Field-deployable CRISPR-based biosensing platform could enable facile, real-time monitoring of marine barometer species and ecosystems
By Benjamin Boettner
(BOSTON) — Oceanic ecosystems are increasingly threatened by global warming which causes coral bleaching, species migration and, through the loss of habitats and biodiversity, food web disruptions on major scales. Also, pollutants such as plastics and other marine debris, wastewater, as well as chemical runoffs, including oil spills, cause major ecosystem disruptions. Importantly, given the interconnectedness of all life on the planet, the deteriorating health of our oceans directly impacts human health and sustenance.
Monitoring so-called "barometer species" can provide critical insights into the pulse of ocean health, reveal both acute local and long-term global trends, and help drive effective climate change policy, remediation and stewardship solutions. However, current marine surveillance methods like, for example, satellite-based ocean imaging or automated robotic systems are limited by their demands for extensive resources and, in many cases, limited spatial and biological resolution. Virtually all ocean or land-based laboratory approaches require sophisticated instruments, trained personnel, and long analysis times to quantify critical barometer species in water samples, which prevents frequent and wide-spread on-site analysis.
Now, addressing the urgent need for advanced ocean health monitoring, a research team at the Wyss Institute at Harvard University and Massachusetts Institute of Technology (MIT), led by Wyss Founding Core Faculty member James Collins , Ph.D. and Wyss Senior Scientist Peter Nguyen , Ph.D. in his group, have developed an inexpensive, laboratory-free approach to be used by many to rapidly quantify marine species and their physiological states on-site. Housed in highly portable, easy-to-handle device, the CRISPR-based biosensing platform has potential to be advanced to enable the prediction of outbreaks in marine communities, and routine monitoring of critically threatened species. The findings are published in Nature Sustainability .
"We aimed to lay the groundwork for more sustainable marine stewardship by developing a CRISPR-based technology platform that has the potential to reduce barriers to routine monitoring of critical oceanic species and to building large, user-driven data collections that can function as early warning systems of ecosystem disruptions," said Collins, who is also the Termeer Professor of Medical Engineering & Science at MIT. "While our focus on three very different barometer species highlights the diverse applicability of this platform, it can be easily adapted for the detection of other species and their physiological states."
From human to ocean health care
Critical for the study's advances were diagnostic capabilities that the Collins group had developed for human health care , as well as their introduction of " smart materials " that are able to sense certain stimuli. Deeply rooted in synthetic biology, the team merged work in different research disciplines for the design of new biological parts and devices that greatly facilitate the detection of infectious and other diseases in relevant home and hospital settings, as well as medically underserved regions of the world. Leveraging their expertise in biomedical diagnostics, the group has turned their attention to the development of "planetary diagnostics" for understanding the dysregulation of ecosystems caused by climate change. In their new study, they succeeded in bringing these accomplishments and technologies to bear on challenges of ocean health care.
"About 90% of the excess heat in the atmosphere caused by global warming has been absorbed by the oceans over the past five decades. This has been disrupting marine communities at an accelerated pace and impacted many natural species and entire ecosystems, the aquaculture industry and, as a consequence, also human health," said Nguyen. "Being able to sense these changes early and in real time through easy-to-handle diagnostic assays that can be performed by almost anyone, and whose results become immediately accessible to a large community of engaged sentinels is a first step toward mitigating arising threats."
To do this, the team harnessed the programmability of CRISPR technology to detect DNA and RNA nucleic acid molecules from key barometer species with high sensitivity and specificity, and integrated the assays into an automated workflow that can be fast and easily performed in a simple, yet sophisticated device.
An ocean of opportunities
"To demonstrate its broad applicability and programmability of our platform, we built biosensors for three climate-linked barometer species that inform about very diverse oceanic threats," said the study's first author Nayoung Kim , Ph.D., who spearheaded the study as a Wyss Technology Development Fellow in Collins' group. In their first application, the team targeted a virulence-factor gene from pathogenic Vibrio spp.bacteria whose populations frequently explode in warming seawater. Vibrio outbreaks can devastate oyster beds and contribute to disease in adult shellfish and coral reefs. Contact with contaminated seawater or consumption of contaminated seafood can also cause vibriosis, a bacterial infection that can cause several illness, particularly in people with liver disease, diabetes and weakened immune systems. As a second target, they selected the microscopic, single-celled Pseudo-nitzschia spp. algae. During the blooming events, these diatoms produce large quantities of a potent neurotoxin which causes the death of shellfish, fish, marine mammals, and seabirds. Humans consuming seafood with this accumulated toxin can develop potentially lethal amnesiac shellfish poisoning (ASP). Finally, to diagnose the physiological state of coral communities under heat stress in warming seawater, the team adapted their biosensors to detect biomarker RNA transcripts produced by the easy-to-sample Caribbean Porites astreoides coral when it experiences thermal stress.
