Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes
| Autor: | Christof Fellmann, Haridha Shivram, Blake McMahon, Kyle E. Watters, Jennifer A. Doudna, Rachel J. Lew |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
anti-CRISPR
Staphylococcus aureus Inverted repeat Staphylococcus Computational biology Biology Genome Bacteriophage 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Bacterial Proteins Genome editing CRISPR-Associated Protein 9 Genetics Humans genome editing CRISPR Amino Acid Sequence Enzyme Inhibitors Cas9 Gene Conserved Sequence 030304 developmental biology Gene Editing 0303 health sciences Prevention Inverted Repeat Sequences Human Genome Bacterial DNA biology.organism_classification 3. Good health HEK293 Cells Emerging Infectious Diseases Infectious Diseases chemistry 5.1 Pharmaceuticals CRISPR-Cas Systems self-targeting Development of treatments and therapeutic interventions Infection 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America, vol 117, iss 12 |
| DOI: | 10.1101/799403 |
| Popis: | Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three new potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14 and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for anti-CRISPR function, and have divergent C-termini that are required in each case for selective inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust and specific inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-15 as unique bifunctional inhibitors of SauCas9. |
| Databáze: | OpenAIRE |
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