Genomic DNA at sgRNA-complementary sites adjacent to a Protospacer Adjacent Motif (CRISPR target DNA)

Genomic DNA at sgRNA-complementary sites adjacent to a Protospacer Adjacent Motif (PAM) serves as the fundamental substrate for CRISPR-Cas9-based genome editing and transcriptional regulation. This target is defined by a specific 20-nucleotide sequence, known as the protospacer, which is complementary to a synthetic guide RNA and located immediately upstream of a PAM sequence (typically 5'-NGG-3' for Streptococcus pyogenes Cas9). In therapeutic contexts, this DNA site is targeted to induce precise double-strand breaks for gene disruption or repair, or in the case of catalytically inactive 'dead' Cas9 (dCas9 or Cas9d), to sterically hinder transcription or deliver epigenetic modifiers to the promoter or enhancer regions (Gilbert et al., 2013, Cell). By targeting these specific genomic loci, CRISPR-based drugs can address the underlying genetic causes of diseases such as sickle cell disease, transthyretin amyloidosis, and various hereditary blindness disorders. The programmable nature of this target allows for highly specific interventions across the human genome, though the potential for off-target effects at similar DNA sequences remains a primary safety consideration (Hsu et al., 2013, Nature Biotechnology).

Other names

ProtospacerCRISPR target sitedCas9 target sitePAM-adjacent DNAsgRNA target sequence

The target DNA is recognized by a ribonucleoprotein (RNP) complex consisting of a Cas9 protein and a single guide RNA (sgRNA). The sgRNA hybridizes with the complementary genomic DNA sequence (protospacer) through Watson-Crick base pairing, while the Cas9 protein (or its catalytically inactive variant, dCas9/Cas9d) recognizes the adjacent Protospacer Adjacent Motif (PAM) (Jinek et al., 2012, Science). This binding facilitates site-specific DNA cleavage by active Cas9 or transcriptional modulation (interference or activation) by dCas9 (Qi et al., 2013, Cell).