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Programmed genome editing by a miniature CRISPR-Cas12f nuclease

Nature Chemical Biology volume 17, pages 1132–1138 (2021)Cite this article

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Abstract

The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5′ T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.

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Fig. 1: AsCas12f1, an RNA-guided nuclease, cleaves dsDNA in a PAM-dependent manner.
Fig. 2: AsCas12f1-mediated genome editing in bacteria.
Fig. 3: AsCas12f1 facilitates genome editing in human cells.
Fig. 4: Mismatch tolerance and genome-wide editing fidelity of AsCas12f1.

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Data availability

NGS data are available at the NCBI Sequence Read Archive (SRA) under accession codes SRX9277304, SRX9277305, SRX9277306, SRX10828545, SRX10828546, SRX10828547 and SRX10839449. Plasmids can be accessed in Addgene under accession codes 171609, 171610, 171611, 171612, 171613 and 171614. Source data are provided with this paper.

Code availability

The custom Python scripts for calculating sequence frequencies in the PAM depletion assay are available at GitHub (https://github.com/atlasbioinfo/midseq). The computational pipeline of CIRCLE-seq to identify genome-wide off-target sites for AsCas12f1 is available at GitHub (https://github.com/tsailabSJ/circleseq).

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Acknowledgements

This work was supported by grants 21922705 (Q.J.), 91753127 (Q.J.) and 2207783 (Q.J.) from the National Natural Science Foundation of China; 19QA1406000 (Q.J.) from the Shanghai Committee of Science and Technology, China; 2017YFA0506800 (Q.J.) from the National Key R&D Program of China; EKPG21-18 (Q.J.) from the Emergency Key Program of Guangzhou Laboratory; and 2019M651627 (Z.W.) from the China Postdoctoral Science Foundation. We thank C. He at The University of Chicago for helpful discussions. We thank R. Liu, X. Huang and J. Li at ShanghaiTech University for kindly providing HEK293 cells and valuable advice regarding cell culture experiments. We thank G. Luo and Z. Ren at Sun Yat-sen University for helpful advice in RNA-seq experimental design. We thank J. Li, Y. Xiong and X. Ren from the Molecular and Cell Biology Core Facility (MCBCF) at the School of Life Science and Technology, ShanghaiTech University, for providing technical support.

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Authors and Affiliations

  1. School of Physical Science and Technology, ShanghaiTech University, Shanghai, China

    Zhaowei Wu, Yifei Zhang, Deng Pan, Yujue Wang, Yannan Wang, Fan Li, Chang Liu, Weizhong Chen & Quanjiang Ji

  2. University of Chinese Academy of Sciences, Beijing, China

    Yifei Zhang, Deng Pan, Yujue Wang & Yannan Wang

  3. Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich, UK

    Haopeng Yu

  4. College of Life Sciences, Northwest A&F University, Yangling, China

    Hao Nan

  5. Guangzhou Laboratory, Guangzhou, China

    Quanjiang Ji

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Contributions

Z.W., Y.Z. and Q.J. conceived the initial study. Z.W. and Yujue Wang performed the PAM depletion assay. Z.W. and H.Y. conducted the small RNA-seq and computational analyses. Z.W., Y.Z., Yannan Wang, F.L., D.P. and W.C. performed plasmid construction, protein purification and biochemical experiments. Z.W., Yujue Wang and Yannan Wang performed the genome editing in bacteria. Z.W., H.Y., D.P. and Yujue Wang performed the genome editing in human cells and NGS data analyses. Z.W., H.N. and D.P. designed the AAV vector and performed the AAV-based genome editing. Z.W. and C.L. analyzed the sequence homology among Cas12 nucleases and built the structure model. Z.W., H.Y. and D.P. performed the CIRCLE-seq. Z.W. and Q.J. prepared the figures and wrote the manuscript. The manuscript was reviewed and approved by all authors.

Corresponding author

Correspondence to Quanjiang Ji.

Ethics declarations

Competing interests

Q.J., Z.W., Y.Z., Yujue Wang and Yannan Wang have filed a patent application (PCT/CN2020/132759) related to this work through ShanghaiTech University. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Optimization of the sgRNA for AsCas12f1.

a, The predicted secondary structure of sgRNA_v1 and the truncation information. The gray shaded tri-guanosine is added to the beginning of sgRNA to maximize the RNA production. b, in vitro dsDNA cleavage assay for different truncations of sgRNA. c, Bacterial genome targeting using different truncations of sgRNA. d, The predicted secondary structure of sgRNA_v2.

Source data

Extended Data Fig. 2 Sequence and structural analysis of AsCas12f1 and its mutations in the RuvC active site.

a, Multiple sequence alignment of Cas12 nucleases. b, Structure model of AsCas12f1 was built using the cryo-EM structure of Cas14a1/sgRNA/dsDNA (PDB ID: 7C7L) as a template using SWISS-MODEL web server with its default parameters. c, D225A and E324A completely abolished the nuclease activity of AsCas12f1, whereas R383A and D401A retained minor NTS nickase activity at 18-nt downstream the PAM.

Source data

Extended Data Fig. 3 AsCas12f1 cleaves ssDNA in a PAM-independent manner.

a, Cleavage pattern for complementary-PAM (cPAM)-containing ssDNA. b, Cleavage pattern for non-cPAM-containing ssDNA. c, Cleavage pattern for PAM-containing staggered substrate. d, Cleavage pattern for non-PAM-containing staggered substrate. e, Non-targeting sgRNA cannot mediate targeted cleavage on short ssDNA substrate. f, AsCas12f1 cannot non-specifically cleave short ssDNA substrate.

Source data

Extended Data Fig. 4 Flow cytometry gating strategies.

a, b, Gates used for the analysis of flow cytometric data shown in Fig. 3c. Gate 1 is used to remove the debris from the cell population, gate 2 specifies single cells from cell aggregates, and gate 3 identifies the cells with GFP fluorescence. Gates were set on the control sample (a) and was then kept constant for the experimental group (b).

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–3.

Reporting Summary

Supplementary Data 1

Primers and substrates.

Supplementary Data 2

Predicted and experimental off-targets

Source data

Source Data Fig. 1

Unmodified gels.

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Unmodified gel.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Unmodified gel.

Source Data Extended Data Fig. 2

Unmodified gels.

Source Data Extended Data Fig. 3

Unmodified gels.

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Wu, Z., Zhang, Y., Yu, H. et al. Programmed genome editing by a miniature CRISPR-Cas12f nuclease. Nat Chem Biol 17, 1132–1138 (2021). https://doi.org/10.1038/s41589-021-00868-6

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