Using a CRISPR-based method, conservation scientists have dramatically shortened the time it takes to genetically identify endangered fish species from days and months to mere minutes.
Researchers have exploited the benefits of CRISPR since the technology went mainstream in 2012—and they’re still only at the tip of the iceberg. The gene-editing technology is so powerful that it can make a difference in a variety of applications from curing diseases and improving food crops to developing sustainable methods for producing fuel.
Now, a study led by researchers from UC Davis, the California Department of Water Resources and MIT Broad Institute says CRISPR can also be a critical conservation and resource management tool.
To accurately identify fish species currently, field researchers rub a swab over the fish to collect a mucus sample or take a fin clip for a tissue sample. They then send that sample to a laboratory with a trained geneticist who performs standard molecular genetic analysis. The process takes days, sometimes even months, and it’s not possible for field work in remote locations.
In a proof-of-concept experiment, the CRISPR-based detection platform SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) was able to genetically distinguish threatened fish species from similar-looking nonnative species in nearly real time, without the need to extract DNA.
SHERLOCK, developed by Feng Zhang from the Broad Institute, combines isothermal amplification with the functional capability of Cas13a to indiscriminately cleave RNA only after it detects a specific target sequence. The signal can then be read with a simple paper strip or as an electrochemical readout using a mobile phone. Zhang and MIT’s James Collins pioneered the technology in 2017 to detect low levels of Zika virus, as well as the presence of pathogenic bacteria.
"But SHERLOCK is well suited for ecology because it is extremely fast (results possible in under 30 minutes), very sensitive (can detect very small amounts of DNA/RNA), specific (can distinguish single base pair differences), and its all done at 37 C (roughly body temperature), which makes field deployment feasible," lead author Melinda Baerwald told Laboratory Equipment.
In her study, recently published in Molecular Ecology Resources, Baerwald and her team engineered SHERLOCK DNA assays to genetically distinguish three similar-looking fish species: the U.S.-threatened and California-endangered Delta smelt, the California-threatened longfin smelt and the nonnative wakasagi.
"We were amazed to find we could just take the mucus swab and put it directly into phosphate buffered saline or the SHERLOCK reaction itself and there was sufficient free DNA in the mucus, so the reaction worked without the need for DNA extraction at all," said Baerwald. "This is a huge advancement. It cuts down on cost, time, the potential for contamination across samples during processing and it greatly simplifies field testing. We can simply swab a fish and put the mucus right into a SHERLOCK reaction and 20 to 30 minutes later we know its species ID."
Real-time identification and knowledge of endangered species is critically important. For example, the U.S. Endangered Species Act places regulations on the number of individuals in an endangered species allowed to be “fished” by humans. Additionally, state and federal water pumping projects must reduce water exports if enough endangered species are sucked into the pumps.
“Rapid species identification ensures accurate data collection in ecological studies and can be critically important for time-sensitive species management and compliance with laws protecting threatened species. Field-deployable genetic assays for these species enable real-time decision-making when evaluating protected species ‘take’,” the researchers write in their paper.
Baerwald said there is no reason SHERLOCK can't be used for any animal species, or even other taxonomic groups such as plants, fungi, bacteria, etc. For their part, she and her team are currently working on optimizing SHERLOCK for environmental DNA (eDNA) applications, developing new assays for other marine species of interest and optimizing the protocol for field deployment and use by non-geneticists.
The study authors say the success of the proof-of-concept experiment moves “CRISPR beyond the realm of geneticists and into the hands of field biologists,” redefining by whom and where genetic identification is possible.
Photo: A Delta smelt is swabbed to be genetically identified through SHERLOCK. Credit: Alisha Goodbla/UC Davis.