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The Innovation > 2024 Vol. 5 > No. 2
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An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants

  • 1.

    Institute of Advanced Biotechnology and School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China

  • 2.

    Bellagen Biotechnology, Jinan 250000, China

  • 3.

    Department of Medical Neuroscience, School of Medicine, Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, Guangdong Province 518055, China

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  • 4These authors contributed equally

  • Corresponding author: zhujk@sustech.edu.cn
  • Received Date:  October 23, 2023
    Accepted Date:  January 01, 2024
    Published Online:  January 08, 2024
The Innovation  5(2),  https://doi.org/10.1016/j.xinn.2024.100564  |  Cite this article
    GRAPHICAL ABSTRACT
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    • PUBLIC SUMMARY
    1. ■ AI-guided prediction of structures and residues interacting with nucleic acids.
    2. ■ Engineering highly active and specific Cas-SF01.
    3. ■ High editing efficiency in animals and plants.
    • ABSTRACT
  • The type Ⅴ-Ⅰ CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure-guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that Cas-SF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.
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    • REFERENCES
  • [1] Wang, J.Y., and Doudna, J.A. (2023). CRISPR technology: A decade of genome editing is only the beginning. Science 379: eadd8643. https://doi.org/10.1126/science.add8643.

    View in Article CrossRef Google Scholar

    [2] Doudna, J.A. (2020). The promise and challenge of therapeutic genome editing. Nature 578: 229–236. https://doi.org/10.1038/s41586-020-1978-5.

    View in Article CrossRef Google Scholar

    [3] Strecker, J., Jones, S., Koopal, B., et al. (2019). Engineering of CRISPR-Cas12b for human genome editing. Nat. Commun. 10: 212. https://doi.org/10.1038/s41467-018-08224-4.

    View in Article CrossRef Google Scholar

    [4] Kleinstiver, B.P., Sousa, A.A., Walton, R.T., et al. (2019). Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37: 276–282. https://doi.org/10.1038/s41587-018-0011-0.

    View in Article CrossRef Google Scholar

    [5] Yan, W.X., Hunnewell, P., Alfonse, L.E., et al. (2019). Functionally diverse type Ⅴ CRISPR-Cas systems. Science 363: 88–91. https://doi.org/10.1126/science.aav7271.

    View in Article CrossRef Google Scholar

    [6] Huang, X., Sun, W., Cheng, Z., et al. (2020). Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2. Nat. Commun. 11: 5241. https://doi.org/10.1038/s41467-020-19072-6.

    View in Article CrossRef Google Scholar

    [7] Zhang, B., Luo, D., Li, Y., et al. (2021). Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1. Nat. Commun. 12: 3476. https://doi.org/10.1038/s41467-021-23876-5.

    View in Article CrossRef Google Scholar

    [8] Chen, Y., Hu, Y., Wang, X., et al. (2022). Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing. Innovation 3: 100264. https://doi.org/10.1016/j.xinn.2022.100264.

    View in Article CrossRef Google Scholar

    [9] Zhang, H., Kong, X., Xue, M., et al. (2023). An engineered xCas12i with high activity, high specificity, and broad PAM range. Protein Cell 14: 538–543. https://doi.org/10.1093/procel/pwac052.

    View in Article CrossRef Google Scholar

    [10] Yang, Y., Liu, S., Cheng, Y., et al. (2016). Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter. Appl. Biochem. Biotechnol. 180: 655–667. https://doi.org/10.1007/s12010-016-2122-8.

    View in Article CrossRef Google Scholar

    [11] Jumper, J., Evans, R., Pritzel, A., et al. (2021). Highly accurate protein structure prediction with AlphaFold. Nature 596: 583–589. https://doi.org/10.1038/s41586-021-03819-2.

    View in Article CrossRef Google Scholar

    [12] Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163: 759–771. https://doi.org/10.1016/j.cell.2015.09.038.

    View in Article CrossRef Google Scholar

    [13] Crooks, G.E., Hon, G., Chandonia, J.M., et al. (2004). WebLogo: a sequence logo generator. Genome Res. 14: 1188–1190. https://doi.org/10.1101/gr.849004.

    View in Article CrossRef Google Scholar

    [14] Tsai, S.Q., Zheng, Z., Nguyen, N.T., et al. (2015). GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33: 187–197. https://doi. org/10.1038/nbt.3117.

    View in Article CrossRef Google Scholar

    [15] Hiei, Y., Ohta, S., Komari, T., et al. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271–282. https://doi.org/10.1046/j.1365-313x.1994.6020271.x.

    View in Article CrossRef Google Scholar

    [16] Aarrouf, J., Castro-Quezada, P., Mallard, S., et al. (2012). Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes. Plant Cell Rep. 31: 391–401. https://doi.org/10.1007/s00299-011-1174-z.

    View in Article CrossRef Google Scholar

    [17] Kereszt, A., Li, D., Indrasumunar, A., et al. (2007). Agrobacterium rhizogenes-mediated transformation of soybean to study root biology. Nat. Protoc. 2: 948–952. https://doi.org/10.1038/nprot.2007.141.

    View in Article CrossRef Google Scholar

    • Cited by

  • Cite this article:

    Zhiqiang Duan, Yafeng Liang, Jialei Sun, Hongjin Zheng, Tong Lin, Pengyu Luo, Mengge Wang, Ruiheng Liu, Ying Chen, Shuhua Guo, Nannan Jia, Hongtao Xie, Meili Zhou, Minghui Xia, Kaijun Zhao, Shuhui Wang, Na Liu, Yongling Jia, Wei Si, Qitong Chen, Yechun Hong, Ruilin Tian, Jian-Kang Zhu. An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants[J]. The Innovation, 2024, 5(2). https://doi.org/10.1016/j.xinn.2024.100564
    Zhiqiang Duan, Yafeng Liang, Jialei Sun, Hongjin Zheng, Tong Lin, Pengyu Luo, Mengge Wang, Ruiheng Liu, Ying Chen, Shuhua Guo, Nannan Jia, Hongtao Xie, Meili Zhou, Minghui Xia, Kaijun Zhao, Shuhui Wang, Na Liu, Yongling Jia, Wei Si, Qitong Chen, Yechun Hong, Ruilin Tian, Jian-Kang Zhu. An engineered Cas12i nuclease that is an efficient genome editing tool in animals and plants[J]. The Innovation, 2024, 5(2). https://doi.org/10.1016/j.xinn.2024.100564

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