A catalogue of biochemically diverse CRISPR-Cas9 orthologs

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. Dooley, Mark Cigan, Clara Alarcon, N. Doane Chilcoat, Greta Bigelyte, Jennifer L. Curcuru, Megumu Mabuchi, Zhiyi Sun, Ryan T. Fuchs, Ezra Schildkraut, Peter R. Weigele, William E. Jack, G. Brett Robb (), Česlovas Venclovas and Virginijus Siksnys ()
Additional contact information
Giedrius Gasiunas: CasZyme
Joshua K. Young: Corteva Agriscience™
Tautvydas Karvelis: Vilnius University
Darius Kazlauskas: Vilnius University
Tomas Urbaitis: CasZyme
Monika Jasnauskaite: CasZyme
Mantvyda M. Grusyte: CasZyme
Sushmitha Paulraj: Corteva Agriscience™
Po-Hao Wang: Corteva Agriscience™
Zhenglin Hou: Corteva Agriscience™
Shane K. Dooley: Iowa State University
Mark Cigan: Corteva Agriscience™
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

Nature Communications, 2020, vol. 11, issue 1, 1-10

Abstract: 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.

Date: 2020
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-19344-1

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DOI: 10.1038/s41467-020-19344-1

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