The RNA-guided dsDNA endonuclease Cas9 plays a crucial role in defending bacteria against phageinfection and has great potential to be harnessed for therapeutic gene editing. The suitability of Cas9 orthologs for therapeutic applications depends on factors such their size, activity, specificity and PAM [Charpentier et al. 2013]. Here, we discuss techniques used to optimize the production of engineered Cas9 orthologues to provide insights into the molecular basis of the modified nuclease specificity and activity by biophysics, X-ray crystallography and cryogenic electron microscopy.
