User-specified genomic DNA locus

The user-specified genomic DNA locus is the programmable site within a genome targeted for modification by the PE3max prime editing system. PE3max represents an optimized iteration of prime editing technology, which employs a Cas9 nickase (H840A) fused to an engineered reverse transcriptase (RT) and utilizes a prime editing guide RNA (pegRNA) for targeting (Chen et al., 2021, Cell). The pegRNA spacer sequence is engineered to be complementary to the target DNA locus, allowing for precise Watson-Crick base pairing and recruitment of the fusion protein (Anzalone et al., 2019, Nature). Upon binding, the system executes a "search-and-replace" mechanism where the RT uses the 3' extension of the pegRNA as a template to synthesize new DNA directly into the target site. This approach allows for the installation of precise genetic changes, including all twelve types of point mutations, small insertions, and small deletions, with high specificity and reduced off-target effects compared to traditional CRISPR-Cas9. Because the target is user-defined, its clinical relevance spans a vast array of genetic diseases, though safety concerns such as off-target activity and unintended indels remain critical considerations for therapeutic application (Kantor et al., 2020, Nature Biomedical Engineering).

Other names

Genomic target siteProtospacerDNA target sequencePrime editing target locus

The target DNA is recognized by the pegRNA spacer via Watson-Crick base pairing, followed by a Cas9 nickase-mediated single-strand break and subsequent reverse transcription of a new DNA sequence into the target site (Anzalone et al., 2019, Nature).