Off-target genomic DNA sites (Pah-directed)

Off-target genomic DNA sites (Pah-directed) refer to unintended locations within the genome that share sequence similarity with the prime editing guide RNAs (pegRNAs) designed to correct mutations in the Phenylalanine hydroxylase (PAH) gene (Böck et al., 2022). These sites are not therapeutic targets but represent a critical safety concern in the development of gene therapies for Phenylketonuria (PKU). When a prime editor complex—comprising a Cas9 nickase fused to a reverse transcriptase—binds to these partially complementary sequences, it can introduce permanent and potentially deleterious genetic modifications (Anzalone et al., 2019). Such off-target activity can result in insertions, deletions, or point mutations that may disrupt essential genes or regulatory elements, potentially leading to oncogenic transformation (Kim et al., 2020). Consequently, the identification and characterization of these sites are vital for assessing the safety profile of PAH-directed interventions. Monitoring these sites using high-throughput sequencing and computational prediction tools is a standard requirement for ensuring the specificity of the editing process. Minimizing off-target effects is essential for the clinical translation of prime editing technologies in metabolic liver diseases.

Other names

Off-target DNA sitesUnintended pegRNA binding sitesPAH off-targetsUnintended genomic modificationsNon-target genomic loci

Unintended binding of the pegRNA-Cas9-RT complex to DNA sequences with partial homology, followed by reverse transcription and integration of the pegRNA-encoded sequence into the genome.