Speaker: Peter Fineran
Subject: Spacer acquisition and regulation of CRISPR-Cas systems
Author: Carolien Bastiaanssen
The Fineran lab is interested in the interactions between mobile genetic elements and bacteria. They study the defense mechanisms that bacteria evolved to protect themselves against invaders such as bacteriophages. Their research focusses on one particular type of defense mechanisms, the CRISPR-Cas adaptive immune systems. In his talk Peter Fineran first gave a short recap of the CRISPR-Cas systems. Next he explained how the CRISPR-Cas systems form their immunological memory and he presented recent work on the regulating role of quorum sensing communication in the CRISPR-Cas systems.
In short the CRISPR-Cas systems work in three stages. The first stage is the acquisition stage where the bacteria forms immunological memory by incorporating part of the foreign DNA into its own genome at the CRISPR locus. Once incorporated, this sequence is referred to as a spacer. The second stage is the expression and processing stage. This involves the transcription of the CRISPR locus leading to the production of CRISPR RNAs (crRNAs). Each crRNA contains a spacer. In the third stage, the interference stage, the crRNAs with the help of their spacer recognize invading DNA as foreign and the invader is degraded. The many different types of CRISPR-Cas systems all work following the adaptation, expression and interference stages.
Figure 1: Stages of the CRISPR-Cas immune systems. In stage 1 foreign DNA is acquired and incorporated as spacers. In stage 2 the CRISPR locus is transcribed and the RNA is processed to form mature CRISPR RNAs (crRNAs). In stage 3 the crRNAs target viral elements with the spacer as a guide. Source: http://rna.berkeley.edu/crispr.html
The questions that intrigued the Fineran lab included how adaptation occurs and what part of the plasmid is taken up by the bacteria. To answer these questions they added plasmids to a population of bacteria and subsequently analyzed the bacterial genomes. Surprisingly there was a lot of acquisition of spacers from the bacteria’s own chromosome. This phenomenon is like an autoimmune defect and it occurs constantly in wild-type cells. However, chromosomal spacers are counter-selected as they typically result in cell death. Fineran and his colleagues found that 93% of the times the last acquired spacer in the CRISPR array before cell death was from the bacterial chromosome. If CRISPR-Cas is so costly, why do bacteria still have this immune system? Does this mean that defense is increased with increased vulnerability? And how do bacteria regulate CRISPR-Cas immunity?
It is known that high cell densities of bacteria are prone to horizontal gene transfer because phages can spread rapidly through a dense population. One way in which bacteria can determine the cell density and regulate gene expression accordingly is through a form of communication called quorum sensing (QS). In low cell density there is are low amounts of the signaling molecules and in high cell density the signaling molecules accumulate, causing an upregulation of certain genes. Fineran and his colleagues set out to study the possible role of QS in regulating CRISPR-Cas immunity. They showed that mutant strains that were unable of QS were more vulnerable for invading plasmids because they had reduced CRISPR-Cas activity. At low cell density there is a repressor, SmaR, that shuts down CRISPR-Cas activity. At high cell density the signaling molecule AHL represses SmaR and CRISPR-Cas activity is no longer repressed. Hence, at high cell density there is increased CRISPR immunity and at low cell density CRSIPR immunity is reduced, leading to less autoimmune effects and thus lower fitness costs.
Figure 2: CRISPR-Cas immunity is regulated through quorum sensing. When the cell density is low the concentration of AHL is also low and SmaR represses CRISPR expression. When the cell density is high the concentration of AHL is high and AHL represses SmaR. As there is no SmaR to repress CRISPR expression, CRISPR expression is upregulated. Thus at high cell densities the cells are in a higher state of defense than at low cell densities, thereby reducing the costs of the CRISPR-Cas system when there is no high threat. Source: Patterson, A. G. et al. Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems. Mol. Cell 64, 1102–1108 (2017).
CRISPR is a hot topic and it the CRISPR-Cas9 system is commonly used as a gene editing technique. Therefore, I find it quite surprising how many aspects of the CRISPR systems are still to be unraveled. The research performed by the Fineran lab provides insights in how bacteria control the rather large fitness costs of the CRSIPR systems. All in all, the talk was clear and a pleasure to attend due to the enthusiasm of the speaker.