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Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

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Guan, Kaoling, Ocampo, Rodrigo Fregoso, Matheus Carnevali, Paula B., Castelle, Cindy J., Gonzalez-Osorio, Liliana, Castanzo, Dominic T., Thomas, Nicole C., Brothers, Molly, Dangerfield, Tyler L., Hooper, Matthew M., West, Madeline S., Appleby, Nathan M., Krudop, Isabella, Lamothe, Rebecca C., Aliaga Goltsman, Daniela S., Alexander, Lisa M., Butterfield, Cristina N., Johnson, Kenneth A., Brown, Christopher T., Taylor, David W.·2026·Nature Structural & Molecular Biology
DOI 10.1038/s41594-026-01788-6

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Abstract

Miniature CRISPR–Cas12f nucleases are attractive candidates for therapeutic genome editing because of their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here we identify and characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Alistipes sp. Cas12f (Al3Cas12f), which supports robust genome editing in human cells. Through structural, biochemical and kinetic analyses, we compare Al3Cas12f to two recently described orthologs, Oscillibacter sp. Cas12f and Ruminiclostridium herbifermentans Cas12f. These orthologs present divergent architectures and regulatory features governing protospacer-adjacent motif recognition, guide RNA (gRNA) binding, dimerization and DNA cleavage. Notably, Al3Cas12f achieves efficient R-loop formation through a stable dimer interface and a naturally optimized gRNA. Leveraging these structural insights, we generate an engineered Al3Cas12f variant (RKK) that increases editing and improves activity across several tested genomic loci. By overcoming locus-dependent variability and an apparent potency threshold, this engineered compact editor seems to expand the feasibility of low-dose, AAV-compatible therapeutic genome editing. Our results elucidate mechanistic determinants of Cas12f activity and offer a framework for engineering compact genome editors that may bear therapeutic potential.

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Research works printed but not linked: 52

