title: DNase H Activity of Neisseria meningitidis Cas9.
abstract: Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.
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 Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3500, USA.
authors.forename: Rakhi
authors.initials: R
authors.lastname: Rajan
authors.affiliation: Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 320 East Superior Avenue, Chicago, IL 60611, USA.
authors.forename: H Steven
authors.initials: HS
authors.lastname: Seifert
authors.affiliation: Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3500, USA.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Mondragón
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
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology / Q000235:genetics*; D059372:Nucleotide Motifs; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000094704:RNA, Guide, CRISPR-Cas Systems; D004706:Endodeoxyribonucleases