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Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

Author

Listed:
  • Minh Thuan Nguyen Tran

    (University of Tasmania)

  • Mohd Khairul Nizam Mohd Khalid

    (University of Tasmania)

  • Qi Wang

    (University of Tasmania)

  • Jacqueline K. R. Walker

    (University of Tasmania)

  • Grace E. Lidgerwood

    (The University of Melbourne
    The University of Melbourne)

  • Kimberley L. Dilworth

    (The University of Sydney)

  • Leszek Lisowski

    (The University of Sydney
    Military Institute of Hygiene and Epidemiology, The Biological Threats Identification and Countermeasure Centre)

  • Alice Pébay

    (The University of Melbourne
    The University of Melbourne)

  • Alex W. Hewitt

    (University of Tasmania
    The University of Melbourne)

Abstract

Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

Suggested Citation

  • Minh Thuan Nguyen Tran & Mohd Khairul Nizam Mohd Khalid & Qi Wang & Jacqueline K. R. Walker & Grace E. Lidgerwood & Kimberley L. Dilworth & Leszek Lisowski & Alice Pébay & Alex W. Hewitt, 2020. "Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-18715-y
    DOI: 10.1038/s41467-020-18715-y
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    Cited by:

    1. Shuqian Zhang & Bo Yuan & Jixin Cao & Liting Song & Jinlong Chen & Jiayi Qiu & Zilong Qiu & Xing-Ming Zhao & Jingqi Chen & Tian-Lin Cheng, 2023. "TadA orthologs enable both cytosine and adenine editing of base editors," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
    2. Shuqian Zhang & Liting Song & Bo Yuan & Cheng Zhang & Jixin Cao & Jinlong Chen & Jiayi Qiu & Yilin Tai & Jingqi Chen & Zilong Qiu & Xing-Ming Zhao & Tian-Lin Cheng, 2023. "TadA reprogramming to generate potent miniature base editors with high precision," Nature Communications, Nature, vol. 14(1), pages 1-12, December.

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