Precision Microbiome Editing To Tackle Methane Emissions

In 2006, two University of California (UC) Berkeley scientists – Professors Jillian Banfield and Jennifer Doudna – met for coffee at the Free Speech Café, located on campus. In the years since, their individual research contributions have shaped the fields of genomics and microbiology, transforming our understanding of how life functions on our planet.

As a collective, they’re now embarking on an adventurous $70 million project at the Innovative Genomics Institute (IGI), part of UC Berkeley. The collaborative project combines two cutting-edge technologies, developed in Doudna and Banfield’s respective labs, to address our world’s greatest challenges using its smallest inhabitants.

The history: A special connection

In the early 2000s, Banfield, a renowned earth scientist, was establishing new technologies at UC Berkeley to sample the full diversity of microorganisms in their natural environment. Known as genome-resolved metagenomic sequencing, this approach allows scientists to search for microbial genomes directly in the environment, rather than having to culture microbes in a lab, which has limited the study of many “hidden” species that are unable to survive in isolation.

In genome-resolved metagenomic sequencing, scientists collect samples comprising an assortment of different microbial communities from which DNA is isolated, broken down into shorter fragments and sequenced. The jumbled DNA fragments are then analyzed by computer algorithms, which piece together overlapping fragments to create long and continuous sequences, used to reconstruct complete or near-complete genomes from the microbes present in the sample.

While sequencing bacterial DNA in the early noughties, Banfield encountered something curious. Between repetitive stretches of DNA, previously named “clustered regularly interspaced short palindromic repeats”, or “CRISPR” for short, were DNA sequences that matched DNA from viruses – also sequenced from the same sample. “We were astonished to see that almost every cell of one type of bacteria sampled had a different set of DNA sequences in between the same CRISPR repeats. This was unexpected, because genomes of closely-related bacteria are usually essentially identical to each other in all parts of their genomes,” Banfield recalls.

Meanwhile at UC Berkeley, Doudna’s laboratory had been studying RNA interference (RNAi), a cellular regulatory mechanism where small RNA molecules bind to messenger RNAs, bringing protein translation to a grinding halt. After reading literature that suggested the CRISPR system might operate in a similar manner to RNAi, Banfield Google searched “RNAi” and “UC Berkeley”. Doudna’s name came up as the first search result.

The two agreed to meet for coffee and, after exchanging research notes, shared a sense of excitement about the possibility of an RNA-guided system existing in bacteria. They committed to pursuing this line of enquiry further. Doudna’s laboratory focused on a group of enzymes, called CRISPR-associated endonucleases, or “Cas” proteins – likened to molecular scissors due to their ability to create breaks in DNA. Banfield continued to explore how bacteria battled against viruses in a fight for survival, and how this shapes microbial communities.

You’re likely familiar with how the next part of the story unfolds in the Doudna lab. A collaboration with Professor Emmanuelle Charpentier pieced together the unknowns of how this acquired immunity system operates in bacteria. The team discovered that a dual RNA complex – comprising CRISPR RNAs (crRNAs) and trans-activating CRISPR RNA – is responsible for guiding Cas9 proteins to create double-strand breaks in DNA at specific sites.

In June 2012, Doudna and Charpentier published their findings in Science. Though the mechanisms of CRISPR-Cas-mediated genome editing had been described – not demonstrated – in the paper, Doudna said “it was a very real possibility” given the mechanism described. Within a year, the CRISPR-Cas9 system was engineered to induce precise DNA edits in human and mouse cells. A new gene-editing tool was born.

Three-dimensional model of a Cas-9 protein system showing how CRISPR genome editing works at the molecular level. Credit: Innovative Genomics Institute, UC Berkeley.

It’s not uncommon for years – even decades – to pass by before a novel molecular tool finds its feet as an established approach in the scientific community. CRISPR-Cas technology is an exception. Just eight years after the Science paper, Charpentier and Doudna received the 2020 Nobel Prize in Chemistry – the only science Nobel ever won by two women – for the development of CRISPR. In a Berkeley press conference discussing the Nobel win, Doudna acknowledged the contribution that Banfield had made in introducing her to the “signature of a bacterial immune system called CRISPR.”

