Genomic DNA off-target sites with partial prime editing guide RNA (pegRNA) complementarity (pegRNA off-target sites)

Genomic DNA off-target sites with partial pegRNA complementarity refer to unintended locations within the genome where prime editing components bind and exert enzymatic activity. Prime editing utilizes a prime editing guide RNA (pegRNA) consisting of a spacer sequence for targeting and a 3' extension containing a primer binding site (PBS) and a reverse transcriptase template (RTT) (Anzalone et al., 2019). Off-target effects occur when these sequences hybridize with non-target DNA regions that possess high sequence similarity, leading to unintended nicks and genetic insertions or deletions (Doman et al., 2022). While prime editing is generally considered more precise than traditional CRISPR-Cas9, these off-target sites represent a significant safety concern in the development of gene therapies. Unintended modifications at these sites can result in genotoxicity, chromosomal rearrangements, or the disruption of essential genes, potentially leading to oncogenesis (Kim et al., 2020). Consequently, rigorous screening and computational modeling are employed to identify and minimize activity at these sites during the design of therapeutic pegRNAs.

Other names

pegRNA off-targetsPrime editing off-target sitesUnintended genomic modificationsNon-specific pegRNA binding sites

Unintended hybridization of the pegRNA spacer or the 3' extension (primer binding site) to non-target genomic DNA sequences, which allows the Cas9 nickase-reverse transcriptase fusion protein to bind and execute unintended reverse transcription or nicking at incorrect loci (Anzalone et al., 2019; Doman et al., 2022).