RNA Therapeutics Institute Publications

Title

DNase H Activity of Neisseria meningitidis Cas9

UMMS Affiliation

RNA Therapeutics Institute; Program in Molecular Medicine

Publication Date

10-15-2015

Document Type

Article

Disciplines

Biochemistry, Biophysics, and Structural Biology | Cell and Developmental Biology | Genetics and Genomics | Therapeutics

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.

DOI of Published Version

10.1016/j.molcel.2015.09.020

Source

Mol Cell. 2015 Oct 15;60(2):242-55. doi: 10.1016/j.molcel.2015.09.020. Link to article on publisher's site

Journal/Book/Conference Title

Molecular cell

Related Resources

Link to Article in PubMed

PubMed ID

26474066

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