Baerwald Melinda R, Funk Emily C, Goodbla Alisha M, Campbell Matthew A, Thompson Tasha, Meek Mariah H, Schreier Andrea D
Division of Integrated Science and Engineering, California Department of Water Resources, Sacramento, California, USA.
Genomic Variation Laboratory, Department of Animal Sciences, University of California Davis, Davis, California, USA.
Mol Ecol Resour. 2025 Jul;25(5):e13777. doi: 10.1111/1755-0998.13777. Epub 2023 Mar 14.
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.
准确的分类鉴定是有效进行物种监测和管理的基础。当视觉鉴定不可行或不准确时，基因方法提供了一种可靠的替代方案。然而，这些方法有时可行性较低（例如，需要近乎实时的结果、偏远地区、资金问题、分子生物学经验不足）。在这些情况下，基于CRISPR的基因工具可以填补一个空白领域，介于实时、低成本但容易出错的视觉鉴定，以及更昂贵或耗时但准确的基因鉴定之间，用于难以或无法通过视觉鉴定的分类单元。在此，我们利用基因组数据开发了基于CRISPR的SHERLOCK检测方法，能够快速（<1小时）、准确（表型和基因型鉴定之间的一致性为94%-98%）且灵敏地（检测到1-10个DNA拷贝/反应）区分加利福尼亚中央谷地列入《濒危物种法》的奇努克鲑鱼洄游群体（冬季和春季洄游群体），以及彼此之间和未列入名录的洄游群体（秋季和晚秋洄游群体）。这些检测方法可以在野外部署，通过微创黏液擦拭取样，无需进行DNA提取（降低成本和劳动力），所需设备最少且成本低廉，并且在检测方法开发后只需极少的培训即可进行操作。这项研究为一个受到保护关注的物种提供了一种强大的基因方法，该物种受益于近乎实时的管理决策，同时也为转变保护科学家和管理者未来对基因鉴定的看法树立了一个先例。一旦开发出来，基于CRISPR的工具可以提供准确、灵敏且快速的结果，可能无需昂贵的专业设备或广泛的分子生物学培训这种过高的要求。进一步采用这项技术将对我们自然资源的监测和保护具有广泛的价值。
