Rapid identification of invasive Brook Trout through CRISPR‐based environmental DNA detection

Abstract

Objective

Invasions of nonnative fish species, such as Brook Trout Salvelinus fontinalis, disrupt ecosystem function and adversely impact native populations through competition, hybridization, and predation. Timely detection of invasive species is crucial for effective management and prevention of further spread. Environmental DNA (eDNA) technology is a powerful tool for monitoring the spread of invasive species, especially in aquatic environments, where direct observation is challenging. While eDNA sampling is sensitive and cost‐effective, real‐time analysis remains a challenge.

Methods

To address this limitation, we developed eDNA detection tools, using CRISPR‐Cas12a technology, that facilitate the rapid identification of invasive Brook Trout and native cutthroat trout (Lahontan Cutthroat Trout Oncorhynchus henshawi, Westslope Cutthroat Trout O. lewisi, Rocky Mountain Cutthroat Trout O. virginalis, or Coastal Cutthroat Trout O. clarkii), without the need for specialized laboratory equipment. We employed this technology in Marsh Creek, a semi‐isolated tributary to the Sultan River in Washington State with no previous documentation of invasive Brook Trout.

Result

We successfully identified Brook Trout and cutthroat trout eDNA during on‐site analysis at Marsh Creek and found that the sensitivity of detecting these species can be increased with improvements in eDNA purification technology. Our results identified that Brook Trout were present in the lower reaches of the stream, particularly in areas of slow‐moving water.

Conclusion

The portability and simplicity of this method offer the potential for streamside eDNA detection of invasive species, and it could become another tool in the fight against the spread of invasive Brook Trout in the western United States.

Impact statement

Brook Trout invasions in the Pacific Northwest highlight continued struggles with managing nonnative species. We deployed a rapid CRISPR‐Cas12a‐based environmental DNA detection protocol to document their presence in a model Pacific Northwest stream.

INTRODUCTION

Invasive species continue to threaten species biodiversity (Dangora et al. 2023) and can contribute to significant economic losses in diverse sectors such as agriculture, forestry, fisheries, power production, and others (Sepulveda et al. 2020). The invasion of nonnative fish species poses a multifaceted threat, disrupting ecosystem function and suppressing native populations through resource competition, hybridization, and predation (Britton et al. 2011). Timely detection of invasive species is critical for managers to implement effective preventive measures and curb further spread, underscoring the importance of proactive strategies in safeguarding ecosystems and mitigating broader ecological impacts.

The invasion history of Brook Trout Salvelinus fontinalis in the Pacific Northwest raises ecological concerns, particularly regarding their impact on native fish species like cutthroat trout (Lahontan Cutthroat Trout Oncorhynchus henshawi, Westslope Cutthroat Trout O. lewisi, Rocky Mountain Cutthroat Trout O. virginalis, or Coastal Cutthroat Trout O. clarkii) (Dunham et al. 2002). Native to eastern North America, Brook Trout were introduced to the western United States in the late 1800s to bolster recreational fishing opportunities (Pister 2001) and have now established populations across the region (Sinnatamby et al. 2023). This invasion has significant repercussions for sensitive native species such as cutthroat trout and Bull Trout Salvelinus confluentus (Nakano et al. 1998; Gunckel et al. 2002; Sinnatamby et al. 2023). Despite general protocols for proper management of invasive species, including rapid detection, assessment, and response (reviewed in Britton et al. 2011; Day et al. 2018), invasive species suppression efforts are often constrained by the speed of detection.

