Latest revision as of 19:43, 1 October 2024

ModF

Introduction

ModF is a forward primer specifically designed to amplify a fragment of Prymnesium parvum genomic DNA in an RPA (Recombinase Polymerase Amplification) reaction by targeting the 5.8S region in the genome.

This primer contains a T7 promoter to enable in vitro transcription of the amplified product into RNA, preparing it for SHERLOCK detection with Cas13 proteins.

Biology and Usage

The ITS2 (Internal Transcribed Spacer 2) region is a non-coding segment of DNA found within the ribosomal RNA (rRNA) gene cluster. In the genome of Prymnesium parvum, the ITS2 region lies between the 5.8S and nuclear large rRNA genes [1].

The ITS regions, including ITS2, are commonly used for species identification because they tend to vary between species while being conserved within a species [2]. This variability makes the ITS2 region an effective target for designing species-specific primers, such as those used to identify Prymnesium parvum [3].

RPA Reaction

Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technique that operates at a temperature range of 37–42°C, distinguishing it from traditional PCR methods that require thermal cycling for denaturation and annealing of DNA [4].

RPA relies on three essential types of proteins: a recombinase, single-stranded DNA binding proteins (SSBs), and a strand-displacing DNA polymerase.

The process begins when the recombinase protein binds to a primer (about 30–35 nucleotides long) that matches the target DNA sequence. This complex then searches for homologous sequences in double-stranded DNA and initiates strand invasion. The SSBs stabilize the displaced strand to prevent primer dissociation, while the strand-displacing DNA polymerase extends the primer, resulting in exponential amplification of the target sequence [5].

SHERLOCK Method

The SHERLOCK platform is a modern synthetic biology tool that utilizes the properties of the Cas13a protein, an enzyme from the Nobel Prize-winning CRISPR-Cas system. The Cas13a protein is guided with high specificity to the target sequence using crRNA.

The crRNA molecule is crucial for the assay's specificity. It consists of a direct repeat (DR) sequence and a spacer sequence that is complementary to the target. The crRNA molecule is designed to uniquely identify the organism by targeting the Internal Transcribed Spacer (ITS) sequence in its genome.

First, the Cas13a protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the Cas13a protein is activated and exhibits a “collateral” RNase activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules [7].

This activity can be used in assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by Cas13, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA).

Part Performance

This part is an integral component of the PrymDetect Toolkit, which is designed for detecting Prymnesium parvum in water samples using the SHERLOCK method. Our team evaluated this part alongside other components of the toolkit to determine the most reliable configurations and make its use easier for future iGEM teams. In our tests we used the LwaCas13a protein.

The SHERLOCK reactions were carried out according to the protocol, which can be accessed here.

Parts Compatible with ModF

The results show that ModF performs best when paired with the GalR primer and the PrymCrRNA1 design. This combination delivers the highest fluorescence readout intensity and provides results the fastest. Another effective option is to use ModF with ModR and PrymCrRNA1. While ModF also works with AltR and PrymCrRNA1, or with GalR and PrymCrRNA2, these combinations yield lower intensity results. Therefore, the ModF and GalR primer pair is our top choice, as it demonstrates strong performance with both crRNAs and achieves the highest fluorescence intensity when combined with PrymCrRNA1 among all the designs we tested.

Primer Concentration Optimization

Since the ModF and GalR primers combined with PrymCrRNA1 proved to be our most effective combination, we decided to fine-tune the primer concentration to see if this adjustment could help us establish a correlation between fluorescence and the initial amount of DNA present in the sample. Our goal was to make the test quantifiable.

An assay was conducted to compare how varying primer concentrations impact the amplification rate and fluorescence intensity in SHERLOCK detection. The method used is based on the approach presented in the Jonathan S. Gootenberg article [9].

RPA reactions were set up as advised in Kellner’s protocol [7], with different primer and input DNA concentrations, alongside a positive control with SynDNA as the target. For template dilutions, a PCR product coming from a Prymnesium parvum culture was used. Dilutions of 200 nM, 2 nM, 20 pM, and 200 fM were tested, alongside the following primer concentrations: 960 nM, 480 nM, 240 nM, and 120 nM.

Table 1: SHERLOCK Reactions Components – RPA Quantification

Results

The SHERLOCK assay was conducted according to Kellner’s protocol on a 384-well plate [7], with one change: the assay was conducted in two instead of four replicates for each sample.

Results Analysis

• Averaging Fluorescence Intensity: Each sample was measured in duplicates to ensure accuracy and reliability. The final fluorescence intensity for each sample was calculated by averaging the duplicate measurements. These averaged results were then used to create representative bar graphs.
• Subtracting Negative Controls: For each sample, the fluorescence intensity of the negative control was subtracted from the average intensity. Based on these adjusted results, logarithmic functions were fitted.

