Periodic Reporting for period 5 - CRISPR-EVOL (The eco-evolutionary costs and benefits of CRISPR-Cas systems, and their effect on genome diversity within populations)
Reporting period: 2024-05-01 to 2025-04-30
CRISPR-Cas systems are defense systems that protect bacteria and archaea (two forms of single-cell organisms that do not have a nucleus in their cells) against selfish elements, such as viruses. Archaea resemble bacteria in cell shape and size, but they are closer in evolutionary relation to nucleus-containing cells, such as those of fungi, plants, or animals than they are to bacteria. Much like our own immune system, when viruses or virus-like elements inject their genomes into such cells, the system can sample a piece of the selfish DNA and the next time it will meet a similar invading DNA it will cut its genome to pieces and thereby protect the cell from infection. Such defense systems are common in bacteria and even more so in archaea, the vast majority of which have them. However, just as countries must incur large costs to have defense forces to keep invaders out so do cells, and CRISPR-Cas systems do have their costs. One such cost is the potential reduction in genetic novelty. Some viruses of archaea and bacteria can actually benefit their hosts by various mechanisms, especially by killing off competitor strains and species, while keeping a “low profile” in their own host cell. If a CRISPR-Cas system is too active and defensive, it can therefore prevent the cell from acquiring beneficial traits. As an example, bacterial strains that have CRISPR-Cas systems that are active tend to have fewer antibiotic resistance genes than their counterparts in which the system has been inactivated or lost. The “borders” that CRISPR-Cas systems create may even be sufficient to reduce the flow of genetic material between cells and ultimately lead to their divergence into different species.
Another potential cost of CRISPR-Cas system is that it may interfere with DNA repair processes in the cell because its components may clash with repair enzymes that help the cell deal with the damages of UV radiation or toxic chemicals that damage DNA. To date, CRISPR-associated proteins have been shown to contribute to DNA-repair In some bacteria and interfere in others, but this has not been studied in archaea. Our aims are to explore the role of CRISPR-Cas systems in shaping gene exchange, speciation, and genetic novelty in archaea and how they interact with DNA repair processes in the cell.
Another potential cost of CRISPR-Cas system is that it may interfere with DNA repair processes in the cell because its components may clash with repair enzymes that help the cell deal with the damages of UV radiation or toxic chemicals that damage DNA. To date, CRISPR-associated proteins have been shown to contribute to DNA-repair In some bacteria and interfere in others, but this has not been studied in archaea. Our aims are to explore the role of CRISPR-Cas systems in shaping gene exchange, speciation, and genetic novelty in archaea and how they interact with DNA repair processes in the cell.
In this grant, we aimed to study the effects of CRISPR-Cas systems on genome diversity and gene transfer in halophilic archaea—microorganisms that thrive in the world's saltiest ecosystems. We also tested the hypothesis that CRISPR-Cas may play an active role in DNA repair in these organisms, which would help explain why this system is maintained even under conditions where no viruses are present, although it can easily be lost.
The project had 5 aims. Aim 1 explored the association between CRISPR-Cas presence/absence and the level of genetic diversity within a lineage: how many different genes can exist in different strains related to one another within a lineage. We discovered that because CRISPR-Cas systems can be suppressed by endogenous viruses, such viruses are often associated with higher genetic diversity. Furthermore, some of these viruses can encode their own CRISPR-Cas systems, and these become active only when the cell is threatened by external viruses.
In Aim 2, we tested the effects of CRISPR-based "autoimmunity" (when the system attacks the host's own genome), which turned out to be surprisingly mild. In Aim 3, we generated strains that attacked each other's genomes using CRISPR-Cas and showed that this led to lower gene exchange between species but higher gene exchange within species, indicating that CRISPR-Cas systems can contribute to speciation in these organisms.
In Aim 4, we showed that CRISPR-Cas targeting increased homologous recombination—a form of DNA exchange that operates well between strains of the same species, but not between different species. In Aim 5, we showed that one of the components of CRISPR-Cas, a protein named Cas3, independently contributes to faster DNA repair processes and faster recovery from DNA damage caused by UV radiation or chemical mutagens. This role may help preserve these systems since it benefits the cells even when no viral threats exist, because halophilic archaea are exposed to strong UV radiation almost every day.
The project had 5 aims. Aim 1 explored the association between CRISPR-Cas presence/absence and the level of genetic diversity within a lineage: how many different genes can exist in different strains related to one another within a lineage. We discovered that because CRISPR-Cas systems can be suppressed by endogenous viruses, such viruses are often associated with higher genetic diversity. Furthermore, some of these viruses can encode their own CRISPR-Cas systems, and these become active only when the cell is threatened by external viruses.
In Aim 2, we tested the effects of CRISPR-based "autoimmunity" (when the system attacks the host's own genome), which turned out to be surprisingly mild. In Aim 3, we generated strains that attacked each other's genomes using CRISPR-Cas and showed that this led to lower gene exchange between species but higher gene exchange within species, indicating that CRISPR-Cas systems can contribute to speciation in these organisms.
In Aim 4, we showed that CRISPR-Cas targeting increased homologous recombination—a form of DNA exchange that operates well between strains of the same species, but not between different species. In Aim 5, we showed that one of the components of CRISPR-Cas, a protein named Cas3, independently contributes to faster DNA repair processes and faster recovery from DNA damage caused by UV radiation or chemical mutagens. This role may help preserve these systems since it benefits the cells even when no viral threats exist, because halophilic archaea are exposed to strong UV radiation almost every day.
We have made several discoveries that we, and a few other research groups, will be following up on in the coming years. Two specific achievements that greatly moved the field forward are
1. We showed that some viruses that can maintain a chronic infection state in haloarchaea can suppress the CRISPR-Cas system and thereby persist in the host while dampening its immune response. Consequently, these viruses that may also contribute to the host under some situations can be maintained in a CRISPR-Cas positive strain, while others cannot. Curiously, even the “immune-compromised” host is still rather resistant to additional infections. Moreover, some of these viruses carry their own CRISPR-Cas systems.
2. CRISPR-Cas systems play a role in DNA repair and enable faster recovery from DNA damage. Therefore, these systems can be potentially useful to the host even when no viral threats are present in the environment, since for organisms that are subject to high solar radiation every day, DNA damage is inevitable.
We also made a discovery that has practical value. We cured a halophilic archaeon of its chronically infecting virus, which resulted in a strain with greatly improved growth properties. This approach has been patented and we hope it will open new markets for haloarchaea in green biotechnology, converting waste products from agriculture and food production into valuable commodities, such as carotenoids.
1. We showed that some viruses that can maintain a chronic infection state in haloarchaea can suppress the CRISPR-Cas system and thereby persist in the host while dampening its immune response. Consequently, these viruses that may also contribute to the host under some situations can be maintained in a CRISPR-Cas positive strain, while others cannot. Curiously, even the “immune-compromised” host is still rather resistant to additional infections. Moreover, some of these viruses carry their own CRISPR-Cas systems.
2. CRISPR-Cas systems play a role in DNA repair and enable faster recovery from DNA damage. Therefore, these systems can be potentially useful to the host even when no viral threats are present in the environment, since for organisms that are subject to high solar radiation every day, DNA damage is inevitable.
We also made a discovery that has practical value. We cured a halophilic archaeon of its chronically infecting virus, which resulted in a strain with greatly improved growth properties. This approach has been patented and we hope it will open new markets for haloarchaea in green biotechnology, converting waste products from agriculture and food production into valuable commodities, such as carotenoids.