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Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Author

Listed:
  • Benjamin P. Kleinstiver

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Center for Computational and Integrative Biology, Massachusetts General Hospital
    Harvard Medical School)

  • Michelle S. Prew

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Center for Computational and Integrative Biology, Massachusetts General Hospital)

  • Shengdar Q. Tsai

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Center for Computational and Integrative Biology, Massachusetts General Hospital
    Harvard Medical School)

  • Ved V. Topkar

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Center for Computational and Integrative Biology, Massachusetts General Hospital)

  • Nhu T. Nguyen

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Center for Computational and Integrative Biology, Massachusetts General Hospital)

  • Zongli Zheng

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Harvard Medical School
    Karolinska Institutet)

  • Andrew P. W. Gonzales

    (Cardiovascular Research Center, Massachusetts General Hospital
    Harvard Medical School
    Broad Institute)

  • Zhuyun Li

    (Cardiovascular Research Center, Massachusetts General Hospital)

  • Randall T. Peterson

    (Cardiovascular Research Center, Massachusetts General Hospital
    Harvard Medical School
    Broad Institute)

  • Jing-Ruey Joanna Yeh

    (Cardiovascular Research Center, Massachusetts General Hospital
    Harvard Medical School)

  • Martin J. Aryee

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Harvard Medical School
    Harvard T.H. Chan School of Public Health)

  • J. Keith Joung

    (Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital
    Center for Computational and Integrative Biology, Massachusetts General Hospital
    Harvard Medical School)

Abstract

CRISPR-Cas9 nucleases are widely used for genome editing, but the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM); here the commonly used Streptococcus pyogenes Cas9 (SpCas9) is modified to recognize alternative PAM sequences, enabling robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9.

Suggested Citation

  • Benjamin P. Kleinstiver & Michelle S. Prew & Shengdar Q. Tsai & Ved V. Topkar & Nhu T. Nguyen & Zongli Zheng & Andrew P. W. Gonzales & Zhuyun Li & Randall T. Peterson & Jing-Ruey Joanna Yeh & Martin J, 2015. "Engineered CRISPR-Cas9 nucleases with altered PAM specificities," Nature, Nature, vol. 523(7561), pages 481-485, July.
    • Handle: RePEc:nat:nature:v:523:y:2015:i:7561:d:10.1038_nature14592
    DOI: 10.1038/nature14592
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    Cited by:

    1. Eleonora Pedrazzoli & Michele Demozzi & Elisabetta Visentin & Matteo Ciciani & Ilaria Bonuzzi & Laura Pezzè & Lorenzo Lucchetta & Giulia Maule & Simone Amistadi & Federica Esposito & Mariangela Lupo &, 2024. "CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    2. Xu Feng & Ruyi Xu & Jianglan Liao & Jingyu Zhao & Baochang Zhang & Xiaoxiao Xu & Pengpeng Zhao & Xiaoning Wang & Jianyun Yao & Pengxia Wang & Xiaoxue Wang & Wenyuan Han & Qunxin She, 2024. "Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    3. Margot Karlikow & Evan Amalfitano & Xiaolong Yang & Jennifer Doucet & Abigail Chapman & Peivand Sadat Mousavi & Paige Homme & Polina Sutyrina & Winston Chan & Sofia Lemak & Alexander F. Yakunin & Adam, 2023. "CRISPR-induced DNA reorganization for multiplexed nucleic acid detection," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    4. Jeremy Vicencio & Carlos Sánchez-Bolaños & Ismael Moreno-Sánchez & David Brena & Charles E. Vejnar & Dmytro Kukhtar & Miguel Ruiz-López & Mariona Cots-Ponjoan & Alejandro Rubio & Natalia Rodrigo Meler, 2022. "Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    5. 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.
    6. Lin Zhao & Sabrina R. T. Koseki & Rachel A. Silverstein & Nadia Amrani & Christina Peng & Christian Kramme & Natasha Savic & Martin Pacesa & Tomás C. Rodríguez & Teodora Stan & Emma Tysinger & Lauren , 2023. "PAM-flexible genome editing with an engineered chimeric Cas9," Nature Communications, Nature, vol. 14(1), pages 1-8, December.
    7. Jian Wang & Yuxi Teng & Ruihua Zhang & Yifei Wu & Lei Lou & Yusong Zou & Michelle Li & Zhong-Ru Xie & Yajun Yan, 2021. "Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    8. 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.
    9. 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.
    10. Annabel K. Sangree & Audrey L. Griffith & Zsofia M. Szegletes & Priyanka Roy & Peter C. DeWeirdt & Mudra Hegde & Abby V. McGee & Ruth E. Hanna & John G. Doench, 2022. "Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    11. 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.
    12. Fang Liang & Yu Zhang & Lin Li & Yexin Yang & Ji-Feng Fei & Yanmei Liu & Wei Qin, 2022. "SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    13. Shiran Abadi & Winston X Yan & David Amar & Itay Mayrose, 2017. "A machine learning approach for predicting CRISPR-Cas9 cleavage efficiencies and patterns underlying its mechanism of action," PLOS Computational Biology, Public Library of Science, vol. 13(10), pages 1-24, October.
    14. Betz, Ulrich A.K. & Arora, Loukik & Assal, Reem A. & Azevedo, Hatylas & Baldwin, Jeremy & Becker, Michael S. & Bostock, Stefan & Cheng, Vinton & Egle, Tobias & Ferrari, Nicola & Schneider-Futschik, El, 2023. "Game changers in science and technology - now and beyond," Technological Forecasting and Social Change, Elsevier, vol. 193(C).
    15. Dalton T. Ham & Tyler S. Browne & Pooja N. Banglorewala & Tyler L. Wilson & Richard K. Michael & Gregory B. Gloor & David R. Edgell, 2023. "A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    16. Zhaohui Zhong & Guanqing Liu & Zhongjie Tang & Shuyue Xiang & Liang Yang & Lan Huang & Yao He & Tingting Fan & Shishi Liu & Xuelian Zheng & Tao Zhang & Yiping Qi & Jian Huang & Yong Zhang, 2023. "Efficient plant genome engineering using a probiotic sourced CRISPR-Cas9 system," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    17. Maarten H. Geurts & Shashank Gandhi & Matteo G. Boretto & Ninouk Akkerman & Lucca L. M. Derks & Gijs Son & Martina Celotti & Sarina Harshuk-Shabso & Flavia Peci & Harry Begthel & Delilah Hendriks & Pa, 2023. "One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    18. 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.
    19. Daniel C. Volke & Román A. Martino & Ekaterina Kozaeva & Andrea M. Smania & Pablo I. Nikel, 2022. "Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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