Doudna recalls the moment she knew CRISPR-Cas technology would have a major impact on scientific research and our planet: “It started with genetic diseases like sickle cell disease: now we had a way to potentially treat, if not cure, thousands of unaddressed diseases,” she said in an interview with Technology Networks. “It could also be a powerful tool for plant breeders. It could help us study the human genome in new, exciting ways.”

Banfield had made tremendous strides in her quest to develop cultivation-independent methods for sequencing microbes. The pioneering techniques developed in her laboratory, first published in Nature back in 2004, are credited as the platform from which the growing field of gut microbiome research in health and disease could emerge, to name just one application. In 2016, Banfield collaborated with over a dozen researchers to publish genomic data from over 1,000 bacteria and archaea using genome-resolved metagenomics. The genome sequences were used to produce a “dramatically expanded” version of the tree of life, which comprises previously “hidden” uncultivatable organisms. “This is the first three-domain genome-based tree to incorporate these uncultivable organisms, and it reveals the vast scope of as yet little-known lineages,” Banfield said. She is recognized as one of the world’s leading microbiologists, having recently won the prestigious 2023 van Leeuwenhoek Medal for her contributions to the field.

Professors Jill Banfield and Jennifer Doudna. Credit: Keegan Houser, UC Berkeley.

In 2022, Doudna and Banfield married two novel, sophisticated technologies from their labs: Environmental Transformation sequencing (ETC-seq) and DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART). They demonstrated – for the first time – that it was possible to create targeted gene edits within complex microbiomes in Nature Microbiology. “It took advances over the past decade to show that, not only was it technically possible to edit the genes of microbes directly in a complex community, but that doing so would open up new avenues to tackle big, real-world problems,” Doudna said. “It was another eye-opening moment for me.”

A new frontier – precision microbiome editing – was born, and with it came the idea for a bold, audacious project.

The project

“Engineering the Microbiome with CRISPR to Improve our Climate and Health” is an IGI project co-led by Doudna and Banfield, conducted alongside collaborators at UC Davis and UC San Francisco, which recently received $70 million in funding from the TED Audacious project.

Over the next seven years, the team of experts intend to develop and apply precision microbiome editing to address two key areas of need that are linked by a root cause, problem-causing microbes; these are childhood asthma and agricultural methane emissions. The researchers hope to discover new microbiome editing and delivery tools, test new strategies for genome engineering in complex systems and conduct clinical and field trials to prove the safety and efficacy of precision microbiome editing.

Technology Networks had the pleasure of meeting several key players from IGI and UC Davis through a series of interviews. For the purpose of this article, we focused on the climate change impact of the project. Here, the end-goal is to develop an orally delivered genome-editing system that can be delivered to cow microbiomes. The system will target and edit the genes found in microbes that are responsible for methane production, with the overall result being an accessible, inexpensive method for combatting global methane emissions, and therefore climate change. While it’s currently a vision, the roadmap to a real-world intervention is carved out and builds on the collective works of a group of world-leading scientists.

The technology: ET-Seq and DART

At the time of the 2022 Nature Microbiology publication, Dr. Benjamin Rubin was a postdoc in Doudna’s lab, and is the first author of the paper. Now, he is an assistant researcher and principal investigator at IGI, where he will be supporting the development of the toolkit used in the project. Technology Networks interviewed Rubin and Dr. Brad R. Ringeisen, executive director of the IGI, to understand how the technologies work and the roadmap to their development.  

Molly Campbell (MC): Can you discuss the methods that were shared in the 2022 paper, and how they will be developed further in this project?  

Benjamin Rubin (BR): The 2022 paper developed two major technologies, one of which was ET-Seq, which is an approach to measuring what can be edited in a microbial community and by what tools.

You can imagine it as though we’re sprinkling non-targeted edits onto a microbiome, and seeing where they are taken up. Through this process, you develop a roadmap of what is editable in that community and by what mechanisms. This allows you to think about how to edit a microbial community without first breaking it down into individual microbes, which has been a previous bottleneck.

