Genomic DNA target site (CRISPR-Cas9) (Protospacer)

The genomic DNA target site is the specific nucleotide sequence within a host's genome that is recognized and modified by the CRISPR-Cas9 system. This target is defined by its complementarity to a synthetic or natural guide RNA (gRNA) and the presence of an adjacent Protospacer Adjacent Motif (PAM), typically 5'-NGG-3' for Streptococcus pyogenes Cas9 [Jinek et al., 2012, Science]. In a therapeutic context, this DNA sequence serves as the substrate for Cas9-mediated double-strand breaks or base editing, allowing for the precise disruption, correction, or regulation of genes associated with disease [Cong et al., 2013, Science]. For example, the drug exagamglogene autotemcel targets the BCL11A enhancer sequence to induce fetal hemoglobin production in patients with sickle cell disease [Frangoul et al., 2021, NEJM]. Beyond hematologic disorders, this target is being utilized in vivo to treat conditions like transthyretin amyloidosis by knocking out the TTR gene in hepatocytes [Gillmore et al., 2021, NEJM]. The primary challenge in targeting these sequences is ensuring high specificity to avoid off-target mutations at similar genomic loci, which could lead to unintended functional consequences or genotoxicity [Fu et al., 2013, Nature Biotechnology]. Consequently, the selection and validation of the genomic target site are critical steps in the development of safe and effective CRISPR-based medicines.

Other names

CRISPR target sequencePAM-adjacent DNATarget DNA locusProtospacer sequence

The target DNA is recognized by a ribonucleoprotein complex consisting of a Cas9 nuclease and a guide RNA (gRNA). The gRNA hybridizes with the complementary protospacer sequence on the DNA, while the Cas9 protein recognizes the Protospacer Adjacent Motif (PAM), typically 5'-NGG-3'. This interaction triggers a conformational change in the nuclease, leading to a site-specific double-strand break (DSB) or other modifications like base editing [Jinek et al., 2012, Science].