Team:SJTU-BioX-Shanghai/Demonstrate
Introduction
Our project was designed to develop a device for off-target detection, including both real-time report and afterward review of the off-target incidents which was further extended into a biostorage system. All team members have thrown themselves into the project, pushing it towards the ultimate goal throughout the summer. Here we are going to illustrate the efforts we had made and what we’ve achieved.
Model
Modeling served as an indispensable part in our project. We built various types of models and they acted much more than facilitating our project design.
In order to simulate off-target event, we needed to find out an appropriate "lure" sequence for a designated small guide RNA(sgRNA) and place it onto our detection device as a BioBrick so that we could recognize when off-target happens. We established a model to simulate the binding-and-cutting process of sgRNA-Cas9 complex and calculated the miscutting probability of different "lure" sequences. Based on this model, we wrote a program which is able to not only search for possible off-target sites of a given sgRNA sequence but at the same time tell us their locations and miscutting probability. See our model1, model2 and program.
What’s more, as our off-target afterward review module was further extended into information storage, we built a codec to increase its efficiency. By encoding our data to Galois field and decoding them with Reed-Soloman algorithm, maximum fault tolerance was achieved with minimal redundancy. See our codec.
Wet-lab
See our result page.Our initial design was for real-time detection of off-target events, so we constructed general vectors containing Nluc-dCas9 (N-terminal of luciferase linked with dCas9) and Cluc-DBD (C-terminal of luciferase linked with DNA binding domain) respectively, and a specific vector contains DBD binding site, sgRNA and its coordinating lure sequence.
As we worked on, we developed new ideas about "memorizing" off-target events and "reviewing" it later to see whether off-target had ever happened in a period of time. Then transcription activation system upon RopA-dCas9's binding was employed and we observed obvious activation of the downstream reporter, a red fluorescent protein gene. If we put LuxI downstream of lure, off-target would result in the expression of LuxI, which is the enzyme for acyl homoserine lactone(AHL) synthesis. We used Lux pR-HS(a mutant of pLuxR) as the promoter in the memorization system so that it can respond to AHL and we confirmed its function.
In addition, CRISPR biostorage was created based on the memorization system so that it can be applied to store all information in binary format. Surprisingly, our experiments showed different results from a literature we referred to, which makes CRISPR biostorage a simpler process. To make the write-and-read process more convenient, we fixed the engineered bacteria into hydrogel where they still performed well as we had expected.
Human practices
We have set up and applied a new model of human practices: "L-M-H-P-E".
Focus on biostorage part of our project, we had academic conmunications with professors in different fields and a visit to biotech company. We adjust our design and finnally develop a real-time recording and reading system.
Since our project is based on CRISPR technology, issues such as safety, ethics, and law have all been taken into consideration. Not only did we reached out to experts from different institutes, but we also carried out a large-scale survey on public's recognition and attitude towards gene editing, including questionnaire and interview. With 1306 questionnaire samples and more than 30000 words of interview records, we conducted qualitative and quantitative statistical analysis and completed an in-depth report. See more about our survey here. We did deep research in humanity on gene editing as well.
According to the survey, a large portion of common people misunderstand gene editing in some way, which reminded us of how important enducation is. Thus, we participated in several activities of public education. These education activities including science carnival, mooc video making, Bio-Art competition and support education in three locations. We hope to promote to the two-way communication between researchers and the public through our survey and education.
Biosafety
There is few biosafety problem to worry about off-target device because it is developed for lab work only. Biostorage part is fixed in hydrogel which is easy to keep so there is few chance that it could be released into the environment and it can be easily destroyed after use by heating. All of our experiments were conducted in accordance with the laboratory regulations and the model organism we used were E.coli strains that are specially designed and widely used in bio-labs. Actually, one of the goals of our project itself is to make CRISPR gene editing more precise and safer. See our Biosafety page and our Safety Form.
SJTU-BioX-Shanghai
Contact us: sjtuigem@gmail.com
Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800