Structural basis for the activity of the type VII CRISPR–Cas system

The newly identified type VII CRISPR–Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA1. However, the RNA cleavage is

executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR–Cas systems. Here we report seven cryo-electron microscopy structures of the

Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5–Cas7 complex in a target RNA-dependent manner. The

N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5–Cas7 complex.

The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5–Cas7 complex to execute individual cleavage

events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a

complementary protospacer flanking sequence at the 5′ end, but not at the 3′ end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference

complex and substrate cleavage in the type VII CRISPR–Cas system, which may help rational engineering of the type VII CRISPR–Cas system for biotechnological applications.

The atomic coordinates and EM maps have been deposited in the Protein Data Bank under accession codes 8Z4L (substrate-engaged state I), 8Z4J (substrate-engaged state II), 8YHD (post-state

I), 8YHE (post-state II), 8Z99 (substrate-engaged state +I), 8Z9C (substrate-engaged state II) and 8Z9E (substrate-engaged state II), and in the Electron Microscopy Data Bank under the

corresponding accession codes EMD-39767, EMD-39766, EMD-39287, EMD-39288, EMD-39857, EMD-39859 and EMD-39861. PDB accessions 2I7V, 3X1L, 4XWT, 6IFU and 6V4X were used for structure analysis.

Uncropped gels and images are provided in Supplementary Figs. 1–3. Source data are provided with this paper.

We thank the Center for Instrumental Analysis and Metrology of Wuhan Institute of Virology for supporting cryo-EM data acquisition and the Core Facility of Research Center of Basic Medical

Sciences in Tianjin Medical University for providing technical assistance. This work was supported by the National Natural Science Foundation of China (32322040 to H. Zhang, 22377091 to J.Y.

and 32300036 to H.Y.), the Natural Science Foundation of Tianjin Municipal Science and Technology Commission (23JCZDJC00410) and the CAS Pioneer Hundred Talents Program (to Z.D.).

These authors contributed equally: Jie Yang, Xuzichao Li, Qiuqiu He, Xiaoshen Wang, Jingjing Tang, Tongyao Wang

State Key Laboratory of Experimental Hematology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), International Joint Laboratory of Ocular Diseases (Ministry of

Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Institute of Immunology, School of Basic Medical Sciences, Tianjin

Medical University, Tianjin, China

Jie Yang, Xuzichao Li, Qiuqiu He, Xiaoshen Wang, Tongyao Wang, Yi Zhang, Shuqin Zhang, Zhikun Liu, Lingling Zhang, Fumeng Liao, Hang Yin & Heng Zhang

Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China

J.Y., Z.D. and H. Zhang conceived the project. J.Y., X.L., Q.H., X.W., J.T., T.W., Y.Z., F.Y., L.Z. and F.L. carried out the experimental work. Data analysis was performed by J.Y., X.L.,

X.W., J.T., S.Z., Z.L., H.Y., H. Zhao, Z.D. and H. Zhang. H. Zhang supervised the work. J.Y. and H. Zhang wrote the manuscript with contributions from all authors.

Nature thanks Chunyi Hu, David Taylor and John van der Oost for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

(a) The EMSA assay with RNP complex, catalytic dead Cas14 (D66A/H67A), or a combination of both. The triangle indicates the increasing concentrations of proteins. Representative gels from

three independent EMSA experiments are shown. (b) In vitro cleavage assay using ssRNA, ssDNA and dsDNA as the substrate. Only the ssRNA substrate was cleaved by the combination of the RNP

complex and WT Cas14. Three biological replicates were performed, and representative gel is shown. (c) In vivo transcriptional repression assays of the type VII candidate system. BL21(DE3)

cells were co-transformed with three plasmids: one encoding the Cas5-Cas6-Cas7 with an mRFP or KanR targeting crRNA array (SpecR), one encoding WT Cas14 or D66A/H67A mutant Cas14 (AmpR), and