A powerful tool for monitoring the spread of invasive species is environmental DNA (eDNA) technology, which can determine an organism's presence from the source DNA that it sheds into the environment from biological material, such as feces, gametes, hair, skin, and mucus (Ficetola et al. 2008; Williams et al. 2019). The efficacy of eDNA in detecting and monitoring invasive species is well established, particularly for aquatic species that are challenging to observe directly (Nathan et al. 2015; Sepulveda et al. 2020; Thomas et al. 2020). For migrating aquatic species like salmonids, eDNA serves as a valuable tool for tracking migration and colonization patterns (Duda et al. 2020). How eDNA is transported within a given ecosystem can vary in space and time, and there is a wealth of research seeking to improve our understanding of the distribution and stability of eDNA under different environmental scenarios (Deiner et al. 2016). Despite its advantages, obtaining real‐time eDNA results is challenging, as postcollection processing usually occurs in dedicated laboratories. The increasing adoption of eDNA monitoring has required molecular laboratories to handle a high volume of samples, resulting in delays of weeks or months in reporting (Blasko and Phelps 2024). Invasive species suppression efforts are one area of fisheries management that benefits from rapid or real‐time information on species distribution to help inform appropriate eradication or suppression efforts. Environmental DNA has an important role to play in the fight against invasive species; however, there is an urgent need for simplified eDNA processing methods, enabling direct utilization by resource managers in coordination or consultation with molecular laboratories to increase the utilization of eDNA for the management of invasive species.

Beyond its application in the genetic engineering field, the genome‐editing technology clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated proteins (Cas), known as CRISPR‐Cas, and has emerged as a potent method for the rapid detection of nucleic acids (Gootenberg et al. 2017; Chen et al. 2018) and is beginning to be applied to eDNA applications (Williams et al. 2019, 2023; Nagarajan et al. 2024). The CRISPR‐Cas diagnostic methods can detect specific DNA or RNA sequences by programming CRISPR nucleases with short CRISPR RNA molecules (crRNA). Once programmed, the Cas endonuclease–crRNA complex recognizes the target sequence and cleaves the strand with high specificity. The subsequent diagnostic step occurs after target cleavage as the specialized CRISPR‐Cas nucleases (e.g., Cas12, Cas13, etc.) proceed to indiscriminately cleave single‐stranded DNA or RNA within the reaction. This cleavage can be harnessed for diagnostic assays by designing single‐stranded DNA or RNA probes that produce a visual signal if cleaved. The probes can be designed for visualization by fluorescence (Rybnicky et al. 2022) or on a lateral flow strip (Mukama et al. 2020). This diagnostic technology can be combined with an isothermal amplification step to enhance sensitivity and portability (Ali et al. 2020). The CRISPR‐Cas detection of DNA and RNA has proven effective for identifying the presence of DNA from aquatic species in mucus and environmental DNA samples (Williams et al. 2019, 2020; Baerwald et al. 2020, 2023, Nagarajan et al. 2024).

Environmental DNA technology has been used with passive water sampling to effectively detect invasive Brook Trout (Nolan et al. 2023). Increased adoption of eDNA technology for Brook Trout could improve monitoring programs by reducing the resources required for sampling and therefore facilitate more in‐depth or expansive stream surveys. The research presented in this manuscript seeks to expand the eDNA toolbox by developing Brook Trout and cutthroat trout CRISPR‐Cas12a eDNA detection technologies that are simple to perform and require minimal equipment such that they may be utilized in the field. The eDNA technology that was developed is sensitive and specific for Brook Trout and cutthroat trout mitochondrial DNA detection and works effectively on eDNA samples. The CRISPR‐Cas12a detection capabilities were evaluated for Brook Trout monitoring on Marsh Creek, a Pacific Northwest stream with no official history of Brook Trout presence and several barriers to invasion but with rumored reports of invasive Brook Trout. Using CRISPR detection technology, we were able to identify invasive Brook Trout and native cutthroat trout in the stream, which was confirmed by physical sampling. Our results highlight the utility of rapid eDNA detection technology for improving invasive species monitoring to enhance fisheries management efforts.

