Genome-wide sgRNA-dependent off-target DNA loci (CRISPR off-targets)

Genome-wide sgRNA-dependent off-target DNA loci refer to unintended genomic sites that are recognized and modified by CRISPR-Cas gene-editing systems (Fu et al., 2013). These sites typically share significant sequence homology with the intended target DNA, allowing the single-guide RNA (sgRNA) to facilitate Cas nuclease binding and cleavage despite the presence of mismatches (Zhang et al., 2015). The occurrence of off-target activity is a primary safety concern in the development of CRISPR-based therapeutics, as it can lead to permanent mutations in functional genes or regulatory elements. Such unintended modifications may result in genotoxicity, including the activation of proto-oncogenes or the inactivation of tumor suppressor genes, potentially leading to cellular transformation and malignancy (Tsai et al., 2015). To ensure therapeutic safety, researchers utilize various genome-wide screening methods and high-fidelity Cas variants to identify and minimize these off-target effects. Regulatory agencies, such as the FDA, require extensive characterization of these loci to assess the risk-benefit profile of any gene-editing drug candidate (FDA Guidance, 2024).

Other names

CRISPR off-target sitessgRNA off-targetsNon-specific cleavage sitesCas9 off-targetsUnintended genomic modificationsOff-target DNA cleavage

Unintended binding and cleavage of DNA by CRISPR-Cas complexes mediated by partial sgRNA complementarity, leading to double-strand breaks and subsequent error-prone repair via non-homologous end joining (NHEJ) or homology-directed repair (HDR).