Proof of concept

Based on our implementation, we have more results to proof our concept.

Click HERE to see out implementation page.

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.

We have proposed a complete pipeline from a certain target sequence to Cas9/dCas9 variants with lower off-target effect. Models both guide the experimental process and evaluate the result of directed evolution. Researchers can use our off-target predictor to detect and select off-target sites with high confidence in experiments. Molecular dynamics method helps to find residues playing critical roles in interactions between DNA and Cas9/dCas9-sgRNA complex. The discovery of these functional positions integrates rational design into the directed evolution experiment. A graphic interpretation is also adopted to measure the mutation distance between different variants. On the other hand, our kinetics model fits the relationship between mismatch condition and off-target probability. The explainable parameters about transition state energy for WT Cas9 and evolved Cas9 can provide reliable inference on the changes in targeting progression after directed evolution.

What’s more, we have built a collection of previous models from iGEM Team Wikis, especially those focusing on hot spots in synthetic biology can develop a convenient method to learn from successful iGEM models for all iGEMers.

Click HERE to see details of our model page.

Wet-lab

We design CRISPRa and CRISPRi systems to evaluate and select dCas9 mutants generated by random mutagenesis in order to obtain THE ONE that has both high on-target level and low off-target level. We also make rational design with the assistance of model.

Since we have verified the CRISPRa and CRISPRi systems to evaluate and select dCas9 mutants generated by random mutagenesis, we are able to obtain dCas9 mutants with higher on-target level and lower off-target level. Our effective workflow to produce expected proteins is now accessible for other research groups. They can apply our reporting system, mutagenesis method and screening strategy to other biological experiment designs. According to our design, we do not need to sequence the plasmids immediately after acquiring the mutant library, but only to verify the success of our mutant library by flow cytometry analysis.

Click to see details of our design and result page.

Human practices

Our human practices work runs through the whole project from the beginning to the end.

Focus on design of our project, we had academic communications with professors in different fields and our partnership friend SYSU-CHINA. We adjust our design and finally develop CRISPRa and CRISPRi systems for evolution screening.

Since our project is based on CRISPR technology, we would like to know its acceptance in public. So we continued to carry out a survey on public's recognition and attitude towards gene editing, including questionnaire and interview. Totally, we collected near 2000 questionnaire samples(including samples our team collected last year), we conducted qualitative and quantitative statistical analysis and completed many visualization diagrams.

Click HERE to see details about questionnaires and interviews.

Acknowledgements

SJTU-BioX-Shanghai

contact us

Contact us: sjtuigem@gmail.com or sjtuigem@gmail.com