scholarly journals Advice to consider when developing a CRISPR-Cas assay for single species detection using eDNA

2021 ◽  
Vol 4 ◽  
Author(s):  
Molly Williams ◽  
Fiona Regan ◽  
Anne Parle-McDermott
Keyword(s):  
In Silico ◽  
Salmo Trutta ◽  
Detection System ◽  
Environmental Dna ◽  
Single Species ◽  
Nucleic Acid Detection ◽  
Target Sequence ◽  
Binding Region ◽  
Sequence Recognition ◽  
Cas Assay

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

scholarly journals A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

2020 ◽  
Author(s):  
Bettina Thalinger ◽  
Kristy Deiner ◽  
Lynsey R. Harper ◽  
Helen C. Rees ◽  
Rosetta C. Blackman ◽  
...  
Keyword(s):  
In Silico ◽  
Meta Analysis ◽  
Environmental Dna ◽  
Single Species ◽  
Future Research ◽  
Individual Species ◽  
General Applicability ◽  
Routine Monitoring ◽  
User Friendly

AbstractThe use of environmental DNA (eDNA) analysis for species monitoring requires rigorous validation - from field sampling to interpretation of PCR-based results - for meaningful application and interpretation. Assays targeting eDNA released by individual species are typically validated with no predefined criteria to answer specific research questions in one ecosystem. Their general applicability, uncertainties and limitations often remain undetermined. The absence of clear guidelines prevents targeted eDNA assays from being incorporated into species monitoring and policy, thus their establishment will be key for the future implementation of eDNA-based surveys. We describe the measures and tests necessary for successful validation of targeted eDNA assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was compiled, consolidated into 14 thematic blocks, such as “in silico analysis”, and arranged on a 5-level validation scale from “incomplete” to “operational”. Additionally, minimum validation criteria were defined for each level. These variables were evaluated for 546 published single-species assays. The resulting dataset was used to provide an overview of current validation practices and test the applicability of the validation scale for future assay rating. The majority (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve this first level. These assays were characterised by minimal in silico and in vitro testing, but their share in annually published eDNA assays has declined since 2014. The total number of reported variables ranged from 20% to 76% and deviated both between and within levels. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future research and routine monitoring, while also enabling appropriate interpretation of results. Finally, it provides guidance on validation and reporting standards for newly developed assays.

scholarly journals Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Ryohei Nakao ◽  
Hideyuki Doi ◽  
Toshifumi Minamoto
Keyword(s):  
Fish Species ◽  
High Throughput Sequencing ◽  
Correlation Coefficients ◽  
Environmental Dna ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Taqman Probe ◽  
Individual Species ◽  
Natural Environments ◽  
Target Sequence

AbstractThe combination of high-throughput sequencing technology and environmental DNA (eDNA) analysis has the potential to be a powerful tool for comprehensive, non-invasive monitoring of species in the environment. To understand the correlation between the abundance of eDNA and that of species in natural environments, we have to obtain quantitative eDNA data, usually via individual assays for each species. The recently developed quantitative sequencing (qSeq) technique enables simultaneous phylogenetic identification and quantification of individual species by counting random tags added to the 5′ end of the target sequence during the first DNA synthesis. Here, we applied qSeq to eDNA analysis to test its effectiveness in biodiversity monitoring. eDNA was extracted from water samples taken over 4 days from aquaria containing five fish species (Hemigrammocypris neglectus, Candidia temminckii, Oryzias latipes, Rhinogobius flumineus, and Misgurnus anguillicaudatus), and quantified by qSeq and microfluidic digital PCR (dPCR) using a TaqMan probe. The eDNA abundance quantified by qSeq was consistent with that quantified by dPCR for each fish species at each sampling time. The correlation coefficients between qSeq and dPCR were 0.643, 0.859, and 0.786 for H. neglectus, O. latipes, and M. anguillicaudatus, respectively, indicating that qSeq accurately quantifies fish eDNA.

scholarly journals Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243319
Author(s):  
Takeshi Hanami ◽  
Tetsuya Tanabe ◽  
Takuya Hanashi ◽  
Mitsushiro Yamaguchi ◽  
Hidetaka Nakata ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Detection System ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Digital Measurement ◽  
Excitation Light ◽  
Target Nucleic Acid ◽  
Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

scholarly journals Airborne environmental DNA metabarcoding detects more diversity, with less sampling effort, than a traditional plant community survey

