Genome-wide sgRNA-independent DNA off-target sites (sgRNA-independent off-targets)

Genome-wide sgRNA-independent DNA off-target sites are unintended genomic modifications that occur during gene editing without being directed by the guide RNA (sgRNA) sequence. These events are primarily associated with the inherent enzymatic activity of gene-editing tools, such as the stochastic deamination of cytosine or adenine by base editors (Zuo et al., 2019; Jin et al., 2019). Unlike traditional off-target effects that rely on sequence homology to the target site, these independent mutations are often randomly distributed across the genome and are difficult to predict using standard computational algorithms (Zuo et al., 2019). The occurrence of these mutations poses a significant safety risk in clinical gene therapy, as they can lead to the disruption of essential genes or the activation of oncogenic pathways (Doman et al., 2020). Consequently, these sites are not therapeutic targets but rather critical safety parameters that must be monitored using sensitive detection methods like GOTI or whole-genome sequencing (Zuo et al., 2019). Reducing guide-independent activity through protein engineering is a major focus for improving the precision and safety of next-generation CRISPR technologies (Kim et al., 2019).

Other names

Cas9-independent off-target sitessgRNA-independent mutationsGuide-independent off-target effectsSpontaneous genomic mutationsBackground gene-editing mutations

Unintended enzymatic modification of DNA (e.g., deamination) or DNA cleavage occurring through stochastic binding or transient interactions of the gene-editing complex with non-target genomic regions (Zuo et al., 2019; Jin et al., 2019).