METHODS

Brook Trout and cutthroat trout CRISPR eDNA detection assay

To increase detection sensitivity, a previously developed loop‐mediated isothermal amplification (LAMP) assay that targets a conserved region of the salmonid mitochondrial D‐Loop was used for the Brook Trout and cutthroat trout eDNA detection reactions (Blasko and Phelps 2024). Laboratory LAMP reactions contained 12.5 μL of LAMP WarmStart 2X Master Mix (New England Biolabs), 2.5 μL of primer master mix, 8 μL of deionized water, and 2 μL of the DNA sample (i.e., 1 ng/μL synthetic DNA, 10 ng/μL target eDNA, or crude field extracted eDNA; information provided in the Supplement in the online version of this article). The primer master mix contained 16 μM FIP and BIP, 2 μM F3 and B3, and 4 μM LF and LB primers (Integrated DNA Technologies; Table S1 available in the Supplement). Streamside reactions contained 12.5 μL of premixed LAMP WarmStart 2X Master Mix and 2.5 μL of primer master mix with 10 μL of extraction solution added in the field (see the Supplement). The reactions were carried out at 65°C for 30 min.

The CRISPR endonuclease Lachnospiraceae bacterium ND2006 Cas12a (L.b.Cas12a; Integrated DNA Technologies) used for diagnostic applications requires a crRNA target site to be located downstream of a TTTV protospacer adjacent motif sequence (PAM; Chen et al. 2018). To identify a target site unique to Brook Trout and cutthroat trout, an alignment of the mitochondrial D‐loop of 10 salmonid species was performed (SnapGene; Figure 1A). Aligned mitochondrial sequences included Chinook Salmon O. tshawytscha (fall type; NC_002980), Coho Salmon O. kisutch (NC_009263), Chum Salmon O. keta (NC_017838), Sockeye Salmon O. nerka (NC_008615), Pink Salmon O. gorbuscha (NC_010959), Rainbow Trout O. mykiss (NC_001717), cutthroat trout (NC_006897), Bull Trout (D‐loop partial, JQ436479), Lake Trout S. namaycush (NC_036392), and Brook Trout (NC_000860). The CRISPR target sites were chosen based on the lack of a PAM site in other species and/or a minimum of 2–3 base pair (bp) changes in the crRNA target sequence between species, preferably in the seed region of the crRNA near the PAM site (Figure 1A). A similar crRNA between Brook Trout and Bull Trout was present with a conserved PAM and seed region; however, 4‐bp differences existed throughout the remaining crRNA sequence. The selected L.b.Cas12a crRNA sequences targeting Brook Trout or cutthroat trout contain a 20‐bp hairpin scaffold sequence followed by 21‐bp crRNA sequence unique to the Brook Trout or cutthroat trout mitochondrial D‐loop sequence found in those species (synthesized as Alt‐R RNA, Integrated DNA Technologies; Figure 1A; Table S1). After the CRISPR target sequences for both species were identified, we performed a secondary National Center for Biotechnology Information (NCBI) blast search alignment of the sequences and were unable to identify any other species that had sufficient sequence similarity to have false positive detection with the CRISPR detection assays.

A CRISPR master mix for the detection reactions was prepared with 14 μL of H₂O, 3 μL of 10X New England Biolabs Buffer 3.1, 5 μL of 1‐μM crRNA, and 1 μL of 2.5‐μM L.b.Cas12a (Integrated DNA Technologies; see Supplement). The crRNA and L.b.Cas12a were complexed for 15 min at 25°C prior to including in the master mix. A single‐stranded fluorescent DNA probe was added to the CRISPR master mix at a final concentration of 16 nM (1 μL of 0.5 μM) for visualizing signal amplification (see Supplement). The fluorescent probe contained a FAM fluorescent molecule on the 5′ end and an Iowa Black FQ quencher on the 3′ end of a random 12‐bp sequence (Integrated DNA Technologies; Table S1). The CRISPR reactions were incubated at 37°C for up to 30 min. Laboratory results during the testing phase were analyzed on a qTower quantitative polymerase chain reaction (qPCR) machine (Analytik Jena), and the field samples were visually identified on a portable transilluminator. Specificity of the Brook Trout and cutthroat trout CRISPR assays were tested against 1 ng/μL of synthesized DNA from Chinook Salmon, Coho Salmon, Chum Salmon, Sockeye Salmon, Pink Salmon, Rainbow Trout, cutthroat trout, Bull Trout, Lake Trout, and Brook Trout added directly to the CRISPR reaction (Figure 1B; Integrated DNA Technologies; Table S1). The sensitivity of each CRISPR assay was determined by spiking a 10‐fold serial dilution of synthetic DNA (3.3 × 10⁷ to 3300 copies per microliter of eDNA before filtering) into eDNA taken from the Palouse River in Pullman, Washington. The eDNA was filtered and extracted with both the laboratory and rapid field extraction protocols to determine the sensitivity of both methods (Figure 1C,D). The region of the Palouse River sampled was selected because it lacked the presence of cutthroat trout and Brook Trout, and therefore the detection limit of the assay could be estimated from the spiked DNA down to the copy number of the target sequence. All specificity and sensitivity experiments were performed in triplicate with results compared to samples containing no DNA.

