Periodic Reporting for period 4 - CRISPR-EVOL (The eco-evolutionary costs and benefits of CRISPR-Cas systems, and their effect on genome diversity within populations)
Berichtszeitraum: 2022-11-01 bis 2024-04-30
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.
We have already isolated and determined the genome sequences of nearly a hundred of archaeal genomes from four sampling sites, and compared CRISPR-containing and CRISPR-free lineages to explore the effects of CRISPR on gene diversity, genome size and, mobile element content. Preliminary results indicate that in the CRISPR-negative strains genome size is more variable than in CRISPR-positive lineages, up to 6% of the genome in strains that are >99.9% identical in their shared gene
We have also discovered that CRISPR-Cas systems may sometimes have opposing roles because they can be encoded by different elements. We knew that most CRISPR-Cas loci in halophilic archaea were encoded on extra-chromosomal pieces of DNA known as plasmids. However, we have more recently discovered that ta least some of the “chromosomal” loci are actually on integrated viruses, some of which are fully functional and can exit the genome and package their DNA in a capsid ready to infect a new strain and provide it with a new CRISPR-Cas system. This raises the possibility that virus-encoded systems may be in conflict with plasmid-encoded ones, because viruses can target plasmids and vice-versa, but because their systems are generally compatible with one another, they can also work together.
Work from this project that we have published in 2019 has shown that not only do CRISPR–Cas decreasing the transfer of genes between different species by cell fusion in archaea and suggested that these adaptive immune systems might influence microbial speciation. We have shown that when two archaeal species mate this induces the systems to acquire new spacers from the other species, probably, because under such situations virus-like elements that were dormant start replicating and try to invade the other cell. Furthermore, when one species was targeted by the CRISPR-Cas system of the other, the mating efficiency was reduced. More recent follow-up work in our lab showed that causing otherwise genetically identical strains to target one another can lead to unexpected results. In contrast to what we have observed when different species target one-another during mating using CRISPR-Cas, within species targeting sometimes resulted in comparable or even higher mating frequency to non-targeted mating, depending on the targeted site in the genome. This implies that some level of CRISPR-Cas activity can actually promote gene exchange within species while reducing gene exchange between different species.
We have also made several key discoveries about the connection between DNA repair and CRISPR-Cas. A key mechanism of DNA repair in archaea and human cells is homologous recombination, a process in which a break in the DNA is repaired by using a template that is still intact from another copy of identical DNA that is still intact. We have observed that when we delete one of the key CRISPR-associated genes, this increases the efficiency of homologous recombination. Moreover, when we have tested resistance we have discovered that mutants that lacked that gene were more resistant, while cells that expressed it at levels higher than normal had higher sensitivity to DNA damage. We are now using a new technology called RADAR-SEQ to gain mechanistic insights and are also generating double mutants in cas genes and in DNA repair genes to reveal the mechanism of interaction.
We have also discovered that CRISPR-Cas systems may sometimes have opposing roles because they can be encoded by different elements. We knew that most CRISPR-Cas loci in halophilic archaea were encoded on extra-chromosomal pieces of DNA known as plasmids. However, we have more recently discovered that ta least some of the “chromosomal” loci are actually on integrated viruses, some of which are fully functional and can exit the genome and package their DNA in a capsid ready to infect a new strain and provide it with a new CRISPR-Cas system. This raises the possibility that virus-encoded systems may be in conflict with plasmid-encoded ones, because viruses can target plasmids and vice-versa, but because their systems are generally compatible with one another, they can also work together.
Work from this project that we have published in 2019 has shown that not only do CRISPR–Cas decreasing the transfer of genes between different species by cell fusion in archaea and suggested that these adaptive immune systems might influence microbial speciation. We have shown that when two archaeal species mate this induces the systems to acquire new spacers from the other species, probably, because under such situations virus-like elements that were dormant start replicating and try to invade the other cell. Furthermore, when one species was targeted by the CRISPR-Cas system of the other, the mating efficiency was reduced. More recent follow-up work in our lab showed that causing otherwise genetically identical strains to target one another can lead to unexpected results. In contrast to what we have observed when different species target one-another during mating using CRISPR-Cas, within species targeting sometimes resulted in comparable or even higher mating frequency to non-targeted mating, depending on the targeted site in the genome. This implies that some level of CRISPR-Cas activity can actually promote gene exchange within species while reducing gene exchange between different species.
We have also made several key discoveries about the connection between DNA repair and CRISPR-Cas. A key mechanism of DNA repair in archaea and human cells is homologous recombination, a process in which a break in the DNA is repaired by using a template that is still intact from another copy of identical DNA that is still intact. We have observed that when we delete one of the key CRISPR-associated genes, this increases the efficiency of homologous recombination. Moreover, when we have tested resistance we have discovered that mutants that lacked that gene were more resistant, while cells that expressed it at levels higher than normal had higher sensitivity to DNA damage. We are now using a new technology called RADAR-SEQ to gain mechanistic insights and are also generating double mutants in cas genes and in DNA repair genes to reveal the mechanism of interaction.
Thus far, we have gone beyond the state of the art by successfully demonstrating that CRISPR-Cas systems in archaea affect gene flow and even speciation processes. We have also shown that CRISPR-Cas has a cost for the host cell and that it reduces survival after DNA damage. We plan on further elucidating the interaction between Cas genes and DNA repair processes in archaea, and obtain more precise mechanistic insights. To go beyond the state of the art in terms of evolutionary implications we will study conflicts and ecological interactions between the genetic elements that encode those systems, and how they affect gene flow. We can study such interactions in the lab using competition assays and other experimental evolution approaches. We will also systematically test the costs of CRISPR-Cas carriage in areas other than DNA damage, using experimental evolution assays where cells evolve under different conditions and interact with other strains with different CRISPR-Cas systems or no such system at all.