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Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

View ORCID ProfileArik Shams, Sean A. Higgins, View ORCID ProfileChristof Fellmann, View ORCID ProfileThomas J. Laughlin, Benjamin L. Oakes, View ORCID ProfileRachel Lew, Maria Lukarska, Madeline Arnold, Brett T. Staahl, View ORCID ProfileJennifer A. Doudna, View ORCID ProfileDavid F. Savage
doi: https://doi.org/10.1101/2020.10.19.344077
Arik Shams
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
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Sean A. Higgins
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
3Scribe Therapeutics, Alameda, CA 94501, USA
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Christof Fellmann
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
4Gladstone Institutes, San Francisco, CA 94158, USA
5Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
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Thomas J. Laughlin
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
6Division of Biological Sciences, University of San Diego, San Diego, CA 92093, USA
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Benjamin L. Oakes
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
3Scribe Therapeutics, Alameda, CA 94501, USA
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Rachel Lew
4Gladstone Institutes, San Francisco, CA 94158, USA
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Maria Lukarska
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
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Madeline Arnold
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
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Brett T. Staahl
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
3Scribe Therapeutics, Alameda, CA 94501, USA
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Jennifer A. Doudna
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
2Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA
4Gladstone Institutes, San Francisco, CA 94158, USA
7Graduate Group in Biophysics, University of California, Berkeley, Berkeley, CA 94720, USA
8Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA
9Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA
10Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
11Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA
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David F. Savage
1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
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  • For correspondence: savage@berkeley.edu
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Abstract

Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

Competing Interest Statement

UC Regents have filed a patent related to this work. S.A.H. is an employee of Scribe Therapeutics. B.L.O. and B.T.S. are co-founders and employees of Scribe Therapeutics. C.F. is a co-founder of Mirimus, Inc.. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribous Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Metagenomi, Mammoth Biosciences, and Inari. J.A.D is a member of the board of directors at Driver and Johnson & Johnson. D.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. All other authors declare no competing interests.

Footnotes

  • https://github.com/savagelab

Copyright 
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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Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
Arik Shams, Sean A. Higgins, Christof Fellmann, Thomas J. Laughlin, Benjamin L. Oakes, Rachel Lew, Maria Lukarska, Madeline Arnold, Brett T. Staahl, Jennifer A. Doudna, David F. Savage
bioRxiv 2020.10.19.344077; doi: https://doi.org/10.1101/2020.10.19.344077
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Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
Arik Shams, Sean A. Higgins, Christof Fellmann, Thomas J. Laughlin, Benjamin L. Oakes, Rachel Lew, Maria Lukarska, Madeline Arnold, Brett T. Staahl, Jennifer A. Doudna, David F. Savage
bioRxiv 2020.10.19.344077; doi: https://doi.org/10.1101/2020.10.19.344077

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