Cryo-EM structure of the RNA-guided ribonuclease Cas12g

Abstract

Cas12g, the type V–G CRISPR–Cas effector, is an RNA-guided ribonuclease that targets single-stranded RNA substrate. The CRISPR–Cas12g system offers a potential platform for transcriptome engineering and diagnostic applications. We determined the structures of Cas12g–guide RNA complexes in the absence and presence of target RNA by cryo-EM to a resolution of 3.1 Å and 4.8 Å, respectively. Cas12g adopts a bilobed structure with miniature REC2 and Nuc domains, whereas the guide RNAs fold into a flipped ‘F’ shape, which is primarily recognized by the REC lobe. Target RNA and the CRISPR RNA (crRNA) guide form a duplex that inserts into the central cavity between the REC and NUC lobes, inducing conformational changes in both lobes to activate Cas12g. The structural insights would facilitate the development of Cas12g-based applications.

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Fig. 1: Overall structure of the Cas12g–sgRNA binary complex.
Fig. 2: Domain structure and active site of Cas12g.
Fig. 3: Overall structure of sgRNA.
Fig. 4: Interactions between Cas12g and sgRNA.
Fig. 5: Target RNA recognition of Cas12g.

Data availability

Cryo-EM reconstructions of Cas12g–sgRNA and Cas12g–sgRNA-target RNA complexes have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-22257 and EMD-22258, respectively. Coordinates for atomic models of Cas12g–sgRNA and Cas12g–sgRNA-target RNA complexes have been deposited in the Protein Data Bank under accession numbers 6XMF and 6XMG, respectively. Source data are provided with this paper.

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Acknowledgements

We thank T. Klose and V. Bowman for help with cryo-EM, and S. Wilson for computation. This work made use of the Purdue Cryo-EM Facility. This work was supported by the NIH grant R01GM138675 and a Showalter Trust Research Award to L.C.

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Authors

Contributions

L.C. supervised the study. H.Z., R.X. and Z.L. prepared samples. Z.L., H.Z. and L.C. collected and processed cryo-EM data. Z.L., H.Z., R.X. and R.H. performed biochemical analysis. Z.L., H.Z. and L.C. prepared figures. All authors analyzed the data. Z.L., H.Z. and L.C prepared the manuscript with input from R.X. and R.H.

Corresponding author

Correspondence to Leifu Chang.

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Extended data

Extended Data Fig. 1 Sample preparation and cryo-EM of the Cas12g-sgRNA binary complex.

a, Purification of Cas12g. Upper: Size exclusion chromatography (SEC) profile. UV absorbance curves at 280 nm and 260 nm are shown in blue and red, respectively. Lower: SDS-PAGE analysis. b, Circular dichroism (CD) spectra of wild-type and mutants of Cas12g used in this study. c, A representative raw cryo-EM micrograph of the Cas12g-sgRNA complex from a total of 2520 micrographs. d, Representative 2D class averages from a total of 100 images. e, Three major classes from 3D classification. f, 3D refinement for Class I with angular distribution of particles. g, Focused classification around the flexible region of the REC1 domain (REC1220–354). h, Cryo-EM map of class I with each domain of Cas12g and sgRNA colored coded. i, Plot of the global half-map FSC (solid red line), map-to-model FSC (solid orange line), and spread of directional resolution values (±1σ from mean, green dotted lines; the blue bars indicate a histogram of 100 such values evenly sampled over the 3D FSC). FSC plot for the reconstruction suggests an average resolution of 3.1 Å. j, Local resolution map for the reconstruction in f. Source data

Extended Data Fig. 2 Detailed cryo-EM density map of the Cas12g-sgRNA complex with atomic model fitted in.

a, Fitting of the REC1 domain, with density of REC1220–354 from focused classification shown in grey mesh. A representative α-helix from the REC1 domain is shown in detail on the right. b, Secondary structure prediction of REC1220–354 using PSIPRED. c, Fitting of the WED domain. d, Fitting of the RuvC domain. e, Fitting of the REC2 domain. f, Fitting of the Nuc domain. g, Fitting of nucleic acids to the corresponding cryo-EM map, with details of a segment shown as indicated on the right. The atomic models are shown in stick with crRNA and tracrRNA regions colored in orange and yellow, respectively. h, Detailed interactions between scaffolding duplex 1 and Cas12g. Cryo-EM density map is shown in mesh.

Extended Data Fig. 3 Structural comparison between Cas12g and Cas12b.

a, Overall structures of the Cas12g-sgRNA and the Cas12b-sgRNA complexes. b-f, Structural comparison of each domain. The structures are aligned by secondary-structure matching (SSM) in COOT.