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mark D. Johnson ◽  
Mohamed Fokar ◽  
Robert D. Cox ◽  
Matthew A. Barnes
Keyword(s):  
Plant Community ◽  
Plant Communities ◽  
Traditional Method ◽  
Environmental Dna ◽  
Single Species ◽  
Community Survey ◽  
Sampling Effort ◽  
Sampling Campaign ◽  
Dna Metabarcoding ◽  
Human Health Impacts

Abstract Background Airborne environmental DNA (eDNA) research is an emerging field that focuses on the detection of species from their genetic remnants in the air. The majority of studies into airborne eDNA of plants has until now either focused on single species detection, specifically only pollen, or human health impacts, with no previous studies surveying an entire plant community through metabarcoding. We therefore conducted an airborne eDNA metabarcoding survey and compared the results to a traditional plant community survey. Results Over the course of a year, we conducted two traditional transect-based visual plant surveys alongside an airborne eDNA sampling campaign on a short-grass rangeland. We found that airborne eDNA detected more species than the traditional surveying method, although the types of species detected varied based on the method used. Airborne eDNA detected more grasses and forbs with less showy flowers, while the traditional method detected fewer grasses but also detected rarer forbs with large showy flowers. Additionally, we found the airborne eDNA metabarcoding survey required less sampling effort in terms of the time needed to conduct a survey and was able to detect more invasive species than the traditional method. Conclusions Overall, we have demonstrated that airborne eDNA can act as a sensitive and efficient plant community surveying method. Airborne eDNA surveillance has the potential to revolutionize the way plant communities are monitored in general, track changes in plant communities due to climate change and disturbances, and assist with the monitoring of invasive and endangered species.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

Design and Implementation of Experimental Consumable Management Device for the Automatic Nucleic Acid Detection System

2016 ◽  
Vol 16 (7) ◽  
pp. 7069-7076
Author(s):  
Jun Yu ◽  
Zhu Chen ◽  
Chao Wang ◽  
Yana Hu ◽  
Hongming Dong ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Detection System ◽  
Nucleic Acid Detection ◽  
Design And Implementation

Mechanical Gripper Design and Force Analysis of Microplates for Automated High-Throughput Nucleic Acid Detection System

2020 ◽  
Vol 20 (3) ◽  
pp. 1401-1408 ◽  
Author(s):  
Chao Wang ◽  
Zhukang Guo ◽  
He Zhu ◽  
Nongyue He ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Detection System ◽  
Nucleic Acid Detection ◽  
Force Analysis ◽  
Control Board ◽  
Absolute Position ◽  
Element Analysis ◽  
Position Data ◽  
Master Control

In the automated high-throughput nucleic acid detection system, we need to grip and transfer consumables frequently when carrying out multichannel nucleic acid detection. In order to ensure the efficiency of experiments and solve problems of the deflection and drop when transferring microplates, we design a self-locking mechanical gripper which consists of a rotary positioning module and a gripping module. The absolute position encoder fixed on the top of the stepper motor can collect the position data of the mechanical gripper in real time and send them to the master control board based on STM32 for processing, which ensures the accuracy of the movement of the mechanical gripper. We used SolidWorks to build models of the mechanical gripper and different microplates, and we carried on finite element analysis of microplates to find the suitable gripping position. Through the force analysis, we obtained the pressure distribution and the deformation of different microplates, and defined the effective gripping areas, which is important to the grip and transfer of microplates.

scholarly journals BchY-Based Degenerate Primers Target All Types of Anoxygenic Photosynthetic Bacteria in a Single PCR

2009 ◽  
Vol 75 (23) ◽  
pp. 7556-7559 ◽  
Author(s):  
Natalya Yutin ◽  
Marcelino T. Suzuki ◽  
Mira Rosenberg ◽  
Denisse Rotem ◽  
Michael T. Madigan ◽  
...  
Keyword(s):  
In Silico ◽  
Photosynthetic Bacteria ◽  
Environmental Dna ◽  
Reaction Centers ◽  
Photosynthetic Reaction Centers ◽  
Degenerate Primers ◽  
Nucleotide Database ◽  
Anoxygenic Photosynthetic Bacteria

ABSTRACT To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the bchY gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.

scholarly journals ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

2010 ◽  
Vol 10 (1) ◽  
pp. 189 ◽  
Author(s):  
Eva Bellemain ◽  
Tor Carlsen ◽  
Christian Brochmann ◽  
Eric Coissac ◽  
Pierre Taberlet ◽  
...  
Keyword(s):  
In Silico ◽  
Dna Barcode ◽  
Environmental Dna
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