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A Cas9 with PAM recognition for adenine dinucleotides

Author

Listed:
  • Pranam Chatterjee

    (Center for Bits and Atoms
    Massachusetts Institute of Technology)

  • Jooyoung Lee

    (University of Massachusetts Medical School)

  • Lisa Nip

    (Center for Bits and Atoms
    Massachusetts Institute of Technology)

  • Sabrina R. T. Koseki

    (Center for Bits and Atoms
    Massachusetts Institute of Technology)

  • Emma Tysinger

    (Center for Bits and Atoms
    Massachusetts Institute of Technology)

  • Erik J. Sontheimer

    (University of Massachusetts Medical School)

  • Joseph M. Jacobson

    (Center for Bits and Atoms
    Massachusetts Institute of Technology)

  • Noah Jakimo

    (Center for Bits and Atoms
    Massachusetts Institute of Technology)

Abstract

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5$$^{\prime}$$′-NAAN-3$$^{\prime}$$′ PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells.

Suggested Citation

  • Pranam Chatterjee & Jooyoung Lee & Lisa Nip & Sabrina R. T. Koseki & Emma Tysinger & Erik J. Sontheimer & Joseph M. Jacobson & Noah Jakimo, 2020. "A Cas9 with PAM recognition for adenine dinucleotides," Nature Communications, Nature, vol. 11(1), pages 1-6, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-16117-8
    DOI: 10.1038/s41467-020-16117-8
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    Cited by:

    1. 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.
    2. 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.

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