Part:BBa_K5087019
PrymCrRNA1 spacer template
Introduction
This part is a spacer template sequence used as a building block to create a functional crRNA molecule through in vitro transcription. The resulting crRNA, named PrymCrRNA1, is specifically designed to detect Prymnesium parvum, a harmful algal species. This spacer targets the ITS2 sequence in the genome of Prymnesium parvum.
Biology
The ITS sequences
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. This variability makes the ITS2 region an effective target for designing species-specific primers [2] and crRNA molecules.
The crRNA
CrRNA (CRISPR-RNA) is the molecule that guides Cas13 proteins of the SHERLOCK [3] (Specific High Sensitivity Enzymatic Reporter Unlocking) platform to their specific targets, making it essential for accurate identification of the targeted sequence.
The crRNA consists of two key components: the DR (direct repeat) loop and the spacer sequence. The DR loop is crucial for attaching the Cas13 protein to the crRNA molecule, while the spacer is a 28-nucleotide programmable sequence complementary to the detection target. It enables the Cas13 protein to be accurately guided to the target for precise identification of the target sequence.
Design
We designed this spacer sequence to build PrymCrRNA1. This spacer targets a 28-base-pair fragment of the ITS2 sequence from Prymnesium parvum, which we obtained through Sanger sequencing. The DNA was extracted from an environmental sample our team collected and cultured in the lab. After isolating the DNA, we confirmed the presence of Prymnesium parvum via PCR using specific primers GalF (BBa_K5087000) and GalR (BBa_K5087001) [2]. The sequencing of the 132 bp PCR product with these same primers gave us the sequence we used as a target for our crRNA spacer.
We designed the crRNA spacers based on our own sequencing data instead of relying on existing databases because, at the time, the ITS2 sequence of Prymnesium parvum from the Oder River wasn't yet available. Given the genetic variability of Prymnesium strains across different regions and the existence of three distinct genetic types (A, B, and C) [4], it was important for us to ensure the crRNAs were perfectly complementary to the ITS sequence of the specific strain that caused the disaster in the Oder River. This precision was crucial, especially because the SHERLOCK method is highly sensitive—even a two nucleotide mismatch between the target and crRNA would result in detection failure [3].
Figure 2. Sequencing data. The fragment of the genomic DNA targeted by the PrymCrRNA1 spacer is highlighted in blue.
Genomic Analysis
As mentioned above, when we first designed our crRNA spacers, there was no data available on the ITS sequence of the Prymnesium parvum Oder strain. To verify our results, we compared the sequences obtained from our sequencing with those available in the databases using an online nucleotide BLAST search. Our search identified the presence of a couple of high-score (Per. Iden 100%) results. Top results included Prymnesium strains:
- KAC39 type B
- UIO223 type B
- ARC140 type B
- SAG18.97 type B
- K0374 type B
- K0081 type B
All of these strains produce type-B prymnesins [4] and all are European strains apart from ARC140. KAC39 originates from a Norwegian sample, while SAG18.97 comes from a German sample, both from countries geographically close to Poland. Notably, KAC39 is identical to SAG19.97 when comparing the available nucleotide sequences.
All six type B Prymnesium strains mentioned above had identical sequences across the entire 672-nucleotide ITS1-5.8S-ITS2 fragment. Since this fragment is the same for all six strains, it was impossible to determine which specific strain we were working with based solely on the 132-nucleotide sequencing data that was contained within this identical 672-nucleotide fragment. From a sequencing standpoint, it didn’t matter which strain we used, as all type B Prymnesium strains were identical in this region. Given this, we decided to proceed with the KAC39 (GenBank: MK091113.1) strain for further experimentation.
However, after rerunning the BLAST search on our sequencing product in August 2024, we obtained an additional result: the ODER1 (GenBank: CP154518.1) strain of Prymnesium, which had its sequence published recently [5] and was added to the NCBI database on 06.07.2024. This came after we had already started developing our test based on the KAC39 sequence.
However, we confirmed that the KAC39 fragment is 100% identical to the ODER1 sequence, proving that our previous analyses were based on solid data. This further confirms that the design of our crRNA spacers to target the Oder strain of Prymnesium parvum, even with the limited data available at the time, was accurate and that our spacers were correctly designed for the Prymnesium parvum strain from the Oder River.
Figure 3. Positioning of crRNA Spacers. A depiction showing how the two crRNA spacers, designed by our team, are positioned to target the ITS2 sequences in the genome of Prymnesium parvum ODER1 (GenBank: CP154518.1) strain.
Detection Capabilities
Our genomic analysis has identified which types of Prymnesium can be detected with our crRNA spacers. This determination is based on the ITS sequence, which corresponds to the specific type of prymnesin toxin produced by the algae [6].
Classification of Prymnesins
Prymnesins are classified into three distinct types based on the structure of their carbon backbone [4]:
- 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: Detection Capabilities of the PrymCrRNA spacers
| crRNA Combination | Target Region | Detected Types |
|---|---|---|
| PrymCrRNA1 | ITS2 | Type A and B |
| PrymCrRNA2 | ITS2 | Type A, B, and some C |
Part Performance
The performance of this basic part is described on the PrymCrRNA1 (BBa_K5087022) composite part page (under the sections "Test: Experimental Validation" and "PrymFlow").
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 15
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
References
[1] White, T.J., Bruns, T., Lee, S., & Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications Academic Press. Published online 1990.
[2] Galluzzi L, Bertozzini E, Penna A, et al. Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol. 2008;46(2):261-266. doi:10.1111/j.1472-765X.2007.02294.x
[3] 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.
[4] 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.
[5] Kuhl H, Strassert JFH, Čertnerová D, et al. The haplotype-resolved Prymnesium parvum (type B) microalga genome reveals the genetic basis of its fish-killing toxins. Curr Biol. 2024;34(16):3698-3706.e4. doi:10.1016/j.cub.2024.06.033.
[6] Jian J, Wu Z, Silva-Núñez A, et al. Long-read genome sequencing provides novel insights into the harmful algal bloom species Prymnesium parvum. Sci Total Environ. 2024;908:168042. doi:10.1016/j.scitotenv.2023.168042.
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