DNA locus complementary to the delivered CRISPR–Cas9 guide RNA (Protospacer) (Protospacer)

The DNA locus complementary to the delivered CRISPR–Cas9 guide RNA is the specific genomic sequence targeted for therapeutic modification. This site, typically 20 nucleotides long and adjacent to a Protospacer Adjacent Motif (PAM), is recognized by the guide RNA (gRNA) through complementary base pairing (Jinek et al., 2012, Science). Upon binding, the Cas9 nuclease induces a double-strand break, which triggers cellular DNA repair pathways like non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Doudna & Charpentier, 2014, Science). These pathways are leveraged to knock out deleterious genes, correct mutations, or modulate gene expression by targeting regulatory elements. In clinical applications, such as the treatment of sickle cell disease with exagamglogene autotemcel, the target locus is carefully selected to ensure high editing efficiency and minimal off-target activity (Frangoul et al., 2021, NEJM). The precision of this interaction is fundamental to the safety and efficacy of CRISPR-based gene therapies, as unintended editing at similar sequences can lead to genotoxicity (Fu et al., 2013, Nature Biotechnology). Furthermore, the choice of target locus determines the therapeutic strategy, whether it be direct gene correction or the disruption of a transcriptional repressor binding site. Safety monitoring typically involves assessing the frequency of insertions and deletions (indels) and potential chromosomal rearrangements at the target site. As a therapeutic target, it is unique because its 'druggability' is determined by the design of the synthetic guide RNA rather than the intrinsic pocket of a protein. This target represents the fundamental unit of precision medicine in the era of genome engineering.

Other names

Target DNA sequenceCRISPR target siteGenomic target locusProtospacer sequence

The guide RNA (gRNA) directs the Cas9 nuclease to the complementary DNA locus via Watson-Crick base pairing, where the enzyme creates a double-strand break (DSB). This break is then repaired by the cell's internal machinery (NHEJ or HDR), resulting in gene disruption, deletion, or precise sequence correction (Jinek et al., 2012, Science).