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TitleComprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.
Journal, issue, pagesNat Commun, Vol. 12, Issue 1, Page 5664, Year 2021
Publish dateSep 27, 2021
AuthorsArik 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 / David F Savage /
PubMed AbstractProteins 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 ...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.
External linksNat Commun / PubMed:34580310 / PubMed Central
MethodsEM (single particle)
Resolution6.2 Å
Structure data

EMDB-22518:
SpCas9 delta4CE Ternary Complex
Method: EM (single particle) / Resolution: 6.2 Å

Source
  • Streptococcus pyogenes (bacteria)
  • Escherichia coli (E. coli)

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