Cas9 / Wk. 3 #3

In this article, researchers wanted to learn about the conformational changes of Cas9. Conformation changes can be caused by binding of single guide RNA (sgRNA). These researchers wanted to figure out which molecular components of sgRNA caused the conformation change in Cas9 from inactive to active. In order to do that, the researchers removed different parts of the sgRNA to see how much it changed the change in conformation. When they removed the entire target recognition sequence, the sgRNA was unable to find the Cas9 and there was no change in conformation. However, when they only removed pieces of the recognition sequence, there was some conformational change. This showed that the target recognition sequence could still work if there was some pieces missing, although it would not work consistently. In addition to that, they tried removing the hairpins at the 3’ end of the sgRNA. When they removed the hairpins, they observed intermediate levels of conformation change. These intermediate levels of conformation change indicate that there was incoverting from closed to open in the Cas9 sample.