Stream survey site at Marsh Creek

Four field sites were chosen within Marsh Creek, a small tributary to the Sultan River in Washington State (Figure 2). The field sites were selected based on a gradient of habitat types and included both deep, slow‐moving sites and shallow, fast‐moving areas (Figure 2). A raised culvert in the stream between site 1 (47.932471°, −121.778579°) and site 2 (47.932080°, −121.778141°) was filled with debris, which inhibited streamflow on the sampling date. Sample site 1 was located below the culvert, while site 2 was located upstream of the culvert (Figure 3). Water was collected from the surface of a shallow riffle at sample site 1 to determine if salmonids were present below the flow‐inhibiting culvert. The riffle flowed at a mellow pace and was lightly turbid. Site 2 was sampled approximately 30‐cm below the surface of a deep, slow‐flowing, murky pool created upstream of the culvert (Figure 3). Site 3 (47.940185°, −121.759142°) was a shallow, clear, fast‐flowing riffle in Marsh Creek upstream of a confluence with an unnamed tributary (Figure 4). Site 4 (47.939792°, −121.760035°) was a shallow, clear, fast‐flowing riffle in the small tributary to Marsh Creek (Figure 4). Sites 3 and 4 were both approximately 15 cm deep at the sampling location. All stream water samples were collected by hand from surface water in the thalweg of the stream at the sampling location. The conditions on the sampling date were overcast with periods of light rain and wind speeds up to 16 km/h.

Environmental DNA field survey of Marsh Creek

A field eDNA water collection kit was assembled containing sterile nitrile gloves, tweezers, whirlpacks, 1.2‐μm mixed cellulose ester (MCE) filter (MilliporeSigma), 1 L of deionized water, and a filter holder for each site. Additional equipment included a hand pump, prepared tubes with nondenatured ethanol for filter storage, 50‐mL conical tubes containing bleach, magnetic tube rack, and deionized water for sterilization. An independent field CRISPR‐Cas12a detection kit was assembled with a portable heat block, power source (Ryobi), P‐20 and P‐200 pipettes and tips, sharpies, magnetic tube block, and a portable UV transilluminator (E‐Gel Safe Imager, Invitrogen). A cooler containing preprepared extraction mix, bead purification tubes, LAMP master mix, CRISPR master mix, and reusable ice packs was packed with a combination of ice and dry ice (total cost of field kit approximately US$1000).

