Automated identification of sequence-tailored Cas9 proteins using massive metagenomic data.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 36309502.
- Also identified by DOI 10.1038/s41467-022-34213-9 and PMC identifier 9617884.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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.
Medical subject headings
- CRISPR-Associated Protein 9
- CRISPR-Cas Systems