Note:: The assay needed to be manually extended, which caused a bump on the graph (fluorescence intensity versus time), but this does not impact the final results.

Data Visualizations

Primer Concentration 960 nM Results

Figure 4: SHERLOCK results - 960 nM

Figure 5: Final fluorescence intensity to Prymnesium parvum DNA concentration - 960 nM

Figure 6: Final fluorescence intensity to log(Prymnesium parvum DNA concentration) - 960 nM - function fit

Primer Concentration 480 nM Results

Figure 7: SHERLOCK results - 480 nM

Figure 8: Final fluorescence intensity to Prymnesium parvum DNA concentration - 480 nM

Figure 9: Final fluorescence intensity to log(Prymnesium parvum DNA concentration) - 480 nM - function fit

Primer Concentration 240 nM Results

Figure 10: SHERLOCK results - 240 nM

Figure 11: Final fluorescence intensity to Prymnesium parvum DNA concentration - 240 nM

Figure 12: Final fluorescence intensity to log(Prymnesium parvum DNA concentration) - 240 nM - function fit

Primer Concentration 120 nM Results

Figure 13: SHERLOCK results - 120 nM

Figure 14: Final fluorescence intensity to Prymnesium parvum DNA concentration - 120 nM

Figure 15: Final fluorescence intensity to log(Prymnesium parvum DNA concentration) - 120 nM - function fit

Conclusions

Based on figures with SHERLOCK plots (Figure 4, 7, 10,13) neither 200 fM nor 20 pM target DNA was detected in 120 nM RPA primer concentration – this is the first primer concentration that we reject because it would not provide sufficient sensitivity.

Among other tested options (240, 480 and 960 nM) the 960 nM emerges as the most favorable option. This conclusion is based on the observation that it exhibited the highest R² value, suggesting that the relationship between initial DNA concentration and fluorescence intensity is more proportional at this concentration compared to the lower concentrations of primers. 

Additionally, while all tested concentrations showed signs of fluorescence signal saturation at 200 nM, the saturation effect was least pronounced at 960 nM. This indicates that the 960 nM concentration may provide a broader dynamic range for fluorescence detection, allowing for more accurate quantification of target DNA concentrations. It is worth noting that although the 200 fM concentration was not detected in the 960 nM set, we decided not to be overly concerned about this result. It is possible that an error occurred during the preparation of the RPA mix, which could have led to the absence of a detectable signal.

It is recommended to repeat this test to rule out any potential operator error. If the results remain inconsistent between runs and lack proportionality, it would be advisable to conduct the test using a primer concentration of 480 nM. Unfortunately, this could not be attempted due to time constraints.

According to the article literature [10], there may be other reasons why RPA is challenging to quantify. The authors suggest, based on their model and laboratory experiments, that commercial RPA kits are optimized for rapid amplification, but the amplified DNA might show a more proportional relationship with the initial target DNA concentration when the kit is diluted (e.g., 2x).

In summary, to achieve quantification with the SHERLOCK assay, the following steps are recommended:

• Evaluate RPA proportionality using direct methods, such as fluorescence dyes [10]:

  • Repeat the evaluation using the same primers and target DNA concentrations as tested before.

  • Test with a 2x-diluted RPA kit. [10]

• Once a proportional relationship between the initial target DNA concentration and post-amplification DNA is confirmed across a range of initial concentrations, proceed with SHERLOCK tests as outlined in “Limit of Detection” section, Attempts 1 and 2.

Limit of Detection

Based on the optimal primer concentration established in the previous assay (960nM), we conducted an additional test to determine the final Limit Of Detection (LOD) for our assay.

The experiment below was conducted to:

1. Determine the final LOD for our test.

2. Establish the range of DNA concentrations for which the relationship between log₂[target DNA] and fluorescence intensity is linear, allowing for quantification of target DNA concentration in the test sample.

3. Test two genomic samples to verify if genomic DNA isolated from the cultures can be detected using the chosen primer-PrymcrRNA pair.

To verify if RPA combined with SHERLOCK can be used for quantitative sample testing, we designed tests in Attempt 1 and Attempt 2 to assess the repeatability of both reactions:

• RPA: In Attempt 2, we used a separate RPA reaction (but on the same algal template) compared to the test from Attempt 1. This was done to check if RPA reactions conducted under the same conditions produce a comparable amount of target DNA for subsequent SHERLOCK reactions.

