title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.
abstract: Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.
authors.affiliation: Institute of Molecular Biophysics, ‡Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.
authors.forename: Tsz Kin Martin
authors.identifier: 0000-0001-5741-1941
authors.initials: TKM
authors.lastname: Tsui
authors.affiliation: Institute of Molecular Biophysics, ‡Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.
authors.forename: Travis H
authors.initials: TH
authors.lastname: Hand
authors.affiliation: Institute of Molecular Biophysics, ‡Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.
authors.forename: Emily C
authors.initials: EC
authors.lastname: Duboy
authors.affiliation: Institute of Molecular Biophysics, ‡Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32306, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D039903:Actinobacteria / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003596:Cytosine / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D003596:Cytosine; D004247:DNA; D004720:Endonucleases
keywords: CRISPR; Cas9; PAM; RNA-guided DNA cleavage