Warring genetic parasites could lead to new defenses against dangerous bacteria

CRISPR-Cas has become something of a superstar over the past decade as a gene-editing tool with breakthrough potential, especially in the health sciences. Originally known as an immune defense in bacteria, natural CRISPR-Cas has proven to be more diverse and versatile than scientific researchers thought. Now, a group of researchers from the Department of Biology at the University of Copenhagen has studied the prevalence of CRISPR-Cas systems in plasmids.

The researchers examined more than 30,000 complete plasmid genomes and found CRISPR-Cas in about 3% of them, a high proportion even compared to bacteria. They also found various representatives of as many as five of the six known CRISPR-Cas types in the plasmid genomes studied. The results demonstrate that CRISPR-Cas systems are both widespread and diverse in plasmids and, interestingly, that the vast majority of them target other plasmids.

“In part, this is exciting because it supports a more recent understanding of plasmids as having a higher degree of autonomy from their host cells, usually bacteria. But also, because in the long run it may open up pathways to combat virulence and resistance in bacteria, which plasmids help spread,” says Rafael Pinilla-Redondo, who is one of the study’s lead researchers and based in the Department of Biology at the University of Copenhagen.

CRISPR acts as a genomic GPS, where a stored memory of foreign DNA fragments can be used to locate a target for Cas proteins, the “genetic scissors”. In the majority of the study results, it was DNA from other plasmids that was found in the immune memory of CRISPR-Cas systems, i.e. placed in the crosshairs.

Part of a paradigm shift

According to the researchers, this suggests a struggle for resources between plasmids, where plasmids serve their own interests by actively working to prevent other plasmids from gaining access to the host bacterium in which they reside. In this battle, they are using CRISPR as a weapon.

The researchers had the opportunity to simultaneously examine host bacteria from the plasmid dataset of over 30,000 for the same CRISPR-Cas sequences. The idea was to study whether the sequences found in the plasmids reflected the CRISPR content in the host cells, but this was generally not the case.

“Our results suggest that plasmids have a high degree of autonomy from the bacteria in which they live. While plasmids are host-dependent, they are also genetically independent entities that serve their own interests. Their CRISPR-Cas content different is a great example of this autonomy,” says Rafael Pinilla-Redondo.

The new research findings will contribute to what researchers see as a paradigm shift in microbiology. In microbiology, gene flow or gene transfer refers to when genetic material moves between cells, through mobile genetic elements. While certain mobile genetic elements are allowed to infiltrate for the benefit of a cell, others are stopped because they are harmful. The common understanding has long been that bacteria control gene flow.

The paradigm shift points to an understanding where bacteria actually play a much smaller role in influencing gene flow.

“What used to be assumed to be bacteria fighting to protect themselves from genetic parasites, like viruses and plasmids, is much more complex. Perhaps we should better understand that parasites fight each other, for example, to find out which ones should be allowed to live behind a cow’s ear,” explains Rafael Pinilla-Redondo.

The possibility of new weapons against antibiotic resistance

New knowledge about how plasmids use CRISPR could impact how we fight dangerous bacteria in the future. Plasmids are essential for the spread of harmful genes between bacteria through what is known as horizontal gene transfer.

The proliferation of genetic material is crucial for the ability of bacteria to adapt to new environments and challenges. From an antibiotic-resistant bacterium, a plasmid can copy itself and transfer this property to surrounding bacteria as part of its own DNA.

As such, battles between plasmids may also help researchers learn more about how to fight them.

“By understanding how plasmids compete with each other, we may be able to learn how to slow them down and thus slow the spread of antibiotic resistance and virulent and harmful properties between bacteria,” says Søren Johannes Sørensen, professor of microbiology and co – author of the research article.

“In the long term, it is possible that we could appropriate the strategies of plasmids and use them as tools. Without borrowing from nature, we would be quite limited. But if we can learn the strengths and weaknesses of the plasmids from themselves, opportunities will arise,” he says.

What does CRISPR-Cas mean?

DNA fragments (CRISPR) and Cas protein scissors (eg, Cas9) can locate specific DNA sequences and cut them.

CRISPR-Cas is expected to play a breakthrough role as a gene editing tool, especially in the health sciences, for the treatment of genetic disorders, among others.

CRISPR systems were originally thought of as an immune system against bacteria, especially against viruses. However, many researchers now see CRISPR-Cas as a “Guns for Hire” tool that can be deployed for a multitude of purposes, by many different actors, including bacteria, plasmids, and humans.

What is a plasmid?

A plasmid is a small, ring-shaped DNA molecule, a so-called mobile genetic element, found in bacteria and some other types of microorganisms.

It is reminiscent of viruses, as both are parasites inside cells. Plasmids can replicate independently of the host cell and often provide benefits to the host cell.

Among other things, they can donate or transfer genetic properties to a bacterium, for example making it resistant to antibiotics or pathogenic, in a process known as horizontal gene transfer.

Plasmids have long been an important tool in molecular biology for, among other things, the cloning of genes and the introduction of genetic material into bacterial cells.

What is horizontal gene transfer?

Horizontal gene transfer occurs when an organism transfers genes to another organism that is not its own offspring.

To a large extent, the ability of bacteria to adapt to new environments and challenges depends on the delivery of new genes in this way.

The phenomenon is responsible for the impending antibiotic resistance crisis, as bacteria rapidly develop antibiotic resistance by acquiring resistant genes. It is very often mediated by plasmids, making plasmid proliferation a global public health problem.

About the study: Researchers created a CRISPR scanner

To study the prevalence of CRISPR-Cas in plasmids, the researchers used the largest collection of fully sequenced plasmid genomes, a dataset compiled by researchers around the world.

To manage the large amounts of data, researchers in the Department of Biology developed software to search for known CRISPR pieces. The program, named CRISPRCasTyper, has since been made freely available to other researchers.