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.

Appendix 2—figure 2—figure supplement 1. Non-target-strand cleavage precedes target-strand cleavage for AsCas12a and Cas12i1.

100 nM AsCas12a or Cas12i1 was incubated with 120 nM cognate crRNA and 2 nM radiolabeled duplex DNA target for 1 hr at 37°C, followed by quenching, denaturing PAGE, and phosphorimaging. For AsCas12a, the reaction was conducted in 5 mM CaCl₂. For Cas12i1, the reaction was conducted in 5 mM MgCl₂. In the duplex diagrams, red shading indicates the presence of a phosphorothioate (PS) tract across the standard cleavage sites. Blue indicates phosphodiester (PO) linkages within the TS. Gray indicates phosphodiester linkages within the NTS. For both AsCas12a and Cas12i1, the nature of the linkages in the TS has no apparent effect on NTS cleavage. However, the presence of phosphorothioates in the NTS inhibits cleavage of both the NTS and the TS. Trace TS cleavage is observed for Cas12i1 in the PS-NTS condition (lane 6)—it is unclear whether this is due to TS cleavage prior to NTS cleavage or to incomplete duplex formation, as the trace cleavage event is shifted to the site cleaved during ssDNA-targeting (lane 7).

Appendix 2—figure 2—figure supplement 2. Cleavage at phosphorothioates can be selectively slowed by substitution of CaCl₂ for MgCl₂.

100 nM AsCas12a and 120 nM crRNA were incubated with 1 nM radiolabeled duplex DNA target at 37°C, followed by quenching (at timepoints 0, 5 s, 15 s, 1 min, 5 min, 10 min, 30 min, 1 hr) and resolution by denaturing PAGE. Substrate diagrams are colored as in Appendix 2—figure 2—figure supplement 1. The top panel shows the experiment done in cleavage buffer with 5 mM MgCl₂. The bottom panel shows the experiment done in cleavage buffer with 5 mM CaCl₂—at the end of each time course on this gel, the 1-hr timepoint of the MgCl₂ experiment is included for visual comparison. ‘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. The plateau value was constrained to 1 for those time courses that did not exceed fraction cleaved = 0.5 by the 1-hr timepoint. Rate constants (with 95% confidence intervals) are shown in the table below the gels. It is unclear why cleavage of a phosphorothioated TS occurs more rapidly than cleavage of a phosphorothioated NTS, although it is conceivable that this is an intrinsic feature of the enzyme cleavage pathway when the chemical transformation is rate-limiting. Considering only effects on the NTS, the calcium substitution decreases the phosphodiester cleavage rate by a factor of 57 and decreases the phosphorothioate cleavage rate by a factor of 730, resulting in a 13-fold increase in selectivity for phosphodiesters over phosphorothioates and yielding kinetics slow enough to resolve by manual pipetting.

Appendix 2—figure 2—figure supplement 3. Interference complexes are stable in the presence of CaCl₂ and with a phosphorothioated DNA target.

Using a filter-binding assay, we assessed the affinity of dAsCas12a/crRNA for cognate DNA targets, either fully phosphodiester (PO) or containing phosphorothioate (PS) linkages across the NTS and TS cleavage sites, in the presence of either 5 mM MgCl₂ or 5 mM CaCl₂. Substrate diagrams are colored as in Appendix 2—figure 2—figure supplement 1. ‘Fraction bound’ is defined as (background-subtracted volume of Protran spot)/(total background-subtracted volume of Protran spot + Hybond N+ spot). The value of ‘fraction bound’ was 0 at [dAsCas12a]=0 for both substrates and both assays (not shown due to the logarithmic x-axis). All data shown are from a representative replicate (n = 3). When appropriate, data were fit to the sum of a hyperbola and a line (y = B_max*x/(K_D+x)+NS*x), where NS describes a non-specific binding mode. It is common to see B_max values below 1 in filter binding assays, in which the process of physical separation can disrupt bound species. K_D for the PO substrate in MgCl₂ was 8.1 nM ±0.8 (SD) (n = 3). Data from the other binding conditions indicated that the K_D was near or below [DNA probe], preventing accurate K_D determination by hyperbolic fitting. For unknown reasons, the Mg²⁺→Ca²⁺ and the PO→PS substitutions stabilized the ribonucleoprotein:DNA interaction both separately and together.

Appendix 2—figure 2—figure supplement 4. Non-target-strand variants used in gap-dependence experiments.

Nucleotides 7–22 of the NTS are shown for each variant. A red circle indicates a phosphorothioate linkage. A gray circle indicates a phosphodiester linkage. A 5′ phosphate was placed on all PAM-distal NTS fragments to recapitulate the end chemistry left by the RuvC-catalyzed hydrolysis reaction.

Appendix 2—figure 2—figure supplement 5. Phosphorimage and quantification of non-target-strand gap-dependence experiments, in CaCl₂.

Experiment was performed as described in legend to Appendix 2—figure 2A. Substrates indicated by a letter are as schematized in Appendix 2—figure 2—figure supplement 4. When a given category has more than one substrate (e.g., 3-nt gap includes substrates M, N, O, and P), the first listed substrate (M) is shown as a blue bar, the second (N) as a red bar, the third (O) as a green bar, and the fourth (P) as a pink bar. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).

Appendix 2—figure 2—figure supplement 6. Phosphorimage and quantification of non-target-strand gap-dependence experiments, in MgCl₂.

