Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa

Sungwon Hwang; Chuxi Pan; Bianca Garcia; Alan R Davidson; Trevor F Moraes; Karen L Maxwell

doi:10.1016/j.jmb.2021.167420

Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4_Hpa

J Mol Biol. 2022 Mar 15;434(5):167420. doi: 10.1016/j.jmb.2021.167420. Epub 2021 Dec 23.

Authors

Sungwon Hwang¹, Chuxi Pan², Bianca Garcia³, Alan R Davidson⁴, Trevor F Moraes⁵, Karen L Maxwell⁶

Affiliations

¹ Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada. Electronic address: https://twitter.com/s1hwang_21.
² Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada.
³ Department of Molecular Genetics, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada.
⁴ Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada; Department of Molecular Genetics, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada. Electronic address: https://twitter.com/ARDavidson_UofT.
⁵ Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada. Electronic address: https://twitter.com/MoraesTrevor.
⁶ Department of Biochemistry, University of Toronto, 661 University Avenue, Suite 1600, Toronto, Ontario M5G 1M1, Canada. Electronic address: karen.maxwell@utoronto.ca.

PMID: 34954237
DOI: 10.1016/j.jmb.2021.167420

Abstract

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4_Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4_Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

Keywords: CRISPR-Cas; Cas9; anti-CRISPR; inhibition; mechanism.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteriophages* / enzymology
CRISPR-Associated Protein 9* / antagonists & inhibitors
CRISPR-Associated Protein 9* / chemistry
DNA Cleavage*
Haemophilus parainfluenzae* / virology
Prophages / enzymology
Protein Domains
Viral Proteins* / chemistry
Viral Proteins* / metabolism

Substances

Viral Proteins
CRISPR-Associated Protein 9

Grants and funding

CIHR/Canada