title: Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.
abstract: The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Joseph
authors.initials: J
authors.lastname: Bondy-Denomy
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Bianca
authors.initials: B
authors.lastname: Garcia
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Scott
authors.initials: S
authors.lastname: Strum
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Mingjian
authors.initials: M
authors.lastname: Du
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: MaryClare F
authors.initials: MF
authors.lastname: Rollins
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Yurima
authors.initials: Y
authors.lastname: Hidalgo-Reyes
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana 59717, USA.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
authors.affiliation: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Maxwell
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Davidson
mesh_terms: D001419:Bacteria / Q000378:metabolism* / Q000821:virology*; D001435:Bacteriophages / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019143:Evolution, Molecular; D011485:Protein Binding; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; D014764:Viral Proteins; D004720:Endonucleases; D004265:DNA Helicases