CRISPR-Cas9 is a system for genome editing which enables researchers to target and cut a particular DNA sequence. The term CRISPR stands for “clustered regularly interspaced short palindromic repeats” and Cas9 refers to the enzyme that cuts DNA. Bacteria first developed CRISPR to defend themselves against viruses, and scientists have since modified this natural defense mechanism into a programmable tool for genetic research.
The two main parts of CRISPR-Cas9 are guide RNA and the Cas9 protein. To begin with, the researchers create a guide RNA that has a sequence matching the DNA site that they want to target. Then, this guide RNA takes Cas9 to that position. Cas9 subsequently looks for a DNA signal nearby known as a PAM, or protospacer adjacent motif. When it has located the right target and PAM, Cas9 cuts both strands of the DNA. The cell then repairs the break, and in doing so may introduce small changes that switch off a gene. Alternatively, the researchers can give the cell a DNA template in order to achieve a more precise repair. The original 2012 study demonstrated that researchers could use CRISPR-Cas9 as a programmable DNA-cutting system.
Why Understanding CRISPR-Cas9 Is Important
By understanding CRISPR-Cas9, students are able to tell the difference between the targeting, cutting, and repairing of DNA. It is the guide RNA that decides where Cas9 will act; nevertheless, it is the cell’s repair process that determines the end result. Since the guide RNA may at times match with similar sequences elsewhere in the genome, researchers have to look for possible off-target effects. For this reason, they sequence the area that has been edited and examine other possible sites before reaching a conclusion.
Moreover, scientists can alter Cas9 according to their various aims. For instance, dCas9 is not capable of cutting DNA but can be used to turn genes on or off. Because of this, CRISPR-based tools are useful in the fields of medicine, agriculture, biotechnology, and education. Yet CRISPR-Cas9 does not ensure a perfectly accurate edit. It is necessary for researchers to take into account accuracy, delivery methods, cellular reactions, and ethical issues. In the end, CRISPR-based tools provides a powerful means of studying genes, but careful design and verification are still essential.
Reference: Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829