4.8 Article

DNase H Activity of Neisseria meningitidis Cas9

Journal

MOLECULAR CELL
Volume 60, Issue 2, Pages 242-255

Publisher

CELL PRESS
DOI: 10.1016/j.molcel.2015.09.020

Keywords

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Funding

  1. American Heart Association
  2. NIH [R01 GM051350, R37 AI033493, R01 AI044239, R01 GM093769]

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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 50 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.

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