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A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo

Author

Listed:
  • Burcu Bestas

    (AstraZeneca)

  • Sandra Wimberger

    (AstraZeneca
    University of Gothenburg)

  • Dmitrii Degtev

    (AstraZeneca)

  • Alexandra Madsen

    (AstraZeneca)

  • Antje K. Rottner

    (AstraZeneca)

  • Fredrik Karlsson

    (AstraZeneca)

  • Sergey Naumenko

    (Harvard Chan School of Public Health)

  • Megan Callahan

    (AstraZeneca)

  • Julia Liz Touza

    (AstraZeneca)

  • Margherita Francescatto

    (AstraZeneca)

  • Carl Ivar Möller

    (AstraZeneca)

  • Lukas Badertscher

    (AstraZeneca)

  • Songyuan Li

    (AstraZeneca)

  • Silvia Cerboni

    (AstraZeneca)

  • Niklas Selfjord

    (AstraZeneca)

  • Elke Ericson

    (AstraZeneca)

  • Euan Gordon

    (AstraZeneca)

  • Mike Firth

    (AstraZeneca)

  • Krzysztof Chylinski

    (Vienna Biocenter (VBC))

  • Amir Taheri-Ghahfarokhi

    (AstraZeneca)

  • Mohammad Bohlooly-Y

    (AstraZeneca)

  • Mike Snowden

    (AstraZeneca)

  • Menelaos Pangalos

    (AstraZeneca)

  • Barrett Nuttall

    (AstraZeneca)

  • Pinar Akcakaya

    (AstraZeneca)

  • Grzegorz Sienski

    (AstraZeneca)

  • Marcello Maresca

    (AstraZeneca)

Abstract

Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9’s off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.

Suggested Citation

  • 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.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-41240-7
    DOI: 10.1038/s41467-023-41240-7
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    as
    1. Giedrius Gasiunas & Joshua K. Young & Tautvydas Karvelis & Darius Kazlauskas & Tomas Urbaitis & Monika Jasnauskaite & Mantvyda M. Grusyte & Sushmitha Paulraj & Po-Hao Wang & Zhenglin Hou & Shane K. Do, 2020. "A catalogue of biochemically diverse CRISPR-Cas9 orthologs," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    2. Jungjoon K. Lee & Euihwan Jeong & Joonsun Lee & Minhee Jung & Eunji Shin & Young-hoon Kim & Kangin Lee & Inyoung Jung & Daesik Kim & Seokjoong Kim & Jin-Soo Kim, 2018. "Directed evolution of CRISPR-Cas9 to increase its specificity," Nature Communications, Nature, vol. 9(1), pages 1-10, December.
    3. 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.
    4. Liyang Zhang & John A. Zuris & Ramya Viswanathan & Jasmine N. Edelstein & Rolf Turk & Bernice Thommandru & H. Tomas Rube & Steve E. Glenn & Michael A. Collingwood & Nicole M. Bode & Sarah F. Beaudoin , 2021. "AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines," Nature Communications, Nature, vol. 12(1), pages 1-15, December.
    5. Fatwa Adikusuma & Sandra Piltz & Mark A. Corbett & Michelle Turvey & Shaun R. McColl & Karla J. Helbig & Michael R. Beard & James Hughes & Richard T. Pomerantz & Paul Q. Thomas, 2018. "Large deletions induced by Cas9 cleavage," Nature, Nature, vol. 560(7717), pages 8-9, August.
    6. Eunji Kim & Taeyoung Koo & Sung Wook Park & Daesik Kim & Kyoungmi Kim & Hee-Yeon Cho & Dong Woo Song & Kyu Jun Lee & Min Hee Jung & Seokjoong Kim & Jin Hyoung Kim & Jeong Hun Kim & Jin-Soo Kim, 2017. "In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni," Nature Communications, Nature, vol. 8(1), pages 1-12, April.
    7. Grégoire Cullot & Julian Boutin & Jérôme Toutain & Florence Prat & Perrine Pennamen & Caroline Rooryck & Martin Teichmann & Emilie Rousseau & Isabelle Lamrissi-Garcia & Véronique Guyonnet-Duperat & Al, 2019. "CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    8. Ha Youn Shin & Chaochen Wang & Hye Kyung Lee & Kyung Hyun Yoo & Xianke Zeng & Tyler Kuhns & Chul Min Yang & Teresa Mohr & Chengyu Liu & Lothar Hennighausen, 2017. "CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome," Nature Communications, Nature, vol. 8(1), pages 1-10, August.
    9. Samuel H. Sternberg & Sy Redding & Martin Jinek & Eric C. Greene & Jennifer A. Doudna, 2014. "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9," Nature, Nature, vol. 507(7490), pages 62-67, March.
    10. Stamatis Papathanasiou & Styliani Markoulaki & Logan J. Blaine & Mitchell L. Leibowitz & Cheng-Zhong Zhang & Rudolf Jaenisch & David Pellman, 2021. "Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing," Nature Communications, Nature, vol. 12(1), pages 1-7, December.
    11. Jennifer A. Doudna, 2020. "The promise and challenge of therapeutic genome editing," Nature, Nature, vol. 578(7794), pages 229-236, February.
    12. Max W. Shen & Mandana Arbab & Jonathan Y. Hsu & Daniel Worstell & Sannie J. Culbertson & Olga Krabbe & Christopher A. Cassa & David R. Liu & David K. Gifford & Richard I. Sherwood, 2018. "Predictable and precise template-free CRISPR editing of pathogenic variants," Nature, Nature, vol. 563(7733), pages 646-651, November.
    13. 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.
    14. Liyang Zhang & John A. Zuris & Ramya Viswanathan & Jasmine N. Edelstein & Rolf Turk & Bernice Thommandru & H. Tomas Rube & Steve E. Glenn & Michael A. Collingwood & Nicole M. Bode & Sarah F. Beaudoin , 2021. "Author Correction: AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines," Nature Communications, Nature, vol. 12(1), pages 1-1, December.
    15. Fuqiang Chen & Xiao Ding & Yongmei Feng & Timothy Seebeck & Yanfang Jiang & Gregory D. Davis, 2017. "Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting," Nature Communications, Nature, vol. 8(1), pages 1-12, April.
    16. Pinar Akcakaya & Maggie L. Bobbin & Jimmy A. Guo & Jose Malagon-Lopez & Kendell Clement & Sara P. Garcia & Mick D. Fellows & Michelle J. Porritt & Mike A. Firth & Alba Carreras & Tania Baccega & Frank, 2018. "In vivo CRISPR editing with no detectable genome-wide off-target mutations," Nature, Nature, vol. 561(7723), pages 416-419, September.
    17. Songyuan Li & Nina Akrap & Silvia Cerboni & Michelle J. Porritt & Sandra Wimberger & Anders Lundin & Carl Möller & Mike Firth & Euan Gordon & Bojana Lazovic & Aleksandra Sieńska & Luna Simona Pane & M, 2021. "Universal toxin-based selection for precise genome engineering in human cells," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
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