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Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

View ORCID ProfileStephen K. Jones Jr, John A. Hawkins, Nicole V. Johnson, Cheulhee Jung, Kuang Hu, View ORCID ProfileJames R. Rybarski, Janice S. Chen, Jennifer A. Doudna, View ORCID ProfileWilliam H. Press, View ORCID ProfileIlya J. Finkelstein
doi: https://doi.org/10.1101/696393
Stephen K. Jones
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USAInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USACenter for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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  • ORCID record for Stephen K. Jones
John A. Hawkins
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USAInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USACenter for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USAOden Institute for Computational Engineering and Science, University of Texas at Austin, Austin, Texas 78712, USA
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Nicole V. Johnson
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USAInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USACenter for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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Cheulhee Jung
Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea
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Kuang Hu
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USAInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USACenter for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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James R. Rybarski
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USAInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USACenter for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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Janice S. Chen
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720, United States
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Jennifer A. Doudna
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720, United StatesUniversity of California, Chemistry, Berkeley, CAHoward Hughes Medical Institute, University of California, Berkeley, CALawrence Berkeley National Laboratory, Physical Biosciences Division, Berkeley, CA
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William H. Press
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USAOden Institute for Computational Engineering and Science, University of Texas at Austin, Austin, Texas 78712, USA
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Ilya J. Finkelstein
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USAInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USACenter for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas 78712, USA
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  • For correspondence: ifinkelstein@cm.utexas.edu
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Abstract

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeq— nuclease digestion and deep sequencing—a massively parallel platform that measures cleavage kinetics and captures the time-resolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a—reported as a more specific nuclease in cells—has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

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  • https://github.com/finkelsteinlab/nucleaseq

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 09, 2019.
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Massively parallel kinetic profiling of natural and engineered CRISPR nucleases
Stephen K. Jones Jr, John A. Hawkins, Nicole V. Johnson, Cheulhee Jung, Kuang Hu, James R. Rybarski, Janice S. Chen, Jennifer A. Doudna, William H. Press, Ilya J. Finkelstein
bioRxiv 696393; doi: https://doi.org/10.1101/696393
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Massively parallel kinetic profiling of natural and engineered CRISPR nucleases
Stephen K. Jones Jr, John A. Hawkins, Nicole V. Johnson, Cheulhee Jung, Kuang Hu, James R. Rybarski, Janice S. Chen, Jennifer A. Doudna, William H. Press, Ilya J. Finkelstein
bioRxiv 696393; doi: https://doi.org/10.1101/696393

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