Off-target genomic loci with partial complementarity to the G6PC guide RNA (G6PC off-target sites)

Off-target genomic loci with partial complementarity to the G6PC guide RNA are unintended DNA sequences that share significant sequence homology with the target site in the Glucose-6-Phosphatase Catalytic Subunit (G6PC) gene (Chou et al., 2018, Nature Reviews Endocrinology). In CRISPR-Cas9 based therapies for Glycogen Storage Disease Type Ia (GSDIa), the guide RNA (gRNA) is designed to direct the Cas9 nuclease to the G6PC locus; however, the enzyme may also bind and cleave these off-target sites due to partial complementarity (Zhang et al., 2015, Science). Such unintended cleavage can lead to permanent mutations, including insertions and deletions (indels) or large-scale chromosomal rearrangements, which may disrupt essential genes or regulatory elements (Fu et al., 2013, Nature Biotechnology). These events pose a substantial safety risk, potentially leading to cellular transformation or loss of function in non-target pathways. Consequently, the identification and characterization of these loci using methods like GUIDE-seq or CIRCLE-seq are mandatory steps in the preclinical evaluation of G6PC-targeting gene editors (Tsai et al., 2015, Nature Biotechnology). Minimizing activity at these loci is a primary goal in the development of high-fidelity Cas9 variants and optimized gRNA architectures for treating metabolic disorders.

Other names

CRISPR off-target sitesNon-specific genomic cleavage sitesG6PC gRNA off-targetsUnintended genomic modifications

Unintended double-strand breaks (DSBs) followed by error-prone DNA repair mechanisms such as non-homologous end joining (NHEJ) or homology-directed repair (HDR) at non-target genomic sites (Fu et al., 2013, Nature Biotechnology).