iGEM2017_Munich constructed the part BBaK2323004 to express LwaCas13a protein, and then characterized the target dependent activity of the protein. This year, on the basis work of iGEM2017_Munich, ZJUT_China_B further characterized the base preference of LwaCas13 protein's collateral cleavage.
We learned from literature that Cas13 protein homologues have different cleavage base preference, and LwaCas13a protein's base preference is poly U/AU. Based on our multivirus detecting goal, we designed four kind fluorescent reporters (Table 1). It has a fluorophore on one end and a quencher on the other, the linking RNA nucleotide are AC, AU, GA, and UC respectively. And the TA and GC are DNA nucleotide which could prevent the influence of nonspecific cleavage on the detection results ( since Cas13 protein could cleave RNA nucleotide only). The fluorophores are FAM, Cy5, Texas-Red-X and VIC respectively and the excitation and emission wavelength are optimized according to the filters to combine with our detecting device. This enables us detecting the corresponding extention of the cleavage. We added those 4 fluorescent reporters in one detecting pot, and got the following data.
Figure 1 The base preference cleavage of LwaCas13 protein upon AC, AU, GA, UC
1. Because our former experiment shows that as the excitation goes on, our fluorescent dyes show a certain degree of photobleaching phenomenon, so we decrease the time of excitation. For details of photobleaching phenomenon,see >>Model<<
2. The "negative" is the group that we didn't add target while the "experiment" is the group where we add our target.
As shown in Figure 1, the results suggest that LwaCas13a protein could cleave AU and AC oligonucleotide even though there is no target in the detecting system which we called "the leakage activity of Cas13 protein ". Obviously, the LwaCas13a protein shows more "leakage" upon AC while the leakage upon GA and UC are quite slight. When we added target sequence in the detecting system, the cleavage of AC obviously increased (the RFU increased 2.04 times at 30min) but the background subtracted fluorescence of AU reporter increased in a cliff like manner (the RFU increased 13. 89 times at 30min), which means that when the target was added, the protein's activity to cleave AU was activated significantly. Due to the preference appears only when the target was added, we defined this as the cleavage base preference of LwaCas13a protein, or more specifically, "the activating associated cleavage base preference " of the protein.
For more information, please click >>BBa_K2323004<<
CRISPR Cas13 technology is a powerful RNA editing technology, which plays an increasingly important role in detection. When we began to learn CRISPR Cas13 technology, it took us a long time to fully understand its principle because it is our first time encountering this technology. And how to design crRNA once became a major obstacle to our project. Although we tried to find relevant databases and literature on how to design crRNA, we only found a website about the gRNA design which is part of CRIPSR Cas9 technology at that time. In the follow-up communication with other teams, we found that not only our team had difficulty in designing crRNA when learning CRISPR Cas13 technology, but some other teams also had difficulties. However, at that time, we had fully figured out how to design crRNA, so we thought about whether our learning results could help other iGEM teams as much as possible (no matter the past or the future). We want to help them learn better and faster with the technology. We searched plenty of information and integrated the crRNA which is corresponding to the pathogen into a database (due to the limited time, there are currently only 39 items). This is an open crRNA sharing platform where iGEMers can find relevant crRNA information and can also upload their designed crRNA to share with other iGEMers. We hope that this platform can help other teams quickly find the crRNA they want. We also compiled a document on how to design crRNA >>click here to see the file<< to help other teams better understand this technology so that they can independently design the corresponding crRNA according to their projects.
SJTU wrote codes to help us render the viralbrary as a web page, all iGEM teams could obtain these codes from github.
As to the final application in the real world, hardware plays an important role in the project application. Our detecting device, "Viralertor, " which was finally determined as our ultimate detecting device has gone through 4 generations. Our leader of dry lab, Yi Fan, made all those devices by way of learning and practice. He made files to document this process and to help other iGEMers learn how to make a detecting device thus help them avoid detours. The file can be acquired below.
Related documents please click>>3D model<<
We uploaded the Arduino program used in our project hoping this could help the future teams for the design of similar programs. This is the first time our college used Arduino as a controller in the iGEM competition, which is a milestone.
Please click >>Arduino program.txt<<
The Program could achieve the following functions:
1. The screen lights up, indicating that the virus detector is ready.
2. Rotate the two filter turntables to place the first corresponding set of filters to the optical path. Turn on the LED light for one second, then take a reading and record the data on the data1 file in the SD card.
3. Rotate the two filter turntables to place the second corresponding set of filters to the optical path. Turn on the LED light for one second, then take a reading and record the data on the data2 file in the SD card.
4. Similarly, we will use the same method to detect the third and the fourth fluorescein, and record the analog value of their signal to the corresponding files in the SD card.
5. Before each reading, we will turn on the LED light for one second, and at other times, the LED light will be turned off, which is to reduce the influence of the photodegradation effect of fluorescein molecules on the experimental results.