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Directed evolution of CRISPR-Cas9 to increase its specificity

Author

Listed:
  • Jungjoon K. Lee

    (Toolgen)

  • Euihwan Jeong

    (Institute for Basic Science (IBS)
    Seoul National University)

  • Joonsun Lee

    (Toolgen)

  • Minhee Jung

    (Toolgen)

  • Eunji Shin

    (Toolgen)

  • Young-hoon Kim

    (Toolgen)

  • Kangin Lee

    (Toolgen)

  • Inyoung Jung

    (Toolgen)

  • Daesik Kim

    (Seoul National University)

  • Seokjoong Kim

    (Toolgen)

  • Jin-Soo Kim

    (Institute for Basic Science (IBS)
    Seoul National University)

Abstract

The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.

Suggested Citation

  • 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.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-05477-x
    DOI: 10.1038/s41467-018-05477-x
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    Cited by:

    1. Dawn G. L. Thean & Hoi Yee Chu & John H. C. Fong & Becky K. C. Chan & Peng Zhou & Cynthia C. S. Kwok & Yee Man Chan & Silvia Y. L. Mak & Gigi C. G. Choi & Joshua W. K. Ho & Zongli Zheng & Alan S. L. W, 2022. "Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    2. Na Zhao & Jian Zhou & Tianfu Tao & Qi Wang & Jie Tang & Dengluan Li & Shixue Gou & Zhihong Guan & Joshua Seun Olajide & Jiejing Lin & Shuo Wang & Xiaoping Li & Jiankui Zhou & Zongliang Gao & Gang Wang, 2024. "Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells," Nature Communications, Nature, vol. 15(1), pages 1-11, December.
    3. 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.
    4. Jianli Tao & Daniel E. Bauer & Roberto Chiarle, 2023. "Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    5. Jian Wang & Ke Wang & Zhe Deng & Zhiyu Zhong & Guo Sun & Qing Mei & Fuling Zhou & Zixin Deng & Yuhui Sun, 2024. "Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    6. 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.
    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. Dmitrii Degtev & Jack Bravo & Aikaterini Emmanouilidi & Aleksandar Zdravković & Oi Kuan Choong & Julia Liz Touza & Niklas Selfjord & Isabel Weisheit & Margherita Francescatto & Pinar Akcakaya & Michel, 2024. "Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    9. Pierre Aldag & Marius Rutkauskas & Julene Madariaga-Marcos & Inga Songailiene & Tomas Sinkunas & Felix Kemmerich & Dominik Kauert & Virginijus Siksnys & Ralf Seidel, 2023. "Dynamic interplay between target search and recognition for a Type I CRISPR-Cas system," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    10. Yanbo Wang & W. Taylor Cottle & Haobo Wang & Momcilo Gavrilov & Roger S. Zou & Minh-Tam Pham & Srinivasan Yegnasubramanian & Scott Bailey & Taekjip Ha, 2022. "Achieving single nucleotide sensitivity in direct hybridization genome imaging," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    11. András Tálas & Dorottya A. Simon & Péter I. Kulcsár & Éva Varga & Sarah L. Krausz & Ervin Welker, 2021. "BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
    12. Akiko Tomita & Hiroyuki Sasanuma & Tomoo Owa & Yuka Nakazawa & Mayuko Shimada & Takahiro Fukuoka & Tomoo Ogi & Shinichiro Nakada, 2023. "Inducing multiple nicks promotes interhomolog homologous recombination to correct heterozygous mutations in somatic cells," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    13. Giulia I. Corsi & Kunli Qu & Ferhat Alkan & Xiaoguang Pan & Yonglun Luo & Jan Gorodkin, 2022. "CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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