The acronym CRISPR has become synonymous with DNA editing in recent years, taking center stage in a molecular geneticist's toolkit as a way to pinpoint genetic code and then precisely chop it up.
In its original function as an immunogen in bacteria, the CRISPR/Cas system searches for known genes of invading viruses and renders them inactivated.
And scientists from the University of Rochester and Cornell University in Ithaca in the US have discovered that the popular gene-editing tool does more in bacteria than just snipping DNA; It also coordinates with other proteins to assemble defenses against invading viruses.
Activation of funnel-shaped proteins called Csx28 disrupts the permeability of the bacteria's membrane, making it difficult for viral DNA to hijack the cell machinery and replicate. The finding, published in Science, "is unexpected and raises all kinds of new questions," says University of Rochester biochemist Marc Dumont, a collaborator on the study.
"Although there is no immediate medical relevance or application, the insights that emerge from this can be very powerful," says Dumont.
The study involved a series of experiments in which Escherichia coli was infected with a virus that infects the bacterium, or phage, called enterobacteria phage λ.
This phage attaches to the surface of the bacteria cell and injects its DNA into the cell to make copies of itself.
coli, using CRISPR technology to identify the threat by matching repetitive DNA sections from previously encountered phages, then using an enzyme called Cas13b to cut the invading DNA into pieces.
The researchers found that the virus replicated slowly when Csx28 was present inside the bacteria.
This protein only works with Cas13b, suggesting that the two were coordinating with each other to disarm the virus.
When both Cas13b and Csx28 were present, the proportion of infected bacteria that released infectious viral particles decreased from about 19% to approximately 3%, and there was a significant decrease in the numbers of phages per milliliter. In other words, the virus was not able to replicate as much as it normally would.
The researchers examined the structure of the Csx28 protein using a technique called cryoelectron microscopy and found that it looked like a funnel with a hole in the center.
This increased the possibility that the protein formed a membrane pore and disrupted the cell's metabolism to make it an unfavorable environment for the virus.
The researchers tested this hypothesis using a technique that causes cells to fluoresce after they lose their membrane potential, which is a small electrical charge caused by the difference in the concentration of ions inside and outside the cell.
They found that the two proteins together caused a depolarization in the membrane, resulting in a rush of charged atoms that radically changed the cell's internal environment. After 90 minutes, the depolarization removed 40% of the bacteria with this method.
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