Cut site selection by the two nuclease domains of the Cas9 RNA-guided endonuclease

Cas9, the RNA-guided DNA endonuclease from the CRISPRCas (clustered regularly interspaced short palindromic repeat-CRISPR-Associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a transactivatingRNA(tracrRNA) and a CRISPRRNA(crRNA) cleaves double-strandedDNAwith a specificity dictatedbythe sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM forLMG18311Cas9.Wealso show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.