• SHERLOCK Reaction: In Attempt 2, we conducted the SHERLOCK assay in two separate runs (test duplicates). Both runs used the same RPA mixes from Attempt 2, so any differences in the fluorescence signal would be due to the kinetics of the SHERLOCK reaction. This test aimed to determine if the average final fluorescence intensity signal corresponding to a particular target DNA concentration can be used to quantify target DNA in test samples, potentially eliminating the need for a standard curve in every test.

Methods:

1. RPA reactions were set up, with different template DNA concentrations: 200 nM, 20 nM, 2 nM, 200 pM, 40 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, 200 fM, 20 fM and 2 fM, as well as genomic DNA isolates, coming from water samples: L1 (80.6 ng/μl) and Szczecin algal culture (7.3 ng/μl). For template dilutions, a PCR product coming from an algal culture was used.

2. The SHERLOCK assay was conducted on the following samples in 2 separate runs (duplicate of the test):

Table 2: SHERLOCK reactions components

Results

Attempt 1

Lower concentrations set of DNA

Figure 16. SHERLOCK results — limit of detection at the lower concentration set of the Prymnesium parvum DNA.

Higher concentrations set of DNA

Figure 17. SHERLOCK results — limit of detection at the higher concentration set of the Prymnesium parvum DNA.

Attempt 2

Lower concentrations set of DNA

Figure 18. SHERLOCK results — limit of detection at the lower concentration set of the Prymnesium parvum DNA.

Higher concentrations set of DNA

Figure 19. SHERLOCK results — limit of detection at the higher concentration set of the Prymnesium parvum DNA.

Conclusions:

1. Determining the Range of DNA Concentrations: Despite optimizing the RPA primer concentration, it is not possible to define a range of target DNA concentrations that are proportional to the final fluorescence intensity.

2. SHERLOCK Reaction Repeatability: The SHERLOCK reaction proved to be unrepeatable. Different fluorescence intensities were observed for samples from the same RPA, and varying limits of detection (LOD) were determined for test duplicates: 1 pM for the first test and 200 fM for the second. This inconsistency undermines the ability to compare fluorescence intensities between both attempts.

Given these observations, the test does not appear suitable for quantitative measurements. If a range of concentrations proportional to fluorescence intensity could be established (point 1), then creating a standard curve for each test would provide reference points for quantifying DNA concentration.

However, we can conclude that the limit of detection (LOD) of our test is 1 pM. This concentration corresponds to 5 × 10¹⁰ Prymnesium parvum cells per liter of water. Additionally, we successfully detected Prymnesium parvum in the genomic sample (without prior PCR amplification) isolated from the “Szczecin” culture. These findings suggest that SHERLOCK can be used for screening the presence of Prymnesium parvum in water with a detection limit of 1 pM. These results indicate that our SHERLOCK assay, with an LOD of 1 pM, is capable of detecting Prymnesium parvum concentrations as low as 5 × 10¹⁰ cells per liter, making it a useful tool for screening water for the presence of this algae.

PrymFlow

Introduction to the Lateral Flow Assay

The SHERLOCK method, like other CRISPR/Cas-based detection techniques, is compatible with a Lateral Flow readout format. The Lateral Flow Test is known for its speed, simplicity, and ease of interpretation. We aimed to adapt the previously optimized SHERLOCK components for use with Lateral Flow dipsticks, resulting in the development of the PrymFlow test for detecting the presence of Prymnesium parvum.

LFA Result Interpretation

If the target is present, the T-line becomes visible, indicating a positive result. Meanwhile, the C-line serves as a control that is less visible when greater amounts of the target sequence are present. Gold nanoparticles (GNPs) with anti-FAM antibodies provide the visual indication of the test result.

WHAT HAPPENS WHEN Prymnesium parvum IS NOT PRESENT IN THE WATER SAMPLE?

Streptavidin, immobilized on the C-line, captures the biotin-labeled ends of the intact reporters. The reporters are captured on the C-line, and the binding of gold nanoparticles (GNPs) conjugated with anti-FAM antibodies makes only the C (control) line visible.

AND WHEN IT IS PRESENT?

The reporters are cleaved by the activated Cas protein. Consequently, gold nanoparticles (GNPs) with anti-FAM antibodies capture the FAM-labeled fragments, which then bind to the anti-anti-FAM antibody immobilized on the test line (T-line), producing a strong signal. The presence of the T-line indicates the presence of Prymnesium parvum. Some intact reporters might remain in the mix (the amount depends on how many target DNA molecules were present in the sample, influencing the ratio of activated to non-activated Cas13 protein and the number of cleaved reporter molecules). As a result, a weak control line (C-line) is visible.

Figure 20. Explanation of the lines visible on the Lateral Flow test during a negative and positive result.

The LFA tests were conducted following the protocol, which can be accessed here.

