Accomplishments by Dr. Doudna and Dr. Charpentier:
- The first accomplishment of the joint research venture led to the successful purification of Cas9, the molecular scissors of the CRISPR system. The purified form of Cas9 was from two different types of bacteria, namely, Streptococcus thermophilus and Streptococcus pyogenes. As a gram-positive bacteria, S. thermophilus is a non-pathogenic harmless bacterium used for start-up cultures of fermented milk products such as yogurt. The second type of bacterial cell, also known as group A streptococci with the acronym of “GAS,” is the causative factor of acute pharyngitis, commonly known as strep throat. The significance of obtaining the purified form of Cas9 is great because it made it possible to test the functional role of the molecular scissors in cutting bacterial DNA in a laboratory Petri dish.
- In addition to Cas9 purification, the two scientists also made an instrumental discovery of how Cas9's DNA-cutting action is regulated. They revealed two regulatory components that work collaboratively to ensure Cas9 cuts out the DNA site needed for anti-phage bacterial immune defense.
- The first component is the proto-spacer adjacent motif (PAM) that resides adjacent to the phage sequence, marking the specific starting nucleotide for cleavage.
- The second component is seed sequence, which is the very same phage DNA target selected for destruction by Cas9.9 Of note: Cas9 is not the only CRISPR molecular scissors, but it is the best characterized. Other Cas molecules include Cas13 and Cpf1.
- The third accomplishment by Dr. Doudna and Dr. Charpentier pertains to seed sequence manipulation. Specifically, by modifying the nucleotide composition of the bacterial phage seed sequence, CRISPR became a potent silencer of genes of interest.
9. Gleditzsch, D., Pausch, P., Müller-Esparza, H., Özcan, A., Guo, X., Bange, G., & Randau, L. (2019). PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA biology, 16(4), 504–517. https://doi.org/10.1080/15476286.2018.1504546
