Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes
- Univ. of California, Berkeley, CA (United States)
- Univ. of California, Berkeley, CA (United States); Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology; Univ. of California, San Francisco, CA (United States)
- Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology
- Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Institute and Innovative Genomics Institute; Gladstone Institutes, San Francisco, CA (United States). Gladstone Institute of Data Science and Biotechnology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
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 potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust 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–AcrIIA15 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–AcrIIA15 as unique bifunctional inhibitors of SauCas9.
- Research Organization:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC); Defense Advanced Research Projects Agency (DARPA); National Science Foundation (NSF); National Institutes of Health (NIH)
- Grant/Contract Number:
- AC02-05CH11231
- OSTI ID:
- 1633267
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, Issue 12; ISSN 0027-8424
- Publisher:
- National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14
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journal | December 2020 |
Machine learning predicts new anti-CRISPR proteins
|
journal | April 2020 |
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