Team:ECUST China/Experiments
Overview:
In order to detect the mutated new coronavirus, we can implement the following two schemes, or even a comprehensive scheme of the two schemes.
1. Design different T strands of DNA walkers to recognize the mutated new coronavirus, produce the same CF double strand, which is recognized by E-CRISPR and output the same MB signal, and react in different lanes of the digital microfluidic device.
2. Design different T strands of DNA walkers to recognize the mutated new coronavirus, and produce different CF double strands corresponding to (T), which are recognized and cut by different Cas9 nickases to generate the same MB signal.
3. Design different T strands of DNA walker to recognize the mutated new coronavirus, and produce different CF double strands corresponding to (T), which are recognized and cut by different Cas9 nickases to generate different electrical signals.
As the serial number increases, the greater the experimentally detectable flux, stability will be a huge challenge.
After comprehensive consideration, in the experiment of the DNA walker part, we hope to verify the following hypotheses:
1. Magnetic beads saturation assembly LABC
2. T can be recognized by DNA walker to release CF double strand
3. Determine the detection limit of DNA walker
4. Measure the time required for the DNA walker reaction
Therefore, we designed the following experiment.
1.1 Target sequence design
First, we use the primers of N-SARS-CoV-2 published by WHO and CDC as the prototype to design the sequence T (trigger seq).
1.2 DNA walker design
Secondly, based on the principle of DNA walker and the guiding significance of the modeling results, we designed a preliminary sequence. After consulting Ma Peiqiang, considering the experimental period and stability of DNA walker and other influencing factors, we decided to use the ABCF sequence as shown for qualitative verification and design the LWT sequence. According to the sequence T, the sequence L and the sequence W which are complementary to its part are designed. The final result is shown in the Table S1.
2. Magnetic beads saturation assembly LABC
Add magnetic beads, AL, and BC into the eppendorf tube, and save the corresponding supernatant for PAGE gel electrophoresis. If AL bands appear in the supernatant AL, it proves that the magnetic beads are saturated to modify AL. If the BC band appears in the supernatant BC, it proves that the magnetic beads are saturated to modify the ABC sandwich structure. If there is no band in the supernatant after assembly and washing, it is proved that the magnetic beads LABC is washed clean.
3. T can be recognized by DNA walker to release CF double strand
When the magnetic beads LABC reacted, T was added to the positive control, and the same amount of ddH2O was added to the negative control. Take the supernatant for PAGE gel electrophoresis. If the positive control has a CF band and is brighter than the negative control CF band, the hypothesis is true.
4. Determine the detection limit of DNA walker
Dilute T gradually, and then add it to the reaction. The rest is the same as the experimental scheme of hypothesis 3. If a CF band is observed, it proves that the detection limit designed by the DNA walker is T.
5. Measure the time required for the DNA walker reaction
When the magnetic beads are added to the T reaction, different time gradients are set for measurement.
Overview： In this module, we hope we can confirm that line G can pair with line C instead of line F, and then, sgRNA can recognize it and lead Cas9 nickase to cut line G successfully, producing a relatively obvious change in electric signals. Therefore, we designed a four-part experiment. At first, we tried to modify line G to the electrode efficiently. We set up experimental groups with different modification times and chose a condition that works best for subsequent experiments. Secondly, we directly added line C to the electrode which has been modified with line G, and then we added sgRNA and Cas9 nickase to cut line G. Next, it should be confirmed that line G, instead of line F, can pair with line C, and the cutting will still happen in this system. Finally, we try to make it work on the electrode plate.
Sequence design
In our E-CRISPR system, the main components are line G, line F, line C, sgRNA, Cas9 nickase. Line C is paired with line F when they come into the E-CRISPR module because they are products of the DNA Walker module. And the sequences of them are also the same as which when they are in the DNA Walker module.
Then, line G, which is modified on the electrode plate, will compete with line F to pair with line C because line G has more complementary bases with line C than line F.
