Genomic DNA locus (gDNA) (gDNA)

The genomic DNA locus targeted by the Cas9–gRNA complex is the precise chromosomal site where CRISPR-Cas9-based therapeutics exert their molecular effect. This target is defined by a specific 20-nucleotide sequence, known as the protospacer, which must be located adjacent to a Protospacer Adjacent Motif (PAM), typically NGG for Streptococcus pyogenes Cas9 (Jinek et al., 2012, Science). The synthetic single-guide RNA (gRNA) directs the Cas9 nuclease to this locus through complementary base pairing, facilitating a site-specific double-strand break (DSB) (Doudna & Charpentier, 2014, Science). This DSB is subsequently repaired by the cell's endogenous machinery, such as non-homologous end joining (NHEJ), which often introduces small insertions or deletions (indels) to knock out a gene, or homology-directed repair (HDR) to introduce specific sequence changes. In clinical practice, this target is utilized to treat a variety of conditions; for instance, Exagamglogene autotemcel targets the BCL11A erythroid-specific enhancer to induce fetal hemoglobin in sickle cell disease (Frangoul et al., 2021, N Engl J Med). Similarly, NTLA-2001 targets the TTR gene in hepatocytes to reduce toxic transthyretin production in patients with amyloidosis (Gillmore et al., 2021, N Engl J Med). The therapeutic success of hitting these DNA loci depends heavily on the precision of the gRNA to avoid off-target effects at unintended genomic sites.

Other names

Target DNA sequenceProtospacerCRISPR target siteGenomic target sequenceCas9 target site

RNA-guided site-specific DNA cleavage followed by endogenous DNA repair mechanisms (NHEJ or HDR) to achieve gene disruption, correction, or regulation.