Tailored identification of Cas9 proteins with specific PAM requirements using a computational prediction pipeline

Abstract The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation as genome editing tools. Here we interrogated a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions, in order to identify novel Cas9s selected for their PAM requirements. As a test bed we selectively isolated a Cas9 which uses a PAM sequence corresponding to a disease-causing mutation, P23H in the RHO gene. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement leading towards a natural PAM-free genome editing toolbox.