The CRISPR-Cas9 system works as the adaptive immune system in bacteria and archaea. When a virus infects the bacteria, this system incorporates segments of the viral DNA into the bacterial genome. Upon a second invasion, transcripts from these sequences direct a nuclease activity to its complementary sequence in the invading virus so as to destroy it. In order to extrapolate this method into eukaryotes in order to develop a gene editing method, a Cas9 protein, a recognition sequence RNA, and a transactivating RNA are required. The fusion of both the recognition sequence specificity CRISPR RNA (crRNA) and transactivating RNA (tracrRNA) is commonly used in experiments and called a single guide RNA (sgRNA). While the Cas9 specificity is believed to be controlled by the 20nt sgRNA and PAM, off-target mutations are still prevalent and could occur with as many as 3-5 base pair mismatches (out of 20) between the sgRNA and the target DNA sequence. Furthermore, sgRNA secondary structures could also affect cleavage of on-target and off-target sites. As mentioned above, sgRNA consists of a sequence (~20nt) which is complementary to the target sequences and this is followed by a PAM sequence which activates the endonuclease activity. While it was shown that 10-12 nt adjacent to PAM (called the “seed sequence”) was enough for Cas9 specificity, Wu et al. showed that in a catalytically dead Cas9 only 1-5 base pairs of seed sequence is required for specificity. A recent study published in Cell by Jinek's laboratory (Pacesa et al, 2022, Cell, 185: 4067-4081.e21), describes the structural basis of the off-target events showed by Cas9 protein. Here you can see one of the structures published in this manuscript, showing Cas9 protein together with the gRNA (red strand) and one of the off-target DNA substrates (blue spheres) (PDB code: 7QQR)
#molecularart ... #immolecular ... #cas9 ... #crispr ... #offtarget ... #complex ... #xray
Structure of the complex rendered with @proteinimaging and depicted with @corelphotopaint
#molecularart ... #immolecular ... #cas9 ... #crispr ... #offtarget ... #complex ... #xray
Structure of the complex rendered with @proteinimaging and depicted with @corelphotopaint
