The rapid rise of antibiotic resistance represents a major global public health threat that has been estimated to endanger millions of lives and cost over $2 billion each year in the U.S. alone. For far too long, researchers have lacked the essential tools to connect the dots between pathogenic genes and phenotypes. Yet now, a team of investigators from the University of Wisconsin-Madison and the University of California, San Francisco may have just turned a corner with their new findings. The researchers developed a new CRISPR tool to study which genes are targeted by particular antibiotics, providing clues on how to improve existing drug compounds or develop new ones. Data from the new study was published recently in Nature Microbiology through an article entitled “Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.”

“What we need to do is to figure out new weaknesses in these bacteria,” noted lead study investigator Jason Peters, PhD a UW-Madison assistant professor of pharmaceutical sciences, who developed the new system.

The new technique, known as Mobile-CRISPRi, harnesses the power of CRISPR interference—which blocks gene expression through the use of a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs—allowing scientists to screen for antibiotic function in a wide range of pathogenic bacteria.

The research team employed bacterial conjugation—the transfer of genetic material between bacterial cells—to allocate Mobile-CRISPRi from common laboratory strains into diverse bacteria, even including a little-studied microbe making its home on cheese rinds. This ease of transfer makes the technique a boon for scientists studying any number of bacteria that cause disease or promote health. Moreover, the system reduces the production of protein from targeted genes, allowing researchers to identify how antibiotics inhibit the growth of pathogens. That knowledge can help direct research to overcome resistance to existing drugs.

“Most people, when they think about CRISPR, think about gene editing,” said Peters. “But that’s not what I do.”

Normally, the CRISPR system gets targeted to a gene where it cuts the DNA in two. The gene can be edited while the cell repairs the damage.

But Peters and his collaborators worked with a defanged form of CRISPR known as CRISPRi. CRISPRi has been engineered to be unable to cut DNA. Instead, it just sits on the DNA, blocking other proteins from gaining access to and turning on a particular gene. The result is a lower expression of the gene and a reduced amount of the protein it codes for.

In the current study, the researchers showed that if they decreased the amount of protein targeted by an antibiotic, bacteria became much more sensitive to lower levels of the drug—evidence of an association between gene and drug. Thousands of genes at a time can be screened as potential antibiotic targets this way, helping scientists learn how antibiotics work and how to improve them.

To make CRISPRi mobile, the researchers developed methods to transfer the system from common lab models like E. coli to disease-causing species, which are often harder to study. Peters’ team turned to one of the natural ways bacteria link up and exchange DNA, a kind of bacterial sex called conjugation.

“You basically mix the bacteria together and it happens,” Peters commented. “It doesn’t get much easier than that.”

Using conjugation, Peters’ team transferred Mobile-CRISPRi to the pathogens Pseudomonas, Salmonella, Staphylococcus, and Listeria, among others.

“What that means is that you can now do studies on how antibiotics work directly in these pathogens,” Peters concluded. “That could give us a better clue about how these drugs work in the different organisms and potentially what we can do to make them better.”

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