After that, we will add sgRNA and Cas9 nickase to this system. The sgRNA recognizing a specific sequence of line G will lead Cas9 nickase to be close to line C and line G. The Cas9 nickase can recognize the PAM site of line C, and cut line G specifically, which conduct the decrease of the signal. It should be noted that there are two PAM sites here, GGG and AGG, so we also designed the experiment to confirm which one is appropriate(refer to “validation on electrode; cut validation of line G”).
The specific sequences of line F, line G, line C, and sgRNA are showed on the blowing picture.
Electrochemical workstation
In the E-CRISPR module, when we conduct the experiments on the electrode plate, we need the help of an electrochemical workstation. It is a small but sensitive equipment that can connect the electrode plate to the computer and read out the change of electric signals. Based on it, we can know that if the competition and cut of line G have happened.
Validation in PCR tube
Competition validation of line G
In this part, we designed six control groups, which perspectively contains line C, line G, line F, line C and line G, line F and line G, and line C and line F, while one experiment group contains line F/G/C.
All of these seven groups were incubated under room-temperature for x hours, and then we verified the result of competition through polyacrylamide gel electrophoresis. The imaging result showed that line G, instead of line F could pair with line C to some extent.
Besides, considering that the competition time is too long, we try to increase the efficiency of competition by heating and conjecture based on this result that the reason for the low-efficiency of competition is the low-efficiency of melting.
As we expect, melting and annealing can make the competition effect reach the same level as long-term incubation in a shorter time. So, solving the melting problem is our plan.
Cut validation of line G
In this part, we designed one experiment group that we directly added sgRNA, Cas9 nickase, and buffer to the system which only contains line G and line C, and three control groups. We perspectively only added sgRNA, Cas9 nickase to two of them, and added nothing to another one. The result, which was verified through polyacrylamide gel electrophoresis, showed that it was successful. In brief, the cut can be realized.
Systematic validation
In this part, we added line F and line C into the system at first, after an hour, we added line G and still incubated the same time. Then, sgRNA and Cas9 nickase was added and reacted for 2 hours. Through polyacrylamide gel electrophoresis, we found that the cut could not be successful anymore. Considering that in the “competition validation” part, line G can compete with line F and pair with line C to some extent, a small amount of line G could be cut by Cas9 nickase. So, we conjecture that the line F solution or buffer were not pure enough. We may use a more pure line F solution and buffer to continue the subsequent experiments.
Validation on the electrode
Optimization of line G-modification time
We first carried out the detection experiment of the optimal modification time of line G on the electrode plate. We designed 8 groups of electrode plate experiments with different modification time of 2hr, 4hr, 6hr, 8hr, 10hr, 12hr, 24hr, and 36hr. The electric signal after the modification was detected. According to the baseline and peak value of the electric signal and considering the significance of the experimental results and the experimental period, we chose 2hr as the modification time for our follow-up experiments.
It can be seen from the figure that with the increase of the modification time, the detected electric signal gradually increased but the tendency is not worthy of prolonging the reaction time. Significant results could be obtained in the experiment, so considering the arrangement of the experiment cycle, we chose 2 hr for follow-up experiments.
The decline of the electric signal
We first used line G to modify for 8 hours, then blocked it with MCH for 45 minutes, eluted, and measured the signal for the first time. Then we continued to measure the electric signal kept in the buffer at regular intervals until the electric signal dropped below 50% of the first measured value.
By measuring the attenuation of the electric signal after modification, it further explained that when the cutting verification was performed. The measured electric signal decreased by more than 10%, which was caused by the successful cutting.
From the figure, it can be seen that at about 54hr, the electric signal dropped below 50% of the first measured result.
Cut validation of line G
We first used line G to modify for 2 hours, then blocked it with MCH for 45 minutes, eluted, and added complementary strands for 1 hr. And we performed the first signal measurement after elution. Then, we added sgRNA and Cas9 to cut the chain and performed the second signal measurement after elution. For some reason, this validation is a partial success, and we tried to draw a conclusion of this outcome.
Systematic validation
The reaction system in this part was the same as the system of systematic validation in the EP tube, but line G was modified on the electrode plate. Through the change of signals, we can estimate whether the cut is successful or not. But we haven’t completed the validation of this part, we will continue to work on this in the future.
For information about the experimental protocol, please click here.