Next-level gene editing with DARTs


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Next-level gene editing with DARTs

CRISPR-Cas9 took the scientific world by storm and transformed genome editing into a precise, quick and cheap technology. The potential of this method seems almost limitless. Now, researchers from the laboratory of Jennifer Doudna, who together with Emmanuelle Charpentier was awarded the Nobel Prize in Chemistry in 2020 for the development of CRISPR-Cas9 technology for genome editing, have taken it to the next level: community editing. The research, published recently in Nature Microbiology, reports two new tools that enable genome editing of microbes within their natural communities.

The troubles of bacterial genome editing
Often, the goal of microbiome studies is to identify key bacteria in a microbial community that can be modified to prevent or resolve disease or to increase crop yield or crop resistance. This requires experimental tools to genetically manipulate isolated bacterial species in a lab environment. However, isolating and successfully growing a microbe in a lab can be quite challenging. In fact, many microbes that are relevant to human health have not yet been successfully grown outside of their native environments. And the challenges don’t end there. 

If successfully isolated, the next step would be to find the optimal means of editing the genome, which could take years or fail altogether. And even if all of this succeeds, chances are that the microbe, once released into its natural community, has adapted to the lab environment and may not behave in the way you engineered it.

The ideal solution to circumvent all these difficulties would be to edit bacterial genomes in their natural environment. And the research team around Jennifer Doudna provided the tools to do just that.


Identify target
Analogous to shooting a weapon, which requires taking aim first, the researchers started by selecting their bacterial targets within a microbial community. They developed ET-seq (environmental transformation sequencing) to determine which microbes in a community are actually susceptible to genome editing. ET-seq uses a transposon, which is a genetic element that can randomly insert into genomes. 

The researchers determined which species incorporated the transposon by sequencing the DNA of the entire microbial community before and after adding the transposon. Those species that had the highest number of insertions were the most susceptible to genome editing. Using ET-seq, the researchers were able to screen the entire microbial community without the need to isolate and test individual species.

Take aim
With the bacterial targets at hand, the researchers took aim. For their editing weapon, they developed a tool they quite fittingly named DART (DNA-editing all-in-one RNA-guided CRISPR–Cas transposase). Similar to the CRISPR-Cas9 system, DART uses a specific RNA sequence to guide the editing machinery to the targeted genomic region. Instead of cleaving the DNA like the Cas9 protein does, DART relies on a CRISPR-associated transposase, which inserts a barcoded transposon at the targeted region. 

The efficiency of these insertions can then be easily quantified using ET-seq. “The new microbial community editing approach leverages CRISPR technology and enables the genetic modification of a specific gene within a specific bacterium,” summarized Deutschbauer, one of the authors of the research paper.

DART (DNA-editing all-in-one RNA-guided CRISPR–Cas transposase) uses a specific guide RNA sequence to guide the CRISPR-Cas transposase to a specific genomic region, where the transposon will be inserted. Using ET-seq, the researchers can then quantify the editing efficiency and specificity.

To demonstrate the power of their tools, the researchers successfully modified individual E.coli strains of a microbial community from a stool sample of an infant. They specifically targeted genes that have been associated with disease.

Only a laboratory weapon – for now
This powerful weapon that allows for precisely editing genes in specific bacteria within microbial communities is still a lab weapon only. Circumventing laborious isolation or engineering of individual species in the lab certainly holds great promise for future applications. 

“Eventually, we may be able to eliminate genes that cause sickness in your gut bacteria or make plants more efficient by engineering their microbial partners,” said Brady Cress, one of the authors of the study. “But likely, before we do that, this approach will give us a better understanding of how microbes function within a community.” Because microbes do not live in isolation. Studying them in their natural setting is much more representative of how they live and function in nature.

Link to the original post: Rubin, B.E., Diamond, S., Cress, B.F. et al. Species- and site-specific genome editing in complex bacterial communities. Nat Microbiol 7, 34–47 (2022).

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