  1. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). · DOI · Paper page
  2. Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR–Cas system. Nat. Biotechnol. 31, 227–229 (2013). · DOI · Paper page
  3. Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229–1232 (2013). · DOI
  4. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). · DOI · Paper page
  5. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013). · DOI · Paper page
  6. Koonin, E. V., Gootenberg, J. S. & Abudayyeh, O. O. Discovery of diverse CRISPR–Cas systems and expansion of the genome engineering toolbox. Biochemistry 62, 3465–3487 (2023). · DOI
  7. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015). · DOI · Paper page
  8. Gier, R. A. et al. High-performance CRISPR–Cas12a genome editing for combinatorial genetic screening. Nat. Commun. 11, 3455 (2020). · DOI
  9. Tang, K. et al. Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering. Nat. Biomed. Eng 9, 1290–1308 (2025). · DOI
  10. Ma, E. et al. Improved genome editing by an engineered CRISPR–Cas12a. Nucleic Acids Res. 50, 12689–12701 (2022). · DOI · Paper page
  11. Fuentes, C. M. & Schaffer, D. V. Adeno-associated virus-mediated delivery of CRISPR–Cas9 for genome editing in the central nervous system. Curr. Opin. Biomed. Eng 7, 33–41 (2018). · DOI
  12. 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). · DOI
  13. Aliaga Goltsman, D. S. et al. Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes. Nat. Commun. 13, 7602 (2022). · DOI
  14. Harrington, L. B. et al. Programmed DNA destruction by miniature CRISPR–Cas14 enzymes. Science 362, 839–842 (2018). · DOI
  15. Bigelyte, G. et al. Miniature type V-F CRISPR–Cas nucleases enable targeted DNA modification in cells. Nat. Commun. 12, 6191 (2021). · DOI
  16. Kong, X. et al. Engineered CRISPR–OsCas12f1 and RhCas12f1 with robust activities and expanded target range for genome editing. Nat. Commun. 14, 2046 (2023). · DOI
  17. Xiao, R., Li, Z., Wang, S., Han, R. & Chang, L. Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease. Nucleic Acids Res. 49, 4120–4128 (2021). · DOI
  18. Takeda, S. N. et al. Structure of the miniature type V-F CRISPR–Cas effector enzyme. Mol. Cell 81, 558–570 (2021). · DOI
  19. Wu, T. et al. An engineered hypercompact CRISPR–Cas12f system with boosted gene-editing activity. Nat. Chem. Biol. 19, 1384–1393 (2023). · DOI
  20. Wu, Z. et al. Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1. Nat. Catal. 6, 695–709 (2023). · DOI
  21. Su, M. et al. Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity. Nat. Chem. Biol. 20, 180–189 (2024). · DOI
  22. Hino, T. et al. An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Cell 186, 4920–4935 (2023). · DOI
  23. Wu, Z. et al. Programmed genome editing by a miniature CRISPR–Cas12f nuclease. Nat. Chem. Biol. 17, 1132–1138 (2021). · DOI
  24. Zhang, X. et al. Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing. Nat. Commun. 16, 3016 (2025). · DOI
  25. Madariaga-Marcos, J. et al. Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f. Nucleic Acids Res. 53, gkaf588 (2025). · DOI
  26. Sharrar, A. et al. Discovery and characterization of novel type V Cas12f nucleases with diverse protospacer adjacent motif preferences. CRISPR J. 6, 350–358 (2023). · DOI
  27. Tordoff, J. et al. Initial characterization of 12 new subtypes and variants of type V CRISPR systems. CRISPR J. 8, 149–154 (2025). · DOI
  28. Stella, S. et al. Conformational activation promotes CRISPR–Cas12a catalysis and resetting of the endonuclease activity. Cell 175, 1856–1871 (2018). · DOI
  29. Zhang, H., Li, Z., Xiao, R. & Chang, L. Mechanisms for target recognition and cleavage by the Cas12i RNA-guided endonuclease. Nat. Struct. Mol. Biol. 27, 1069–1076 (2020). · DOI
  30. Kurihara, N. et al. Structure of the type V-C CRISPR–Cas effector enzyme. Mol. Cell 82, 1865–1877.e4 (2022). · DOI
  31. Gong, S., Yu, H. H., Johnson, K. A. & Taylor, D. W. DNA unwinding is the primary determinant of CRISPR–Cas9 activity. Cell Rep. 22, 359–371 (2018). · DOI
  32. Liu, M.-S., Gong, S., Yu, H.-H., Taylor, D. W. & Johnson, K. A. Kinetic characterization of Cas9 enzymes. Methods Enzymol. 616, 289–311 (2019). · DOI
  33. Johnson, K. A. Fitting enzyme kinetic data with KinTek Global Kinetic Explorer. Methods Enzymol. 467, 601–626 (2009). · DOI
  34. Johnson, K. A., Simpson, Z. B. & Blom, T. FitSpace Explorer: an algorithm to evaluate multidimensional parameter space in fitting kinetic data. Anal. Biochem. 387, 30–41 (2009). · DOI
  35. Ocampo, R. F. et al. DNA targeting by compact Cas9d and its resurrected ancestor. Nat. Commun. 16, 457 (2025). · DOI
  36. Hibshman, G. N. et al. Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9. Nat. Commun. 15, 3663 (2024). · DOI
  37. Liu, M.-S. et al. Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA. Nat. Commun. 11, 3576 (2020). · DOI
  38. Bravo, J. P. K. et al. Structural basis for mismatch surveillance by CRISPR–Cas9. Nature 603, 343–347 (2022). · DOI
  39. Pacesa, M. et al. R-loop formation and conformational activation mechanisms of Cas9. Nature 609, 191–196 (2022). · DOI
  40. Li, D., Liu, C.-M., Luo, R., Sadakane, K. & Lam, T.-W. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31, 1674–1676 (2015). · DOI
  41. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010). · DOI
  42. Menzel, P., Ng, K. L. & Krogh, A. Fast and sensitive taxonomic classification for metagenomics with Kaiju. Nat. Commun. 7, 11257 (2016). · DOI
  43. Alexander, L. M. et al. Novel and engineered type II CRISPR systems from uncultivated microbes with broad genome editing capability. CRISPR J. 6, 261–277 (2023). · DOI
  44. Passaro, S. et al. Boltz-2: towards accurate and efficient binding affinity prediction. Preprint at bioRxiv https://doi.org/10.1101/2025.06.14.659707 (2025). · DOI
  45. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005). · DOI
  46. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). · DOI · Paper page
  47. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). · DOI
  48. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010). · DOI · Paper page
  49. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in PHENIX. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019). · DOI · Paper page
  50. Cianfrocco, M. A., Wong-Barnum, M., Youn, C., Wagner, R. & Leschziner, A. COSMIC2: a science gateway for cryo-electron microscopy structure determination. In Proc. Practice and Experience in Advanced Research Computing 2017: Sustainability, Success and Impact (ed. Hart, D.) (ACM, 2017). · DOI
  51. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D Struct. Biol. 74, 519–530 (2018). · DOI
  52. Sonn-Segev, A. et al. Quantifying the heterogeneity of macromolecular machines by mass photometry. Nat. Commun. 11, 1772 (2020). · DOI

Metadata

Authors
Guan, K., Ocampo, R. F., Matheus Carnevali, P. B., Castelle, C. J., Gonzalez-Osorio, L., Castanzo, D. T., Thomas, N. C., Brothers, M., Dangerfield, T. L., Hooper, M. M., West, M. S., Appleby, N. M., Krudop, I., Lamothe, R. C., Aliaga Goltsman, D. S., Alexander, L. M., Butterfield, C. N., Johnson, K. A., Brown, C. T., & Taylor, D. W.
Affiliations
  • The University of Texas at Austin (US)
  • University of Nevada, Las Vegas (US)
Corresponding author
Taylor, David W.
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
Springer Science and Business Media LLC
Published
2026-04-13
Volume
33
Issue
5
Pages
756-767
ISSN
  • 1545-9985 (ISSN-L)
  • 1545-9993
DOI
10.1038/s41594-026-01788-6
OpenAlex
W4413348174
Cited by
5
References
3
Citations by year
2026: 5
FWCI
10.33
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Structural & Molecular Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
Funders
  • University of Texas at Austin
  • National Institutes of Health (F-1938)
  • National Institute of General Medical Sciences (R35GM138348, R35GM158551)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Health Sciences › Medicine › Oncology › CAR-T cell therapy research
Keywords
genome editing efficiency, R-loop formation, therapeutic genome editing
MeSH terms
Eubacteriales, Gene Editing, R-Loop Structures, RNA, Guide, CRISPR-Cas Systems, Humans, Models, Molecular, HEK293 Cells, CRISPR-Cas Systems, CRISPR-Associated Proteins

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