Engineering the MmeFz2-ωRNA system for efficient genome editing through an integrated computational-experimental framework

1. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

   Google Scholar 

2. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

   Google Scholar 

3. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

   Google Scholar 

4. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).

   Google Scholar 

5. Fellmann, C., Gowen, B. G., Lin, P. C., Doudna, J. A. & Corn, J. E. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nat. Rev. Drug Discov. 16, 89–100 (2017).

   Google Scholar 

6. Chen, K., Wang, Y., Zhang, R., Zhang, H. & Gao, C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70, 667–697 (2019).

   Google Scholar 

7. Wang, J. Y. & Doudna, J. A. CRISPR technology: a decade of genome editing is only the beginning. Science 379, eadd8643 (2023).

   Google Scholar 

8. Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358–378 (2019).

   Google Scholar 

9. Harrington, L. B. et al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362, 839–842 (2018).

   Google Scholar 

10. Karvelis, T. et al. PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage. Nucleic Acids Res. 48, 5016–5023 (2020).

    Google Scholar 

11. Takeda, S. N. et al. Structure of the miniature type V-F CRISPR-Cas effector enzyme. Mol. Cell 81, 558–570.e553 (2021).

    Google Scholar 

12. Wu, Z. et al. Programmed genome editing by a miniature CRISPR-Cas12f nuclease. Nat. Chem. Biol. 17, 1132–1138 (2021).

    Google Scholar 

13. Wang, Y. et al. Guide RNA engineering enables efficient CRISPR editing with a miniature Syntrophomonas palmitatica Cas12f1 nuclease. Cell Rep. 40, 111418 (2022).

    Google Scholar 

14. Kong, X. et al. Engineered CRISPR-OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing. Nat. Commun. 14, 2046 (2023).

    Google Scholar 

15. Pausch, P. et al. CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science 369, 333–337 (2020).

    Google Scholar 

16. Pausch, P. et al. DNA interference states of the hypercompact CRISPR-CasΦ effector. Nat. Struct. Mol. Biol. 28, 652–661 (2021).

    Google Scholar 

17. Carabias, A. et al. Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3. Nat. Commun. 12, 4476 (2021).

    Google Scholar 

18. Chen, W. et al. Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors. Mol. Cell 83, 2768–2780.e2766 (2023).

    Google Scholar 

19. Karvelis, T. et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature 599, 692–696 (2021).

    Google Scholar 

20. Nakagawa, R. et al. Cryo-EM structure of the transposon-associated TnpB enzyme. Nature 616, 390–397 (2023).

    Google Scholar 

21. Sasnauskas, G. et al. TnpB structure reveals minimal functional core of Cas12 nuclease family. Nature 616, 384–389 (2023).

    Google Scholar 

22. Li, Z. et al. Engineering a transposon-associated TnpB-ωRNA system for efficient gene editing and phenotypic correction of a tyrosinaemia mouse model. Nat. Commun. 15, 831 (2024).

    Google Scholar 

23. Xiang, G. et al. Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors. Nat. Biotechnol. 42, 745–757 (2024).

    Google Scholar 

24. Altae-Tran, H. et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science 374, 57–65 (2021).

    Google Scholar 

25. Schuler, G., Hu, C. & Ke, A. Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9. Science 376, 1476–1481 (2022).

    Google Scholar 

26. Han, D. et al. Development of miniature base editors using engineered IscB nickase. Nat. Methods 20, 1029–1036 (2023).

    Google Scholar 

27. Yan, H. et al. Assessing and engineering the IscB-ωRNA system for programmed genome editing. Nat. Chem. Biol. 20, 1617–1628 (2024).

    Google Scholar 

28. Han, L. et al. Engineering miniature IscB nickase for robust base editing with broad targeting range. Nat. Chem. Biol. 20, 1629–1639 (2024).

29. Xue, N. et al. Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos. Mol. Cell 84, 3128–3140.e3124 (2024).

    Google Scholar 

30. Xiao, Q. et al. Engineered IscB-ωRNA system with expanded target range for base editing. Nat. Chem. Biol. 21, 100–108 (2025).

31. Saito, M. et al. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature 620, 660–668 (2023).

    Google Scholar 

32. Jiang, K. et al. Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses. Sci. Adv. 9, eadk0171 (2023).

    Google Scholar 

33. Xu, P. et al. Structural insights into the diversity and DNA cleavage mechanism of Fanzor. Cell 187, 5238–5252.e20 (2024).

    Google Scholar 

34. Jiang, K. et al. Rapid in silico directed evolution by a protein language model with EVOLVEpro. Science 387, eadr6006 (2025).

