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Developmental Biology - CRISPR

New Anti-CRISPR Proteins Found

Seven new anti-CRISPR proteins are discovered in soil and the human gut...


Scientists from the Novo Nordisk Foundation Center for Biosustainability (DTU) have found four new anti-CRISPR proteins distributed across different natural environments. Their new study published in Cell Host & Microbe suggests anti-CRISPR proteins are more widespread in nature than expected, and may potentially be useful in regulating activity in CRISPR-Cas9 systems.
CRISPR systems are bacterial immune systems that enable bacteria to fight off infecting viruses (phages).

Due to their programmable nature, CRISPR systems and in particular Cas9 are widely used in the life science industry for their potential to deliver breakthrough gene and malaria therapies, and perhaps new antibiotics.

Interestingly, phages evolved anti-CRISPR proteins to overcome bacterial CRISPR systems in the evolutionary arms race between viruses and bacteria. These anti-CRISPR proteins quickly inhibit a host bacteria's defences leaving it vulnerable to infection.

In spite of their significant biological importance, only a few anti-CRISPR proteins have been discovered in a very specific subset of bacteria. Anti-CRISPR proteins are not abundant in nature. When identified, its been through the study of DNA in phages that harbor bacteria infected with CRISPR-Cas9. According to Ruben Vazquez Uribe, Postdoc at the Novo Nordisk Foundation Center for Biosustainability (DTU): "We focused on anti-CRISPR functional activity rather than DNA sequence similarity. This enabled us to find anti-CRISPRs in bacteria that can't necessarily be cultured or infected with phages. These results are very exciting."

Researchers identified anti-CRISPR genes by using the total DNA from four human fecal samples, two soil samples, one cow fecal sample and one pig fecal sample.

The DNA was chopped into smaller pieces and randomly expressed on a plasmid within a bacterial cell. This cell now contained a gene circuit for selecting anti-CRISPR activity. This means cells containing a plasmid with a potential anti-CRISPR gene can become resistant to a specific antibiotic. Cells in which the plasmid did not confer anti-CRISPR-activity die. With this system, researchers could easily detect and select DNA with anti-CRISPR activity, tracing it back to its origin.
Using this metagenomic approach, scientists identified eleven DNA fragments that circumvent Cas9 activity.

Further characterisation confirmed activity of four new anti-CRISPRs. Analysis revealed genes identified in fecal samples are present in bacteria found in multiple environments: bacteria living in insects' gut, seawater and food. This shows these newly discovered genes are in many bacterial branches throughout the tree of life, and in some cases, evidence that some of these genes were horizontally transferred numerous times during evolution.
"The fact that the anti-CRISPRs we discovered are so abundant in nature suggests they are very useful and have significance from a biological perspective."

Morten Sommer PhD, Scientific Director, Professor, Novo Nordisk Foundation Center for Bio-sustainability (DTU).

Earlier studies in this field demonstrated that anti-CRISPR proteins can be used to reduce errors, such as cutting DNA at off-target sites, when doing genome editing in the laboratory. Morten Sommer explains: "Today, most researchers using CRISPR-Cas9 have difficulties controlling the system and its off-target activity. Anti-CRISPR systems are very important, as you want to be able to turn your system on and off. Therefore, these new proteins could become very useful."
These findings suggest anti-CRISPRs could be playing a much bigger role in the interplay between phage and host than previously suggested.

Highlights
A genetic circuit designed to functionally select anti-CRISPR genes from metagenomes
Four protein families, AcrIIA710, that inhibit Cas9 in vivo and in vitro were identified
AcrIIA710 are widely distributed across seven bacterial phyla
Dissemination patterns of AcrIIA710 suggest interphylum horizontal gene transfer

Summary
CRISPR-Cas systems in bacteria and archaea provide immunity against bacteriophages and plasmids. To overcome CRISPR immunity, phages have acquired anti-CRISPR genes that reduce CRISPR-Cas activity. Using a synthetic genetic circuit, we developed a high-throughput approach to discover anti-CRISPR genes from metagenomic libraries based on their functional activity rather than sequence homology or genetic context. We identified 11 DNA fragments from soil, animal, and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain. Further in vivo and in vitro characterization of a subset of these hits validated the activity of four anti-CRISPRs. Notably, homologs of some of these anti-CRISPRs were detected in seven different phyla, namely Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Spirochaetes, and Balneolaeota, and have high sequence identity suggesting recent horizontal gene transfer. Thus, anti-CRISPRs against type II-A CRISPR-Cas systems are widely distributed across bacterial phyla, suggesting a more complex ecological role than previously appreciated.

Authors
Ruben V. Uribe, Eric van der Helm, Maria-Anna Misiakou, Sang-Woo Lee, Stefan Kol and Morten O.A. Sommer.

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Feb 19, 2018   Fetal Timeline   Maternal Timeline   News  




Some anti-CRISPRs were detected in seven different phyla: Firmicutes, Proteobacteria,
Bacteroidetes, Actinobacteria, Cyanobacteria, Spirochaetes, and Balneolaeota,
with a high sequence identity suggesting recent horizontal gene transfer.


Phospholid by Wikipedia