关键词: CRISPR/Cas系统, 环境DNA, 核酸快速检测, 生物监测, 水生生态系统

Abstract

Aims: Aquatic biomonitoring provides an essential foundation for biodiversity conservation, aquatic ecosystem health assessment, and ecological risk early warning. Traditional aquatic biomonitoring methods primarily rely on morphological identification, which is time-consuming, labor-intensive, and requires specialized expertise. Therefore, there is an urgent need to explore novel biomonitoring approaches to meet the growing demands of aquatic biomonitoring. 

Progresses: With advances in molecular biology, environmental DNA (eDNA) technology has been introduced into aquatic biomonitoring and applied to species distribution surveys, biodiversity assessment, and ecosystem conservation. However, its heavy reliance on laboratory equipment, high-throughput sequencing, and bioinformatics expertise has constrained its routine application. In recent years, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) systems have been increasingly applied to eDNA detection. While maintaining high sensitivity, optimized CRISPR/Cas-based detection systems can achieve single-nucleotide resolution, enabling the accurate discrimination of closely related species. Moreover, their targeted detection strategies eliminate the need for time-consuming high-throughput sequencing procedures and, when combined with isothermal amplification, enable rapid detection at a constant temperature under field conditions. These advances have facilitated the development of CRISPR-based environmental biosurveillance (CRISPR-eBx), providing a promising technological approach for routine aquatic biomonitoring. 

Review Results: This review first introduces CRISPR-eBx technology and systematically describes its detection workflow, highlighting key considerations in water sample collection, eDNA extraction, nucleic acid amplification, CRISPR/Cas-based detection, and result readout. It then summarizes current applications in four areas: conservation of endangered and rare species, early warning of invasive and harmful species, aquatic ecosystem health assessment, and monitoring of aquatic pathogens. Challenges associated with practical applications are also discussed. Finally, the advantages and limitations of CRISPR-eBx technology are summarized. Its major advantages include: (1) improving the sensitivity and accuracy of biomonitoring; (2) enabling rapid on-site detection under field conditions; and (3) enhancing the timeliness of ecological monitoring and the capacity for ecological risk early warning. Nevertheless, several limitations remain: (1) temporal and spatial uncertainties in eDNA sources may increase the uncertainty of detection results; (2) false-negative and false-positive results may occur in practical applications; (3) limited quantitative capability makes it difficult to accurately assess the actual occurrence and abundance of target species in the environment; and (4) insufficient standardization of detection procedures hinders comparisons of results across different studies. 

Going Forward: To promote the routine application of CRISPR-eBx technology in aquatic biomonitoring, future research should focus on four aspects: (1) improving the portability and field deployability of existing detection technologies to gradually achieve integrated on-site operations from environmental sample collection to result readout; (2) conducting large-scale field validation across different waterbody types, seasons, and environmental conditions, while establishing unified quality control systems and result interpretation criteria to improve the reliability and comparability of detection data across studies and platforms; (3) strengthening the integration of CRISPR-eBx with emerging technologies to further enhance detection efficiency and expand its functionality; and (4) promoting the complementary application of CRISPR-eBx and conventional eDNA monitoring technologies to achieve more comprehensive, accurate, and multiscale monitoring of aquatic ecosystems.

Key words: CRISPR/Cas system, environmental DNA (eDNA), rapid nucleic acid detection, biomonitoring, aquatic ecosystems