To make targeted edits, we have largely been using a tool called DART, which stands for DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase, a CRISPR-Cas-based editing system that we developed. We apply this to our roadmap created through ET-Seq. The DART system is wonderful because, unlike traditional CRISPR-Cas systems, it has all the machinery necessary to find a particular microbe in a community and change its DNA in a desired way. We’ve demonstrated the use of this system on human gut microbiomes in the lab, showing that you can use it as a method to edit a particular community member, gifting it with genetic advantages over other members of the community and increasing its abundance.

Pairing ET-Seq with DART allows the researchers to access and edit microbial species that are incredibly challenging – or even impossible – to culture in a laboratory. While the system took several years to develop, the entire process can now be run on a new microbial community in approximately two to three weeks.  

MC: Can you discuss the efficiency of the toolkit?

BR: We want to edit a broader diversity of organisms, and we want to do it with higher efficiency than we currently can. You can break down the levels of editing that enable certain outcomes into a few categories. The category in which we’re operating at right now is between 0.1–1% editing efficiency. At this level of efficiency, you can’t do much to change the function of a microbiome, but you can learn more about that microbiome by making an edit and exploring how this affects the organism.

Then there are editing efficiencies between 1–10%, which start to allow you to add in an edit and change the function of a community. We would love to reach an editing efficiency between 10–100%. At this level, you can start to remove certain functions that have adverse effects or change functions in a microbial community. I think the big hurdle is being able to get to that higher editing efficiency, and to be able to do it in more microbes.

Brad R. Ringeisen (BRR): Regardless of the editing efficiency, what’s important is that the edit itself can be controlled. If the efficiency is lower than desired, the right set of tools can enhance its effect, increasing the abundance of microbes carrying the edit such that they make up a greater proportion of the overall microbial community.

Plating bacterial cultures in the BIOME lab at the Innovative Genomics Institute. Credit: Innovative Genomics Institute, UC Berkeley.

The target: Methane-emitting microbes

While often used interchangeably, climate change and global warming are not synonyms; the former is a product of the latter. Global warming describes a long-term increase in the temperature of the Earth’s atmosphere – which has risen by 1.1 °C  since the Industrial Revolution – causing changes to the planet’s natural climate and weather patterns, with disastrous consequences.

This exponential rise in temperature is attributed to an enhanced greenhouse gas effect – an intensification of the naturally occurring greenhouse gas effect through human activities. Greenhouse gases in the atmosphere trap heat that is radiated from the Earth’s surface. This creates an overall warming effect, a process that is central to our survival. Without it, the planet would be too cold to sustain life. But human activities are leading to an excess of greenhouse gases in the atmosphere, exacerbating the warming effect and driving climate change. Methane (CH₄) is one example of a greenhouse gas, which is incredibly potent in the atmosphere.

Approximately 50–65% of total methane emissions are attributed to human activities, including oil and gas production, agriculture and livestock management and landfill management. These are known as anthropogenic emissions – of which 35–40% of are caused by livestock – and they are forecast to exponentially climb in the coming years.

MC: The ultimate goal of the project’s climate component is to create a genome-editing system that targets methane-producing genes in cow microbes, ultimately reducing the methane emissions from livestock. Can you talk more about the microbes and the initial steps in the project?

BRR: The target is methane-emitting microbes, or methanogens, that live in the rumen of the cow. A large focus of our team’s work will be understanding how these methanogens operate in their complex microbial communities. Then, we hope to target genes or microbes that alter the composition and metabolic activity of the microbiome to shut down methane production right at the source. Our editing system could target several of the microbes that make up the metabolic environment in which the methanogens live; each one of those individual microbes could be a new opportunity for us to effect change in the total microbial environment, creating a low- or no-methane microbiome in the gut of cows.

BR: A huge first step will be bringing the microbes into the lab, and characterizing model versions of the microbiomes that are essential to methane production. The next step is to develop the editing and delivery tools – and by this, I mean microscale delivery – the ability to deliver editing tools into the target organisms individually.

We need to develop methods for controlling the edits once they are in the microbiomes, which may involve increasing the abundance of low efficiency edits and developing containment measures to prevent edits from dispersing into the environment. We will then test all these systems in the lab-grown microbiomes to make sure the process works and decreases methane emissions in this environment. What’s great is that the research team at UC Davis has shown that you can move research directly from a bioreactor to the cow – i.e., if manipulation of the microbiome achieves a specific end-goal in a bioreactor, we can expect the same result in cows. This means we have a direct pathway to move from the laboratory to a real-world effect, and quickly. We are exploring the utility of lipid nanoparticles as a delivery mechanism.

