The Summary
After several iterations of the engineering design cycle, we finally designed six pairs of primers based on verified hydatid DNA sequences and six sgRNAs based on PAM sequences of CRISPR-Cas12a. We mixed six primers to detect echinococcosis sequences by RPA and CRISPR coupling. The experimental results show that the method can accurately detect free echinococcosis DNA with a high detection rate.
Iteration of the engineering design cycle
We designed 16 primer pairs for the characteristic fragments of Echinococcus and confirmed that 14 pairs can amplify the characteristic fragments in subsequent experiments. Considering whether the corresponding fragments can design sgRNA and amplification effect, we finally selected 6 effective primers to perform multiple primer amplification, and explored the stable system of multiple primer RPA amplification, namely to ensure a single each primer concentration of 0.3 μ M.
Fig 1. RPA amplification results of 16 primer pairs
For the amplification products of the characteristic Echinococcus fragments, we designed 13 sgRNA, and the sgRNAs corresponding to mgs4, mgs5, mgs6, mgs9, mgs10 and mgs14 fragments were highly active.
Since the cfDNA in the patient's blood often exists in small, randomly broken fragments, we hope to improve the true-positive rate by detecting multiple sequences simultaneously.
In our project, it is divided into AND and OR two parts.
Fig 2. CRISPR-Cas12a shear fluorescent reporter
And
Our AND partially consists of the RPA system and the CRISPR Cas12a system. After repeating practical experiments, we have achieved the first multi-primer pairs isothermal amplification and multiple sgRNA-guided CRISPR Cas12a detection in the iGEM project since 2020.
1.RPA system
| Agentia |
Dosage/μl |
| Buffer R2.1(10×） |
0.6 |
| DEPC water |
3.6 |
| Cas protein (10 μM) |
0.3 |
| sgRNA (250 nM) |
1.5 |
| 37 ℃ 15 min |
| 15 nt probe (10 μM) |
4 |
| add 9μl after mixing |
| substrate |
1 |
| Buffer R2.1(1×） |
40 |
| 37 ℃ 30 min |
2.Crispr-Cas12 system
| Agentia |
Dosage/μl |
| Buffer R2.1(10×） |
1.2 |
| DEPC water |
7.2 |
| Cas protein (10 μM) |
0.6 |
| sgRNA (250 nM) |
3.0 |
| 37 ℃ 15 min |
| 15 nt probe (10 μM) |
3.0 |
| add 9μl after mixing |
| substrate |
1 |
| Buffer R2.1(1×） |
40 |
| 37 ℃ 30 min |
If the substrate was E gene,add 1μl sgRNA and 4.1μl DEPC water
The RPA and CRISPR methods shown in the table are the results of our improved system. We continued to explore the reaction time, ratio and temperature of the system, and after asking medical workers who had actually used RPA for nucleic acid detection, we developed a preincubation method for amplification.
After several experiments, the method can significantly improve the success rate and accuracy of RPA, so that the results meet the experimental expectation. It also has a significant effect on subsequent CRISPR-CAS cutting.
By coupling RPA with CRISPR, we were able to detect cfDNA at room temperature without instrument dependence.
Or
In the conventional amplification technique, only one pair of primers was selected. In order to improve the detection rate and accuracy as high as possible under the condition of room temperature without instruments, we screened out 6 pairs of primers with basically no interaction, and then tested them after mixing them in an appropriate proportion.
Our OR part consists of 6 primer pairs and the sgRNA targeting the corresponding sequence, and the simultaneous detection of multiple sequences increases our true-positive rate from 40% to 70%.
As long as any pair of primers are successfully combined with the target sequence, they can be reported in the subsequent system to achieve low requirements, high coverage and high detection.
