We report the first application of CRISPR‐Cas technology to single species detection from environmental DNA (eDNA). Organisms shed and excrete DNA into their environment such as in skin cells and faeces, referred to as environmental DNA (eDNA). Utilising eDNA allows noninvasive monitoring with increased specificity and sensitivity. Current methods primarily employ PCR‐based techniques to detect a given species from eDNA samples, posing a logistical challenge for on‐site monitoring and potential adaptation to biosensor devices. We have developed an alternative method; coupling isothermal amplification to a CRISPR‐Cas12a detection system. This utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA. We used the target species Salmo salar as a proof‐of‐concept test of the specificity of the assay among closely related species and to show the assay is successful at a single temperature of 37°C with signal detection at 535 nM. The specific assay, detects at attomolar sensitivity with rapid detection rates (<2.5 hr). This approach simplifies the challenge of building a biosensor device for rapid target species detection in the field and can be easily adapted to detect any species from eDNA samples from a variety of sources enhancing the capabilities of eDNA as a tool for monitoring biodiversity.
Molecular techniques offer sensitive, specific, noninvasive monitoring of target species from a variety of environmental samples. We recently developed a CRISPR‐Cas‐based eDNA assay for rapid single‐species detection as a route to a simple, cost‐effective biosensor device. CRISPR‐Cas‐based diagnostic assays use isothermal conditions in combination with a highly specific sequence recognition system. This CRISPR‐Cas assay was designed to target Salmo salar, and we previously demonstrated its utility in eDNA samples from sites in Ireland. The aim of this study was to validate our assay in two larger sample sets from Canada (n = 16/n = 63) in comparison with an independent S. salar qPCR assay. We demonstrate that overall, the CRISPR‐Cas assay performs similarly to qPCR for assessing the presence or absence of S. salar from eDNA and provides a viable alternative approach where qPCR assay design and application have proven to be challenging.
Isothermal molecular techniques offer an alternative to single taxa monitoring using environmental DNA (eDNA). Methodologies such as Loop-Mediated Amplification (LAMP) (Notomi et al., 2000) and Recombinase Polymerase Amplification (RPA) (Piepenburg et al., 2006) have been readily adopted for point-of-care diagnostics (Craw & Balachandran, 2012;Oliveira et al., 2021), but use in environmental monitoring remains limited. Despite this, previous studies have shown successful detection of, for example, Dreissena sp. in the Great Lakes using LAMP (Williams et al., 2017) and harmful algae using RPA (Toldrà et al., 2019). Moreover, such isothermal amplification methods have the potential to be coupled to fluorescencebased detection such as through incorporation of an exo-probe (TwistAmp® exo RT kit) (Li et al., 2018) for real-time RPA or through coupling with a secondary technique such as CRISPR-Cas as reviewed in Kaminski et al. (2021).Both LAMP and RPA have been coupled with CRISPR-Cas technology to increase detection specificity (Broughton et al., 2020;
Development of simple and rapid techniques to monitor species of conservation importance is vital to further the capabilities of environmental DNA. Conventional methods for eDNA detection pose a logistical challenge for on-site monitoring due to the need for high temperatures and thermal cycling. To circumvent this, we recently adapted an isothermal CRISPR-Cas based detection assay for single-species assessment of Salmo salar as a route to a simple, cost-effective biosensor device (Williams et al., 2019). CRISPR-Cas for detection (rather than genome editing) was first developed for clinical diagnostic applications. The variety of Cas nucleases allow detection of either RNA or DNA with attomolar sensitivity (Chen et al., 2018; Gootenberg et al., 2017). This detection approach is versatile and has recently been adopted for the detection of SARS-CoV-2 (Broughton et al., 2020). The CRISPR-Cas detection system consists of two main elements; a guide RNA specific to the target and an effector Cas12a nuclease. The Cas12a nuclease will only cleave at the target site when a specific protospacer adjacent motif (PAM) is present downstream. The requirement to recognise two separate sequences supports a highly specific recognition system that can distinguish closely related species. However, although its use is expanding rapidly for the detection of pathogens, it is yet to be fully embraced for eDNA detection. The RPA-CRISPR-Cas methodology we have developed utilises the isothermal recombinase polymerase amplification and CRISPR-Cas12a detection, leading to four unique sequence recognition elements, which require stringent design and in-lab testing to ensure assay specificity. Development of our published S. salar CRISPR-Cas assay (Williams et al., 2019), and subsequent assays for Salmo trutta and Salvelinus alpinus, highlighted critical steps to consider and pitfalls to avoid when designing such isothermal assays. 1) Only the target sequence should contain the required PAM site. In version 1 of our assay, both S. salar and S. trutta contained the PAM site; we were unable to distinguish them. In version 1 of our assay, both S. salar and S. trutta contained the PAM site; we were unable to distinguish them. 2) An RPA primer screen is essential. Multiple forward and reverse primers are screened up-/down-stream of the gRNA binding region to select the optimum primer pair. Multiple forward and reverse primers are screened up-/down-stream of the gRNA binding region to select the optimum primer pair. 3) Specificity tests should be carried out on tissue from the target species and other species present in the sampling environment. In silico design is not sufficient to ensure assay specificity. In silico design is not sufficient to ensure assay specificity. References Broughton, J. P., Deng, X., Yu, G., Fasching, C. L., Servellita, V., Singh, J., … Chiu, C. Y. (2020). CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology, 38(7). https://doi.org/10.1038/s41587-020-0513-4 Chen, J. S., Ma, E., Harrington, L. B., Costa, M. Da, Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360(6387), 436–439. https://doi.org/10.1126/SCIENCE.AAR6245 Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … Zhang, F. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356(6336), 438–442. https://doi.org/10.1126/science.aam9321 Williams, M. A., O’Grady, J., Ball, B., Carlsson, J., de Eyto, E., McGinnity, P., … Parle-McDermott, A. (2019). The application of CRISPR-Cas for single species identification from environmental DNA. Molecular Ecology Resources, 19(5). https://doi.org/10.1111/1755-0998.13045
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