Genomic DNA at sgRNA-specified Cas9 target sites (Cas9 target site)

Genomic DNA at sgRNA-specified Cas9 target sites refers to the specific sequences within the human genome targeted by the CRISPR-Cas9 system for therapeutic intervention. These sites are identified by a 20-nucleotide sequence complementary to a single guide RNA (sgRNA) and must be immediately followed by a protospacer adjacent motif (PAM), typically NGG for Streptococcus pyogenes Cas9 (Jinek et al., 2012). Upon binding, the Cas9 nuclease induces a double-strand break, which the cell repairs via non-homologous end joining (NHEJ) or homology-directed repair (HDR), leading to gene knockout or precise sequence correction (Doudna & Charpentier, 2014). This target is central to modern gene-editing therapies, such as exagamglogene autotemcel, which targets the BCL11A erythroid enhancer to treat sickle cell disease and beta-thalassemia (Frangoul et al., 2021). Other applications include targeting the TTR gene in the liver to treat transthyretin amyloidosis (Gillmore et al., 2021). The primary clinical challenges associated with these targets include off-target cleavage at unintended genomic loci and the potential for large-scale chromosomal rearrangements (Kosicki et al., 2018). Consequently, rigorous bioinformatic prediction and experimental validation of target specificity are essential for patient safety.

Other names

CRISPR-Cas9 target sequenceProtospacersgRNA-binding siteGenomic target locusCas9-targeted genomic DNA

The mechanism involves the formation of a ribonucleoprotein (RNP) complex where the sgRNA guides the Cas9 nuclease to a complementary genomic DNA sequence. Once the PAM is recognized and the DNA is unwound, Cas9 creates a site-specific double-strand break (DSB). This break triggers cellular DNA repair mechanisms: non-homologous end joining (NHEJ) often results in small insertions or deletions (indels) that disrupt gene function, while homology-directed repair (HDR) can be used with a donor template to introduce specific genetic changes (Jinek et al., 2012; Doudna & Charpentier, 2014).