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Welcome to the proof of concept page!

We implemented the technology to accurately and rapidly cleave individual bases and generate fluorescent proteins in the wet lab, and developed matching hardware based on the results we achieved in the wet lab. As a result, we have successfully accomplished our goal of developing a portable, single-base, amplification-free nucleic acid detection platform based on CRISPR/cas proteins with shackle sequences. You will see how we accomplished this proof of concept on this page.

Overview

Due to the tolerance of Cas13a and Cas14a proteins to 1-2 base nucleotide polymorphisms in the target sequence, the efficiency of cleavage of Cas proteins in the detection system is greatly reduced, and incorrect recognition and cleavage will also lead to "false positives" in the detection. Thus, we can assume that if the detection system contains a certain concentration of SNPS target sequences, it will be difficult to distinguish whether the detection signal comes from the target fragment that we want to track (the difference is a thousand miles). Therefore, it is essential to accurately and effectively "block" the interference of the mutated nucleic acid fragment of the target gene.

We will improve the accuracy of target sequence detection and enhance the specificity of the detection system by artificially designing Peptide nucleic acids (PNA) to base complementary pair with single base mutated target sequences to achieve single base recognition detection. This solution is an improvement to the existing nucleic acid detection methods of CRISPR/Cas systems (Cas13a and Cas14a), and additionally the csm6 protein is capable of signal amplification for detection of Cas13a and Cas14a proteins [1].

We will show some facts about the proof of concept based on the project implementation plan and matching experiments with corresponding results. The validation of expectations by these experimental results ensures the reliability and feasibility of our project.

Expression and purification of protein

The Cas13a1 and Cas14a1 proteins, as well as the csm6 protein used to release fluorescent signals, were expressed by plasmid-transformed E. coli BL21 (DE3) and purified for our experiments. After our repeated experiments, we successfully obtained the target proteins using BL21 (DE3) expression, and successfully purified Cas13a, Cas14a and csm6 proteins using the protein purification instrument belonging to the State Key Laboratory of Marine Resources Utilization in the South China Sea.

The two graphs show the protein purification real-time monitoring data and the csm6 protein gel electrophoresis results respectively (same for Cas13a and Cas14a proteins)

Protein activity validation

Activity validation of cas13a1, cas14a1 protein, and csm6 protein for signal reporter

After successful purification of the proteins, we determined that the activities of all three proteins were at high levels (Fig.3a,Fig.3b), and the enzymatic cleavage activities of the two proteins were also tested, and the results also met our expectations for the proteins (Fig.3c).

Confirmation of “false positive” characterization

Validation of false-positive characterization due to Cas13a1, Cas14a1's own misidentification of nucleic acid sequences

To verify the false-positive characterization of Cas13a1 protein, we designed the single nucleotide polymorphic sequence of Target RNA (Fig.2a) to simulate the false-positive recognition characterization generated in the real situation. The recognition and cleavage of the artificially designed RNA by the Cas13a1 protein, and thus the trans cleavage activity exhibited, and thus the fluorescence signal reported, we show as Relative Fluorescence unit (RFU). We can observe that each detected sequence exhibits a different fluorescence signal intensity, and RM8, RM15 and RM19 are significantly larger than the target sequences. This indicates that false positives do exist and have different effects on the specificity of the detection depending on the mutation site (Fig.2b).

We used the same method to verify to the same false-positive recognition characterization of the Cas14a1 protein in recognition of the sequence (Fig 3c, Fig 3d).

Verification of clamp effect

Clamp is a system we envisioned to assist Cas13a and Cas14a proteins to avoid "false positive" characterization caused by single base mutation sequences by artificially designing complementary PNAs (peptide nucleic acids) of single base mutation sequences to shield them from interference in the assay system.

Taking Cas14a1 as an example, based on the experimental results of Step 2 (Confirmation of "false positive" characterization), sequences with mutation sites at 5'-5, 7, 20-3' bases were selected from a series of sequences with single-base mutations. 5'-5, 7, 20-3' bases, which showed high RFU in the false positive mock assay, were selected as typical sequences to demonstrate the role of the shackle system (clamp).

Initially we used the designed complementary DNA as clamp as a pre-experiment to verify the preliminary role of Clamp. As illustrated by the experimental results presented in Fig 4a and Fig 4 b: the Relative fluorescence unit (RFU) of the experimental group with the addition of DNA-clamp was significantly lower than that of the control group, and combined with the Delta-RFU analysis, Clamp did reduce the interference of single-base mutant sequences on the assay results.

However, DNA as clamp alone is not enough for the goal of our project. So we discovered peptide nucleic acids (PNA), a class of DNA analogs with a peptide backbone replacing the sugar phosphate backbone, as nucleic acid sequence-specific reagents by reviewing the literature [2]. We designed PNA as clamp and validated it in the same way(Fig 4c, Fig 4d). We also compared the control group with DNA-clamp and PNA-clamp to make the data more reliable (Fig 4e). The above two experiments also demonstrated that our idea of designing "Clamp" to avoid the shortcoming of Cas13a and Cas14a in detecting the target sequences due to similar mutated sequences, which leads to misidentification, can be realized.