Over at UC Davis, co-principal investigators of the project include Professor Ermias Kebreab, associate dean for global engagement in the College of Agricultural and Environmental Sciences and director of the World Food Center, and Associate Professor and microbiologist Matthias Hess.

Hess will co-develop the microbial tools and biocontainment methods in the laboratory with the researchers at IGI. His data will then be passed to Kebreab, who will take the research out into the field for testing. Their overall vision is that one day, the oral genome-editing treatment could be delivered to calves. By intervening with the calves’ microbiome at an early stage, they hope that methane production could be limited for the rest of their lifetime.

Kebreab brings extensive experience in sustainable agriculture and animal science to the team. The IGI project builds on his previous research demonstrating that food additives such as seaweed can effectively alter cow microbiomes and reduce methane production. 

MC: Can you tell us about your previous research using seaweed as a food additive to reduce methane production in cattle? 

Ermias Kebreab (EK): We have published two experiments exploring the use of seaweed in this context, which build on in vitro research conducted in Australia. The team had conducted an in vitro study screening seaweed, where they discovered that Asparagopsis interacts with methanogens and disrupts their production of methane.

After replicating the in vitro data from Australia, we conducted the first study in dairy cattle, once again replicating the data but this time in vivo. We had no idea how much of the material we would need to include in the diet, so we started small and ramped it up, eventually reaching 1% of the food intake. This, we discovered, was too high – there were issues with palatability and the animals didn’t want to eat the food. But the research was a proof-of-concept.

We then had a lot of questions, such as whether the microbes in the rumen would adapt over time, resulting in the methane production reversing back to normal levels. We conducted a second study in beef cattle over a period of five months, where we found no adverse adaptation. We also reduced the dose by half to 0.5%, which in beef cattle is equivalent to approximately 50g per day. We found that, at this level, methane production reduced by approximately 80%. Other institutes in Australia have demonstrated reductions of up to 98%. This research is ongoing across the globe, and we have also tested other additives – the majority of which haven’t worked. For me, the priority here is to focus on multiple different solutions for reducing methane production, not just one.

MC: How has this research led to your involvement in the IGI project?

EK: When we conducted our research using methanogen inhibitors such as seaweed, we realized that the resulting decrease in methane production wasn’t caused by a decline in the methanogen populations. Rather, it was caused by a downregulation of genes that are responsible for methane production. This identified a mechanism that we could potentially target and intervene with externally, but that would require tools that enable us to study the entire microbial population and edit the genes of individual microbes. Of course, through the project, we are now working with the IGI team to develop and test these tools.  

Previous research suggests that when you administer food additives at the beginning of life before the rumen is fully developed, and then you stop administering the additives, the reduced methane output continues. It’s like reprogramming the rumen, so that’s the basis of our idea. It is extremely exciting research – a high-risk and high-reward project.

Professor Ermias Kebreab. Credit: Innovative Genomics Institute, UC Berkeley.

We’re frequently told that to reduce methane emissions we should be consuming less meat. Kebreab emphasizes that, should the project be successful, this is still the case – it’s not an excuse to eat meat at high volumes “because we can.” Rather, the project recognizes that a growing population inevitably means the demand for meat as a protein source will continue, but takes action to offset some of the industry’s contributions to climate change.

Challenges, impact and the future

The $70 million funding provided by TED’s Audacious project is the largest to have ever been awarded to a scientific project, perhaps indicative of just how impactful this research could be on a global scale if successful. The IGI team are excited and encouraged by the recognition, while acknowledging the challenges that likely lie ahead. Much of the microbiome remains unchartered territory, and the regulatory landscape surrounding food products that are subjected to genome-editing is tricky to navigate – a significant amount of variation exists across countries. It won’t be an easy feat; but ground-breaking science never is. Technology Networks asked Ringeisen to share his vision for the future of the project, how he envisions the team will overcome the inevitable hurdles they encounter and why public awareness will be so important.