Experiment was performed as described in legend to Appendix 2—figure 2A, except 5 mM MgCl₂ was used instead of CaCl₂, and the reaction was quenched after only 10 s at 37°C. Substrates indicated by a letter are as schematized in Appendix 2—figure 2—figure supplement 4. When a given category has more than one substrate (e.g., 3-nt gap includes substrates M, N, O, and P), the first listed substrate (M) is shown as a blue bar, the second (N) as a red bar, the third (O) as a green bar, and the fourth (P) as a pink bar. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).

Appendix 2—figure 2—figure supplement 7. Phosphorimage and quantification of non-target-strand gap-dependence experiments, in MgCl₂, with radiolabeled trans substrate.

Extent of trans cleavage by wild type AsCas12a in the presence of various DNA activator variants, as resolved by denaturing PAGE. Cas12a ternary complex (final concentrations: 100 nM AsCas12a, 120 nM crRNA, pre-hybridized 120 nM TS/240 nM NTS) was assembled with each of the indicated NTS variants and combined with 2 nM (final concentration) radiolabeled non-specific trans ssDNA target in cleavage buffer (5 mM MgCl₂). These reactions were then incubated for 30 min at 37°C prior to quenching and resolution by denaturing PAGE. Control lanes on the left contain some combination of intact NTS/TS with phosphodiesters (PO) or phosphorothioates (PS) across the standard cleavage sites; ‘pc’ stands for pre-cleaved (only PAM-proximal cleavage products: NTS truncated after nt 13, TS truncated after nt 22). Reactions without NTS contained 120 nM of a non-specific DNA oligonucleotide to account for substrate competition. All lanes indicated by a letter (C–Y) contained an NTS variant (see Appendix 2—figure 2—figure supplement 4) along with a TS containing phosphorothioates across the standard cleavage sites. When a given category has more than one substrate (e.g., 3-nt gap includes substrates M, N, O, and P), the first listed substrate (M) is shown as a blue bar, the second (N) as a red bar, the third (O) as a green bar, and the fourth (P) as a pink bar. ‘Fraction cleaved’ is defined as (sum of the volume of all bands below the uncleaved band)/(total volume in lane).

Appendix 2—figure 2—figure supplement 8. Phosphorimages and quantification of pre-gapped non-target-strand experiments.

Experiment was performed as described in legend to Appendix 2—figure 2B. ‘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. The plateau value was constrained to 1 for those time courses that did not exceed fraction cleaved = 0.5 by the 1-hr timepoint. The exponential decay constant k is reported as k_obs in Appendix 2—figure 2B. The data shown here are from a representative replicate (n = 3).

Appendix 2—figure 2—figure supplement 9. Affinity measurements for RNA-guided interaction of AsCas12a mutants with dsDNA.

The affinity of AsCas12a/crRNA for a cognate DNA target was assessed by a filter-binding assay, with three variants of AsCas12a. AsCas12a was titrated in a solution with fixed [crRNA] (750 nM) and [DNA probe] (100 pM), in binding buffer containing MgCl₂, followed by separation of protein-bound DNA from free DNA on membranes. ‘Fraction bound’ is defined as (volume of Protran spot)/(total volume of Protran spot + Hybond N+ spot). The value of ‘fraction bound’ was 0 at [AsCas12a]=0 for all protein variants (not shown due to the logarithmic x-axis). All data shown are from a representative replicate (n = 3). Data were fit to the sum of a hyperbola and a line (y = B_max*x/(K_D+x)+NS*x), where NS describes a non-specific binding mode. It is common to see B_max values below 1 in filter-binding assays, in which the process of physical separation can disrupt bound species. K_D for AsCas12a D908A was 11 nM ±3 (SD) (n = 3). K_D for AsCas12a R1226A was 2.7 nM ±0.2 (SD) (n = 3). K_D for AsCas12a WT was 1.86 nM ±0.04 (SD) (n = 3).

Appendix 2—figure 2—figure supplement 10. Non-target-strand cleavage product mapping for AsCas12a R1226A.

The evolution of the pattern of cis cleavage over time for AsCas12a R1226A was assayed analogously to that of WT AsCas12a, as in Appendix 2—figure 1. At the 1-hr timepoint, AsCas12a R1226A had made at least one cut in ~70% of the assayed NTS molecules, which matches the average fraction cleaved by WT AsCas12a at the 5-s timepoint. The cleavage site distributions in each of these experiments can be compared by calculating the probability density of each cleavage product as: (volume of band corresponding to cleavage at phosphodiester X/Y)/(total volume of all cleaved products), such that the area under each distribution is 1. Unlike the WT distribution, for which the 5′-mapped and 3′-mapped distributions are mostly non-overlapping, the R1226A distributions have significant overlap at dinucleotides 14/15, 15/16, and 16/17. In our ensemble biochemical experiment, this overlap is consistent with (although not uniquely explainable by) the presence of a large population of molecules that have been cleaved exactly once (i.e., have not undergone gap formation). If this explanation is correct, these data point to an accumulation of AsCas12a R1226a complexes with a once-cut NTS, supporting the model that gap formation is disproportionately slow (and perhaps rate-limiting for TS cleavage) for this mutant.

Appendix 2—figure 2—figure supplement 11. Cleavage product mapping for Cas12i1.

The evolution of the pattern of cis cleavage over time for Cas12i1 was assayed analogously to that of WT AsCas12a, as in Appendix 2—figure 1. The pattern of cleavage is largely similar to that of AsCas12a, although cleavage kinetics are dramatically slower, and disproportionately so for TS cleavage. The NTS gap of Cas12i1 is wider (8 nt) than that of AsCas12a (5 nt) at 1 hr. No DNA cleavage was detected on either strand when a RuvC-inactivated Cas12i1 mutant (D647A) was used.