Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data
Matteo Ciciani, Michele Demozzi, Eleonora Pedrazzoli, Elisabetta Visentin, Laura Pezzè, Lorenzo Signorini, Aitor Blanco‐Míguez, Moreno Zolfo, Francesco Asnicar, Antonio Casini, Anna Cereseto, Nicola Segata
Abstract
The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation in genome editing. Here we develop a computational pipeline that was used to interrogate a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions. This procedure allows the identification and isolation of sequence-tailored Cas9 nucleases by using the target sequence as bait. As proof of concept, starting from the disease-causing mutation P23H in the RHO gene, we find, isolate and experimentally validate a Cas9 which uses the mutated sequence as PAM. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement.