Closing system

Bacteria scrambled by the CRISPR system

Call it a CRISPR conundrum.

Bacteria use CRISPR-Cas systems as adaptive immune systems to resist attacks from enemies like viruses. These systems have been adapted by scientists to delete or cut and replace specific genetic code sequences in a variety of organisms.

But in a new study, researchers at North Carolina State University show that viruses engineered with a CRISPR-Cas system can thwart bacterial defenses and make selective changes to a targeted bacterium, even when other bacteria are nearby.

“Viruses are very good at delivering payloads. Here we are using a bacterial virus, a bacteriophage, to deliver CRISPR to bacteria, which is ironic because bacteria normally use CRISPR to kill viruses,” said Rodolphe Barrangou, Todd R. Klaenhammer Emeritus Professor of Food Science. , bioprocessing and nutrition at NC. Status and corresponding author of a paper describing the research published today in Proceedings of the National Academy of Sciences. “In this case, the virus targets E. coli by delivering DNA to it. It’s like using a virus as a syringe.

NC State researchers deployed two different modified bacteriophages to provide CRISPR-Cas payloads for targeted editing of E. coli, first in a test tube and then in a synthetic soil environment created to mimic soil – a complex environment that can harbor many different types of bacteria.

The two modified bacteriophages, called T7 and lambda, managed to find and then deliver payloads to the host E. coli on the lab bench. These payloads expressed fluorescent bacterial genes and manipulated the bacteria’s resistance to an antibiotic.

The researchers then used lambda to deliver a so-called cytosine base editor to the E. coli host. Rather than the sometimes harsh cleavage of CRISPR DNA sequences, this base editor only changed one letter of E. coli, showing the sensitivity and accuracy of the system. These modifications inactivated certain bacterial genes without making other modifications to E. coli.

“Here we used a base editor as a sort of programmable on-off switch for the genes of E. coli. Using a system like this, we can make very precise, single-letter changes to the genome without the double-stranded DNA break commonly associated with CRISPR-Cas targeting,” said Matthew Nethery, former PhD holder. Ph.D. from NC State. student and lead author of the study.

Finally, the researchers demonstrated in-situ editing through the use of a fabricated ecosystem (EcoFAB) loaded with a synthetic soil medium of sand and quartz, as well as liquid, to mimic a soil environment. The researchers also included three different types of bacteria to test if the phage could specifically locate E. coli in the system.

“In a lab, scientists can oversimplify things,” Barrangou said. “It’s better to model environments, so rather than soup in a test tube, we wanted to look at real environments.”

The researchers inserted lambda into the fabricated ecosystem. It has shown good efficiency in finding E. coli and making the targeted genetic modifications.

“This technology will allow our team and others to uncover the genetic basis of key bacterial interactions with plants and other microbes in highly controlled laboratory environments such as EcoFABs,” said Trent Northen, a scientist at the Department of Energy’s Lawrence Berkeley National Laboratory. (Berkeley Lab) which collaborates with Barrangou.

“We see this as a mechanism to help the microbiome. We can make a change to a particular bacteria and the rest of the microbiome remains unscathed,” Barrangou said. “This is a proof-of-concept that could be used in any complex microbial community, which could result in improved plant health and improved gastrointestinal tract health – important environments for human development. diet and health.

“Ultimately, this study represents the next chapter in CRISPR delivery – the use of viruses to deliver CRISPR machines in a complex environment.”

The researchers plan to continue this work by testing the CRISPR phage technique with other soil-associated bacteria. Importantly, this illustrates how soil microbial communities can be manipulated to control the composition and function of plant-associated bacteria in engineered ecosystems to understand how to enhance plant growth and promote plant health. which is of great interest for sustainable agriculture.

Reference: Nethery MA, Hidalgo-Cantabrana C, Roberts A, Barrangou R. CRISPR-based engineering of phages for in situ bacterial base editing. PNAS. 2022;119(46):e2206744119. doi:10.1073/pnas.2206744119

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