Conformational dynamics of CasX (Cas12e) in mediating DNA cleavage revealed by single-molecule FRET

Abstract

CasX (also known as Cas12e), a Class 2 CRISPR-Cas system, shows promise in genome editing due to its smaller size compared to the widely used Cas9 and Cas12a. Although the structures of CasX-sgRNA-DNA ternary complexes have been resolved and uncover a distinctive NTSB domain, the dynamic behaviors of CasX are not well characterized. In this study, we employed single-molecule and biochemical assays to investigate the conformational dynamics of two CasX homologs, DpbCasX and PlmCasX, from DNA binding to target cleavage and fragment release. Our results indicate that CasX cleaves the non-target strand and the target strand sequentially with relative irreversible dynamics. The two CasX homologs exhibited different cleavage patterns and specificities. The dynamic characterization of CasX also reveals a PAM-proximal seed region, providing guidance for CasX-based effector design. Further studies elucidate the mechanistic basis for why modification of sgRNA and the NTSB domain can affect its activity. Interestingly, CasX has less effective target search efficiency than Cas9 and Cas12a, potentially accounting for its lower genome editing efficiency. This observation opens a new avenue for future protein engineering.

Graphical Abstract

Figure 1.

Conformational dynamics of CasX cleavage captured by single-molecule FRET. (A) Diagram of the experimental design. The 3′ end of the sgRNA is labeled with Cy3 (green dots). The TS 34th nucleotide from the PAM is labeled with Cy5 (red dots). The process from adding CasX-sgRNA complex to the release of cleavage products is captured. (B) Representative single-molecule FRET trajectories of DpbCasX-sgRNAv2 on cognate DNA. The black curve represents the apparent FRET efficiency, while the orange lines represent Hidden Markov Modeling of FRET. The spontaneous appearance of Cy3 and FRET signals indicates the formation of CasX ternary complexes on immobilized dsDNAs (left blue arrow), and the disappearance of FRET indicates the dissociation of cleaved DNA fragments (right blue arrow). Three distinct FRET states are identified and assigned as S1, S2 and S3, in order of increasing values. (C) Time-dependent FRET probability density plots are synchronized at the appearance of FRET (t = 0, left panel) and at the disappearance of FRET (t = 0, right panel), enabling examining FRET evolution after ternary complex formation and before the release of cleaved DNA, respectively. N represents the number of events. Three independent experiments show consistent results. (D) Transition density plot displays the transition frequencies between different FRET states, initial and final FRET values for each transition event are accumulated into a 2D histogram. (E) FRET histograms of individual states were extracted from the original FRET trajectories after Hidden Markov Modeling classification. FRET values for each state are obtained through single Gaussian fitting. Each histogram is normalized by the total sum of all histograms.

Figure 2.

Dynamics and cleavage of DpbCasX. (A) Schematic of mismatched DNA. (B) The number of dDpbCasX-sgRNAv2 complexes bound on immobilized cognate or mismatched dsDNAs before and after 1 hour of incubation in the dark (top) and the FRET dwell time of dDpbCasX on immobilized mismatched dsDNAs after correcting for photobleaching (bottom). The original dwell time distributions without correction are shown in Supplementary Figure S3B. (C) Effective target search rates of dDpbCasX or DpbCasX on dsDNA to form stable or transient stable complex (dwell time 1.5 s or longer). The distributions of appearance time are shown in Supplementary Figure S3C and S4E. The CasX RNP concentration was 3 nM. The search rate of DpbCasX on 14m-DNA is beyond our detection limit (< 0.3 μM⁻¹s⁻¹). (D) Cleavage activity of DpbCasX. The cleavage rates for 20m- (2.6 ± 0.1 min⁻¹), 19m- (2.6 ± 0.3 min⁻¹), 18m- (1.6 ± 0.2 min⁻¹), 17m- (0.43 ± 0.03 min⁻¹), 16m- (0.14 ± 0.02 min⁻¹) and 15m-DNA (0.020 ± 0.002 min⁻¹) were fitted by a single exponential decay curve. (E-F) FRET histograms (E) and dwell time (F) of individual states for different DNAs. (G) Effective target search rates of DpbCasX on 16m-DNA, 16m-Nick in TS DNA, and 16m-Nick in NTS DNA. The appearance time are shown in Supplementary Figure S5A. The CasX RNP concentration was 3 nM. (H) Cleavage activity of DpbCasX on 16m-DNA (0.15 ± 0.04 min⁻¹) and 16m-Nick in NTS DNA (1.4 ± 0.1 min⁻¹) using Cy5-labeled TS. Cleavage activity of DpbCasX on 16m-DNA (0.16 ± 0.06 min⁻¹) and 16m-Nick in TS DNA (0.20 ± 0.05 min⁻¹) using Cy5-labeled NTS. Three repeats were performed. Mean and SEM are shown where appropriate.

Figure 3.

