Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
| Autor: | Rachel J. Lew, Christof Fellmann, Thomas G. Laughlin, Arik Shams, Benjamin L. Oakes, Maria Lukarska, David F. Savage, Jennifer A. Doudna, Brett T. Staahl, Shin Kim, Sean Higgins, Madeline L. Arnold |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
CRISPR-Cas9 genome editing
Genetic enhancement Science General Physics and Astronomy Computational biology Biology General Biochemistry Genetics and Molecular Biology Article 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genome editing CRISPR-Associated Protein 9 Cell Line Tumor DNA-binding proteins CRISPR Humans Protein Interaction Domains and Motifs 030304 developmental biology Gene Editing 0303 health sciences Modularity (networks) Multidisciplinary Effector Cas9 Cryoelectron Microscopy RNA General Chemistry DNA Single Molecule Imaging chemistry Protein design CRISPR-Cas Systems 030217 neurology & neurosurgery RNA Guide Kinetoplastida |
| Zdroj: | Nature Communications, Vol 12, Iss 1, Pp 1-11 (2021) Nature Communications |
| ISSN: | 2041-1723 |
| Popis: | 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. Proteins evolve through the modular rearrangement of domains. Here the authors introduce MISER, a minimization by iterative size-exclusion and recombination method to make all possible deletions of a protein, uncovering functions for Cas9 domains involved in DNA binding. |
| Databáze: | OpenAIRE |
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