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Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA

Author

Listed:
  • Mu-Sen Liu

    (Department of Molecular Biosciences
    Institute for Cellular and Molecular Biology)

  • Shanzhong Gong

    (Department of Molecular Biosciences
    Institute for Cellular and Molecular Biology)

  • Helen-Hong Yu

    (Department of Molecular Biosciences
    Institute for Cellular and Molecular Biology)

  • Kyungseok Jung

    (Department of Molecular Biosciences)

  • Kenneth A. Johnson

    (Department of Molecular Biosciences
    Institute for Cellular and Molecular Biology)

  • David W. Taylor

    (Department of Molecular Biosciences
    Institute for Cellular and Molecular Biology
    University of Texas at Austin
    Dell Medical School)

Abstract

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

Suggested Citation

  • Mu-Sen Liu & Shanzhong Gong & Helen-Hong Yu & Kyungseok Jung & Kenneth A. Johnson & David W. Taylor, 2020. "Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA," Nature Communications, Nature, vol. 11(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17411-1
    DOI: 10.1038/s41467-020-17411-1
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    Cited by:

    1. Péter István Kulcsár & András Tálas & Zoltán Ligeti & Sarah Laura Krausz & Ervin Welker, 2022. "SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    2. Sundaram Acharya & Asgar Hussain Ansari & Prosad Kumar Das & Seiichi Hirano & Meghali Aich & Riya Rauthan & Sudipta Mahato & Savitri Maddileti & Sajal Sarkar & Manoj Kumar & Rhythm Phutela & Sneha Gul, 2024. "PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics," Nature Communications, Nature, vol. 15(1), pages 1-23, December.
    3. Grace N. Hibshman & Jack P. K. Bravo & Matthew M. Hooper & Tyler L. Dangerfield & Hongshan Zhang & Ilya J. Finkelstein & Kenneth A. Johnson & David W. Taylor, 2024. "Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    4. Evan A. Schwartz & Tess M. McBride & Jack P. K. Bravo & Daniel Wrapp & Peter C. Fineran & Robert D. Fagerlund & David W. Taylor, 2022. "Structural rearrangements allow nucleic acid discrimination by type I-D Cascade," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    5. Péter István Kulcsár & András Tálas & Zoltán Ligeti & Eszter Tóth & Zsófia Rakvács & Zsuzsa Bartos & Sarah Laura Krausz & Ágnes Welker & Vanessza Laura Végi & Krisztina Huszár & Ervin Welker, 2023. "A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    6. Pascal D. Vos & Giulia Rossetti & Jessica L. Mantegna & Stefan J. Siira & Andrianto P. Gandadireja & Mitchell Bruce & Samuel A. Raven & Olga Khersonsky & Sarel J. Fleishman & Aleksandra Filipovska & O, 2022. "Computationally designed hyperactive Cas9 enzymes," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    7. Burcu Bestas & Sandra Wimberger & Dmitrii Degtev & Alexandra Madsen & Antje K. Rottner & Fredrik Karlsson & Sergey Naumenko & Megan Callahan & Julia Liz Touza & Margherita Francescatto & Carl Ivar Möl, 2023. "A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    8. Jeong Moon & Changchun Liu, 2023. "Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA," Nature Communications, Nature, vol. 14(1), pages 1-11, December.

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