Fragment for gRNA1
The CRISPR/Cas13a system is the first step of our diagnostic approach, as it can be designed for the specific recognition of our microRNA biomarkers. It consists of two parts: Cas13a nuclease and CRISPR RNA (crRNA). The crRNA directs Cas13a to single-stranded RNA targets with high specificity through sequence complementarity, making the CRISPR/Cas13a system suitable for the recognition of our microRNA biomarkers. When Cas13a binds its RNA target, it becomes activated. Activated Cas13a not only cleaves the target RNA but also performs “collateral” cleavage of nearby non-target RNAs. This property allows the generation of detectable signals even at low biomarker concentrations. It also enables real-time monitoring and increases the sensitivity of detection. (1) To maximize sensitivity of our approach, we selected a Cas13a ortholog with high collateral cleavage activity. Through literature research, we found that among fifteen Cas13a orthologs, the ortholog from Leptotrichia wadei (LwCas13a) was the most active with high trans-cleavage activity. (2)
To design crRNAs for our CRISPR/Cas13a system, we based our approach on both our selected microRNA biomarker sequences and existing literature. Each crRNA consists of two key regions:
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC (3)
We constructed the plasmid for the expression of our CRISPR/Cas13a system by using as vector Cas_variants_hLwCas13a from the iGEM Distribution Kit 2025. This vector contained the gene of a variant of Cas13a, a high copy origin of replication and a gene for antibiotic resistance to Chloramphenicol. We designed the two fragments, that contained the gRNA genes each:
BBa_J23101 and BBa_J23100: strong, constitutive promoters for the continuous expression of gRNAs and hLwCas13a protein respectively.
Bacterial terminator for the end of gRNA genes transcription
gRNA genes
Ribosome binding site downstream BBa_J23100 promoter for better Cas13a expression
6xHis Tag for subsequent protein purification
After cloning, we inserted our plasmid into Rosetta 2(DE3)pLysS cells for the expression of our CRISPR/Cas13a system. This cell line is derived from E.coli BL21 and is suitable for the expression of proteins that contain codons rare for E.coli, thanks to the extra tRNAs encoded on a chloramphenicol-resistant plasmid (pRARE2). Since our expression vector also carries chloramphenicol resistance, the antibiotic selection pressure ensures maintenance of both the expression plasmid and the rare-tRNA helper plasmid. 24 hours after transformation we isolated DNA from single colonies to verify whether our plasmid was right.
We expressed our system by inoculating bacterial colonies with our plasmid into liquid LB. We performed one small starter culture, expecting overnight growth. The day after, we added the culture into bigger LB quantity to ensure higher protein yield. The use of constitutive promoters allowed for continuous expression of LwaCas13a without the need for induction.
By adding a 6xHis affinity tag to the N-terminal end of our Cas13a protein we aimed to ensure purification by using HisPur Ni-NTA columns. A 3 amino acid linker between the His Tag and the protein was added for subsequent cleavage of the His Tag after purification using a TEV protease. The Ribosome Binding site ensures that translation of Cas13a will occur in the right place. We verified the results of the purification with a 10% SDS PAGE electrophoresis.
In the Catalytic Hairpin Assembly participate two partially complementary DNA hairpins, DNA hairpin probe 1 (H1) & DNA hairpin probe 2 (H2) and one single-stranded oligonucleotide (Initiator). The hairpin motif of H1 contains three concatenated domains and each domain has a special nucleation site, called toehold.
The hairpin motif of H2 contains two continuous domains, each of them has also a toehold. These domains are designed to be complementary to two of the H1 domains: *=complementary
a* domain with toehold at* (complementary to H1 domain a)
b* domain with toehold bt* (complementary to H1 domain b)
The hairpin probes in our system were designed based on the initiator sequence. The initiator is a single-stranded oligonucleotide that serves to trigger the Catalytic Hairpin Assembly (CHA) reaction. More specifically, the initiator is part of a non-specific DNA hairpin 0 for CRISPR/Cas13a and is released after its activation upon biomarker recognition and the subsequent collateral cleavage activity. Hairpin H0 differs from H1 and H2. It has a DNA stem and an RNA loop that serves as a collateral cleavage site for Cas13a. To maximize Cas13a activity, the loop contains a poly-U region, as LwCas13a collateral cleavage activity is enhanced on UU base motifs (5).
The initiator is released after cleavage of H0 in the polyuracil region inside its loop. The initiator binds to the complementary a domain of H1 via its at* toehold. This nucleation triggers a branch migration that opens H1.The opened H1 has now the domains b and c exposed,along with the toeholds bt and ct. The bt toehold can then serve as binding site for hairpin H2.A disassembly reaction occurs when single-stranded domain a* of H2 initiates a branch migration that displaces the initiator from H1. (3)
The sequence of H2 probe was designed based on H1, with two complementary domains (a* and b* domain of H2 complementary to a and b domain of H1). Domain a* overlaps with the initiator-binding site on H1, ensuring that once H2 binds, the initiator is displaced and recycled. The H1–H2 duplex is designed to be more stable than the ternary Initiator–H1–H2 complex, ensuring efficient turnover. (6)
GC content and nucleotide lengths were tuned to balance thermodynamic stability and complementarity, rather than strictly following conventional design rules. (6)
Toehold: 9nt, <40% GC content
Stem: 11nt, close to the optimal of 12nt. It has a high GC content > 60%
Loop: 10nt, <40% GC content
Modification: 3’ sulfide group for Au electrode immobilization
Toehold: 7nt, < 40% GC content
Stem: 10nt
Loop: 11nt
Modification: 5’ methylene blue (MB) label
Each hairpin probe was analysed with NUPACK software to check the stability and thermodynamic properties. READ MORE
The CRISPR/Cas13a reaction was performed in a reaction buffer at 37 °C for 1 hour. The system included the Cas13a protein, H0 hairpin, and the target miRNA biomarker. Two separate reactions were conducted: one targeting miR-150-5p and the other miR-486-5p. Following the reaction, the resulting mixture was transferred into a solution containing H1 and H2 hairpin probes, allowing the Catalytic Hairpin Assembly (CHA) to proceed and form H1–H2 duplexes.
To assess the efficiency of both the CRISPR/Cas13a reaction and the Catalytic Hairpin Assembly (CHA), we performed 3% agarose and native PAGE electrophoresis. These analyses allowed us to verify whether the H1–H2 duplexes were formed following initiator release. In the presence of the target miRNAs, Cas13a activation cleaves the H0 hairpin, releasing the initiator that triggers CHA and drives H1–H2 duplex formation. The H1–H2 duplex appears as a distinct band at approximately 80 nucleotides, while unreacted hairpins (H1, H2, and H0) migrate at around 40 nucleotides. In the absence of the target miRNAs, no initiator is released, and no duplex band should be observed confirming the specificity of the CRISPR/Cas13a-CHA system.
We analyzed the electrophoresis gels using Fiji software to quantify the band intensities corresponding to the H1–H2 duplexes. A strong duplex band indicates efficient CHA amplification and successful CRISPR/Cas13a activation. By comparing the band intensities in reactions with and without target miRNAs, we assessed both the specificity and efficiency of our system. A significant increase in the 80-nt duplex band intensity in the presence of target miRNAs confirms that the Cas13a-mediated initiator release effectively triggered the Catalytic Hairpin Assembly. Additionally, analyzing the relative duplex band intensity provides insight into the quantitative response of the system toward different miRNA concentrations.