Off-target genomic loci with partial complementarity to the CLN2-targeting sgRNA (CLN2 sgRNA off-targets)

Off-target genomic loci with partial complementarity to the CLN2-targeting sgRNA represent unintended DNA sequences that may be cleaved by CRISPR-Cas9 systems designed to treat CLN2 disease, also known as Batten disease. CLN2 disease is a neurodegenerative disorder caused by mutations in the TPP1 gene, which leads to a deficiency in the tripeptidyl-peptidase 1 enzyme (Mole et al., 2015). Gene editing strategies for this condition utilize a single guide RNA (sgRNA) to direct the Cas9 nuclease to the TPP1 locus; however, the nuclease can occasionally bind to and cut genomic regions that are similar but not identical to the intended target (Fu et al., 2013). These off-target effects are a major safety concern in clinical translation, as they can result in permanent mutations, chromosomal translocations, or the disruption of essential genes (Zhang et al., 2015). Rigorous identification of these loci using methods like GUIDE-seq or CIRCLE-seq is required to assess the genotoxic risk of the therapeutic intervention (Tsai et al., 2015). Minimizing activity at these sites through the use of high-fidelity Cas9 variants or optimized sgRNA design is critical for ensuring the safety of gene-editing therapies in patients.

Other names

CRISPR off-target sitesNon-specific genomic cleavage sitesUnintended genomic modificationssgRNA off-targetsCas9 off-target loci

Unintended DNA double-strand breaks caused by Cas9 nuclease binding to sequences with high homology to the guide RNA, followed by error-prone DNA repair (Fu et al., 2013).