Overview

Our wetlab activities could be divided into two parts: RT-qPCR and our MB-ERC2 (miRNA biomarker-based exponential RCA Cas12a/CRISPR) system. The RT-qPCR trials were performed as comparison groups for the EXTRA-CRISPR groups. Moreover, through examining the results yielded by RT-qPCR, the golden standard for miRNA detection, we selected miR-223, which gave significantly better performance, as our final target biomarker. The Cas12a-based assay trials demonstrated the efficiency and accuracy of our detection method. Through single-variable experiments, we optimised the reaction system and verified that our standard reaction system has the best reaction kinetics.

RT-qPCR

RT-qPCR (Reverse Transcription Quantitative Real-time Polymerase Chain Reaction), combining RT-PCR and qPCR, involves the detection and quantification of RNA. The procedure involves the enzyme reverse transcriptase converting total RNA or messenger RNA (mRNA) to complementary DNA (cDNA). This cDNA is then amplified and used in quantitative PCR (qPCR) or real-time PCR to detect specific targets. This PCR uses a number of fluorescent chemicals to quantify the amount of DNA at each cycle in real-time. Moreover, the RT-qPCR method has become an essential tool of detecting MicroRNA (miRNA), which is the key indicator for detecting the presence of COPD. Our first RT-qPCR trial starts with the miRNA biomarkers: miR-223 and miR-1274a. They are extracted from a fridge, then undergo centrifugation for 3000r 3min, and dilute with water, and pre-heated in an incubator. Then 4 PCR tubes are labelled miR-223 and miR-1274a. The next step is to add mRQ buffer and mRQ enzymes as well as miRNA samples to respective PCR tubes, all operating on ice. The tunes are then transferred to the incubator for body temperature, an hour. Lastly, they are taken out and placed in a drying oven, with addition of DEPC-treated water, and then stored in the fridge for later usage.

After preparing cDNA, RT-qPCR could then be prepared by 8 PCR tubes (4 each for miR-223 and miR-1274a), with mRQ buffer each tube, miRNA sample respectively and mRQ enzyme each tube. All the tubes are then controlled at 37ºC, 1 h in an incubator. Finally, they are sent to a driving oven, with addition of DEPC-treated water.

RCA

Padlock Design

The ends of the padlock are the miRNA binding sites. They are complementary to our target miRNAs (miR-223 and miR-1274a). The influence of mismatches was investigated with padlocks M1-14 in the Padlock Probe Optimization section below. A cas12a detection zone is also located on the padlock. The detection module contains sequences complementary to the crRNA used in the synthesis of RNP. An optimization of the location of the detection modules was performed with padlocks 2-4 in the Padlock Probe Optimization section.

SplintR Ligation

While the conventional T4 ligase also ligases DNA-RNA hybridization, SplintR is far more efficient in terms of reaction kinetics. Thus, after a comparative trial (see Reaction System Optimization section), we chose SplintR as the ligase used in the system.

RCA Process

The first step in RCA is the annealing of the miRNA to the padlock's miRNA binding sites. Then, SplintR ligase circularises the originally linear-bent padlock. Phi29 DNA polymerase binds to the padlock-miRNA duplex and initiates the amplification of a DNA chain complementary to the padlock (the amplicon).

Cas12a

The enzyme Cas12a is a component of the CRISPR-Cas system, a potent tool for precise genetic modification. Unlike Cas9, Cas12a locates its target DNA sequence using a single RNA guide molecule. Cas12a is used for recognizing the domain, binding to target DNA, and carrying out cleavage. In our detection method, Cas12a is coupled with CRISPR-RNA (crRNA) to form the Cas12a-crRNA ribonucleoprotein complex (RNP). The role of the crRNA is to guide the cas12a enzyme to the target sequence (the RCA amplicon). The RNP ‘scans’ the DNA in order to find a match with the crane sequence, after which it undergoes a conformational change to activate its nuclease.

Trans-Cleavage

The trans-cleavage of Cas12a is nonspecific. Thus, it cleaves both the RCA amplicon, the padlock, and the fluorophore-quencher complex which will be explained shortly. When there is an excess of padlocks or RCA amplicon, trans-cleavage would preferentially cut these instead of reporters. When there is an excess of reporters, false positives may also occur. Therefore it is important to regulate the reporter-padlock ratio.

Cis-Cleavage

Cis-cleavage of cas12a is specific, thus it only cleaves the RCA amplicon. Cis-cleavage amplicons are complementary to the padlocks, and thus could serve as primers for initiating secondary RCA (i.e. the same process as described in the RCA section, only with amplicons in place of miRNAs). Digestion of longer amplicon sequences (long cuts) into shorter amplicons (short cuts) are primarily due to cis-cleavages.

Intermediary Assays

Both the three-step and two-step assays are tandem combinations of RCA and Cas12a’s fluorogenic readout. However, in the three-step method, ligation and rolling circle amplification (RCA) are separate, and the dye SYBR Green II is added (commonly used to stain RNA/ssDNA for qPCR analysis). In the two-step method, RCA and ligation steps are combined, and the fluorogenic readout is solely from the fluorophore-quencher reporting system.

Cis-Cleavage

Cis-cleavage of cas12a is specific, thus it only cleaves the RCA amplicon. Cis-cleavage amplicons are complementary to the padlocks, and thus could serve as primers for initiating secondary RCA (i.e. the same process as described in the RCA section, only with amplicons in place of miRNAs). Digestion of longer amplicon sequences (long cuts) into shorter amplicons (short cuts) are primarily due to cis-cleavages.