Figure 21. Sample description by strip number: 5. Negative control for ModF-ModR + PrymCrRNA1, 6. Negative control for ModF-ModR + PrymCrRNA2, 7. Prymnesium parvum DNA ModF-ModR + PrymCrRNA1, 8. Prymnesium parvum DNA ModF-ModR + PrymCrRNA2, 11. Positive control.

Both test samples containing Prymnesium parvum DNA, amplified using the ModF and ModR (BBa_K5087003) primer pair, yielded positive results (two lines visible). Sample (7), where the Mod primers were paired with PrymCrRNA1, displayed a more intense band compared to Sample (8), which used PrymCrRNA2.

Note: The remaining LFA strips pertain to other BioBrick parts or are not relevant to the conclusions presented here.

Figure 22. Sample description by strip number: 2. Prymnesium parvum DNA ModF-GalR + PrymCrRNA1, 14. Negative control for ModF-GalR + PrymCrRNA1/2.

The test sample containing Prymnesium parvum DNA, amplified using the ModF and GalR (BBa_K5087001) primer pair, yielded positive results (two lines visible), while negative control was indeed negative confirming effective operation of the ModF-GalR primer pair with PrymCrRNA1.

Note: The remaining LFA strips pertain to other BioBrick parts or are not relevant to the conclusions presented here.

PrymFlow — Limit of Detection

The purpose of this experiment was to determine the final Limit of Detection (LOD) for our LFA test.

The Limit of Detection (LOD) was determined as the lowest concentration of PCR-amplified Prymnesium gDNA for which the test result remained positive, indicated by the presence of a visible T-line. The RPA primers chosen were ModF and GalR and the crRNA used was PrymCrRNA1.

Conclusions:

• Tests 1-13 were conducted using progressively decreasing amounts of Prymnesium parvum DNA, as reflected by the corresponding decrease in test band intensity. By comparing these with test 16, a negative control, it can be estimated that the limit of detection (LOD) for our Sherlock assay is approximately 10 pM, as indicated by test 7. However, this conclusion is somewhat subjective, given that band intensity was not quantified. To obtain a more precise and reliable LOD determination, this test should be repeated.

• The synDNA positive control (stripe 17) yielded a positive result. However, the test performed on older RPA samples previously provided a more definitive positive result (C-line was not visible). This suggests there may be inefficiencies in the RPA procedure or potential contamination of its reagents, which could be affecting the consistency of the results.

• The tests for both the water DNA isolation and culture samples returned negative results, indicating that Prymnesium parvum DNA was not detected in these samples. This is likely due to unidentified issues within the procedure, possibly linked to the RPA step or sample handling.

Detection of Prymnesium Types through Genomic Analysis

Through genomic analysis, we have determined which types of Prymnesium our primer and crRNA combinations can detect.

Classification of Prymnesins

Prymnesins are classified into three distinct types based on the structure of their carbon backbone [8]:

• A-type Prymnesins: Have the largest carbon backbone with 91 carbon atoms. They are the most potent, exhibiting significant ichthyotoxic (fish-killing) properties.
• B-type Prymnesins: Feature a carbon backbone of 85 carbon atoms. They lack certain ring structures found in A-types and are generally less potent, though still toxic.
• C-type Prymnesins: Possess the shortest carbon backbone with 83 carbon atoms and show significant structural diversity. Their toxicity is variable and less well-characterized compared to A- and B-types.

Table 3: Primer Combinations with ModF and Their Detection Capabilities

Table 4: crRNA Combinations and Their Detection Capabilities

Sequence

Primer and crRNA Collection Binding Sites

Here, we illustrate the positioning of all primers and crRNAs in our PrymDetect Toolkit on the ribosomal cistron of Prymnesium parvum genomic DNA.

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

Sequence Source and Design

This sequence was introduced by Luo et al. [6] and is intended for ITS sequencing of Prymnesium parvum. ModF is an unmodified version of the PR-RPA-4-F primer described in their publication. According to the authors, PR-RPA-4-F and its complementary PR-RPA-4-R primer were chosen as the best options for RPA reactions.

Resources

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• [6] Luo N, Huang H, Jiang H. Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick. Frontiers in Marine Science, 9. Available from: https://doi.org/10.3389/fmars.2022.1032847
• [7] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
• [8] Binzer SB, Svenssen DK, Daugbjerg N, et al. A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum. Harmful Algae. 2019;81:10-17. doi:10.1016/j.hal.2018.11.010
• [9] Jonathan S. Gootenberg et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6.Science360,439-444(2018).DOI:10.1126/science.aaq0179
• [10] P. Valloly and R. Roy, “Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification,” Anal Chem, vol. 94, no. 40, pp. 13897–13905, Oct. 2022, doi: 10.1021/acs.analchem.2c02810