Genomic DNA target site for CRISPR-Cas9

The genomic DNA target site for CRISPR-Cas9 is a specific sequence within the genome defined by a 20-nucleotide protospacer and an adjacent Protospacer Adjacent Motif (PAM), typically 5'-NGG-3' for Streptococcus pyogenes Cas9 (SpCas9) (Jinek et al., 2012, Science). This site serves as the substrate for the Cas9 ribonucleoprotein (RNP) complex, where the single guide RNA (sgRNA) hybridizes with the DNA strand to facilitate site-specific cleavage. In therapeutic applications, this target is exploited to induce double-strand breaks that are subsequently repaired by cellular mechanisms, leading to gene disruption or gene correction. Clinical examples include the targeting of the BCL11A enhancer in exagamglogene autotemcel to treat sickle cell disease and beta-thalassemia (FDA, 2023). Other therapies, such as NTLA-2001, target the TTR gene in the liver to treat transthyretin amyloidosis (Gillmore et al., 2021, NEJM). The primary challenge in targeting these sequences is ensuring high specificity to avoid off-target effects, which could lead to unintended mutations or chromosomal rearrangements (Fu et al., 2013, Nature Biotechnology). Additionally, the presence of the PAM sequence is a strict requirement for Cas9 binding, limiting the range of targetable sites within the genome. Successful engagement of this target results in permanent genomic modifications that can provide long-term therapeutic benefits for patients with monogenic disorders.

Other names

ProtospacerCas9 target sitesgRNA-targeted DNA sequenceNGG-adjacent DNACRISPR-Cas9 genomic target

The Cas9-sgRNA complex binds to the target DNA sequence via a Protospacer Adjacent Motif (PAM) and RNA-DNA hybridization, inducing a site-specific double-strand break to facilitate gene editing (Jinek et al., 2012, Science).