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Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease

Nature Structural & Molecular Biology volume 27, pages 1069–1076 (2020)Cite this article

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Abstract

Cas12i is a recently identified type V CRISPR-Cas endonuclease that predominantly cleaves the non-target strand of a double-stranded DNA substrate. This nicking activity of Cas12i could potentially be used for genome editing with high specificity. To elucidate its mechanisms for target recognition and cleavage, we determined cryo-EM structures of Cas12i in multiple functional states. Cas12i pre-orders a seven-nucleotide seed sequence of the crRNA for target recognition and undergoes a two-step activation through crRNA–DNA hybridization. Formation of 14 base pairs activates the nickase activity, and 28-bp hybridization promotes cleavage of the target strand. The atomic structures and mechanistic insights gained should facilitate the manipulation of Cas12i for genome editing applications.

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Fig. 1: Structure of the Cas12i–crRNA–dsDNA ternary complex.
Fig. 2: Pre-crRNA processing, substrate recognition and cleavage by Cas12i.
Fig. 3: Mechanism of Cas12i activation.
Fig. 4: The lid in Cas12 endonucleases.

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Data availability

Cryo-EM reconstructions of Cas12i(E894A)–crRNA–dsDNA, Cas12i–crRNA and the I1 complexes have been deposited in the Electron Microscopy Data Bank under accession nos. EMD-21541, EMD-21551 and EMD-21552, respectively. Coordinates for atomic models of Cas12i(E894A)–crRNA–dsDNA, Cas12i–crRNA and the I1 complexes have been deposited in the Protein Data Bank under accession nos. 6W5C, 6W62 and 6W64, 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, S. Wilson for computation and J. Tesmer and C. Gabel for critical reading of the manuscript. This work was supported by NIH grant no. R01GM138675 and a Showalter Trust Research Award to L.C.

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  1. These authors contributed equally: Heng Zhang, Zhuang Li, Renjian Xiao.

Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

    Heng Zhang, Zhuang Li, Renjian Xiao & Leifu Chang

  2. Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN, USA

    Leifu Chang

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Contributions

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

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Correspondence to Heng Zhang or Leifu Chang.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz and Anke Sparmann were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Sample preparation and cryo-EM for the Cas12i(E894A)-crRNA-dsDNA complex.

a, Purification of Cas12i. Upper: Size exclusion chromatography (SEC) profile of Cas12i. UV absorbance curves at 280 nm and 260 nm are shown in blue and red, respectively. Lower: SDS-PAGE analysis of the elution fractions from SEC as indicated. b, Urea-PAGE analysis of pre-crRNA before and after processing by Cas12i. RNA markers indicate that the mature crRNA is 51nt. c, A representative raw cryo-EM micrograph of the Cas12i-crRNA-dsDNA ternary complex. d, Representative 2D class averages. e, Two major classes from 3D classification. The two maps are similar, with class 2 at better quality. f, 3D auto-refinement using particles from class 2. Angular distribution of particles is shown on the right. g, Procedures used for improving the resolution of the PI domain. Focused refinement followed by focused 3D classification using a soft mask (in pink mesh) resulted in a 3D class from 13% particles with improved density in the PI domain. h, 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). i, Local resolution map of the final reconstruction. Uncropped images for panels a and b are available as source data online.

Source data

Extended Data Fig. 2 Detailed cryo-EM density map of the Cas12i(E894A)-crRNA-dsDNA complex with final atomic model fitted in.

a, b, Fitting of nucleic acids to the corresponding cryo-EM map. The atomic models are shown in stick with crRNA, the target strand and the non-target strand colored in orange, magenta and cyan, respectively. Cryo-EM density from the sharpened map (a) or the unsharpened map (b) is shown in mesh. c, Fitting of the REC1 domain. A representative α-helix from the REC1 domain is shown in details on the right. d, Fitting of the WED domain. A representative β-strand is shown in details on the right. e, Fitting of the PI domain. f, Fitting of the RuvC domain. g, Fitting of the REC2 domain. h, Fitting of the Nuc domain. i, Fitting of the substrate DNA.

