Off-target genomic DNA loci (SpRY-mediated) (Off-target sites)

This entry refers to unintended genomic locations where the engineered CRISPR-Cas9 variant SpRY may bind and exert activity. SpRY is a "near-PAMless" variant of the Streptococcus pyogenes Cas9 (SpCas9) protein, designed to overcome the restriction of the NGG protospacer adjacent motif (PAM) requirement (Walton et al., 2020, Science). While this expansion allows for targeting nearly any site in the genome, it significantly increases the risk of "off-target" effects at loci that possess partial complementarity to the single-guide RNA (sgRNA). Because SpRY can recognize a much wider array of PAM sequences, it can potentially cleave or modify DNA at numerous unintended sites across the genome (Kleinstiver et al., 2016, Nature). These off-target events are a major safety concern in gene therapy, as they can lead to permanent mutations, genomic instability, or the disruption of essential genes (Fu et al., 2013, Nature Biotechnology). Monitoring and predicting these sites is critical for the development of safe CRISPR-based therapeutics using high-throughput sequencing methods like GUIDE-seq (Tsai et al., 2015, Nature Biotechnology).

Other names

Off-target effectsUnintended cleavage sitesSpRY off-targetsNon-specific genomic lociPermissive PAM off-targets

Unintended DNA binding and cleavage by the SpRY-sgRNA complex at genomic sites with partial sequence homology and compatible PAM sequences, leading to double-strand breaks or other modifications.