|United States Of America||Issued Patent||11,208,638||12/28/2021||2015-060|
The CRISPR-Cas9 system can be used to quickly and specifically target and cleave DNA at sites defined by engineered single-guide RNAs (sgRNAs) and has led to its adoption as a robust and versatile platform for genome engineering. Cas9 contains two nuclease active sites that function together to generate DNA double-strand breaks (DSBs) at sites complementary to the guide RNA sequence and adjacent to a protospacer adjacent motif.
Structural studies of the Streptococcus pyogenes Cas9 showed that the protein exhibits a bilobed architecture comprising the catalytic nuclease lobe and the α-helical lobe of the enzyme and interactions between the two lobes seem to be mediated primarily through contacts with the bound nucleic acid rather than direct protein–protein contacts.
UC Berkeley researchers have developed a heterodimeric Cas9 system whose assembly and function is regulatable by the sgRNAs. The enzymatic activity of the split-Cas9 also closely mimics that of WT Cas9. Such a system enables analysis of the functionally distinct properties of each Cas9 structural region and offers a unique mechanism for controlling active protein assembly.