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New anti-CRISPR proteins discovered in soil and human intestine



CRISPR-associated protein Cas9 (white) from Staphylococcus aureus based on Protein Database ID 5AXW. Credit: Thomas Splettstoesser (Wikipedia, CC BY-SA 4.0)

Researchers from the Novo Nordisk Foundation's Center for Biosustainability (DTU) have found four new anti-CRISPR proteins that are distributed in different environments. The new study published in Cell Host & Microbe suggests that some anti-CRISPR proteins are more prevalent in nature than previously expected. These anti-CRISPRs can potentially be used to better regulate CRISPR-Cas9 system activity in the future.


CRISPR systems are bacterial immune systems that allow the bacterium to fight infectious viruses (phages) in a targeted manner.

Due to their programmable nature, CRISPR systems, and especially Cas9, is currently widely used in the life science industry with the potential to provide breakthrough generations, new antibiotics and malaria preparations.

Subjects have developed interest in developing anti-CRISPR proteins to overcome bacterial CRISPR systems in the evolutionary armament between viruses and bacteria. These proteins rapidly inhibit the host bacterial defense system, leaving the bacterium vulnerable to infection.

Despite their significant biological significance, only a few anti-CRISPR proteins have been found so far in a very specific subset of bacteria. Current anti-CRISPR proteins are not abundant in nature and have been identified by studying DNA & apos; s one of the subjects capable of infecting bacteria containing CRISPR-Cas9. By this method, one depends on being able to grow bacteria and on phage capable of infecting and avoiding monitoring of the endogenous CRISPR Cas9 system.

"We used a different approach that focused on anti-CRISPR functional activity rather than DNA sequence similarity. This approach allowed us to find anti-CRISPRs in bacteria that may not necessarily be cultured or infected with subjects. really exciting, ”says Ruben Vazquez Uribe, Postdoc at the Novo Nordisk Foundation's Center for Biosustainability (DTU).

Fecal samples contained anti-CRISPRs

The researchers identified the anti-CRISPR genes using the assembled DNA from four human fecal samples, two soil samples, a coke sample, and a swine calf sample. The DNA was cut into smaller pieces and randomly expressed on a plasmid in a bacterial cell. This cell contained a genetic circuit for selecting anti-CRISPR activity. In short, this meant that cells containing a plasmid with a potential anti-CRISPR gene would become resistant to a particular antibiotic. On the contrary, cells where the plasmid did not give anti-CRISPR activity would die.

With this system, the researchers could easily detect and select DNA with anti-CRISPR activity and trace it back to its origin. Using this metagenomic library approach, the researchers could identify eleven DNA fragments that bypassed Cas9 activity.

Further characterization could then confirm the activity of four new anti-CRISPRs. Phylogenetic analysis showed that the genes identified in the fecal samples are present in bacteria found in several environments, e.g. In bacteria that live in insects ̵

1; intestine, seawater and food.

This shows that the newly discovered genes are spread in many bacterial branches of the tree of life and, in some cases, with evidence that some of these genes have been transmitted horizontally several times during evolution.

"The fact that the anti-CRISPR's we discovered is so abundant suggests that they are very useful and of great importance from a biological perspective," says Morten Sommer, Scientific Director and Professor at the Novo Nordisk Fund for Biosustainability.

These results suggest that anti-CRISPRs are likely to play a much greater role in phage and host interaction than previously suggested.

Useful switch for by editing

Previous studies in this field have shown that anti-CRISPR proteins can be used to reduce errors, such as cutting DNA at off-target sites, when performing genome research in the laboratory.

"Today, most researchers using CRISPR-Cas9 have problems controlling the system and off-target activity. Therefore, anti-CRISPR systems are very important because you will be able to turn on and off The system for testing the activity, therefore, these new proteins can be very useful, "says Morten Sommer.

Indeed, the researchers discovered that the four new anti-CRISPR proteins appear to have different properties and properties. Looking ahead, it will be very exciting to investigate further.


Explore further:
An anti-CRISPR for gene editing

More information:
Ruben V. Uribe et al., Discovery and Characterization of Cas9 Inhibitors Disseminated Over Seven Bacterial Phyla, Cell Host & Microbe (2019). DOI: 10,1016 / j.chom.2019.01.003

Journal reference:
Cell Host & Microbe

Provided by:
Technical University Denmark


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