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Mol Cell. 2015 Oct 15;60(2):242-55. doi: 10.1016/j.molcel.2015.09.020.

DNase H Activity of Neisseria meningitidis Cas9.

Author information

1
RNA Therapeutics Institute, Program in Molecular Medicine, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA 01605-2324, USA.
2
Department of Molecular Biosciences, Northwestern University, 2205 Tech Drive, Evanston, IL 60208-3500, USA.
3
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 320 East Superior Avenue, Chicago, IL 60611, USA.
4
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.

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.

PMID:
26474066
PMCID:
PMC4609032
DOI:
10.1016/j.molcel.2015.09.020
[Indexed for MEDLINE]
Free PMC Article

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