The field sites were sampled in order, starting from the most downstream location to avoid contaminating downstream locations during upstream sampling. At each site, three 1‐L stream water samples were collected as well as a control sample containing 1 L of deionized water. The water samples were collected in whirlpack bags and vacuumed through MCE filters using a hand pump. After filtration, half of each filter was processed on site at a portable field station for CRISPR‐Cas12a analysis, while the other half was preserved in nondenatured ethanol to be brought back to the laboratory for analysis. Streamside eDNA extraction was carried out according to protocols developed by Blasko and Phelps (2024). The field extraction protocol consisted of incubating the filter for 30‐min in a proteinase‐K solution (200 μL of 1X New England Biolabs Buffer r2.1 and 6 U of proteinase‐K; New England Biolabs) before performing a magnetic bead purification of the samples, following the manufacturers protocols (CleanNGS; Bulldog Bio). For magnetic bead purification, the protease‐digested samples are incubated 1:1 with the magnetic beads to capture the DNA. After removal of the supernatant and a wash with 80% ethanol, water was used to elute the DNA from the beads for use in the downstream CRISPR reactions. After field extraction, the sample eDNA was added to the LAMP master mix and incubated at 65°C for 30 min. During this incubation, the CRISPR master mix was located in the lid of the tube with a reusable ice pack placed on the lid to reduce potential RNA degradation. After amplification, the tubes were flicked to mix the amplified DNA with the CRISPR master mix and the sample was incubated at 37°C for 30 min. Results were analyzed on a portable UV transilluminator. In addition to the stream water samples and the deionized water control for each location, a second unfiltered water‐only negative control was also used to ensure no contamination occurred postextraction. A positive control sample, which consisted of using 10 ng of synthesized DNA for the target species, was also included for each site. Physical hook‐and‐line sampling using fly fishing gear with floating line and a barbless dry fly was performed at sites 1 and 2 after eDNA was collected, as a secondary survey method because the depth of the water limited visual examination of the water bodies. For sites 3 and 4, the size and clarity of the water facilitated visual stream surveys and prevented hook‐and‐line sampling. To compare the accuracy of streamside CRISPR‐Cas12a eDNA analysis to laboratory CRISPR‐Cas12a analysis, the unused half of the filter that was preserved in ethanol was brought back to the lab and analyzed following the identical CRISPR‐Cas12a protocols, but instead, eDNA samples were column‐purified according to an established eDNA extraction workflow (DNeasy Blood and Tissue, Qiagen; Goldberg et al. 2015).

RESULTS

Development of rapid CRISPR eDNA assay for Brook Trout and cutthroat trout detection

The CRISPR‐Cas12a assays developed for Brook Trout and cutthroat trout displayed rapid fluorescent detection in the presence of the target sequence (Figure 1B). The single‐species specificity of the Brook Trout and cutthroat trout CRISPR‐Cas12a assays was confirmed by performing the assay on high concentrations of synthesized DNA from nine salmonid species (Figure 1B). No fluorescent detection was observed in these trials, showing that the assay does not cross‐react with other closely related species (Figure 1B). The sensitivity of the reaction was evaluated on eDNA samples spiked with a serial dilution of synthesized DNA from both species. We determined that the CRISPR eDNA assays could detect Brook Trout and cutthroat trout eDNA starting with an initial water concentration (i.e., prefiltration) down to 3.3 × 10³ and 3.3 × 10⁵ copies/μL using the laboratory and field eDNA extraction protocols, respectively (Figure 1C,D).

Detection of invasive Brook Trout and native cutthroat trout in Marsh Creek

On‐site CRISPR‐Cas12a eDNA analysis at Marsh Creek sites 1 and 2 detected the presence of cutthroat trout in one of the replicate samples (Figures 2, 3). Site 1 identified one positive detection of Brook Trout, while site 2 identified two positive Brook Trout samples (Figure 3). Field sites 3 and 4 were both negative for the presence of Brook Trout and cutthroat trout eDNA while assayed in the field (Figure 4). Hook‐and‐line sampling at sites 1 and 2 was able to identify invasive Brook Trout in the system (Figure 3) but was not practical at sites 3 and 4. Visual stream surveys at sites 3 and 4 were unable to identify any fish in the system. The portability of the kit and rapid processing of results allowed for all sites to be sampled and analyzed in a single day, with the results obtained in less than 2 h after sampling. Laboratory analysis confirmed the presence of both Brook Trout and cutthroat trout at sites 1 and 2 in all three samples at both sites (Figure 3). Site 3 also showed one positive sample for Brook Trout and two positive samples for cutthroat trout (Figure 4). Site 4 was negative for Brook Trout and cutthroat trout (Figure 4).