a third encoding mRFP or KanR (KanR). The transformed cells were induced with 0.5 mM IPTG and plated on solid LB agar containing three antibiotics and IPTG. Representative images from four

independent assays are shown. (d) The percentage of mRFP-positive colonies (left panel) and the number of colonies formed on plates containing three antibiotics (right panel) were determined

by manual colony counting. Bar plots show mean ± standard deviation (s.d.) with individual data points. P-values were calculated from four biological replicates (N = 4) using an unpaired

two-tailed t-test with Welch’s corrections. (e) Scheme of Cas14 domain truncations, related to Fig. 1f. (f) Input for the strep pull-down assay, related to Fig. 1f. The gel is representative

of three biological replicates. For gel source data, see Supplementary Figs. 1–3.

(a) Overall structure of Cas5-Cas7 filaments in the types VII (upper panel, this study), III-A (middle panel, PDB ID: 6IFU) and III-B (lower panel, PDB ID: 3X1L) systems. (b) Structural

alignment of Cas5 in the type VII system (green) and Cmr3 in the type III-B system (wheat, PDB ID: 3X1L). The Cas5 protein adopts a ferredoxin-like fold, similar to Csm4/Cmr3 proteins from

the type III-A/B systems. Despite the structural similarities between Cas5 and its counterparts Csm4/Cmr3 of type III systems, the crucial regions of Csm4/Cmr3 responsible for the

interaction with Cas10 protein are partially absent in Cas5, which is in line with the lack of Cas10 in the type VII system. (c) Structural alignment of Cas7 in the type VII system (cyan)

and Csm3 in the type III-A system (wheat, PDB ID: 6IFU). The single Cas7 unit, structurally analogous to Csm3/Cmr4 proteins, possesses a conserved thumb-palm-finger architecture, with the

thumb region flanked by the palm and finger segments from the adjacent Cas7. In the type III systems, Csm3/Cmr4 proteins feature a catalytic loop containing an aspartic acid residue

responsible for target RNA cleavage. However, the equivalent position in Cas7 of the type VII system is occupied by an asparagine residue (Asn36), rendering Cas7 catalytically inactive. (d)

Electrostatic surface representation of Cas5-Cas7 filament in the type VII system. The crRNA-target RNA duplex binds to the positively charged channel of this filament. The EM density

corresponding to the crRNA-target RNA duplex traverses through the positively charged channel of the Cas5-Cas7 helical filament. (e) Interaction interface between the 5′-end repeat segment

of crRNA and Cas5-Cas7 filament. The 5′-end repeat segment displays an S-shaped conformation, with the -1U and (-3A)–(-6A) of the repeat sequence fully exposed, whereas -7U and -8U are

buried within Cas5.

(a) Size-exclusion chromatography coupled with multi-angle light scattering (SEC–MALS) analysis of Cas14 variants. The theoretical molecular weights of the tetrameric full-length Cas14

protein, tetrameric NTD + MLD protein and the dimeric NTD protein (284 kDa, 231 kDa and 99 kDa, respectively) are consistent with their experimental results. (b) Structural alignment of the

NTDs in Cas14.1 and Cas14.3 (left). Ribbon representation of the MβL and β-CASP subdomains of NTD-Cas14.1 (right). (c) Structural alignment of the MLDs from the four Cas14 subunits

(Cas14.1-14.4) (top). Ribbon representation of the MLD (bottom). (d) Structural alignment of the CTDs from the four Cas14 subunits (Cas14.1–14.4) (left). Ribbon representation of the CTD

(right). (e) Structural comparison of CTD-Cas14 and CTD-Cas10 or Cas11 subunits in the type III CRISPR–Cas systems. Left panel: Superimposition of CTD-Cas14 (light blue) and CTD-Cas10

(white, PDB ID: 6IFU) in the type III-A system with a root mean squared deviation (r.m.s.d.) of 3.33 Å. Middle panel: Superimposition of CTD-Cas14 (light blue) and Csm2 subunit (orange, PDB

ID: 6IFU) in the type III-A system with a root mean squared deviation (r.m.s.d.) of 5.79 Å. Right panel: Superimposition of CTD-Cas14 (light blue) and Cmr5 subunit (yellow, PDB ID: 3X1L) in

the type III-B system with a root mean squared deviation (r.m.s.d.) of 5.67 Å.