    Google Scholar 

35. Hennig, J. Structural biology of RNA and protein-RNA complexes after alphaFold3. Chembiochem 26, e202401047 (2025).

    Google Scholar 

36. Su, M. et al. Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity. Nat. Chem. Biol. 20, 180–189 (2024).

    Google Scholar 

37. Kim, D. Y. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat. Biotechnol. 40, 94–102 (2022).

    Google Scholar 

38. Gao, Z., Herrera-Carrillo, E. & Berkhout, B. Delineation of the exact transcription termination signal for type 3 polymerase III. Mol. Ther. Nucleic Acids 10, 36–44 (2018).

    Google Scholar 

39. Zhang, X. et al. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain. Nat. Cell Biol. 22, 740–750 (2020).

    Google Scholar 

40. Yin, S. et al. Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies. Mol. Ther. 31, 744–759 (2023).

    Google Scholar 

41. Yang, C. et al. HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing. Nat. Commun. 14, 2430 (2023).

    Google Scholar 

42. Ding, X. et al. Improving CRISPR-Cas9 genome editing efficiency by fusion with chromatin-modulating Peptides. CRISPR J. 2, 51–63 (2019).

    Google Scholar 

43. Yin, J. et al. Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing. Nat. Commun. 13, 1204 (2022).

    Google Scholar 

44. Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Google Scholar 

45. Roberts, T. C., Wood, M. J. A. & Davies, K. E. Therapeutic approaches for Duchenne muscular dystrophy. Nat. Rev. Drug Discov. 22, 917–934 (2023).

    Google Scholar 

46. Flanigan, K. M. et al. Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum. Mutat. 30, 1657–1666 (2009).

    Google Scholar 

47. Min, Y. L., Bassel-Duby, R. & Olson, E. N. CRISPR correction of Duchenne muscular dystrophy. Annu. Rev. Med. 70, 239–255 (2019).

    Google Scholar 

48. Lin, J. et al. Adenine base editing-mediated exon skipping restores dystrophin in humanized Duchenne mouse model. Nat. Commun. 15, 5927 (2024).

    Google Scholar 

49. Bao, W. & Jurka, J. Homologues of bacterial TnpB_IS605 are widespread in diverse eukaryotic transposable elements. Mob. DNA 4, 12 (2013).

    Google Scholar 

50. Altae-Tran, H. et al. Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12. Proc. Natl. Acad. Sci. USA 120, e2308224120 (2023).

    Google Scholar 

51. Dong, C., Gou, Y. & Lian, J. SgRNA engineering for improved genome editing and expanded functional assays. Curr. Opin. Biotechnol. 75, 102697 (2022).

    Google Scholar 

52. Wu, Z. et al. Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1. Nat. Catal. 6, 695–709 (2023).

    Google Scholar 

Download references

Author notes

1. These authors contributed equally: Shangpu Li, Kun Xu, Guoling Li, Henan Jiang, Zhenmin Wu, Pengfei Gao.

Authors and Affiliations

1. International Joint Agriculture Research Center for Animal Bio-Breeding of Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China

   Shangpu Li, Kun Xu, Henan Jiang, Zhenmin Wu, Pengfei Gao, Yang Yue, Yufei Chen, Zujiang Liu, Bingqian Zhou, Yulin Chen, Xiaolong Wang & Yinghui Wei

2. Hainan Institute of Northwest A&F University, Sanya, Hainan, China

   Shangpu Li, Kun Xu, Zhenmin Wu, Yulin Chen, Xiaolong Wang & Yinghui Wei

3. Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, Guangdong, China

   Guoling Li

4. HuidaGene Therapeutics Co. Ltd., Shanghai, China

   Guoling Li

5. Life Science Research Core Services, Northwest A&F University, Yangling, Shaanxi, China

   Min Zhou

6. Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China

   Xu Liu

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

   Zhaowei Wu

Contributions

Y.W., Z.W.W., K.X., and X.W. conceived the project. Y.W., Z.W.W., K.X., and X.L. designed the experiments. Y.W., S.L., P.G., and G.L. performed data analysis. Z.W.W. conducted the structural prediction analysis. S. L., P.G., Z.M.W., Y.Y., H.J., Y.F.C., Z.L., B.Z., and M.Z. performed cell transfection and FACS. Y.W. and G.L. performed animal experiments. Y.W. and Z.W.W. wrote the manuscripts. Y.W., X.W., Z.W.W., Y.L.C., K.X., and X.L. supervised the project.

Corresponding authors

Correspondence to Xu Liu, Xiaolong Wang, Zhaowei Wu or Yinghui Wei.

Competing interests

The authors declare no competing interests.

Peer review information

Nature Communications thanks Chunyi Hu, Jun-Jie (Gogo) Liu and Hidetoshi Sakurai for their contribution to the peer review of this work. A peer review file is available.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions