Off-target genomic DNA loci (HPD guide RNA) (HPD off-targets)

Off-target genomic DNA loci with partial complementarity to the HPD guide RNA are unintended sites in the human genome where CRISPR-Cas9 or other gene-editing complexes may bind and induce modifications. These sites possess sequence similarity to the guide RNA designed to target the 4-hydroxyphenylpyruvate dioxygenase (HPD) gene, which is a therapeutic target for treating Hereditary Tyrosinemia Type 1 (HT1) [Intellia Therapeutics, 2024]. Because the CRISPR system can tolerate a limited number of mismatches between the gRNA and the DNA template, it may create double-strand breaks at these non-target locations [Nature Communications, 2019]. Such off-target activity poses significant safety risks, including the potential for insertional mutagenesis, large deletions, or chromosomal translocations that could lead to oncogenic transformation [Science, 2013]. In the context of HPD-directed therapies, identifying and characterizing these loci is essential for evaluating the benefit-risk profile of the drug candidate. Sophisticated genomic assays like GUIDE-seq or CIRCLE-seq are typically used to map these sites and ensure that the editing remains highly specific to the HPD locus [NIH, 2023]. Minimizing these interactions is a primary goal in the engineering of high-fidelity Cas enzymes and optimized guide RNA designs. The presence of these loci represents a major hurdle in the regulatory approval process for CRISPR-based therapeutics.

Other names

CRISPR off-target sitesUnintended genomic cleavage sitesHPD-gRNA off-targetsNon-target DNA lociHPD-mismatched genomic sites

Unintended double-strand breaks or base editing at genomic sites with sequence homology to the HPD-targeting guide RNA