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Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9

Author

Listed:
  • Shuliang Gao

    (Tufts University)

  • Benson Weng

    (Tufts University)

  • Douglas Wich

    (Tufts University)

  • Liam Power

    (Tufts University)

  • Mengting Chen

    (Tufts University)

  • Huiwen Guan

    (Tufts University)

  • Zhongfeng Ye

    (Tufts University)

  • Chutian Xu

    (Tufts University)

  • Qiaobing Xu

    (Tufts University)

Abstract

Domain expansion contributes to diversification of RNA-guided-endonucleases including Cas9. However, it remains unclear how REC domain expansion could benefit Cas9. In this study, we identify an insertion spot that is compatible with large REC insertion and succeeds in enlarging the non-catalytic REC domain of Streptococcus pyogenes Cas9. The natural-evolution-like giant SpCas9 (GS-Cas9) is created and shows substantially improved editing precision. We further discover that enlarging the REC domain could enable regulation of the N-terminal adenine deaminase TadA8e tethered to the Cas9 scaffold, which contributes to substantially reducing unexpected editing and improving the precision of the adenine base editor ABE8e. We provide proof of concept for evolution-inspired expansion of Cas9 and offer an alternative solution for optimizing gene editors. Our study also indicates a vast potential for engineering the topological malleability of RNA-guided endonucleases and base editors.

Suggested Citation

  • Shuliang Gao & Benson Weng & Douglas Wich & Liam Power & Mengting Chen & Huiwen Guan & Zhongfeng Ye & Chutian Xu & Qiaobing Xu, 2025. "Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57154-5
    DOI: 10.1038/s41467-025-57154-5
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    1. Kiran Musunuru & Alexandra C. Chadwick & Taiji Mizoguchi & Sara P. Garcia & Jamie E. DeNizio & Caroline W. Reiss & Kui Wang & Sowmya Iyer & Chaitali Dutta & Victoria Clendaniel & Michael Amaonye & Aar, 2021. "In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates," Nature, Nature, vol. 593(7859), pages 429-434, May.
    2. Hongzhi Zeng & Qichen Yuan & Fei Peng & Dacheng Ma & Ananya Lingineni & Kelly Chee & Peretz Gilberd & Emmanuel C. Osikpa & Zheng Sun & Xue Gao, 2023. "A split and inducible adenine base editor for precise in vivo base editing," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    3. Makoto Saito & Peiyu Xu & Guilhem Faure & Samantha Maguire & Soumya Kannan & Han Altae-Tran & Sam Vo & AnAn Desimone & Rhiannon K. Macrae & Feng Zhang, 2023. "Fanzor is a eukaryotic programmable RNA-guided endonuclease," Nature, Nature, vol. 620(7974), pages 660-668, August.
    4. Shouyue Zhang & Ao Sun & Jing-Mei Qian & Shuo Lin & Wenjing Xing & Yun Yang & Han-Zhou Zhu & Xin-Yi Zhou & Yan-Shuo Guo & Yun Liu & Yu Meng & Shu-Lin Jin & Wenhao Song & Cheng-Ping Li & Zhaofu Li & Sh, 2024. "Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity," Nature, Nature, vol. 630(8016), pages 484-492, June.
    5. Yajing Liu & Changyang Zhou & Shisheng Huang & Lu Dang & Yu Wei & Jun He & Yingsi Zhou & Shaoshuai Mao & Wanyu Tao & Yu Zhang & Hui Yang & Xingxu Huang & Tian Chi, 2020. "A Cas-embedding strategy for minimizing off-target effects of DNA base editors," Nature Communications, Nature, vol. 11(1), pages 1-9, December.
    6. Yuan Zhou & Chen Zhang & Weidong Xiao & Roland W. Herzog & Renzhi Han, 2024. "Systemic delivery of full-length dystrophin in Duchenne muscular dystrophy mice," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    7. Michael Elowitz & Wendell A. Lim, 2010. "Build life to understand it," Nature, Nature, vol. 468(7326), pages 889-890, December.
    8. Janice S. Chen & Yavuz S. Dagdas & Benjamin P. Kleinstiver & Moira M. Welch & Alexander A. Sousa & Lucas B. Harrington & Samuel H. Sternberg & J. Keith Joung & Ahmet Yildiz & Jennifer A. Doudna, 2017. "Enhanced proofreading governs CRISPR–Cas9 targeting accuracy," Nature, Nature, vol. 550(7676), pages 407-410, October.
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