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

Arik Shams, Sean A. Higgins, Christof Fellmann, Thomas G. Laughlin, Benjamin L. Oakes, Rachel Lew, Shin Kim, Maria Lukarska, Madeline Arnold, Brett T. Staahl, Jennifer A. Doudna and David F. Savage ()
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Arik Shams: University of California, Berkeley
Sean A. Higgins: University of California, Berkeley
Christof Fellmann: University of California, Berkeley
Thomas G. Laughlin: University of California, Berkeley
Benjamin L. Oakes: University of California, Berkeley
Rachel Lew: Gladstone Institutes
Shin Kim: University of California, Berkeley
Maria Lukarska: University of California, Berkeley
Madeline Arnold: University of California, Berkeley
Brett T. Staahl: University of California, Berkeley
Jennifer A. Doudna: University of California, Berkeley
David F. Savage: University of California, Berkeley

Nature Communications, 2021, vol. 12, issue 1, 1-11

Abstract: Abstract Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be 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) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in 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.

Date: 2021
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DOI: 10.1038/s41467-021-25992-8

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