DISCUSSION

A new CRISPR‐Cas12a eDNA technology was developed for on‐site detection of Brook Trout and cutthroat trout. The eDNA assay was used to identify the presence of these two species in a stream with no previous documentation of invasive Brook Trout. The CRISPR‐Cas12a‐based DNA detection technology was able to rapidly identify Brook Trout and cutthroat trout mitochondrial DNA and was highly specific to the target species, even at high concentrations of mitochondrial DNA from nontarget salmonids (i.e., salmon, trout, and char species). Combining this high specificity with isothermal amplification enabled a simple, sensitive, and highly portable technology that translates well to eDNA monitoring applications.

The CRISPR detection technology was employed for Brook Trout eDNA identification in Marsh Creek in Washington State (Figure 2) to demonstrate its potential for invasive species monitoring. The positive detection of Brook Trout by both eDNA and physical sampling at sites 1 and 2 is the first known documentation of these invasive fish in Marsh Creek. While we were unable to physically sample cutthroat trout in the system, the positive eDNA detection of cutthroat trout in the creek is important because it shows that the two species co‐occupy a habitat where only cutthroat trout would naturally be present. This highlights some advantages of using eDNA technology for stream surveys, since the accurate assessment of species presence or absence using invasive methods such as hook and line, netting, or backpack electrofishing can be challenging and resource intensive (Meador et al. 2003). It is important to note that the CRISPR‐Cas12a assays used in this research are highly sensitive to small genetic changes in or around the CRISPR PAM sequence, and therefore, while unlikely, local variation in this region could influence the detection capabilities of the test. Improvement of the technology to include a quantitative assessment of the CRISPR results with fluorescent photoreceptors instead of visual identification could enhance the confidence of the results, particularly with samples that are near the limit of detection.

Potential vectors of Brook Trout invasion into Marsh Creek include the nearby Cascade Mountains that contain many lakes historically stocked with Brook Trout, a privately stocked lake adjacent to Marsh Creek, or potential private efforts to stock the creek for sporting opportunities. Previous stream surveys of the creek have identified the presence of resident cutthroat trout (Washington Department of Fish and Wildlife's SalmonScape, LLID: 1217959479336). Although the Sultan River receives annual migrations of Pacific salmon Oncorhynchus spp. and has resident native salmonid populations, a large waterfall at the mouth of Marsh Creek prevents upstream migration. The isolation of Marsh Creek is believed to have limited the potential for the invasive spread of Brook Trout.

The presence of both Brook Trout and cutthroat trout suggests the possibility of competition in Marsh Creek, something that is known to limit native cutthroat trout abundance (Peterson et al. 2004). Brook Trout have been shown to impact native species through increased competition, displacement, predation, hybridization, and transmission of diseases (Sinnatamby et al. 2023). Our eDNA detection results showed a strong signal for Brook Trout and cutthroat trout at sites 1 and 2. Abundance estimates are challenging to determine with eDNA, and have not been validated with CRISPR eDNA assays, and therefore we cannot make any conclusion about the abundance of fish at these field sites. The presence of two positive samples for cutthroat trout and a single positive sample for Brook Trout at site 3 could reflect differences in the aquatic environment at site 3 compared with sites 1 and 2. The distribution and retention of eDNA in the environment has been extensively studied, and it is clear that environmental conditions have a significant impact on eDNA detection (Barnes and Turner 2016). Sites 3 and 4 were shallow flowing water, which contrasted significantly with the deep pools at site 1 and 2. A large marsh upstream of site 2, possibly created by the raised culvert at that location, produced deep pools that together formed a large pond during lower flow periods. This may help produce the ideal Brook Trout habitat in the system and may retain eDNA longer than sites with flowing water. Further work to verify more areas of Brook Trout presence using traditional sampling or more fine‐scale eDNA sampling would give managers insight into where eradication efforts should take place. The Marsh Creek waterfall is a barrier to upstream and downstream migration of fish in this system. This makes Marsh Creek a potential candidate for invasive species biocontrol methods, such as the introduction of a Trojan Y Chromosome program (i.e., YY male Brook Trout; Schill et al. 2016).

