Off-target genomic DNA sites with partial sgRNA complementarity and compatible NRCH PAMs

Off-target genomic DNA sites with partial sgRNA complementarity and compatible NRCH PAMs are unintended loci in the genome that are susceptible to modification by CRISPR-Cas systems. These sites are characterized by a DNA sequence that closely resembles the intended target sequence, allowing for mismatch tolerance during guide RNA hybridization (Fu et al., 2013, Nature Biotechnology). The NRCH designation refers to a specific Protospacer Adjacent Motif (PAM) recognized by engineered Cas9 variants, such as SpCas9-NRCH, which expand the targeting range of traditional CRISPR tools (Kim et al., 2020, Nature Communications). While these variants offer greater flexibility, they also increase the likelihood of interacting with non-target sites that possess compatible PAM sequences (Walton et al., 2020, Science). The interaction between the Cas nuclease and these off-target sites can lead to double-strand breaks, resulting in insertions, deletions, or chromosomal translocations (Kosicki et al., 2018, Nature Biotechnology). Such unintended modifications pose significant safety risks in clinical applications, potentially leading to the disruption of tumor suppressor genes or the activation of oncogenes. Therefore, these sites are not therapeutic targets but are instead critical safety liabilities that must be rigorously identified and minimized during drug development. Advanced sequencing techniques like GUIDE-seq and CIRCLE-seq are employed to map these sites and assess the specificity of gene-editing therapies (Tsai et al., 2015, Nature Biotechnology).

Other names

CRISPR off-target sitesNon-specific genomic cleavage sitesOff-target effectsNRCH-compatible off-targets

Unintended endonuclease activity or base modification at genomic loci with sequence homology to the guide RNA and a compatible PAM sequence (Kim et al., 2020, Nature Communications).