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Microbiome Editing with CRISPR/Cas9 Has Research, Diagnostic Potential


NEW YORK (GenomeWeb) – Two of the bacterial kingdom's greatest assets, the CRISPR "immune" system and horizontal gene transfer of plasmids by conjugation, could be exploited by humans to wipe out specific communities of bacteria or even turn them into useful reporters.

CRISPR/Cas9 provides a way to kill off bacterial populations according to the genetic sequences in their genomes, according to a paper published in Nature Biotechnology in September. The authors showed that they could selectively eliminate populations of bacteria harboring antibiotic resistance genes by targeting them with Cas9 nucleases informed by guide RNAs that corresponded to those genes. They did so by creating plasmids packing the Cas9 enzyme and relevant guide RNAs.

"We can selectively cleave DNA in bacteria and by doing that, we can remove plasmids [carrying antibiotic resistance genes] or kill the bacteria," Timothy Lu, a senior author of the study and a professor of synthetic biology at the Massachusetts Institute of Technology, told GenomeWeb. Simply cleaving DNA can in many cases be enough to kill the bacterium, he added, and in bacteria that didn't have those genes, the plasmids could remain and could be passed on to other bacteria through conjugation, a method of direct plasmid exchange between organisms.

It's a sneaky way to attack bacteria. "This is how they pass antibiotic resistance already. They trade these genes with their brethren. Now we can take advantage of this," Lu said. "Why not use the same machinery to spread good stuff?"

While the most obvious application might be in fighting antibiotic-resistant infections in a search-and-destroy fashion, Lu sees plenty of other ways to employ this idea.

For years, Lu has been working on using synthetic biology to manipulate complex microbial communities. His lab started with transcription activator-like effector nucleases and zinc finger nucleases, but just about dropped everything and started using CRISPR when he recognized it as a viable genome editing technology.

One of CRISPR's biggest advantages is its specificity. Antibiotics, which are broad-spectrum and go after conserved proteins or mechanisms in many bacteria, can't discriminate between good and bad bacteria, Lu said, but CRISPR/Cas9 can. Guide RNAs can help these nucleases home in on a specific piece of DNA, which becomes a mark for whether the bacteria will be killed or not.

That selectivity could enable some interesting research on microbial communities and diagnostic applications for human health both directly and indirectly tied to those communities. 

Human gut microbiome studies are showing that microbial populations can affect human health, even influencing mood and diet. However, currently there aren't ways to precisely manipulate it to see what happens when one species disappears. "Our ways to manipulate the microbiome are coarse," Lu said, referring to fecal transplants and antibiotics. "So what we think we need is something like RNAi, where can you do bacterial knockdowns in a targeted way." Because Lu's plasmids could target species-specific segments of DNA, he could have CRISPR determine what lives and what dies.

While Lu's CRISPR plasmids are most efficiently delivered via phages, he has also engineered benign bacteria to carry them and distribute them in the microbiome via conjugation.

Another targeted genome editing system that the Lu lab pioneered, called SCRIBE, has proved useful to different facet of Lu's research, turning bacteria into biological computing devices. In a paper published in November in Science, his lab showed that living cell genomes can be engineered to have the ability to compute and store information using gene circuits, as opposed to circuits of silicon-based transistors. "There are all sorts of microbial sensors you can build that will sense the environment and then remember that information," Lu said. Though SCRIBE doesn't use CRISPR/Cas9, Lu said CRISPR/Cas9 could also be used to build similar gene circuits.

He envisions using synthetic gene circuits in benign bacteria to create non-invasive diagnostics that could detect inflammation, bleeding, infections, and metabolite and nutrient levels in the human gut. He says it's not too far-fetched to imagine eating a yogurt packing engineered lactobacilli that could detect early signs of colon cancer. "That's very far out," Lu conceded, "but the basic technologies are there."

Lu is already taking advantage of applications for the technologies developed in his lab. He's formed a company called Sample6 with fellow MIT scientist James Collins that's starting out in the food pathogen detection market. In October 2013 the company raised $11 million in a Series B financing round.

Sample6's diagnostics rely on phages to detect major food pathogens, including ListeriaEscherichia coli, and Salmonella. What CRISPR could add is the ability to detect antibiotic resistance, which would be an important step towards offering a clinical diagnostic for infectious pathogens.

A CRISPR/Cas9 system that targets the DNA sequences of antibiotic resistance genes, like the one Lu's lab has already designed, could be linked to a fluorescent reporter or some other detectable output. One way to do this would be to exploit the SOS response in the cell, a pathway that detects DNA damage in bacteria. One specific marker in that pathway is LexA, a transcription factor. The diagnostic circuit could be engineered to produce a fluorescent protein via a promoter that responds only to LexA. If the antibiotic resistance gene is present in target cells, the engineered CRISPR/Cas9 system would cleave it, leading to the production of the fluorescent reporter protein.

However, since it is in its infancy, Sample6 has to be selective about the markets it operates in, Lu said. Clinical diagnostics would require a lot more money and a lot more time, but could be in the firm's future.

"It's not too hard to think about expanding this to other pathogens people care about," Lu said.