The use of CRISPR‐Cas detection technology for on‐site nucleic acid detection has been demonstrated in Chinook Salmon (Baerwald et al. 2023; Blasko and Phelps 2024) and Delta Smelt Hypomesus transpacificus (Nagarajan et al. 2024) and has also been used for a wide variety of disease diagnostics (Talwar et al. 2021; Chen et al. 2023). In this study, CRISPR‐Cas12a field detection was less sensitive than when the identical reactions were performed on laboratory‐extracted eDNA (Figures 1, 3, 4). This is expected, as field eDNA extraction methods are designed to be quick and crude, which reduces the initial input eDNA concentration and purity. Improvements in field eDNA extraction methods should significantly enhance the sensitivity of streamside CRISPR‐Cas eDNA detection. Despite the lower sensitivity in the field, CRISPR‐Cas12a technology was able to identify both Brook Trout and cutthroat trout present in Marsh Creek. Streamside eDNA analysis may not need to be as sensitive as laboratory‐based analysis if it can enable more routine sampling because the chance of encountering the target organism is greater. Our results suggest that CRISPR‐Cas detection technologies could be used as a complement to current invasive species eradication efforts, helping to identify hot spots in real time. The use of CRISPR‐Cas eDNA technology may also be valuable for remote field research where on‐site data could be used to guide the research direction in ways not possible if the results had to wait until the samples were analyzed back in the laboratory.

While reliable streamside eDNA detection would be a significant benefit to the ecology and resource management fields, the financial benefits of large‐scale CRISPR‐Cas eDNA deployment is difficult to determine. The CRISPR reagent costs can vary in price by supplier and volume, with raw reaction costs being similar to that of standard qPCR‐based eDNA detection reactions (around $8–10 per reaction in raw reagents). The use of CRISPR‐Cas eDNA technology, however, does not require the purchase and maintenance of expensive equipment as is needed for qPCR diagnostics, which can reduce overall reaction costs. The true advantage of employing CRISPR‐Cas technology for eDNA monitoring is the time and financial benefits that come from not having to outsource collected samples to molecular laboratories for processing, which can cost more than $100 per sample, due to the specialized expertise required for analysis. Coordination with molecular ecology labs would still be highly advantageous and often essential, but the use of simplified eDNA analysis protocols by field biologists could help break the backlog of eDNA samples.

The CRISPR‐Cas eDNA technology is an emerging field that is rapidly expanding with new innovations and demonstrated applications (Hoenig et al. 2023; Wei et al. 2023; Williams et al. 2023). However, the technology is still in its infancy and further development is needed to improve confidence in the technology to the level of current qPCR‐based eDNA assays. It will be important to compare our CRISPR‐Cas12a eDNA technologies to well‐established Brook Trout and cutthroat trout eDNA protocols (Wilcox et al. 2013, 2015) to understand the strengths and weaknesses of each method. Through further technical development, CRISPR‐Cas eDNA technologies, such as the ones developed in this manuscript, may help open the door to increased utilization of eDNA for environmental monitoring of invasive species, an area that is vitally important to protecting ecosystems and organisms threatened by invasive species.

ACKNOWLEDGMENTS

The authors would like to thank Snohomish County Public Utility District biologist K. Legare for contributions regarding the history, ecology, and geography of Marsh Creek. The authors recognize the contributions of the many undergraduate and graduate students in the Phelps Lab, with special recognition to Shubhankar Sircar. The authors acknowledge that laboratory work was completed on the ancestral homelands of the Nez Perce Tribe and Palus People and fieldwork was completed on the ancestral homelands of the Tulalip Tribe. Funding for this project was provided by the Foundation for Food and Agricultural Research on competitive grant #FF‐NIA21—0000000050.

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest to declare.

DATA AVAILABILITY STATEMENT

All data from the manuscript can be found in the supplemental data files available in the online version of this article.

ETHICS STATEMENT

Most of the described research utilized synthesized DNA and eDNA extracted from the water to avoid handling fish where possible. The limited sampling of fish was conducted during cool stream conditions using hook and line. The fish were only briefly removed from the water for imaging before being released to reduce handling stress.

REFERENCES