(a) Structure of the Cas14 tetramer. The NTD dimer interface and MLD tetramer interface are indicated by dashed boxes. The color scheme is the same as in Fig. 2. (b) Detailed view of the

interactions between the NTDs from Cas14.1 and Cas14.3. Key residues in the NTD dimer interface are shown in stick representation. (c) Close-up view of the tetramer interface formed by MLDs

of Cas14.1–Cas14.4. The MLD, which is dispensable for Cas14 binding to the Cas5-Cas7 complex, may serve a role in delivering the NTD and CTD to facilitate their correct orientations. (d)

SEC–MALS analysis of full length-Cas14 (F447A/P454A/F458A) and NTD-Cas14 (Y388A/Y391A/R416A). The theoretical monomeric molecular masses of these two variants are 71 and 51 kDa,

respectively. The experimental molecular weight of Y388A/Y391A/R416A mutant is ~ 52 kDa, suggesting a monomer in solution. However, the F447A/P454A/F458A mutant protein is not homogeneous

and exhibits a broad range of calculated molar masses, highlighting that tetramerization is crucial for Cas14 assembly and function. (e) In vitro target RNA cleavage assay with Cas14

variants harboring mutations in the dimer and tetramer interfaces. The gel is representative of three biological replicates. For gel source data, see Supplementary Fig. 1.

(a) The interdomain contacts for Cas14. The α15 and α16 helices of the CTD are tethered to the NTD through a combination of hydrophobic and polar contacts. In particular, Lys543 of the CTD

wedges between the MβL and β-CASP subdomains, while two other lysine residues, Lys540 and Lys547 of the CTD, stack against the MβL and β-CASP subdomains, respectively. The MβL and β-CASP

subdomains from Cas14.1 are highlighted in purpleblue and green, respectively. The CTD from Cas14.1 is colored in light blue. The zoomed-in view illustrates the detailed interdomain contacts

between the NTD and CTD (right panel). Key residues involved in the interdomain interaction are shown as sticks. (b) In vitro target RNA cleavage assay of WT and mutant Cas14 proteins. The

K540A/K543A/K547A mutation showed compromised nuclease activity. Representative gel image is from three biological replicates. (c) The NTD and MLD-involved contacts with Cas7 or target RNA

are indicated by black (NTD-target RNA), red (NTD-Cas7) and blue (MLD-Cas7) rectangles. (d–f) The zoomed-in view of NTD-target RNA (d), NTD-Cas7 (e) and MLD-Cas7 (f) interaction interfaces.

Key interaction residues are represented as sticks. (g) In vitro target RNA cleavage using Cas14 mutants. The gel is representative of three biological replicates. For gel source data, see

Supplementary Fig. 1.

(a) Schematic representation of the crRNA and target RNAs. The 5′-FAM labeled target RNA was used for cleavage. (b) Fluorescence mapping of the initial cleavage site. In vitro 5′-labeled RNA

cleavage assay results showed that a 4-nt product was produced. The left lane is the alkaline hydrolysis ladder, and the right lane is RNase T1 digestion products. Marker sizes are labeled

in yellow. The sizes of the cleavage products are labeled in pink. Representative gel is from three independent experiments. (c) Structural superposition of the NTD-Cas14 (purpleblue and

green, this study) and RNaseJ (pink, PDB ID: 4XWT). (d) Detailed view of the catalytic pockets in the aligned structures of NTD-Cas14 and RNase J. Catalytic residues are shown as sticks, and