Extended Data Fig. 4 Collateral cleavage and stoichiometric titration assays for Cas12g.

a, Collateral cleavage of unrelated ssDNA by Cas12g. The results shown are representative of three experiments. b, Collateral cleavage of unrelated ssRNA by Cas12g. The results shown are representative of three experiments. c, Stoichiometric titration assay indicates that Cas12g is a multiple turnover enzyme towards RNA substrate. Each point represents the cleavage ratio after 30 minutes at 37 °C. Error bars represent mean ± SD, where n = 3 replicates. Source data

Extended Data Fig. 5 Comparison of the REC1 domain in Cas12g and Cas12b, and crRNA guide mismatch assay for Cas12g.

a, The tracrRNA scaffolding duplex 1 is bound to the concave surface of the REC1 domain in the Cas12g-sgRNA binary complex. b, The crRNA-target DNA duplex is bound to the concave surface of the REC1 domain in the Cas12b-sgRNA-DNA complex. c, The tracrRNA scaffolding duplex 1 is bound to the concave surface of the REC1 domain in the Cas12g-sgRNA-target RNA complex. d, RNA cleavage assay using wild-type and mutant target RNAs. The results shown are representative of three experiments. Source data

Extended Data Fig. 6 Cryo-EM data processing for the Cas12g-sgRNA-target RNA complex.

a, A representative raw cryo-EM micrograph of the Cas12g-sgRNA-target RNA complex from a total of 987 micrographs. b, Representative 2D class averages from a total of 100 images. c, 3D classification. Three major classes were observed. Classes II and III did not result in good reconstructions and likely represent damaged particles. d, 3D refinement for Class I. Angular distribution is shown on the right. e, Plot of the global half-map FSC (solid red line), map-to-model FSC (solid orange line), and spread of directional resolution values (±1σ from mean, green dotted lines; the blue bars indicate a histogram of 100 such values evenly sampled over the 3D FSC). FSC plot for the reconstruction suggests an average resolution of 4.8 Å. f, Local resolution map in two views.

Extended Data Fig. 7 Structural and biochemical analysis of the Cas12g-sgRNA-target RNA complex.

a-b, Structural elements involved in crRNA-target RNA duplex recognition. Three such structural elements are indicated by red lines, including 1) an N-terminal extension enriched in arginines within the WED domain, 2) a loop within the REC2 domain (608-AKKATLQP-615), and 3) REC1220–354. c, Substrate RNA cleavage assay using wild-type Cas12g and Cas12g with mutations in two loops involved in the recognition of the crRNA-target RNA duplex. The results shown are representative of three experiments. d-e, Comparison of the REC1 and REC2 domains for substrate recognition between Cas12g and Cas12b. Source data

Extended Data Fig. 8 Binding assay between the Cas12g-sgRNA complex and target nucleic acids.

Upper: SEC profiles for the Cas12g-sgRNA complex incubated with different targets including target RNA, target RNA with mismatches at positions 16 and 17, target RNA with mismatches at positions 18 and 19, and target ssDNA. UV absorbance curves at 280 nm are shown. Lower: Urea-PAGE (15%) analysis of the elution fractions from SEC as indicated. The results shown are representative of three experiments. Source data

Extended Data Fig. 9 Cryo-EM analysis for the Cas12g-sgRNA-target DNA complex.

a, A representative raw cryo-EM micrograph of the Cas12g-sgRNA-target DNA complex from a total of 1083 micrographs. b, Representative 2D class averages from a total of 100 images. c, 3D classification. Classes 2-6 did not result in good reconstructions and likely represent damaged particles. d, 3D refinement for Class 1 with angular distribution. e, Plot of the global half-map FSC (solid red line), and spread of directional resolution values (±1σ from mean, green dotted lines; the blue bars indicate a histogram of 100 such values evenly sampled over the 3D FSC). FSC plot for the reconstruction suggests an average resolution of 5.8 Å. f, Local resolution map. g, Comparison of the rigid-body fitting between the Cas12g-sgRNA complex and the Cas12g-sgRNA-target RNA complex. Red squares indicate the fitting of the REC1 domain, which shows that the model of the Cas12g-sgRNA complex fits better to the map (left panel). h, Schematic model for Cas12g activation. Target DNA fails to form fully assembled duplex required for Cas12g activation.

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Li, Z., Zhang, H., Xiao, R. et al. Cryo-EM structure of the RNA-guided ribonuclease Cas12g. Nat Chem Biol (2021). https://doi.org/10.1038/s41589-020-00721-2

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