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Bacteria’s defences reveal ways to improve personalised medicine

Researchers have uncovered how proteins keep an updated log of viruses, allowing bacteria to defend against attack. The results could advance work on ‘DNA recorders’ which, by tracking cellular activity, could lead to personalised medicine, including against infections such as COVID-19.

Health

All living organisms use their immune systems to fight off constant attack by viruses. Bacteria allocate part of their genomes to store fragments of virus DNA, creating a log of potential invaders. When a virus injects its DNA into the bacterial cell, the immune system recognises the DNA and destroys it. This system is known as clustered regularly interspaced short palindromic repeats (CRISPR) immunity. Viruses have evolved several countermeasures. Some express anti-CRISPR proteins that can block the function of CRISPR proteins. Others hijack host CRISPR proteins for their own purposes. The genetic and biochemical insights that have followed the discovery of CRISPR have led to Cas9, the game-changing genome engineering tool. Yet, knowledge gaps remain about CRISPR’s underlying adaptation mechanism. The Anti-CRISPR project, undertaken with the support of the Marie Skłodowska-Curie Actions and hosted by the Bionanoscience Department of TU Delft, set out to discover precisely how bacteria form these virus ‘logbooks’. Their discoveries could help develop DNA recording techniques. “This arms race between host bacterial CRISPR systems and anti-CRISPR viral genes is evolution in action. Being able to record this could lead to more targeted treatment, including for infections such as COVID-19 and flu,” explains Sungchul Kim, lead researcher. The work has already been featured in ‘Nature’.

Watching the host-virus arms race

The molecular process of CRISPR adaptive immunity consists of three main steps. The first is adaptation, known as spacer acquisition, where bacteria store viral information in a CRISPR array in what can be thought of as a log. The second and third stages are expression and interference, which produce CRISPR-Cas proteins to destroy the virus. The team set out to first investigate how the cooperating CRISPR proteins, Cas1-Cas2, select suitable viral DNA fragments, so-called pre-spacers, to discriminate between self and non-self. Secondly, they explored how pre-spacers are then trimmed into precise lengths to be integrated into the CRISPR array. Interactions between the Cas1-Cas2 proteins and viral DNA fragments were visualised with a real-time imaging technique – single-molecule fluorescence at nanoscale resolution. The team found that the C-terminal tail of Cas1-Cas2 is critical for self and non-self discrimination. They also discovered that DNA polymerase III was the enzyme responsible for pre-spacer trimming. This enzyme is also known for its role in removing errors as part of DNA replication. “We didn’t expect a DNA synthesising enzyme to be involved in defence. This implies that many biological machineries have evolved for multiple processes,” says Kim. Additionally, the team revealed that the binding of the Cas1-Cas2 C-terminal tail to the virus DNA signature, known as a protospacer adjacent motif, allows the bacteria to store viral DNA fragments in their CRISPR logbook. “Keeping this updated is crucial against viruses that have mutated their DNA in order to escape immunity,” adds Kim.

DNA recording devices

The project’s insights will be valuable for the development of new CRISPR-based DNA recording techniques for live cells. “Temporal DNA recording systems in mammalian cells, including human, have proven unsuccessful. Our findings help outline how to design DNA sequences and architectures to build a system in which information about cellular processes can automatically be stored in that cell’s DNA,” explains Chirlmin Joo, principle investigator. As the Cas1 and Cas2 proteins store information in chronological order, diseases or infections could then be reverse-engineered. For example, by taking some respiratory epithelial cells, a doctor could consult the cells’ ‘biological logbook’ to determine how tissue infected by, for example COVID-19 or flu, has developed. They can then use this diagnostic data for tailor-made treatment plans.

Keywords

Anti-CRISPR, genome, virus, bacteria, protein, Cas9, Cas1, Cas2, DNA, immune, personalised medicine, COVID-19, flu, infection

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