Genomic DNA (CRISPR-Cas9 target site) (gDNA)

Genomic DNA at an sgRNA-specified protospacer adjacent to an NGG PAM represents the precise molecular target for CRISPR-Cas9-based gene editing technologies (Jinek et al., 2012). In this system, a synthetic single guide RNA (sgRNA) is engineered to be complementary to a specific 20-nucleotide sequence, known as the protospacer, within the host genome. The Cas9 nuclease, typically derived from Streptococcus pyogenes, recognizes the NGG protospacer adjacent motif (PAM) and facilitates the unwinding of the DNA duplex to allow RNA-DNA hybridization (Cong et al., 2013). Once bound, the Cas9 enzyme generates a site-specific double-strand break, which is subsequently repaired by the cell's endogenous machinery via non-homologous end joining (NHEJ) or homology-directed repair (HDR). This mechanism is utilized therapeutically to disrupt deleterious genes, correct pathogenic mutations, or insert functional genetic sequences. For instance, exagamglogene autotemcel (Casgevy) targets the BCL11A erythroid-specific enhancer to reactivate fetal hemoglobin production in patients with sickle cell disease and beta-thalassemia (FDA, 2023). While highly precise, the primary challenges include minimizing off-target activity at similar genomic sequences and ensuring efficient delivery to the relevant tissue types (Fu et al., 2013).

Other names

ProtospacerCRISPR target sequenceTarget DNA locusPAM-adjacent genomic sequenceCas9 target site

The target serves as a specific genomic substrate for the CRISPR-Cas9 complex, where the sgRNA hybridizes to the protospacer sequence, allowing the Cas9 endonuclease to induce a double-strand break (DSB) or chemical modification at a site immediately upstream of the NGG protospacer adjacent motif (PAM) (Jinek et al., 2012; Cong et al., 2013).