In designing their CRISPR-based biosensing assays, the team employed the CRISPR–Cas12a enzyme that can be guided to target DNA by complementary guide RNA (gRNA) molecules – in this case genes or reverse-transcribed transcripts from barometer species. Upon recognition of the target, Cas12a unleashes an indiscriminate "collateral" cleavage activity toward nearby single-stranded DNA (ssDNA) molecules. By offering the activated Cas12a enzyme ssDNA reporter molecules labeled with two binding moieties compatible with lateral-flow strips, the researchers implemented a colorimetric, lateral-flow assay (LFA)-based CRISPR biosensing platform. This approach is analogous to LFAs commonly used in infectious disease diagnostics and pregnancy tests. The appearance of a target-specific colored band on a paper strip enables simple, intuitive, instrument-free readout, making LFAs particularly much more suitable for field-deployable devices.
"In establishing accurate biosurveillance for these three barometer species, we had to pay close attention to both sensitivity and specificity," said Kim. "It is essential to detect low levels of target nucleic acids that may signal early population disruption, while also discriminating among highly similar nucleic acid sequences from related species that have lower, different or no impact on ocean health – especially in the case of Vibrioand Pseudo-nitzschia species. For heat-stressed corals, it is also critical to capture early physiological changes through sensitive and specific detection of biomarker RNA transcripts associated with stress." By systematically screening multiple sensor component designs, including gRNAs, the team developed CRISPR biosensors that are capable of selectively detecting miniscule amounts of target DNA or RNA molecules within about 40 min. "We demonstrated robust performance of our biosensors at ambient temperatures and in the presence of seawater, supporting their use in field settings," said Kim. "The programmability of CRISPR allows this approach to be readily adapted for detecting a wide range of other marine species."
However, while highly effective biosensors are a prerequisite for the detection of potentially harmful or harmed marine species, only their instrumentation in a practical device could provide a field-ready marine biosurveillance platform. "An important problem we had to solve for on-site marine monitoring was preparing samples and performing assays without laboratory instruments," said Kim. "We drew inspiration from conventional ocean microbial sampling approaches, which involve passing liters of seawater through membranes to collect and concentrate organisms on filters. Rather than transporting these filters to centralized laboratories, we engineered low-cost, portable, 3D-printed devices that enable instrument-free sample processing and detection directly on site."
Filters containing microbial or shed animal cells are loaded into the disposable, 3D-printed processor, where cells are lysed and target DNA or RNA is amplified directly on the filter in a single 30-minute step. Processed samples are then recovered by simple hand squeezing and detected using optimized CRISPR biosensors with LFA readouts. Reaction activity and lysis efficiency are supported by the inexpensive, insulated 3D-printed incubator warmed with an off-the-shelf, battery-powered hand warmer. "All temperature-sensitive reagents are provided in lyophilized form for shelf-stable deployment, and fluid volumes are optimized for dropper-based handling, allowing simple, in-field operation through squeezing and applying droplets," said Kim. "Using this platform, we successfully demonstrated a fully field-deployable pipeline with live Vibriopathogens spiked into unfiltered natural seawater collected from three distinct ocean sites, using minimal contamination controls."
The researchers think that their field-deployable system could provide a low-cost, portable solution for marine ecosystem monitoring in the hands of a wide range of users, including ecologists, marine biologists, citizen scientists, conservationists, coastal communities, aquaculture farmers, park rangers, and biosecurity officers. In some of these applications, individual measurements could be uploaded via smartphone apps into large data bases that with the help of analysts and AI could be used to synthesize more global trends and ring early alarms at critical moments.
"This truly empowering environmental diagnostic technology embodies how the Wyss Institute is leveraging its diverse engineering strengths, as well as internal collaborations between its Sustainable Futures and Diagnostics for Human and Planetary Health efforts, to pave the way towards protecting our oceans, our planet, and the health of humans world-wide," said Wyss Founding Director Donald Ingber , M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children's Hospital, and the Hansjörg Wyss Professor of Biologically Inspired Engineering at Harvard John A. Paulson School of Engineering and Applied Sciences.
Other authors on the study are Daniel Collins, Nina Gonghia, Benjamin Miller, Hani Sallum, Silvi Lybbert, Elena Perini, and James Niemi. The study was funded by the Wyss Institute at Harvard University.
