duplex (as in Figure 3—figure supplement 3). For the time series that bear asterisks, the DNA target contained a bubble across the region of crRNA:TS complementarity (as illustrated in Figure 3B), which stabilized the R-loop. In the top panel, the NTS was 5'-radiolabeled. In the bottom panel, the TS was 5'-radiolabeled.

Supplemental Figure 9 from paper

Figure 3—figure supplement 5 - Determinants of altered target-strand cleavage kinetics and position.


100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM duplex DNA target radiolabeled on the 5' end of the target strand at 37°C for 0 s, 15 s, 1 min, 4 min, 15 min, or 1 hr, prior to quenching and resolution by denaturing PAGE. The 20-nt target sequence immediately adjacent to the PAM is shown below the crRNA spacer sequence used in each experiment. Red letters indicate TS:crRNA mismatches. Green letters indicate compensatory changes in the crRNA to restore a 20-nt match. The final timepoint of each reaction is reproduced in the gel on the right, for side-by-side comparison of the cleavage site distributions. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y = (y0-plateau)*exp(-k*x)+plateau), with y0 constrained to 0 and the plateau value constrained to ≤1. A representative replicate is shown. The value of kobs for each time course is as follows: A (0.12 s−1), B (0.0093 s−1), C (0.0094 s−1), D (0.16 s−1), E (0.015 s−1). The precise value of kobs for A and D should be interpreted with caution due to poor sampling of informative timepoints.

Supplemental Figure 10 from paper

Figure 3—figure supplement 6 - Non-target-strand cut-site distribution with a shrinking R-loop.


Final timepoint (1 hr) of each time series in the non-target-strand gel shown in the top panel of Figure 3—figure supplement 4, shown side-by-side for visual comparison—analogous to the final timepoints for the target strand shown in Figure 3B.

Supplemental Figure 11 from paper

Figure 3—figure supplement 7 - Kinetics of target-strand cleavage in DNA targets with various sequences in the R-loop flank.


Experiment performed as described in legend to Figure 3C. 100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM of DNA target at 25°C for 0 s, 15 s, 30 s, 1 min, 2 min, 4 min, or 10 min, prior to quenching and resolution by denaturing PAGE. All DNA targets were 5'-radiolabeled on the TS. The NTS was pre-gapped from positions 14–18 but complementary to the TS at positions 1–13 and 19–20. In each lane, the DNA target was varied to contain different sequences in the R-loop flank, which either formed a perfect duplex (substrates A, C, and E) or contained a 3-bp NTS:TS mismatch (substrates B, D, and F). ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane). Data were fit to an exponential decay (y=(y0-plateau)*exp(-k*x)+plateau), with y0 constrained to 0. A representative replicate (n = 3) is shown. The value of kobs ± SD for each time course is as follows: A [0.092 ± 0.012 s−1], B [0.145 ± 0.007 s−1], C [0.0059 ± 0.0006 s−1], D [0.137 ± 0.002 s−1], E [0.024 ± 0.002 s−1], F [0.061 ± 0.013 s−1]. The rate constants for B and D should be interpreted with caution due to poor sampling of informative timepoints.

Supplemental Figure 12 from paper

Figure 4—figure supplement 1 - Permanganate reactivity of the A/T tract in R-loops formed by dCas12a or dCas9.


Permanganate experiments were conducted as in Figure 2B (2 minutes, 30°C). ‘Pre-gapped’ indicates the presence of a 5-nt gap in the non-target strand (see Appendix 2) (the NTS gap in the dCas9 target is unrelated to the cut that would normally be formed by a nuclease-active Cas9—instead, it was designed to be analogous to the NTS gap formed by AsCas12a in an R-loop of the opposite topology, at positions 14–18). ‘Pre-unwound’ indicates the presence of a stretch of NTS:TS mismatches in the DNA substrate (20-nt bubble throughout the region of RNA complementarity); asterisks highlight the constructs that contain these NTS:TS mismatches. The sequence of protospacer 5 is identical to that of protospacer 2 except for a change in the PAM, which is not expected to affect conformational dynamics at the A/T tract (besides in permitting dCas9 binding). See Materials and methods for description of the parameters plotted on the y-axis. Columns and associated error bars indicate the mean and standard deviation of three replicates. Experiments A, B, E, F, G, and J are equivalent to the data shown in Figure 4A.

Supplemental Figure 13 from paper

Figure 4—figure supplement 2 - dCas9 binds tightly to pre-gapped DNA targets.


