دورية أكاديمية
Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.
| العنوان: | Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes. |
|---|---|
| المؤلفون: | Watters KE; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720., Shivram H; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720., Fellmann C; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.; Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA 94158.; Department of Cellular and Molecular Pharmacology, School of Medicine, University of California, San Francisco, CA 94158., Lew RJ; Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA 94158., McMahon B; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720., Doudna JA; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; doudna@berkeley.edu.; Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA 94158.; Department of Chemistry, University of California, Berkeley, CA 94720.; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720.; Innovative Genomics Institute, University of California, Berkeley, CA 94720.; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720. |
| المصدر: | Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2020 Mar 24; Vol. 117 (12), pp. 6531-6539. Date of Electronic Publication: 2020 Mar 10. |
| نوع المنشور: | Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. |
| اللغة: | English |
| بيانات الدورية: | Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, DC : National Academy of Sciences |
| مواضيع طبية MeSH: | CRISPR-Cas Systems*/genetics, Bacterial Proteins/*metabolism , CRISPR-Associated Protein 9/*antagonists & inhibitors , Enzyme Inhibitors/*metabolism , Staphylococcus/*genetics, Amino Acid Sequence ; Bacterial Proteins/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Protein 9/metabolism ; Conserved Sequence ; DNA/metabolism ; Gene Editing ; Genome, Bacterial/genetics ; HEK293 Cells ; Humans ; Inverted Repeat Sequences ; Staphylococcus/chemistry ; Staphylococcus aureus/enzymology |
| مستخلص: | 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. Competing Interests: Competing interest statement: The Regents of the University of California have patents pending for CRISPR technologies on which the authors are inventors. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Felix Biosciences, Mammoth Biosciences, and Inari. J.A.D. is a Director at Johnson & Johnson and has sponsored research projects supported by Pfizer and Biogen. (Copyright © 2020 the Author(s). Published by PNAS.) |
| References: | Mol Cell. 2019 Apr 18;74(2):296-309.e7. (PMID: 30850331) Front Microbiol. 2018 Jul 26;9:1591. (PMID: 30093886) Nature. 2013 Jan 17;493(7432):429-32. (PMID: 23242138) Cell. 2017 Sep 7;170(6):1224-1233.e15. (PMID: 28844692) Nat Commun. 2018 Jul 25;9(1):2919. (PMID: 30046034) Nat Struct Mol Biol. 2019 Apr;26(4):315-321. (PMID: 30936531) Cell Host Microbe. 2019 Feb 13;25(2):233-241.e5. (PMID: 30737174) mBio. 2014 Apr 15;5(2):e00896. (PMID: 24736222) Animal Model Exp Med. 2019 May 29;2(2):69-75. (PMID: 31392299) Cell. 2019 Jan 10;176(1-2):254-267.e16. (PMID: 30633905) Cell. 2017 Jan 12;168(1-2):150-158.e10. (PMID: 28041849) Elife. 2018 Jan 05;7:. (PMID: 29303478) J Mol Biol. 2018 Jul 20;430(15):2237-2243. (PMID: 29258817) Nature. 2015 Apr 9;520(7546):186-91. (PMID: 25830891) Nat Microbiol. 2018 Apr;3(4):461-469. (PMID: 29507349) CRISPR J. 2018 Oct;1:304-305. (PMID: 31021273) CRISPR J. 2019 Feb;2(1):23-30. (PMID: 31021234) Biotechnol Bioeng. 2017 Sep;114(9):2137-2141. (PMID: 28475211) Mol Cell. 2018 Jan 04;69(1):146-157.e3. (PMID: 29304331) Science. 2018 Aug 31;361(6405):866-869. (PMID: 30166482) Elife. 2019 Sep 10;8:. (PMID: 31502535) J Bacteriol. 2016 Apr 28;198(10):1481-6. (PMID: 26929301) Nat Commun. 2018 Aug 17;9(1):3313. (PMID: 30120228) Cell Rep. 2013 Dec 26;5(6):1704-13. (PMID: 24332856) Cell Rep. 2019 Nov 12;29(7):1739-1746.e5. (PMID: 31722192) Cell Host Microbe. 2020 Jul 8;28(1):31-40.e9. (PMID: 32325050) Elife. 2019 Aug 09;8:. (PMID: 31397669) Cell. 2019 Oct 3;179(2):448-458.e11. (PMID: 31564454) Nucleic Acids Res. 2016 Jul 8;44(W1):W16-21. (PMID: 27141966) Cell. 2016 Dec 15;167(7):1829-1838.e9. (PMID: 27984730) Nucleic Acids Res. 2016 May 5;44(8):e75. (PMID: 26740581) Nat Microbiol. 2017 Oct;2(10):1374-1380. (PMID: 28785032) Sci Adv. 2017 Jul 12;3(7):e1701620. (PMID: 28706995) Biochemistry. 2007 May 22;46(20):5904-10. (PMID: 17444659) Virus Res. 2018 May 02;250:21-30. (PMID: 29625148) Nucleic Acids Res. 2015 Jan;43(Database issue):D48-53. (PMID: 25378302) Cell. 2019 Sep 5;178(6):1452-1464.e13. (PMID: 31474367) J Mol Biol. 2019 Jul 12;431(15):2894-2899. (PMID: 31071326) Nat Microbiol. 2016 Jun 13;1(8):16085. (PMID: 27573108) Science. 2018 Oct 12;362(6411):240-242. (PMID: 30190308) mBio. 2018 Dec 4;9(6):. (PMID: 30514786) Science. 2018 Oct 12;362(6411):236-239. (PMID: 30190307) Nat Struct Mol Biol. 2019 Apr;26(4):308-314. (PMID: 30936526) |
| معلومات مُعتمدة: | K99 GM118909 United States GM NIGMS NIH HHS; R00 GM118909 United States GM NIGMS NIH HHS; United States HHMI Howard Hughes Medical Institute |
| فهرسة مساهمة: | Keywords: CRISPR; Cas9; anti-CRISPR; genome editing; self-targeting |
| المشرفين على المادة: | 0 (Bacterial Proteins) 0 (Enzyme Inhibitors) 9007-49-2 (DNA) EC 3.1.- (CRISPR-Associated Protein 9) |
| تواريخ الأحداث: | Date Created: 20200312 Date Completed: 20200810 Latest Revision: 20240328 |
| رمز التحديث: | 20240329 |
| مُعرف محوري في PubMed: | PMC7104187 |
| DOI: | 10.1073/pnas.1917668117 |
| PMID: | 32156733 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1091-6490 |
|---|---|
| DOI: | 10.1073/pnas.1917668117 |