DNase H Activity of Neisseria meningitidis Cas9

Yan Zhang, Rakhi Rajan, Hank Seifert, Alfonso Mondragon, Erik J. Sontheimer*

*Corresponding author for this work

Research output: Contribution to journalArticle

34 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)242-255
Number of pages14
JournalMolecular cell
Volume60
Issue number2
DOIs
StatePublished - Oct 15 2015

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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