Cas9 is a programmable RNA-guided DNA endonuclease [1; 2]. It recognises a short DNA sequence, known as a protospacer-adjacent motif (PAM), and cleaves DNA at a fixed position in accordance to PAM when there is full complementarity between DNA and RNA strands. This feature has made the Cas9 protein a highly attractive and popular molecular tool for genome editing. Nevertheless, there are still gaps in full understanding of the Cas9 biological role in prokaryotic cells. Cas9 belongs to II-type CRISPR-Cas systems and is essential for all three stages of the CRISPR-Cas mechanism (adaptation, RNA biogenesis and interference). During adaptation, viral DNA is selected, cut and integrated as a spacer into the CRISPR region in a bacterial chromosome. The precise mechanism of adaptation in II-A subtype CRISPR-Cas systems is yet to be determined. It has been shown that during adaptation Cas9 aids in viral DNA selection by recognising the PAM sequence [3]. In some way Cas9 has to convey the information of PAM recognition to macromolecular Cas1-Cas2-Csn2 adaptation complexes, which then finalise the adaptation stage [4]. When viral DNA is inserted into the CRISPR region, it is transcribed by RNA polymerase and processed by Cas9 and RNase III into CRISPR RNA (crRNA), which is hybridized with trans-activating crRNA (tracrRNA) forming a functional guide RNA. In the final interference stage, Cas9 uses guide RNA to find the target DNA molecules and induce DNA double-strand breaks. In this way, CRISPR-Cas system confers hereditary and adaptive immunity against viruses.
Here, we report the structure of Streptococcus thermophilus Cas9 in complex with guide RNA and target DNA (interference mode). We also present the structure of Streptococcus thermophilus Cas9-RNA bound to non-target DNA, with the PAM-adjacent target DNA strand non-complementary to the guide RNA. We show that Cas9-RNA adopts different conformations, which might be exploited for the distinct roles of Cas9 in adaptation and interference.