Although we have verified that PNA as clamp has better effect than DNA as clamp, however, we are not yet sure to what extent the shielding effect of using PNA as clamp on single base mutation false sequences can be achieved, and it is not clear what effect different concentrations of PNA have on the effect of target sequence detection. So, we set a certain amount of PNA (100nM) and gradient concentrations of target and mutant sequences in combination, and found that the interference of PNA on mutant sequences was significantly greater than that of target sequences, and even after the concentration of mutant sequences was less than 100nM, its RFU dropped in a precipitous manner. When PNA was relatively saturated in the mutant sequence, it was able to play an almost complete shielding role. In the absence of PNA addition, the magnitude of RFU change of both was not as obvious as when PNA was added (Fig 4f).

Through further experimental design and verification, we found that the shielding effect of PNA on mutant sequences at a certain concentration of PNA (100 nM) was more obvious at lower concentrations (<100 nM), and the shielding effect of PNA gradually diminished as the concentration of mutant sequences increased (Fig 4g). It can be seen that the interference rate of PNA on mutant sequences is gradually reduced with the increase of the latter concentration until it tends to 0 (Fig 4h).

Validation of Csm6-cas13 tandem assay

Due to the impact of the new crown epidemic, we were unable to complete the wet experimental validation of CSM6 in tandem with CAS13 in the laboratory. Therefore, we simulated the process of CSM6 reacting in tandem with CAS13 and releasing the fluorescence effect in our modeling, and simulated the effect of shielding the mutant chain with PNA shackles. (Only a brief elaboration is given here, please jump to the model page for more details:Click here to jump to the model)

The diagram below shows the schematic diagram of the tandem reaction system of CSM6 and CAS13.

PNA was added to the reaction system to verify the shielding effect of the shackle PNA on mutateRNA. As shown in the figure below, the final interfering term mutateRNA was gradually shielded by PNA with the change of time.

Based on the modeling, we tried various values of the pre-RNA coefficients. As shown below, n=1 and n=2 are consistent with the actual situation, and it can be seen that the fluorescence signal curve is a convex function with decreasing growth rate with time.

Testing Instruments

After determining the study of drug-resistant microorganisms in the ocean, we designed a portable marine drug-resistant microorganism detector for this field, taking into account several aspects such as cost, portability and easy operation process. We also developed a hardware system specifically adapted to our needs for this detector. The system is relatively small, capable modules, mainly divided into a temperature regulation module, an optical path detection module, and an Android screen display module.

1.Temperature regulation module:

In order to monitor the culture temperature inside the heat-conducting aluminum in real time, the special heat-conducting aluminum, the heating film and the temperature sensor are physically connected. Use stm32f103c8t6 master control chip, with pid algorithm to control the temperature rise and final stabilization.

2.Light path detection module:

We use green light source led lamp beads, optical fiber to transmit the light path to the culture room directly below, and use special structural components to isolate each way light source to avoid interference from the bypass; and receive the use of photodiodes.

3.Android screen display module:

To make the workflow clear, we developed a software interface.

Click here to learn more details about our hardware section

Summary

1. After expressing the target proteins using BL21 (DE3) and successfully purifying Cas13a, Cas14a and csm6 proteins, we determined the starting activity and the enzymatic activity of two of them and designed crRNAs, as well as target DNA/RNAs, mismatched DNA/RNAs simulated in the laboratory.

2. We verified the target DNA/RNA with 21 single base mutant strands at different positions and confirmed that Cas13a and Cas14a would cause false positives due to misidentification of nucleic acid sequences,

and the interference effect of their misidentification would be even greater than the signal generated by the recognition of the target sequence.

3.We have completed the simulation of the sampling test in a realistic environment in the laboratory,and designed complementary PNAs of single base mutant sequences to protect them from the interference of the analysis system, thus helping Cas13a and Cas14a proteins to avoid the "false positive" characterization caused by single base mutant sequences. The results showed that the PNA concentration was high enough to shield almost 100% of the single-base mutant sequences, and there was almost no interference with the target sequences to be detected. The experimental results are as expected, and we successfully confirmed the success of the wet experiment part of the project.

4. We achieved accurate and rapid cleavage of single bases and generation of fluorescent proteins in the wet experiment, however, in terms of instrumentation, we also needed a hardware device capable of detecting fluorescent signals. However, traditional enzyme markers are large, heavy and costly, and the human-machine interface is mostly connected to a computer screen and then operated, which is extremely unportable. Therefore, we completed the circuit design and successfully developed a small portable enzyme marker by 3D printing to make our project faster, better and easier to be used.

In summary, we implemented the technology to accurately and rapidly cleave individual bases and produce fluorescent proteins in the wet lab, and based on the results we achieved in the wet lab, we developed the hardware to match - a small portable zymograph. As a result, we have successfully accomplished our goal of developing a portable, single-base, amplification-free nucleic acid detection platform based on CRISPR/cas proteins with shackle sequences.

Outlook

After we successfully completed all our designs for the project, we achieved our goal of a highly accurate and easy-to-use tool for nucleic acid bioassays based on the Crispr/cas method. In the future, we will use it for research on nucleic acid detection technology and for studying drug-resistant microorganisms in the ocean to address microbial disease control, to reduce the occurrence of bioresistant pathogens, and to protect human health and life safety; in terms of exploration, we plan to establish partnerships with school institutions, such as local high schools, to create an extracurricular practice platform to stimulate further exploration of CRISPR In terms of instrumentation, we will further optimize, for example, the shape and temperature sensors.

Reference

[1]Liu, T.Y., Knott, G.J., Smock, D.C.J. et al. Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol 17, 982–988 (2021). https://doi.org/10.1038/s41589-021-00842-2

[2]Egholm M, Buchardt O, Christensen L, et al. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules[J]. Nature, 365(6446): 566-568(1993).