Genomic double-stranded DNA (CRISPR-Cas9 target site) (dsDNA (CRISPR target))

Genomic double-stranded DNA (dsDNA) at a specific protospacer sequence serves as the molecular target for CRISPR-Cas9-based therapeutic interventions. This target is defined by a 20-nucleotide sequence complementary to a synthetic single guide RNA (sgRNA) and must be immediately followed by a Protospacer Adjacent Motif (PAM), which for the widely used Streptococcus pyogenes Cas9 (SpCas9) is the sequence 5'-NGG-3' (Jinek et al., 2012). The Cas9 protein utilizes the sgRNA to scan the genome, and upon identifying the PAM and achieving successful base-pairing with the protospacer, it induces a site-specific double-strand break (DSB) (Hsu et al., 2013). This targeted disruption allows for the permanent modification of the genome through endogenous repair pathways like non-homologous end joining (NHEJ), which can knock out genes, or homology-directed repair (HDR), which can correct mutations. Clinically, this target is utilized in therapies such as exagamglogene autotemcel (Casgevy) to disrupt the BCL11A erythroid-specific enhancer, thereby inducing fetal hemoglobin production in patients with sickle cell disease (Frangoul et al., 2021). The precision of this interaction is critical, as unintended binding to similar sequences elsewhere in the genome can lead to off-target mutations and potential genotoxicity (Fu et al., 2013).

Other names

CRISPR target siteProtospacerCas9 target DNAPAM-adjacent DNA sequencesgRNA-programmed DNA site

RNA-guided site-specific DNA cleavage followed by endogenous DNA repair (NHEJ or HDR), or precise nucleotide modification via base/prime editing.