Engineered CRISPR plasmid spreads through bacteria to erase antibiotic resistance — with an added off switch

In a laboratory at the University of California San Diego, a handful of engineered bacteria did what many of the world’s strongest drugs can no longer do: they spread through a dense bacterial community and stripped away the genes that made their neighbors resistant to antibiotics.

The experiment, described this month in npj Antimicrobials and Resistance, showcases a self-propagating CRISPR system that moves from bacterium to bacterium on a mobile DNA element and erases a key resistance gene as it goes. In controlled tests, the tool cut drug-resistant cells by orders of magnitude and offers a glimpse of a future in which doctors and engineers do not just slow the rise of “superbugs,” but try to reverse it.

A gene-drive-like approach for bacteria

Researchers call the system “gene-drive-like,” borrowing a concept first developed in mosquitoes and other insects. Instead of biasing inheritance in an animal’s offspring, this platform uses a conjugative plasmid—a circular piece of DNA that bacteria naturally pass to one another—to drive a CRISPR payload through a population.

“With pPro-MobV we have brought gene-drive thinking from insects to bacteria as a population engineering tool,” said senior author Ethan Bier, a professor of cell and developmental biology at UC San Diego and science director of the Tata Institute for Genetics and Society, in a university statement.

The work targets one of medicine’s most pressing threats. Antibiotic-resistant infections are estimated to directly cause about 1.27 million deaths worldwide each year, with projections rising to more than 10 million annual deaths by 2050 if current trends continue, according to widely cited global health analyses. Many of the genes that shield bacteria from antibiotics ride on plasmids that shuttle between species in hospitals, farms and sewage systems.

Targeting a common resistance gene

The new study focuses on a single, well-known resistance factor: a beta-lactamase gene called bla that allows Escherichia coli to withstand the drug ampicillin.

The researchers built a roughly 65-kilobase plasmid, named pPro-MobV, that combines three capabilities:

• Broad host replication, allowing it to copy itself across a range of bacterial hosts
• Conjugative transfer, enabling it to move cell-to-cell
• A CRISPR-Cas9 “active genetics” cassette programmed to home in on bla

In their experiments, donor E. coli cells carrying pPro-MobV were mixed with recipient strains harboring a separate plasmid that encoded bla and other markers. Over 72 hours in liquid culture at 30°C, the engineered plasmid successfully transferred to about 40% of the recipients, the team reported.

When the scientists added arabinose, a sugar that switches on the plasmid’s CRISPR machinery, the effect was stark. Guided by a bespoke RNA sequence, the Cas9 enzyme cut the bla gene on the target plasmid. Because the CRISPR cassette itself was flanked by DNA segments matching bla, the cell’s repair systems often used those homology arms to insert the cassette into the cut site, disrupting the resistance gene. In other cases, repair deleted most of bla altogether.

Across different tests, activating the system reduced the number of ampicillin-resistant bacteria by about 1,000-fold compared with uninduced controls.

Earlier versions of the same “Pro-Active Genetics,” or Pro-AG, system achieved up to 100,000-fold reductions in similar setups, and the new conjugative design was about 10 times more efficient than non-mobile plasmids carrying the same cassette, the authors found.

“With this new CRISPR-based technology we can take a few cells and let them go to neutralize [antibiotic resistance] in a large target population,” Bier said.

Taking the fight into biofilms

The team also pushed the system into a more realistic setting: biofilms. These slimy, surface-attached bacterial communities help shield microbes from immune attacks and drugs and are implicated in chronic lung infections, urinary tract infections, contaminated hospital plumbing and industrial pipes.

“The biofilm context for combating antibiotic resistance is particularly important since this is one of the most challenging forms of bacterial growth to overcome in the clinic or in enclosed environments such as aquafarm ponds and sewage treatment plants,” Bier said. “If you could reduce the spread from animals to humans you could have a significant impact on the antibiotic resistance problem since roughly half of it is estimated to come from the environment.”

The study’s other senior author, evolutionary biologist Justin R. Meyer of UC San Diego, emphasized how different the strategy is from existing tools that merely slow resistance.

“This technology is one of the few ways that I’m aware of that can actively reverse the spread of antibiotic-resistant genes, rather than just slowing or coping with their spread,” Meyer said.

A built-in reversal mechanism

Scientists have previously used CRISPR-Cas9 to cut and sometimes eliminate resistance plasmids from bacteria, and to block incoming plasmids from establishing themselves. Other groups have used CRISPR interference—enzymes that bind DNA without cutting it—to temporarily shut down resistance genes. Most of those systems, however, rely on direct delivery methods such as transformation or on non-self-propagating plasmids.

One of the most unusual aspects of the UC San Diego work is its attention to safeguards. During the experiments, the team observed that when Cas9 cut within a region flanked by short, repeated DNA sequences, the cell often repaired the break by recombining the repeats and precisely deleting the intervening stretch.

The researchers named this process homology-based deletion (HBD) and realized it could be harnessed as an “off switch.”

By designing resistance genes and inserted cassettes with appropriate repeats, they showed that a separate engineered plasmid—or even a modified bacteriophage λ virus—could deliver Cas9 and a guide RNA that triggers HBD. In lab tests, this approach cleanly excised a 1.2-kilobase insert from the target plasmid, restoring the original gene sequence in nearly all examined colonies.

The authors say this kind of reversibility could, in principle, be used as a mitigation tool if a Pro-AG element spreads farther than intended. But they acknowledge that moving from controlled flasks of E. coli to real-world settings will require not only more biology, but also policy decisions.

Ethical, regulatory and technical hurdles ahead

Any attempt to release a self-spreading genetic construct into open environments—such as sewage treatment plants, fish farms or livestock operations—would trigger national biosafety reviews and likely draw scrutiny under international biodiversity agreements. Debates over insect gene drives at United Nations meetings have already highlighted questions about cross-border spread, community consent and the difficulty of recalling a construct once it is loose.

The current pPro-MobV plasmid is also not designed for direct deployment. For laboratory tracking, it carries its own antibiotic resistance markers—chloramphenicol and gentamicin—alongside the Cas9 machinery. Future versions built for clinical or environmental use would need to be stripped of such markers, narrowed in host range and equipped with additional containment features, researchers and regulators say.

There are scientific hurdles as well. The new study targets a single resistance gene in one species of E. coli, while real-world infections and waste streams contain diverse bacteria carrying multiple resistance determinants, often on different plasmids or chromosomes. Bacteria also evolve quickly: under strong selection to avoid being edited, they could mutate CRISPR target sites, acquire anti-CRISPR proteins or change the way they accept foreign plasmids.

A shift in the anti-superbug toolkit

Even with those caveats, the work highlights a broader shift in how scientists are thinking about antibiotic resistance. Instead of relying solely on new chemical compounds, laboratories are increasingly exploring genetic and ecological interventions: bacteriophages that prey on bacteria, engineered probiotics that block pathogens from colonizing the gut, and CRISPR systems that alter or erase resistance genes.

For now, the latest results remain a proof of concept. But by showing that a self-amplifying CRISPR cassette can hitch a ride on a naturally mobile plasmid, spread through biofilms and carry an experimentally validated “undo” button, the study places a new kind of technology on the table in the long fight against resistant microbes.

If antibiotics were the blunt instruments of the last century, tools like pPro-MobV hint at a next phase in which the battle moves inside the genome of the bacteria themselves—a strategy that may demand as much attention from lawmakers and ethicists as it does from microbiologists.