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.Cas9 proteins require a target-adjacent sequence, the PAM, in order to cleave DNA. Here the authors develop a pipeline to accurately predict PAM sequences in order to facilitate the identification of Cas9s targeting specific sequences, including mutations.

Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data / Ciciani, Matteo; Demozzi, Michele; Pedrazzoli, Eleonora; Visentin, Elisabetta; Pezzè, Laura; Signorini, Lorenzo Federico; Blanco-Miguez, Aitor; Zolfo, Moreno; Asnicar, Francesco; Casini, Antonio; Cereseto, Anna; Segata, Nicola. - In: NATURE COMMUNICATIONS. - ISSN 2041-1723. - 13:1(2022), pp. 64741-64748. [10.1038/s41467-022-34213-9]

Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data

Ciciani, Matteo;Demozzi, Michele;Pedrazzoli, Eleonora;Visentin, Elisabetta;Pezzè, Laura;Signorini, Lorenzo Federico;Blanco-Miguez, Aitor;Zolfo, Moreno;Asnicar, Francesco;Casini, Antonio;Cereseto, Anna;Segata, Nicola
2022-01-01

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.Cas9 proteins require a target-adjacent sequence, the PAM, in order to cleave DNA. Here the authors develop a pipeline to accurately predict PAM sequences in order to facilitate the identification of Cas9s targeting specific sequences, including mutations.
2022
1
Ciciani, Matteo; Demozzi, Michele; Pedrazzoli, Eleonora; Visentin, Elisabetta; Pezzè, Laura; Signorini, Lorenzo Federico; Blanco-Miguez, Aitor; Zolfo,...espandi
Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data / Ciciani, Matteo; Demozzi, Michele; Pedrazzoli, Eleonora; Visentin, Elisabetta; Pezzè, Laura; Signorini, Lorenzo Federico; Blanco-Miguez, Aitor; Zolfo, Moreno; Asnicar, Francesco; Casini, Antonio; Cereseto, Anna; Segata, Nicola. - In: NATURE COMMUNICATIONS. - ISSN 2041-1723. - 13:1(2022), pp. 64741-64748. [10.1038/s41467-022-34213-9]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11572/388611
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