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Vizgen Cofounder Adapts MERFISH Method for Spatial Transcriptomics in Bacteria

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NEW YORK – Researchers from Boston Children's Hospital have developed a method that unlocks the use of high-resolution spatial transcriptomics in bacterial samples.

In a paper published last month in Science, researchers led by Jeffrey Moffitt, a researcher at Boston Children's and an assistant professor at Harvard Medical School, described their use of multiplexed, error-robust fluorescence in situ hybridization (MERFISH) along with expansion microscopy to offer the resolution needed to capture RNA molecules in bacteria, where cell sizes are tiny and RNA density is high. Moffitt is a cofounder of Vizgen, a Harvard University spinout that is commercializing MERFISH with the Merscope platform.

In proof-of-concept studies, he and his colleagues showed they could expand the volume of Escherichia coli cells by a factor of 1,000 and obtain information on hundreds of operons — clusters of genes that are expressed together in bacteria.

"They got to about 80 percent of the whole compendium of genes this organism can express," said Anna Kuchina, an expert on bacterial transcriptomics at the Institute for Systems Biology. "The most amazing thing to me is that this is done retaining the spatial information."

While bacterial behavior at the single-cell level has mostly been studied in the test tube, "now you'd be able to grow cells in a more natural environment or even take them from their natural environment," she said.

The authors also demonstrated being able to determine where in a bacterial cell operons are localized, as well as showing RNA expression of both bacterial and host cells in cases of both infection and coexistence.

Even without the aid of single-cell sequencing technologies, bacteriologists had "made it very clear that you can have a tremendous amount of heterogeneity in bacterial responses, even in uniform growth conditions in a test tube," Moffitt said. "Understanding that type of single-cell heterogeneity is quite important, both to understand gene regulation and basic homeostasis in bacteria, but also in a wide variety of clinical applications."

For example, small populations of bacteria can spontaneously enter into antibiotic resistant states and be a major source of recurrent infection after antibiotic treatment.

Moffitt has been working on bacterial MERFISH since he started his own lab in late 2018, wanting to focus on commensal microbial communities. Prior to that, he had helped develop MERFISH while working as a postdoc in Xiaowei Zhuang's lab at Harvard. "It was very clear from back-of-the-envelope calculations that the density of RNAs in bacteria was going to be orders of magnitude higher than in eukaryotic systems, and so the kind of single-molecule imaging that underlies MERFISH was just not going to be possible unless we came up with ways to address that," he said.

Expansion microscopy, which includes methods such as swelling samples with a hydrogel to preserve their spatial organization, was the answer. The lab has built its own protocols "heavily inspired" by other labs that have worked on this technology, including the group of Ed Boyden at MIT, who has formed a spinout called Expansion Technologies.

Once a sample is expanded, the approach uses the same MERFISH protocols that can be run for eukaryotic samples, with "a few minor optimizations," Moffitt said, noting that he has not applied for additional IP around these modifications.

In addition to basic work showing bacterial MERFISH works in E. coli, the authors applied it to look at a single bacterial species in the mouse gut. "We show that this single species will fine-tune its gene expression over microns, so remarkably short distances, in the spatially complex and structured environment of the mouse colon," Moffitt said. "And it's very easy to imagine being able to do those measurements in so many other contexts."

Single-cell methods can provide information on gene expression in host cells that have been invaded by bacteria, but they're "blind to differences within different bacteria within the same human cell," Moffitt said. "The advantage these image-based methods have is that we can really look within an infected cell. We could see individual bacteria. We could profile what those bacteria are doing, if there are differences between different bacteria in the same cell. And because we're targeted, we don't have to worry about this massive imbalance between the eukaryotic mRNA and the bacteria RNA."

Environmental samples are another possible application for bacterial MERFISH. Kuchina noted, for example, that biofilms could be an attractive target for this method.

Though she called the new method a "milestone" in bacterial transcriptomics, she is likely to stick with single-cell sequencing-based methods she has developed, including microSPLiT, a split-pool barcoding approach she published in 2020 in collaboration with two cofounders of Parse Biosciences.

Implementing both expansion microscopy and MERFISH are a tall order for her young lab. "The learning curve is pretty high," she said, adding that probe-based methods like MERFISH can only measure what one expects to be there. "For many organisms, nucleic acids are less well known," she said, and can only be discovered using untargeted approaches.

To gain wider acceptance, bacterial MERFISH will need to expand beyond the two species in the proof-of-concept study. Each species technically needs its own probe set, though similar species may be able to share probes. According to Moffitt, this will not be a big impediment, though. "Probe design is trivial and we developed probe synthesis methods that leverage complex oligo pools," he said, so probes to any species can be made for relatively little cost.

The bigger challenge is expanding to different types of bacteria that may require novel approaches to digesting their cell wall. "There's enough really solid evidence out there, both for Gram-negative and Gram-positive bacteria, as well as many specific bacterial examples, that cell wall digestion is going to be relatively straightforward with some optimization from bacteria to bacteria," he said. "It's just a matter of identifying these protocols, testing them, and validating the quality of the measurements that we get."