Rapid and accurate species identification for ecological studies and monitoring using CRISPR‐based SHERLOCK

Abstract

Accurate species identification is essential for ecological research and environmental monitoring. Some species are easy to identify visually, while identification of others is more challenging due to cryptic speciation (Hubert et al., 2008) and phenotypic plasticity (Pinzón et al., 2013). In these cases, as well as for more refined taxonomic discrimination (e.g., populations), genetic methods are often considerably more accurate (Benjamin et al., 2018; Vrijenhoek, 2009). To date, genetic identification has required a trained geneticist to receive the sample, conduct molecular methods (usually in a laboratory), analyse results and report the findings back to their field collaborators. This process can require days, and possibly even months, thus delaying the progression of research, conservation, and management actions based on the findings. In addition, laboratory facilities may not be available for genetic species identification

Early diagnosis of rabies virus infection by RPA-CRISPR techniques in a rat model

“…Most recently, RPA-CRISPR was applied to the diagnosis of infection with the betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [36][37][38]. Some extended applications have been described, such as field-deployable diagnostics, single-nucleotide polymorphism (SNP) detection, miRNA quantification, and species identification in ecological studies [24,27,39,40]. In this study, an RPA-CRISPR method was developed for the early diagnosis of RABV infection in a rat model.…”

CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

“…Following, or in concert with, nucleic acid amplification, enzymes from the clustered regularly interspaced short palindromic repeats (CRISPR) platform have been employed for detection/diagnostics. CRISPR/Cas12 and Cas13 have nucleic acid cleavage abilities such that target nucleic acid interrogation and recognition leads to ectopic strand breaks of a secondary reporter molecule that can be detected by lateral flow assays (LFA) or fluorometry [ 8 , 9 , 10 , 11 , 12 , 13 ]. CRISPR/Cas9 has also been developed for diagnostics using solid-state nanopores, microfluidics, and LFA [ 14 , 15 , 16 , 17 , 18 ].…”

“…Other studies have employed the nuclease properties of Cas proteins to generate fluorescence signals upon encountering target nucleic acid(s) [ 8 , 9 , 10 , 11 , 12 , 13 , 26 , 27 , 28 ]. We built off these principles by developing a sensitive fluorescence-based assay to detect the SARS-CoV-2 ORF8a gene sequence.…”

The Promise and Pitfalls of Environmental DNA and RNA Approaches for the Monitoring of Human and Animal Pathogens from Aquatic Sources

“…Although it is extremely sensitive (eliminating much of the uncertainties associated with traditional rapid diagnostics), easy to use, and resource efficient (i.e., time, money, and expertise), the optimization of such technologies for the environmental detection of human pathogens has not yet been reported (Lall 2020 , Sheridan 2020 ). However, with recent optimizations to use such technology in the fields of ecology and conservation biology, CRISPR-based SHERLOCK methodologies are already capable of efficient species identification in the field (via corporeal fluids such as mucus), expanding the future possibilities of this technology to be adapted for eDNA detection or species identification in similar aquatic environments in which these bodily fluids are secreted or shed (Baerwald et al 2020 , UCDAVIS 2020 ). With the persistence of the COVID-19 pandemic and technologies that have been rapidly investigated during a time of immense scientific advancement, there has never been a more promising time to apply what has been learned during this pandemic to monitor and mitigate future epidemics and pandemics in both human and wildlife populations.…”