The affinity of dSpCas9/sgRNA for a cognate DNA target was assessed by an electrophoretic mobility shift assay (EMSA) and a filter-binding (FB) assay. dSpCas9 was titrated in a solution with fixed [sgRNA] (750 nM) and [DNA probe] (100 pM), followed by separation of protein-bound DNA from free DNA. The EMSA indicated that the binding conditions yield one major protein-bound species. ‘Fraction bound’ is defined as (background-subtracted volume of upper band)/(total background-subtracted lane volume) for the EMSA and (background-subtracted volume of Protran spot)/(total background-subtracted volume of Protran spot + Hybond N+ spot) for the filter-binding assay. The value of ‘fraction bound’ was 0 at [dSpCas9]=0 for both substrates and both assays (not shown on plot due to the logarithmic x-axis). All data shown are from one representative replicate (n = 1 for EMSA, n = 3 for FB). Protospacer 6 is identical to protospacer 1 (used for AsCas12a), except the PAM has been substituted with a SpCas9 PAM. Protospacer 5 is a modified version of protospacer 6, with differences highlighted in red (equivalent to the protospacer-1-to-protospacer-2 modifications). The ‘intact’ protospacer is as shown in the sequence schematic. The ‘pre-gapped’ protospacer is missing nt 14–18 of the NTS (as measured from the PAM). The NTS gap is unrelated to the cut that would normally be formed by a nuclease-active Cas9—instead, it was designed to be analogous to the NTS gap formed by AsCas12a in an R-loop of the opposite topology, at positions 14–18. This DNA substrate binds tightly to dSpCas9. Thus, the failure of dSpCas9 to significantly distort the R-loop flank in Figure 4A is due to a fundamental difference in conformational dynamics and not to a failure to bind.

Supplemental Figure 14 from paper

Figure 4—figure supplement 3 - Permanganate reactivity of the A/T tract in protein-free R-loops of various sequences.


Permanganate experiments were conducted as in Figure 2B (2 minutes, 30°C). See Materials and methods for description of the parameters plotted on the y-axis. Columns and associated error bars indicate the mean and standard deviation of three replicates. In all schematics, RNA molecules are outlined in orange, and DNA molecules are outlined in black. Circled ‘P’ indicates a 5′-phosphate. All sequences, when read right-side up, go from 5′ on the left to 3′ on the right. The terms ‘Cas12a-like’ and ‘Cas9-like’ are descriptors only of each substrate’s R-loop topology (the end of the RNA next to the boundary of interest is a 3′ end or a 5′ end, respectively)—both kinds of substrates contain a Cas12a PAM and a Cas12a-like NTS gap. These results imply that the asymmetry in R-loop-flank stability is a fundamental feature of R-loop structure and not a peculiarity of the original tested sequence.

Supplemental Figure 15 from paper

Figure 4—figure supplement 4 - Effect of RNA end chemistry on permanganate reactivity of the A/T tract in protein-free R-loops.


Permanganate experiments were conducted as in Figure 2B (2 minutes, 30°C), varying only the nature of the RNA molecule added to the pre-gapped/pre-unwound DNA substrate. See Materials and methods for description of the parameters plotted on the y-axis. Columns and associated error bars indicate the mean and standard deviation of three replicates. In both schematics, RNA molecules are outlined in orange, and DNA molecules are outlined in black. Circled ‘P’ indicates a 5′-phosphate. All sequences, when read right-side up, go from 5′ on the left to 3′ on the right. ‘OH’ indicates a hydroxyl. ‘Phos.’ indicates a phosphate. ‘Cyc. phos.’ indicates a 2′/3′-cyclic phosphate. ‘IVT/rz’ indicates that the RNA oligo was synthesized in an enzymatic in vitro transcription reaction, with ribozymes on both ends that cleaved to yield homogeneous termini. ‘Chem.’ indicates that the RNA oligo was chemically synthesized by a commercial source. The terms ‘Cas12a-like’ and ‘Cas9-like’ are descriptors only of each substrate’s R-loop topology (the end of the RNA next to the boundary of interest is a 3′ end or a 5′ end, respectively)—both kinds of substrates contain a Cas12a PAM and a Cas12a-like NTS gap.

Supplemental Figure 16 from paper

Figure 4—figure supplement 5 - Asymmetry in R-loop flank stability is also a feature of intact R-loops.


Permanganate experiments were conducted as in Figure 2B (2 minutes, 30°C). See Materials and methods for description of the parameters plotted on the y-axis. Columns and associated error bars indicate the mean and standard deviation of three replicates. In all schematics, RNA molecules are outlined in orange, and DNA molecules are outlined in black. All sequences, when read right-side up, go from 5′ on the left to 3′ on the right. These experiments provide the strongest point of comparison between the Cas12a-like and Cas9-like R-loop architecture, as the only component varied across conditions is which strand of an identical DNA bubble is hybridized to RNA (compare the cleaved R-loops, in which the position of the gap must be moved to the opposite strand, yielding slightly different baseline permanganate reactivity).

