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

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 comprehe...

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Bibliographic Details
Published in:Nature communications 2022-10, Vol.13 (1), p.6474-8, Article 6474
Main Authors: 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
Format: Article
Language:English
Subjects:
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Accuracy
Automation
Baits
Computer applications
CRISPR
CRISPR-Associated Protein 9 - genetics
CRISPR-Associated Protein 9 - metabolism
CRISPR-Cas Systems - genetics
Datasets
Deoxyribonucleic acid
DNA
Endonucleases - metabolism
Exploitation
Gene Editing - methods
Genome editing
Genomes
Humanities and Social Sciences
Identification
Metagenome
Metagenomics
Methods
multidisciplinary
Mutation
Nuclease
Nucleotide sequence
Proteins
Rho protein
RNA, Guide, CRISPR-Cas Systems
Science
Science (multidisciplinary)
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Description
Summary: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.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-022-34213-9