zinc ions are represented as spheres. (e) Proposed catalytic mechanism of Cas14 two-metal-ion catalysis. Two Zn2+ ions are depicted as gray circles, and the putatively involved water

molecule is shown in blue. Hydrogen bonds and zinc coordination are represented by dashed lines. Asp66, along with the water molecule, facilitates the nucleophilic attack of the scissile

phosphate. His373 functions as a general acid to protonate and stabilize the reaction products. (f) In vitro RNA cleavage assay showed that the zinc chelator (1,10-phenanthroline) prevents

target cleavage, and this inhibition cannot be reversed by the addition of Mg2+ or Mn2+. Representative gel is from three independent replicates. (g) Structural superposition of the

NTD-Cas14 dimer (purpleblue and deepolive, this study) and RNase J dimer (pink and cyan, PDB ID: 4XWT). (h) Size-exclusion chromatography analysis of NTD-Cas14 proteins in the presence of

Mg2+, Mn2+, EDTA and 1,10-phenanthroline, respectively. For gel source data, see Supplementary Fig. 1.

(a) Schematic representation of the crRNA-target RNA duplex. (b) Mapping of target RNA cleavage products by Cas14. Lane 1: RNase T1 hydrolysis ladder; Lane 2: 60-min cleavage products; Lane

3: 3′-Cy3 labeled 49-, 46-, 43-, 39-, 35-nt RNA oligos; Lane 4: 3-min cleavage products; Lane 5: 3′-Cy3 labeled 48- and 45-nt RNA oligos. The sizes of the RNA are indicated by numbers in

yellow or pink. The gel is representative of three biological replicates. For gel source data, see Supplementary Fig. 1.

(a-b) Cryo-EM structures (left panels) and cartoon representations (middle panels) of Cas14-bound complexes at post-states I (a) and II (b). Right panels: Structural comparisons between

post-state I and substrate-engaged state I (a), or between post-state II and substrate-engaged state II (b).

(a) Superposition of substrate-engaged states I and II. Vector lengths represent the movement of Cas14 domains. (b) Mutations of key residues that mediate the interactions between CTD-Cas14

and Cas7 failed to cleave the target RNA. The gel is representative of three biological replicates. (c) Model of Cas14 binding and target RNA cleavage. Upon CTR binding, the Cas14 proteins

interact with the CTR-bound Cas5–Cas7 complex in two states (I and II), cleaving the CTR at two separate sites. When binding to NTR, the elongated Cas5–Cas7 filament enables the Cas14

proteins to bind at three different sites, resulting in the formation of three major cleavage products (+I, I, and II). The Cas14 probably disassembles from the interference complex, and the

Cas14-mediated cleavage products, such as the major cleavage I product, might also serve as a substrate for subsequent cleavage rounds (dashed arrows). Unlike the type III system, the type

VII system possesses an additional 3′-end repeat in its crRNA besides the 5′-end repeat. On the other hand, target RNA cleavage in the type VII system is affected by a complementary 5′-PFS

but not the 3′-PFS, contrasting with the observations in the type III system. (d) Structural superposition of the NTD-Cas14 (purpleblue and green, this study) with apo CPSF-73 (white, PDB

ID: 2I7V) and target RNA-bound CPSF-73 (brown, PDB ID: 6V4X). Bottom: The arrow indicates the conformational changes between apo CPSF-73 and target RNA-bound CPSF-73. For gel source data,

see Supplementary Fig. 1.

Recognition of cognate target RNA (CTR) by the Cas5–Cas7 complex and the subsequent recruitment of Cas14 for target cleavage. Two substate-engaged states (I and II) are shown, and a

zoomed-in view highlights the catalytic pocket located in the N-terminal region of the Cas14 protein.

Recognition of non-cognate target RNA (NTR) by the Cas5–Cas7 complex and the subsequent recruitment of Cas14 for target cleavage. Three substate-engaged states (+I, I and II) are shown, and

a zoomed-in view highlights the catalytic pocket located in the N-terminal region of the Cas14 protein.

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