Supplemental Figure 17 from paper

Figure 4—figure supplement 6 - Effect of overhanging non-target-strand nucleotides on permanganate reactivity of the A/T tract in protein-free R-loops.


Permanganate experiments were conducted as in Figure 2B (2 minutes, 30°C). See Materials and methods for description of the parameters plotted on the y-axis. Columns and associated error bars indicate the mean and standard deviation of three replicates. In all schematics, RNA molecules are outlined in orange, and DNA molecules are outlined in black. Circled ‘P’ indicates a 5′-phosphate. All sequences, when read right-side up, go from 5′ on the left to 3′ on the right. These experiments probe the role of the 2-nt NTS overhang (immediately adjacent to the R-loop flank) in distortion of the A/T tract. When present, this dinucleotide results in a ‘flapped’ R-loop flank (RLF) terminus, and when absent, the RLF terminus is ‘flush.’ The terms ‘(Cas)12a-like’ and ‘(Cas)9-like’ are descriptors only of each substrate’s R-loop topology (the end of the RNA next to the boundary of interest is a 3′ end or a 5′ end, respectively)—both kinds of substrates contain a Cas12a PAM and a Cas12a-like NTS gap. These results show that the presence of the overhang can affect the absolute magnitude of distortion, but the nature of the asymmetry is unaffected.

Supplemental Figure 18 from paper

Figure 4—figure supplement 7 - 2-aminopurine fluorescence measurements confirm asymmetry in conformational dynamics of R-loop flanks.


Columns and error bars show mean and standard deviation of three replicates. In all schematics, RNA molecules are outlined in orange, and DNA molecules are outlined in black. Circled ‘P’ indicates a 5′-phosphate. All sequences, when read right-side up, go from 5′ on the left to 3′ on the right. The terms ‘Cas12a-like’ and ‘Cas9-like’ are descriptors only of each substrate’s R-loop topology (the end of the RNA next to the boundary of interest is a 3′ end or a 5′ end, respectively)—both kinds of substrates contain a Cas12a PAM and a Cas12a-like NTS gap. The absolute values of 2-AP fluorescence intensity have a wide range across different DNA probes, likely due to local sequence context or, in the case of the ssDNA control in the bottom right panel, perhaps the stable population of a conformation that enhances 2-AP fluorescence. Given this variation, it is important to use the perfect DNA duplex (C) and the pre-gapped/pre-unwound DNA bubble (D) conditions as benchmarks—on the continuum from C to D, where does E lie? For condition E in the Cas12a-like topologies, the 2-AP fluoresces as if the RNA were absent. For condition E in the Cas9-like topologies, 2-AP fluorescence is quenched and approaches the intensity of the fully duplex control.

Supplemental Figure 19 from paper

Figure 5—figure supplement 1 - Thermal stability determination for nicked dumbbell substrates and their constituent hairpins.


Data from representative replicates of refolding experiments (small black dots) are overlaid on a best-fit curve (thick blue line) comprising a Boltzmann sigmoid with inclined baselines (y = (m*x+b)+((n*x+c)-(m*x+b))/(1+exp((Tm-x)/slope))). Because the dumbbells contain two separate duplexes that can, in principle, fold and unfold independently of each other, each of these molecules likely has more than two states. Thus, while there is no obvious visual sign of multiple transitions in the refolding curves, we did not attempt to extract thermodynamic parameters from the slope, and the Tm (written in the center of each plot) should only be used as a point of comparison rather than as a determinant of a defined conformational ensemble. While the two RNA:DNA hairpins have slightly different Tm values, the much larger discrepancy in Tm of the nicked dumbbells is probably mostly due to the nature of duplex juxtaposition.

Supplemental Figure 20 from paper

Figure 5—figure supplement 2 - Molecular dynamics simulations of the Cas12a-like and Cas9-like interhelical junctions, Sequence 2.


Schematics and data are presented as described in the legend to Figure 5B, which depicted simulation of Sequence 1. For Sequence 2, the nucleobases probed at the 3' R-loop boundary frequently exhibited unstacking or poorly stacked conformations (although to a lesser extent than in Sequence 1), while those probed at the 5' R-loop boundary were stably stacked over the course of the simulation. These results suggest that the difference in stacking instability detected for the interhelical junctions (with Sequence 1 or Sequence 2) are due to the difference in junction topology rather than the identities of the monitored nucleobases (pyrimidine/pyrimidine versus purine/purine).