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A catalogue of biochemically diverse CRISPR-Cas9 orthologs

Author

Listed:
  • Giedrius Gasiunas

    (CasZyme)

  • Joshua K. Young

    (Corteva Agriscience™)

  • Tautvydas Karvelis

    (Vilnius University)

  • Darius Kazlauskas

    (Vilnius University)

  • Tomas Urbaitis

    (CasZyme
    Vilnius University)

  • Monika Jasnauskaite

    (CasZyme)

  • Mantvyda M. Grusyte

    (CasZyme)

  • Sushmitha Paulraj

    (Corteva Agriscience™)

  • Po-Hao Wang

    (Corteva Agriscience™
    Inari Agriculture)

  • Zhenglin Hou

    (Corteva Agriscience™)

  • Shane K. Dooley

    (Iowa State University)

  • Mark Cigan

    (Corteva Agriscience™
    Genus plc)

  • Clara Alarcon

    (Corteva Agriscience™)

  • N. Doane Chilcoat

    (Corteva Agriscience™)

  • Greta Bigelyte

    (Vilnius University)

  • Jennifer L. Curcuru

    (New England Biolabs)

  • Megumu Mabuchi

    (New England Biolabs)

  • Zhiyi Sun

    (New England Biolabs)

  • Ryan T. Fuchs

    (New England Biolabs)

  • Ezra Schildkraut

    (New England Biolabs)

  • Peter R. Weigele

    (New England Biolabs)

  • William E. Jack

    (New England Biolabs)

  • G. Brett Robb

    (New England Biolabs)

  • Česlovas Venclovas

    (Vilnius University)

  • Virginijus Siksnys

    (CasZyme
    Vilnius University)

Abstract

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Suggested Citation

  • 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.
  • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-19344-1
    DOI: 10.1038/s41467-020-19344-1
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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. Daniela S. Aliaga Goltsman & Lisa M. Alexander & Jyun-Liang Lin & Rodrigo Fregoso Ocampo & Benjamin Freeman & Rebecca C. Lamothe & Andres Perez Rivas & Morayma M. Temoche-Diaz & Shailaja Chadha & Nata, 2022. "Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    3. 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.
    4. 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.
    5. 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.
    6. Matteo Ciciani & Michele Demozzi & Eleonora Pedrazzoli & Elisabetta Visentin & Laura Pezzè & Lorenzo Federico Signorini & Aitor Blanco-Miguez & Moreno Zolfo & Francesco Asnicar & Antonio Casini & Anna, 2022. "Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data," Nature Communications, Nature, vol. 13(1), pages 1-8, December.

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