Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease [Gene Regulation]

March 14th, 2014 by Chen, H., Choi, J., Bailey, S.

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 LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleave 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 present of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that the both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.