BACKGROUND AND SIGNIFICANCE OF STUDY

We have developed a novel ecological monitoring approach using CRISPR technology, which is widely utilized in the biomedical field for viral diagnostics, but is only starting to expand into other disciplines such as conservation biology. This CRISPR-Cas13a-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking) platform is a highly sensitive, specific, and rapid method to discriminate between species in the field. Traditional genetic species identification requires sampling tissue from the organism and several days of processing in a molecular biology lab. SHERLOCK, however, can be completed in an hour or less without DNA extraction. Being able to make real-time management decisions based on accurate identifications in the field is critical when protecting threatened species, particularly in California’s watersheds.

In this study we focused on three morphologically similar smelt species co-occurring in the San Francisco Estuary, the U.S. threatened and California endangered Delta Smelt, the California threatened Longfin Smelt, and the non-native Wakasagi. These three species can be challenging to identify in the field, particularly as juveniles, which can lead to incorrect abundance and distribution estimates. Using a simple mucus swab for DNA input, we were able to accurately distinguish these three smelts using the SHERLOCK platform. This technique can be utilized by non-molecular biologists with minimal training and can be applied to an expansive range of organisms. SHERLOCK has the potential to revolutionize the field of molecular ecology, including monitoring and management practices.

RESULTS

Using a simple mucus swab for DNA input, we were able to rapidly and accurately distinguish these three osmerids using the SHERLOCK platform. Our protocol produced results in less than an hour, without DNA extraction. We found that a portable fluorescence reader can be used in the field (e.g. on a boat or shoreline) to produce easy-to-interpret results. We have also demonstrated that results can be detected using lateral flow (like a pregnancy test) where no equipment is needed for visualization.

Figure 1.SHERLOCK schematic. Fish mucus is collected via swab which is then swirled in 1X PBS buffer. 2ul of DNA input is transferred into a SHERLOCK one-pot reaction tube. In a single reaction, the DNA is converted to amplified target RNA that binds to target-specific crRNA. Activated Cas13a then collaterally cleaves an RNA reporter, causing fluorescence. 

 

Figure 2. Species specificity for each osmerid assay demonstrated by SHERLOCK fluorescence after one hour. For each target species, mucus swabs placed directly in 300 µl PBS were tested against all three species-specific crRNAs. Boxplots display median and interquartile range for each DNA and crRNA assay combination. DSM = Delta Smelt, LFS = Longfin Smelt, WAG = Wakasagi. Median and interquartile range are shown for each boxplot. For target species, N=10 (DSM), N=7 (LFS), and N=39 (WAG) and ranged from 3 – 10 for each non-target species.

 

Figure 3. a. Schematic of SHERLOCK instrument-free detection using a FAM- and biotin-labeled RNA oligonucleotide reporter and commercial lateral flow strips. Uncleaved reporter accumulates as anti-FAM antibody/gold nanoparticle conjugates at the control (Streptavidin) line. If target DNA is present, the reporter is cleaved by Cas13a, resulting in conjugate binding at the antibody capture line. b. For each species-specific assay, on-target synthetic gBlock, tissue DNA, and mucus swabs (in 300 µL PBS) were detected with lateral flow. Off-target tissue DNA and no template reactions were included as negative controls.

FUTURE WORK

We are currently developing new SHERLOCK assays to identify species of interest in the Bay-Delta ecosystem. These include non-native invasive species, such as Zebra and Quagga Mussels, as well as Chinook Salmon run-type assignment. We are also exploring how SHERLOCK could improve environmental DNA detections.

PUBLICATIONS

Baerwald, M.R., Goodbla, A., Nagarajan, R.P., Gootenberg, J.S., Abudayyeh, O.O., Zhang, F., & A.D. Schreier. 2020. Rapid and Accurate Species Identification for Ecological Studies and Monitoring using CRISPR-based SHERLOCK. Molecular Ecology Resources. https://doi.org/10.1111/1755-0998.13186

FUNDING SOURCE

This study was funded with support from the California Department of Water Resources (Contract # 4600012328).

PROJECT TEAM

Emily Funk, Associate Specialist, University of California, Davis

Melinda Baerwald, Environmental Program Manager, California Department of Water Resources

Sean Canfield, Postdoctoral Researcher, University of California, Davis

Alisha Goodbla, Research Associate, University of British Columbia

Ravi Nagarajan, Assistant Project Scientist, University of California, Davis

Leigh Sanders, Graduate Student, University of California, Davis

Diana Muñoz, Graduate Student, University of California, Davis

Anderson Tate, Graduate Student, University of California, Davis

Andrea Schreier, Principal Investigator, Adjunct Assistant Professor, University of California, Davis

Omar Abudayyeh, McGovern Institute Fellow, Massachusetts Institute of Technology

Jonathan Gootenburg, McGovern Institute Fellow, Massachusetts Institute of Technology

Mariah Meek, Assistant Professor, Michigan State University