These engineered viruses are delivering DNA to E.coli instead of killing it- here's why

In an ironic twist, researchers used viruses engineered with the CRISPR-Cas system to alter bacterial defense mechanisms and edit their genomes selectively in complex environments. Significantly, the novel approach may help address the pressing issue of antibiotic resistance.

The CRISPR Conundrum

CRISPR is a gene-editing tool that allows scientists to make precise cut-and-paste edits to the genomes of living cells. In nature, the CRISPR system evolved as a bacterial defense mechanism against viruses. Upon encountering a viral infection, the bacterium uses this machinery to chop up the invading viral DNA. This fragment is stored as genetic memory to detect and combat future infections.

In the new study, North Carolina State University researchers turned the tables. They engineered two bacteria-eating viruses, called bacteriophages (or phages), to deliver CRISPR-Cas payloads for targeted gene editing of E coli bacteria.

'The virus, in this case, targets E.coli by delivering DNA to it'

"Viruses are very good at delivering payloads. Here, we use a bacterial virus, a bacteriophage, to deliver CRISPR to bacteria, which is ironic because bacteria normally use CRISPR to kill viruses. The virus, in this case, targets E. coli by delivering DNA to it. It's like using a virus as a syringe," said Professor Rodolphe Barrangou, the study's corresponding author.

In a lab test, the engineered bacteriophages, called T7 and lambda, were tasked to deliver fluorescent genes to E. coli and alter their resistance to an antibiotic. And sure enough, the bacteria glowed and showed altered antibiotic resistance.

Next, researchers used lambda phage to deliver a cytosine base editor to the bacterial host. This editor tool doesn't cut the target's DNA as done by CRISPR but changes just one letter in the sequence of DNA. By strategically introducing point mutation, researchers showed the sensitivity and precision of the system. These changes inactivated specific bacterial genes without making other changes to E. coli.

Going beyond common obstacles associated with conventional CRISPR-Cas targeting

"We used a base editor here as a kind of programmable on-off switch for genes in E. coli. Using a system like this, we can make highly precise single-letter changes to the genome without the double-strand DNA breakage commonly associated with CRISPR-Cas targeting," said Matthew Nethery, lead author of the study.

In the final test, researchers tested the CRISPR-Cas system in a simulated natural environment. They used a fabricated ecosystem (EcoFAB) by loading a tank with synthetic soil made of sand and quartz, some liquid, and three different types of bacteria, including E. coli.

The goal was to test how well the phages could hunt down their targets in a more realistic environment and whether they could single out the E. coli from the other species.

When lambda phage was introduced to the fabricated ecosystem, it showed decent efficiency in finding E. coli and making the targeted genetic changes. The team reported the editing efficiencies from 10% to 28% across the bacterial population.

The next chapter in CRISPR

"This is a proof of concept that could be employed in any complex microbial community, which could translate into better plant health and better gastrointestinal tract health—environments of importance to food and health," said Professor Barrangou.

Researchers believe the study shows the next chapter of CRISPR-Cas delivery in complex environments. With further work, the technique could eventually find use in sustainable agriculture by manipulating large-scale gene editing in soil bacteria.

The study is published in the Proceedings of the National Academy of Sciences.

Abstract:

Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal (lacZ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.