Extended Data Fig. 3 Structural comparison of Cas12i and Cas12b.

a, Overall structures of the Cas12i-crRNA-dsDNA and the Cas12b-gRNA-dsDNA complexes. The structures are aligned by secondary-structure matching (SSM) in COOT. b–f, Structural comparison of each domain. Secondary structures in each domain are labeled. The PI domain and the extended α16 and α17 helix pair are indicated by circles and a box, respectively. g, Sequence alignment of the crRNA repeat adjacent to the spacer-derived segment between Cas12i and Cas12b.

Extended Data Fig. 4 Schematic of nucleic acid recognition in the Cas12i(E894A)-crRNA-dsDNA complex.

Intermolecular contacts between Cas12i and nucleic acids, including the target and non-target strands and the substrate DNA, are shown by solid lines. The Cas12i residues are colored based on the domain architecture.

Extended Data Fig. 5 Structure and mutagenesis analysis for the recognition of the crRNA-target DNA heteroduplex.

a, Structure of the Cas12i-crRNA-DNA complex with selected key residues involved in the recognition of the heteroduplex shown as sticks. b, Close-up view of the interactions shown in a with cryo-EM map shown in mesh. c, Substrate cleavage assay using wild-type Cas12i and Cas12i with single mutations on the key residues shown in a. The results shown are representative of three experiments. d, Substrate cleavage assay using poly-A, -T, -C, and G as substrates. The results shown are representative of three experiments. Uncropped images for panels c and d are available as source data online.

Source data

Extended Data Fig. 6 Cryo-EM data processing for the wild-type Cas12i-crRNA-dsDNA complex.

a, A representative raw cryo-EM micrograph of the wild-type Cas12i-crRNA-DNA complex. b, Representative 2D class averages. c, 3D classification. Three major classes are observed, representing the Cas12i-crRNA binary complex, the intermediate state (I1 state, 14-15 bp heteroduplex), and the fully assembled Cas12i-crRNA-DNA complex (26-28 bp heteroduplex). Densities corresponding to the crRNA and target DNA are colored in yellow. The Cas12i-crRNA map is comparable to a reconstruction using biochemically purified Cas12i-crRNA complex (data not shown). d, 3D auto-refinement for the three classes shown in c. Angular distribution of each reconstruction is shown below. e, Local resolution map of the final reconstructions in d. f, 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).

Extended Data Fig. 7 Structural analysis of the seed sequence.

a, Superposition of the seed sequence of Cas12i (orange) with the same sequence simulated in an A-form geometry (blue). b, Structure of the seed sequence with Cas12i domains shown in surface representation.

Extended Data Fig. 8 Conformational changes of recognition loops in Cas12i upon heteroduplex formation.

a, Structure of the Cas12i-crRNA binary complex with cryo-EM density shown in mesh. Loop 726-737 is indicated. b, Structure of the I1 state with cryo-EM density shown in mesh. c, Structure of the Cas12i(E894A)-crRNA-DNA ternary complex with cryo-EM density shown in mesh. d, Structure showing three loops from the REC1 and REC2 domains of Cas12i that are likely involved in heteroduplex recognition. e, Substrate cleavage assay using wild-type Cas12i and Cas12i with each of the three loops shown in d deleted or mutated. The results shown are representative of three experiments. f, Sequence alignment of the lid region among Cas12 orthologs using ClustalW program (Thompson, J. D., Gibson, T. J. & Higgins, D. G. Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2, Unit 2 3, doi:10.1002/0471250953.bi0203s00 (2002).). Uncropped image for panel e is available as source data online.

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Zhang, H., Li, Z., Xiao, R. et al. Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease. Nat Struct Mol Biol 27, 1069–1076 (2020). https://doi.org/10.1038/s41594-020-0499-0

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