AltR

Part of the PrymDetect Toolkit

Introduction

AltR is a reverse primer specifically designed to amplify a fragment of Prymnesium parvum genomic DNA in an RPA (Recombinase Polymerase Amplification) reaction. It targets the 5.8S rRNA region, binding to the end of this sequence in the genome.

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].

Figure 1. The RPA (Recombinase Polymerase Amplification) Mechanism

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 [6].

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).

Figure 2. The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) Method Mechanism

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.

Figure 3: Fluorescence readout results demonstrating the performance of various part combinations from our toolkit, with the AltR primer serving as the connecting component. For simplicity, the negative controls for both crRNAs are not shown, as they were all negative as anticipated.

Parts Compatible with AltR

AltR performs best when paired with the ModF primer and the PrymCrRNA1 molecule. While it also functions with the AltF primer, its effectiveness is somewhat reduced in comparison.

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 4. 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 5. 1. Negative control for AltF-AltR + PrymCrRNA1, 3. Prymnesium parvum DNA AltF-AltR + PrymCrRNA1, 11. Positive control.

The test sample (3) containing Prymnesium parvum DNA, amplified using the AltF and AltR primer pair in combination with PrymCrRNA1, yielded a positive result (two lines visible).

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

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 [7]:

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 1: Primer Combinations with AltR and Their Detection Capabilities

Primer Combination	Target Regions	Detected Types
Mod_F & Alt_R	5.8S & ITS2	Type B
Alt_F & Alt_R	5.8S & ITS2	Type B
Gal_F & Alt_R	ITS2 & ITS2	Type B

Table 2: crRNA Combinations and Their Detection Capabilities

crRNA Combination	Target Region	Detected Types
PrymCrRNA1	ITS2	Type A and B
PrymCrRNA2	ITS2	Type A, B, and some C

Sequence

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 3
1000
COMPATIBLE WITH RFC[1000]

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.

Figure 6. Positioning of primers and crRNAs from the 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

The term "Alt" stands for "alternative": This sequence was developed by our team on the basis of the KAC39 sequence fragment [8] to create alternative primers to those described by Luo et al [9]. This primer was designed in the Primer-BLAST online tool using directions from TwistAmp® DNA Amplification Kits Assay Design Manual [10].

Resources

[1] White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications. Academic Press.
[2] Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L., & Sereno, D. (2017). Leishmania infections: Molecular targets and diagnosis. *Molecular Aspects of Medicine, 57*, 1-29. https://doi.org/10.1016/j.mam.2016.11.012
[3] Galluzzi, L., Bertozzini, E., Penna, A., et al. (2008). Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. *Letters in Applied Microbiology, 46*(2), 261-266. https://doi.org/10.1111/j.1472-765X.2007.02294.x
[4] Lobato, I. M., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. Trends in analytical chemistry : TRAC, 98, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
[5] Ruichen Lv, Nianhong Lu and Junhu Wang et al. Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens: An Overview. Zoonoses. 2022. Vol. 2(1). DOI: 10.15212/ZOONOSES-2022-0002
[6] 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
[7] 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
[8] Prymnesium parvum strain KAC39 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence GenBank: MK091113.1
[9] 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. 2022;9. Available from: https://doi.org/10.3389/fmars.2022.1032847
[10] TwistDx. TwistAmp® Assay Design Manual. Rev 1, V3. December 2023. Available from: https://www.twistdx.co.uk/wp-content/uploads/2023/12/twistamp-assay-design-manual_rev1_v3.pdf

Part:BBa_K5087005