Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Background: The Cas9 RNA-guided endonuclease has been adapted for genome manipulation and regulation. Results: We have characterized target recognition and cleavage by Streptococcus thermophilus LMG18311 Cas9. Conclusion: The two nuclease domains of Cas9 select their cleavage sites by different mechanisms. Significance: These findings contribute to the molecular basis of Cas9-mediated DNA cleavage. Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (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 trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the 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 for LMG18311 Cas9. We also 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.