CRISPR-Cas9 genomic target DNA sequence (Cas9 target site)

The target refers to specific, user-defined sequences within the double-stranded genomic DNA that are recognized and bound by the CRISPR-Cas9 system. Recognition is mediated by a single guide RNA (sgRNA) containing a ~20-nucleotide spacer sequence that is complementary to the target DNA (Jinek et al., 2012). For the Streptococcus pyogenes Cas9 (SpCas9) enzyme to bind, the target sequence must be immediately followed by a 5'-NGG-3' Protospacer Adjacent Motif (PAM) (Hsu et al., 2013). Upon binding, the Cas9 endonuclease induces a site-specific double-strand break (DSB) in the DNA (Doudna & Charpentier, 2014). This break triggers cellular DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR), which can be harnessed to knock out genes or correct mutations. In a therapeutic context, this target is utilized to treat genetic disorders by modifying disease-causing genes or regulatory elements, such as the BCL11A enhancer in sickle cell disease (Frangoul et al., 2021). Drugs targeting these sites are typically delivered as ribonucleoprotein complexes or via viral vectors and lipid nanoparticles. Clinical efficacy is often measured by the frequency of insertions and deletions (indels) at the target site using next-generation sequencing. A primary safety concern involves "off-target" effects, where the Cas9 enzyme cleaves genomic sites with high sequence similarity to the intended target (Fu et al., 2013). Ongoing research focuses on increasing the specificity of these interactions to minimize unintended genomic alterations.

Other names

ProtospacersgRNA target siteCRISPR target sequenceGenomic target locusCas9 binding site

Site-specific DNA cleavage followed by cellular DNA repair (NHEJ or HDR) to achieve gene knockout, insertion, or correction.