Genomic DNA target sequence (CRISPR-Cas system) (gDNA target)

Genomic double-stranded DNA at a guide-RNA–specified protospacer adjacent to a compatible protospacer adjacent motif (PAM) is the precise molecular target for CRISPR-based gene editing technologies. This target is defined by a specific nucleotide sequence (the protospacer) that is complementary to a synthetic guide RNA (gRNA), located immediately upstream of a short, conserved PAM sequence required for nuclease recognition [1, 2]. Upon binding, the CRISPR-Cas complex (such as Cas9 or Cas12a) facilitates the unwinding of the DNA duplex and induces a site-specific double-strand break or chemical modification [2]. This mechanism is leveraged therapeutically to permanently modify the human genome to treat genetic diseases by disrupting pathogenic genes or restoring functional ones [3, 4]. For example, exagamglogene autotemcel targets a specific DNA sequence within the BCL11A enhancer to treat sickle cell disease [3]. Safety considerations primarily involve off-target effects, where the gRNA-Cas complex binds and cleaves DNA at unintended sites with sequence similarity, potentially leading to oncogenic mutations or chromosomal rearrangements [4].

Other names

CRISPR target siteProtospacerPAM-adjacent sequenceTarget DNA locusgRNA-targeted DNA

RNA-guided site-specific DNA cleavage followed by endogenous DNA repair (NHEJ or HDR), base editing, or prime editing.