Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Abstract

CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

Fig. 1. Cas12j3 target dsDNA cleavage and unspecific ssDNA degradation.

a representative dsDNA cleavage pattern generated by Cas12j3 wild type (WT). T-strand (TS) and NT-strand (NTS) products are marked, showing a cut at positions −13, −14 and −15 of the NT-strand, while the T-strand is cleaved at position +23. The sequence of the double-labelled duplex is shown below, marking the position of the cut (triangles), and the size of the labelled products. b Unspecific ssDNA degradation after activation with a specific target ssDNA of different lengths (Oligonucleotides T-AAG-3 to T-AAG-30 in Supplementary Table II). c Unspecific ssDNA degradation after activation with a specific dsDNA activator of different lengths (Oligonucleotides T-AAG-3/NT-TTC-3 to T-AAG-30/NT-TTC-30 in Supplementary Table II). d Schematic cartoon of the results shown in b) and c). Activation of the unspecific ssDNA cleavage is observed between 12–30 nt. (i) The RuvC domain of Cas12j3 RNP is inhibited. Full activation of the unspecific cleavage is observed when using an ssDNA or dsDNA activator pairing with the crRNA between 12–18 nt (ii and iv). The use of longer oligos as ssDNA(iii) or dsDNA (v) results in a reduction of the cleavage efficiency, likely due to a steric occlusion of the catalytic site by the T-strand and NT-strand. e Unspecific ssDNA degradation time course by Cas12j3 activated by 18 and 30 nt, ss- and dsDNA targets. The DNA markers indicate the size of the products in nucleotides. The experiments are shown in a–c and e are representative of three independent experiments with similar results.

Fig. 2. Cryo-EM structure of Cas12j3 R-loop complex after target DNA cleavage.

a Domain architecture of Cas12j3 comprising the T-strand and NT-strand PAM interacting domains (TPID, NPID), the RNA-handle binding domain (RBD), the bridge helices (BH-I and BH-II), the RuvC domain including the insertion and the stop (STP) domain. b Schematic diagram of the R-loop formed by the crRNA and the target DNA. Triangles represent phosphodiester cleavage positions in the T- and NT-strands; the bold font nucleotides represent those visualized in the structure. The PAM distal products are separated by a dashed line to illustrate the post-catalytic state (see Supplementary Fig. 7c–g). c cryo-EM map of the Cas12j3/R-loop complex at 2.7 Å resolution. The cartoon depicts the relative orientation of the domains and the N- and C-terminal regions of the protein. The map and the explicative cartoon are coloured according to Fig. 2a. d View of the R-loop structure and the dinucleotide in the catalytic site (polypeptide omitted). e Overview of the Cas12j3–RNA–target-DNA ternary complex (Supplementary Figs. 3–5, Supplementary Table 1).

Fig. 3. Cas12j3/R-loop atomic model and cryo-EM maps.

Comparison of the three cryo-EM maps (Supplementary Fig. 3d, Supplementary Fig. 4 and Supplementary Table 1). The top left ribbon diagram and the central cartoon are included to facilitate the comparison between the maps. The maps indicate the high flexibility of the NPID, RuvC and STP domains and extra density from the T-strand in map2 (Supplementary Movie 1).

Fig. 4. Cas12j3 PAM recognition, uncoupling of the Watson–Crick dA-1:dT+1 pair and unzipping.

a Surface representation of Cass12j3–R-loop complex. The white dashed arrow shows the predicted path of the NT-strand to the nuclease site after dG-2 (Supplementary Fig. 6). b Detailed view of the PAM nucleotides recognition and dsDNA unwinding depicting the conserved K26, K30, Q123, and Q197 residues (Supplementary Fig. 5c). c Zoom of the dT+1/dA-1 pair uncoupling, phosphate inversion and unzipping (Supplementary Fig. 5d). d dsDNA cleavage assays using Cas12j3 wild type and PAM unwinding, activation and catalytic mutants. Oligonucleotides 3F-T-AAG-30 and 5F-NT-TTC-30 were used as substrates (Supplementary Table II). T-strand (TS) and NT-strand (NTS) products are marked. DNA markers are shown in nucleotides. e Quantification of the activity based on the cleavage experiments as shown in d. Bars represent mean ± SD. n > 3 independent experiments.

Fig. 5. Assembly of the crRNA/DNA hybrid and activation of the RuvC pocket.

a View of the hybrid showing the interaction of the crRNA with residues in the RuvC insertion (Supplementary Fig. 5d). b Inset depicting the hydrophobic interaction between the turn of the RuvC insertion and the and cavity in the STP domain (Supplementary Fig. 5f). c dsDNA cleavage assays using Cas12j3 wild type and PAM unwinding, activation and catalytic mutants. Oligonucleotides 3F-T-AAG-30 and 5F-NT-TTC-30 were used as substrates (Supplementary Table II). T-strand (TS) and NT-strand (NTS) products are marked. DNA markers are shown in nucleotides. d Quantification of the activity based on the cleavage experiments as shown in c Bars represent the mean ± SD. n > 3 independent experiments. e Detailed view of the catalytic site containing a dinucleotide and a divalent metal. The D708 side chain and the associated distances are shown for visualization purposes only (see Supplementary Fig. 5f).

Fig. 6. Structural comparison of Cas12j3 RuvC domain with other Cas nucleases.

A structural homology search of Cas12j3 against the PDB was performed using DALI. Only the RuvC domain displays homology with other Cas nucleases. a The top panel shows the superposition of Cas12j3 with Cas12f and Cas12g. Both Cas12f and Cas12g present a Zn²⁺ atom coordinated by 4 conserved cysteines as Cas12j3. The rest of the domain is different to the TNB domain in Cas12f and Cas12g. Bottom panel Superposition of Cas12j3 with and Cas12i. Both Cas12b and Cas12i present DNA in the catalytic site and the Nuc domain inserted in the RuvC. b Detailed comparison of Cas12j3 and Cas12f after superposition in the RuvC domain. One of the monomers of the dimeric Cas12f is shown in surface representation for clarity. c Homology modelling of Cas12j1 and Cas12j using Cas12j3 and superposition of the three Cas12j family members. The inset shows the differences in the STP domain.

Fig. 7. Model of Cas12j3 PAM-dependent DNA recognition, unwinding and cleavage.

Cartoon model depicting the stages of Cas12j3 nuclease staggered target DNA cleavage (see Discussion). The T-strand and NT-strand are illustrated in green and black, with the PAM colored in pink. In step (i) the RBD, BH, STP, and RuvC domains are represented as an oval (see Discussion).