Rapid CRISPR‐Cas13a genetic identification enables new opportunities for listed Chinook salmon management

Abstract

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real‐time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR‐based genetic tools can fill an unoccupied niche between real‐time, inexpensive, but error‐prone visual identification and more expensive or time‐consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR‐based SHERLOCK assays capable of rapidly (<1 h), accurately (94%–98% concordance between phenotypic and genotypic assignments), and sensitively (detects 1–10 DNA copies/reaction) distinguishing ESA‐listed Chinook salmon runs (winter‐ and spring‐run) from each other and from unlisted runs (fall‐ and late fall‐run) in California's Central Valley. The assays can be field deployable with minimally invasive mucus swabbing negating the need for DNA extraction (decreasing costs and labour), minimal and inexpensive equipment needs, and minimal training to conduct following assay development. This study provides a powerful genetic approach for a species of conservation concern that benefits from near real‐time management decision‐making but also serves as a precedent for transforming how conservation scientists and managers view genetic identification going forward. Once developed, CRISPR‐based tools can provide accurate, sensitive, and rapid results, potentially without the prohibitive need for expensive specialty equipment or extensive molecular training. Further adoption of this technology will have widespread value for the monitoring and protection of our natural resources.

FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection

“…Likely due to the ssRNA degradation properties of Cas13a that would leave ssDNA primers intact, Baerwald et al. (2023) were able to design a ‘one‐pot’ SHERLOCK assay that was as sensitive as qPCR and the two‐step FINDeM assay, detecting single‐digit copies of synthetic target DNA. However, Baerwald et al.…”

“…However, Baerwald et al. (2023) did not test the sensitivity of their ‘one‐pot’ assay on biological samples, which may contain proteins and chemicals that hinder or even entirely inhibit molecular reactions. Therefore, it is difficult to say if the ‘one‐pot’ SHERLOCK approach would exhibit the same sensitivity as the FINDeM protocol, which was able to definitively detect single‐digit DNA copies from amphibian swabs in a lab setting (Figure 6b; PA31, PA24) and inconclusively detect double‐digit copies from amphibian swabs in the field (Figure 2b; 2‐03).…”

“…Likely due to the ssRNA degradation properties of Cas13a that would leave ssDNA primers intact, Baerwald et al (2023) were able to design a 'one-pot' SHERLOCK assay that was as sensitive as qPCR and the two-step FINDeM assay, detecting single-digit copies of synthetic target DNA. However, Baerwald et al (2023) did not test the sensitivity of their 'one-pot' assay on biological samples, which may contain proteins and chemicals that hinder or even entirely inhibit molecular reactions.…”

“…Likely due to the ssRNA degradation properties of Cas13a that would leave ssDNA primers intact, Baerwald et al (2023) were able to design a 'one-pot' SHERLOCK assay that was as sensitive as qPCR and the two-step FINDeM assay, detecting single-digit copies of synthetic target DNA. However, Baerwald et al (2023) did not test the sensitivity of their 'one-pot' assay on biological samples, which may contain proteins and chemicals that hinder or even entirely inhibit molecular reactions. Therefore, it is difficult to say if the 'onepot' SHERLOCK approach would exhibit the same sensitivity as the FINDeM protocol, which was able to definitively detect single-digit DNA copies from amphibian swabs in a lab setting (Figure 6b; PA31, PA24) and inconclusively detect double-digit copies from amphibian swabs in the field (Figure 2b; 2-03).…”

CRISPR‐based environmental DNA detection for a rare endangered estuarine species

“…In addition, CRISPR-Dx methods can be used to detect aquatic eDNA (Williams et al, 2019(Williams et al, , 2021(Williams et al, , 2022Wei et al, 2023; reviewed in Williams et al, 2023). We previously applied SHERLOCK for rapid, non-invasive identification of three smelt species (Baerwald et al, 2020) and determination of Chinook salmon (Oncorhynchus tshawytscha) migratory run type (Baerwald et al, 2023). For both applications, we demonstrated successful detection using both extracted DNAs and mucus swabbing of "fish in hand" without DNA extraction.…”