title: Naturally Occurring Off-Switches for CRISPR-Cas9.
abstract: CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT.
authors.affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
authors.forename: April
authors.initials: A
authors.lastname: Pawluk
authors.affiliation: RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA.
authors.forename: Nadia
authors.initials: N
authors.lastname: Amrani
authors.affiliation: RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
authors.forename: Bianca
authors.initials: B
authors.lastname: Garcia
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
authors.forename: Yurima
authors.initials: Y
authors.lastname: Hidalgo-Reyes
authors.affiliation: RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA.
authors.forename: Jooyoung
authors.initials: J
authors.lastname: Lee
authors.affiliation: RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA.
authors.forename: Alireza
authors.initials: A
authors.lastname: Edraki
authors.affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
authors.forename: Megha
authors.initials: M
authors.lastname: Shah
authors.affiliation: RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA. Electronic address: erik.sontheimer@umassmed.edu.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Sontheimer
authors.affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada. Electronic address: karen.maxwell@utoronto.ca.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Maxwell
authors.affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. Electronic address: alan.davidson@utoronto.ca.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Davidson
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002460:Cell Line; D000072669:Gene Editing; D006801:Humans; D009345:Neisseria meningitidis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins
keywords: CRISPR-Cas; Cas9; Neisseria meningitidis; anti-CRISPR; genome editing; phage