A new approach to CRISPR-Cas adaptation and the discovery of host accessory-proteins

Bacteria provide many health benefits and eco-environmental services. Although physically simple, they have a sophisticated immune system, CRISPR-Cas. Immunization occurs when the bacterium picks up and stores a sample of viral DNA, which it uses to recognize and remember the virus. Although we already understand how the viruses are destroyed, the mechanistic details of immunization are elusive. In this project we will address fundamental questions about the genes required for CRISPR immunization and the evolutionary battle raging between bacteria and their viruses.

The human immune system uses antibodies to fight new infections. Afterwards, it stores a memory of the battle, which provides long-lasting immunity against repeat encounters. This is known as an ’adaptive’ immune system because the antibodies do not exist in the body beforehand but are generated during the infection. This sophisticated system exists only in our close evolutionary relatives, the jawed vertebrates, which include the sharks, fishes, birds and mammals. All other creatures, including some with very sophisticated bodies, rely entirely on their innate immunity, provided by barrier-functions and chemical defenses. The recent discovery that bacteria, the simplest of organisms, have an adaptive immune system was therefore very surprising and exciting for microbiologists. The bacterial adaptive immune system (CRISPR-Cas) defends the bacteria from viral attack. CRISPR is the name of the location in the genome where the memories of past encounters are stored. The Cas proteins fight viral infections and help to create the immunological memory.

Many of the protective Cas proteins (effectors) are already quite well understood because their genes are closely linked to the CRISPR locus, where they were easily discovered. In contrast, the mechanism of immunization that records the identity of the pathogen, and directs the Cas proteins against it, has been more elusive. The final step is catalyzed by Cas1-Cas2, which are the only proteins universally linked to all functional CRISPR systems. Most, or all, of the preceding steps are performed by unknown proteins. These have been difficult to identify because their genes are not linked to the CRISPR locus. Presumably, they perform other unrelated tasks in the cell and their genes are scattered throughout the chromosome.

To gain insight into CRISPR immunization we will identify DNA and protein factors that alter its rate. In addition to host factors that stimulate the reaction, we are interested in finding viral proteins that suppress the system. Many viruses are already known to express proteins to protect themselves from attack by the CRISPR-Cas immune effectors. In contrast, we will search for viral systems that prevent the bacteria from becoming immunized against the attacker in the first place. To date no such system has been discovered.