MB-ERC2

Schematics

The miRNA biomarker-based exponential RCA Cas12a/CRISPR system combines rolling circle amplification with Cas12a cleavages. The overall reaction schematics are shown below.

Detection Mechanism

Our reporter is composed of a fluorophore linked to a quencher molecule. While the two are connected, the quencher absorbs energy from the fluorophore, thus preventing it from emitting fluorescence. But when cas12a trans-cleavage acts on the quencher-fluorophore and severs their link, the fluorophore would start giving out fluorescence. Thus, the fluorescence level is used to quantify the concentration of miRNA biomarkers in the sample tested.

Reaction System Optimization

• Ribonucleoprotein (RNP) Concentration: Cas12a cleavages significantly influence the outcome of the reactions. Cis-cleavages are important for the initiation of secondary RCAs, thus promoting exponential amplification. Trans-cleavages are vital in terms of expression of the fluorescence signals. Thus, it is important to determine the optimal amount of RNP required to best enhance reaction kinetics. Our standard RNP concentration is 1 nM, thus, we tested the MB-ERC2 system with varied RNP concentrations of NC, 0.5 nM, 2.5 nM, 4 nM, and 5 nM.
• Phi 29 Polymerase Concentration: Phi29 polymerase is important in terms of facilitating the amplification in RCA (i.e. synthesis of the DNA strand complementary to the padlock). On the basis of our standard concentration (0.1 U/µl), we tested NC, 0.05 U/µl, 0.15 U/µl, 0.2 U/µl, and 0.5 U/µl.
• Padlock Probe (PLP) Concentration: as previously mentioned in the Cas12a section, PLP-reporter ratio is vital to the experiment outcome. Padlocks are essential in the step of rolling circle amplification (RCA). Starting from the standard 100 nM, we set up trials with PLP concentrations of NC, 50 nM, 75 nM, 150 nM, and 200 nM.
• Reporter Concentration: PLP-reporter ratio is essential to the results of the assay reaction, moreover, reporters generate the fluorescence signals on which our analytical hardware is solely based. We investigated concentrations of 0.5 µM, 1 µM, 2 µM, and 5 µM (standard is 10 µM).
• Padlock Probe Sequence: in order to determine the PLP sequence that yields the best reaction kinetics, we designed: 1. padlocks with the crRNA recognition site at different locations, and; 2. padlocks with single, double, and triple mismatches at the miRNA binding sites. We have 18 padlocks for miR-223 in total.
• BSA Concentration: different amounts of BSA were added to the reaction system. We used 0.1 mg/mL, 0.3 mg/mL, and 0.4 mg/mL (standard 0.2 mg/mL).
• Synthetic short-cuts: according to He et al. (2019), RCA amplicons of different lengths have different effects on reaction kinetics when put into the reaction system to initiate secondary RCA. This is partly due to the fact that longer amplicons could form linear duplexes with un-ligated padlocks. We created a synthetic short-cut (short amplicon), and put the short-cuts in place of miRNA into the reaction system. Results show that short-cuts do in fact enhance secondary RCA, for short-cut results are markedly higher than ordinary MB-ERC2 fluorescence.
• SplintR and T4 Ligase: According to Jin et al. (2016), SplintR ligase yields significantly better performance than T4 ligase. Thus, we designed this experiment, comparing 5 U/20 µl SplintR with 40 U/20 µl T4 ligase.
• SplintR ligase concentration: SplintR ligase is extremely important for initiating the RCA process. We investigated concentrations of 10 U/20 µl, 20 U/20 µl, 25 U/20 µl, and 50 U/20 µl (standard 5 U/20 µl).
• Buffer selection: we investigated the following buffer combinations: 1. Buffer 2.1 (NEB) + BSA; 2. SplintR buffer + BSA, and; 3. Phi29 buffer + BSA. The SplintR/BSA combination yielded the best results.

Methods

Polyadenylation and Reverse Transcription

We prepared a cDNA synthesis reaction for each RNA sample that can be analysed by PCR. Also, to determine the absolute level of an miRNA using a standard curve, it’s required to generate cDNA from dilutions prepared from a known concentration of synthetic miRNA.

In an RNase-free tube, combine the following reagents:

In a thermal cycler, incubate the tube for 1 hour at body temperature, then terminate at 85ºC for 5 minutes.Moreover, add DEPC water to control the volume so that the cDNA would be ready for miRNA quantification protocols.

The portions are then transferred into 8-row tube distribution. The remaining cDNA is stored in a -20ºC fridge.

Three Step Assay

80 ºC 5 min, 26 ºC 1 min

PCR machine, 37 ºC 2h & 65 ºC 10 min, 10 µl system

37 ºC 2h, 65 ºC 10 min

Add 5 µl RNP & 2 µl reporter:

RNP Preparation

Two Step Assay

80 ºC 5 min, 26 ºC 1 min, 5 µl system

Then transfer to ligation-RCA system (a total of 20µl system) mix 2:

37 ºC 2h & 65 ºC 10 min, 20 µl system

Add 5 µl RNP & 2 µl reporter (10µM):

MB-ERC2(EXTRA-CRISPER)

80 ºC 5 min, 26 ºC 1 min

References

Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection method using SplintR Ligase. Nucleic Acids Research, 44(13), e116. https://doi.org/10.1093/nar/gkw399

He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-023-01033-1