DNase H Activity of Neisseria meningitidis Cas9
RNA Therapeutics Institute; Program in Molecular Medicine
Biochemistry, Biophysics, and Structural Biology | Cell and Developmental Biology | Genetics and Genomics | Therapeutics
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
Mol Cell. 2015 Oct 15;60(2):242-55. doi: 10.1016/j.molcel.2015.09.020. Link to article on publisher's site
Zhang Y, Rajan R, Seifert HS, Mondragon A, Sontheimer EJ. (2015). DNase H Activity of Neisseria meningitidis Cas9. RNA Therapeutics Institute Publications. https://doi.org/10.1016/j.molcel.2015.09.020. Retrieved from https://escholarship.umassmed.edu/rti_pubs/23