Off-target genomic DNA loci (Prime Editing) (Off-target sites)

Genomic DNA loci with partial complementarity to the PE3max pegRNA or nicking sgRNA refer to unintended sites in the genome where prime editing components may bind and exert activity. Prime editing (PE) utilizes a Cas9 nickase fused to a reverse transcriptase, guided by a prime editing guide RNA (pegRNA) and often an auxiliary nicking sgRNA in PE3 or PE3max systems to install precise genetic changes (Anzalone et al., 2019, Nature). Although prime editing is generally considered more precise than standard CRISPR-Cas9, the guide RNAs can still hybridize with off-target sequences that possess high sequence homology to the intended target (Kim et al., 2020, Nature Biotechnology). Interaction at these loci can result in permanent, unintended mutations, including insertions, deletions, or substitutions, which may disrupt tumor suppressor genes or activate oncogenes (Chen et al., 2021, Cell). These off-target effects represent a significant hurdle in the development of safe gene therapies, as they can lead to unpredictable cellular phenotypes or long-term genotoxicity. Monitoring these loci using high-throughput sequencing methods is essential for assessing the specificity of a prime editing therapeutic (Doman et al., 2020, Nature Biotechnology). Consequently, identifying and characterizing these loci is a fundamental requirement for the clinical development of prime editing therapies to ensure genomic integrity and patient safety.

Other names

Off-target genomic locipegRNA off-targetsNicking sgRNA off-targetsUnintended genomic modificationsPrime editing off-target effects

Unintended hybridization of guide RNAs to genomic sequences with partial complementarity, leading to Cas9-mediated DNA nicking and subsequent reverse transcription or DNA repair-mediated mutagenesis at non-target sites.