Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope

Yanan Huo; Jiale Mei; Dan Zhang; Bing Yan; Dexin Zhang; Chao Dong; Shuming Yin; Meizhen Liu; Xinyan Wang; Dan Chen; Yuting Guan; Gaojie Song; Bing Du; Yongming Wang; Zongli Zheng; Hong Liu; Dali Li; Lei Yang; Liren Wang

doi:10.1038/s41467-026-69678-5

Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope

Yanan Huo (Co-first Author)
, Jiale Mei (Co-first Author)
, Dan Zhang (Co-first Author)
, Bing Yan
, Dexin Zhang
, Chao Dong
, Shuming Yin
, Meizhen Liu
, Xinyan Wang
, Dan Chen
, Yuting Guan
, Gaojie Song
, Bing Du
, Yongming Wang
, Zongli Zheng
, Hong Liu
, Dali Li^*
, Lei Yang^*
, Liren Wang^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

Abstract

The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering. © The Author(s) 2026.

Original language	English
Article number	2918
Number of pages	14
Journal	Nature Communications
Volume	17
Online published	19 Feb 2026
DOIs	https://doi.org/10.1038/s41467-026-69678-5
Publication status	Published - 2026

Funding

We thank Y. Zhang from the Flow Cytometry Core Facility of School of Life Sciences at ECNU and support from the ECNU Public Platform for innovation (011). This work was partially supported by grants from the National Key R&D Program of China (2023YFE0209200 and 2023YFC3403400 to D.L., 2022YFC3400200 to Y.G.), National Natural Science Foundation of China (32025023, 32230064 and 32311530111 to D.L., U24A20677 to L.W.), the Agriculture Science and Technology Major Project,\u00A0Innovation Program of Shanghai Municipal Education Commission (2025GDZKZD03 to D.L.), Shanghai Municipal Commission for Science and Technology (24J22800400 to D.L.).D.L. is a Shanghai Academy of Natural Sciences Exploration Scholar.

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This full text is made available under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/

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Huo, Y., Mei, J., Zhang, D., Yan, B., Zhang, D., Dong, C., Yin, S., Liu, M., Wang, X., Chen, D., Guan, Y., Song, G., Du, B., Wang, Y., Zheng, Z., Liu, H., Li, D., Yang, L., & Wang, L. (2026). Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope. Nature Communications, 17, Article 2918. https://doi.org/10.1038/s41467-026-69678-5

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abstract = "The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91\% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering. {\textcopyright} The Author(s) 2026.",

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AU - Huo, Yanan

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AU - Yin, Shuming

AU - Liu, Meizhen

AU - Wang, Xinyan

AU - Chen, Dan

AU - Guan, Yuting

AU - Song, Gaojie

AU - Du, Bing

AU - Wang, Yongming

AU - Zheng, Zongli

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AU - Li, Dali

AU - Yang, Lei

AU - Wang, Liren

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Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope

Abstract

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