Genomic DNA locus specified by the sgRNA protospacer and PAM (CRISPR target site)

The genomic DNA locus specified by the sgRNA protospacer and PAM is the precise molecular target for CRISPR-based genome editing technologies. This target is defined by the sequence complementarity between a synthetic single guide RNA (sgRNA) and a 20-nucleotide genomic sequence, known as the protospacer, which must be immediately followed by a Protospacer Adjacent Motif (PAM) for recognition by the Cas enzyme (Jinek et al., 2012, Science). Upon binding, the CRISPR-Cas complex facilitates site-specific modifications, such as double-strand breaks, which are subsequently repaired by the cell's endogenous machinery to alter gene function (Doudna & Charpentier, 2014, Science). This targeting mechanism is the basis for approved therapies like exagamglogene autotemcel, which modifies the BCL11A enhancer to treat hemoglobinopathies (Frangoul et al., 2021, NEJM). The primary therapeutic challenge associated with this target is ensuring high specificity to avoid off-target editing at similar genomic sequences, which could lead to unintended mutations or chromosomal instability (Fu et al., 2013, Nature Biotechnology). Efficacy is typically monitored by quantifying the frequency of insertions and deletions (indels) or specific sequence changes at the target locus using high-throughput sequencing techniques (Zischewski et al., 2017, Biotechnology Advances).

Other names

ProtospacerCRISPR-Cas target sequencesgRNA-binding sitePAM-adjacent genomic locusTarget DNA sequence

The mechanism involves the RNA-guided recognition of a specific genomic DNA sequence (protospacer) adjacent to a Protospacer Adjacent Motif (PAM), followed by site-specific DNA cleavage or modification by a Cas nuclease or derivative (e.g., base editor). This induces cellular DNA repair pathways such as non-homologous end joining (NHEJ) or homology-directed repair (HDR) to achieve therapeutic gene disruption, correction, or insertion.