Dynamics and cleavage of PlmCasX. (A) Representative single-molecule FRET trajectories of PlmCasX-sgRNAv2 complex on cognate DNA. Two distinct FRET states are indicated and assigned as S1 and S2 from low to high values. (B) Time-dependent FRET probability density plots of PlmCasX on cognate DNA. (C) Time-dependent FRET probability density plots of dPlmCasX and dDpbCasX on DNA with NTS-Nick at positions 12, 13 or NTS-Nick at positions 14, 15. (D) Cleavage sites of DpbCasX and PlmCasX on NTS and TS. Cleavage reaction was terminated 15 s after mixing CasX-sgRNA complex and target DNA. (E) The number of dPlmCasX-sgRNAv2 complexes bound on immobilized 20m- or 19m-dsDNAs before and after 1 h of incubation in the dark (top) and the FRET dwell time of dPlmCasX on immobilized dsDNAs after correcting for photobleaching (bottom). The original dwell time distributions without correction are shown in Supplementary Figure S6D, E. (F) Effective target search rates of dPlmCasX. The appearance time are shown in Supplementary Figure S6F. The CasX RNP concentration was 3 nM. (G) The cleavage activity of PlmCasX. Cleavage rates of 20m- (1.0 ± 0.1 min⁻¹), 19m- (0.88 ± 0.09 min⁻¹), 18m- (0.54 ± 0.09 min⁻¹), 17m- (0.081 ± 0.004 min⁻¹), 16m- (0.03 ± 0.01 min⁻¹) and 15m-DNA (0.008 ± 0.001 min⁻¹) were fitted by a single exponential decay curve. Three repeats were performed. Mean and SEM are shown where appropriate.

Figure 4.

The conformational dynamics of PlmCasX affected by sgRNA and the nonspecific binding of CasX to DNA. (A) Distributions of dwell time of dPlmCasX-sgRNAv1 on cognate DNA with an exposure time of 2 s. The dwell time was fitted by a double exponential decay curve. (B) Dwell times of dPlmCasX-sgRNAv2 and dPlmCasX-sgRNAv1 on cognate DNA. (C) Effective target search rates of dPlmCasX-sgRNAv1 and dPlmCasX-sgRNAv2 on cognate DNA. The appearance time are shown in Supplementary Figure S7A. The CasX RNP concentration was 3 nM. (D) Time-dependent FRET probability density plots of PlmCasX-sgRNAv1 and dPlmCasX-sgRNAv2 on cognate DNA and corresponding DNA cleavage. The disappearance of fluorescent spots indicates Cy5-labeled DNA cleavage after CasX addition. (E) Schematic of CasX nonspecific binding and a representative single-molecule fluorescence trace. CasX-sgRNA complex was injected into a chamber containing dsDNA without a PAM sequence. The sudden appearance of Cy3 signal indicated the transient binding of CasX-sgRNA to DNA. The time from injection to Cy3 signal appearance was recorded as appearance time, and the duration of Cy3 signal was recorded as dwell time. (F) Distributions of appearance time of DpbCasX-sgRNAv2 and PlmCasX-sgRNAv2 on DNA without a PAM sequence. The exposure time was 2 ms/frame, and the CasX RNP concentration was 10 nM. (G) Nonspecific binding rates and effective target search rates of different Cas proteins (details in Methods). Data for CasX and correlated rates for Cas9 and Cas12a are shown in Supplementary Table S3 (29,35,36). (H) Effective target search efficiency of different Cas proteins defined as the ratio of the effective target search rate to the nonspecific binding rate shown in (G). Three repeats were performed. Mean and SEM are shown where appropriate.

Figure 5.

NTSB domain modulates DNA unwinding, cleavage and specificity. (A) Structure of DpbCasX–sgRNA–dsDNA (PDB: 6NY2) with the NTSB domain shown in red. (B) Cleavage activity of DpbCasXΔNTSB on cognate DNA, bulged DNA and bulged DNA with mismatches compared to DpbCasX on cognate DNA. (C–E) Time-dependent FRET probability density plots of DpbCasXΔNTSB-sgRNAv2 on cognate DNA (C), Bulge 3–6 DNA (D), and Bulge 11–20 DNA (E). (F) Cleavage activity of DpbCasX and DpbCasXΔNTSB on ssDNA. (G) Effective target search rates of DpbCasXΔNTSB-sgRNAv2 and DpbCasX-sgRNAv2 on ssDNA. The appearance time are shown in Supplementary Figure S9B. The CasX RNP concentration was 10 nM. (H) Left, three residues within the NTSB domain interact closely with the TS in both the NTS-cleavage and TS-cleavage states of DpbCasX. Right, the cleavage activity of DpbCasX mutants on DNAs with varying matches to sgRNA. (I) Diagram of GFP disruption assay in cells (left). Normalized GFP disruption using WT PlmCasX or PlmCasX-K188A, PlmCasX-Q191A targeting site 1–5 within GFP (right). GFP disruption percentage was normalized to their corresponding values of cognate target. Three repeats were performed. Mean and SEM are shown where appropriate.

Figure 6.

Proposed model for CasX binding and cleavage of DNA. After nonspecific binding to DNA, CasX’s efficiency in recognizing PAM and initiating R-loop formation is 5–8 times lower than that of SpCas9, AsCas12a and LbCas12a. After PAM recognition, sufficient PAM-proximal matches are required to sequentially drive R-loop formation, NTS cleavage and TS cleavage. The NTSB domain plays an important role in DNA unwinding and R-loop formation via its interactions with sgRNA and DNA. Insufficient matches between sgRNA and the spacer hinder NTS cleavage and promote the disassembly of the NTS pre-cleavage state. Thus, NTS cleavage acts as a critical checkpoint, after which TS cleavage and DNA fragment release occur rapidly.