This year, NUS Singapore-Sci has sought to find out the myths and misconceptions about gene editing (#CasAsks) among the public, to identify which aspect of gene editing we should focus on for our project. Through surveys and interviews with various stakeholders, we identified that the main concerns were the permanent nature of DNA editing and ethical considerations. This made us decide to focus on RNA editing as our project. We have also attempted to debunk the myths and misconceptions via school outreaches (#CasTeaches) and educational videos (#CasTalks) after speaking to scientists and people in the field. With all the knowledge on hand, we have summarized and compiled all the interviews into #CasWrites. You can hop over to our Human Practices to find out how all of these are integrated into our project!

We sought to improve BBa_K2083010, a GFP with a mutated start codon from ATG to ACG through our own dual color reporter system BBa_K2807013 which is made up of mCherry and eGFP connected by a T2A linker containing a mutated start codon of ACG. It not only allows a robust OFF-TO-ON switch from the ACG mutant to wild type (ATG), but also demonstrates strong linear correlation between EGFP and mCherry fluorescence, allowing normalization of transfection and expression levels. More information can be found in here, detailing on how the improvements were made.

We had three separate approaches to model various parts of our project. First, we attempted to verify the functionality of our fusion protein with different linker sequences using a protein threading server, following by optimization with molecular dynamics. Second, an algorithm known as Ensemble Kalman Filter was coded to infer difficult-to-measure concentrations, such as gRNAs, for our kinetics model through fluorescence reading. Lastly, Least-Squares Regression algorithm was adopted for our kinetics model, which could potentially be used in the future to predict mRNA editing efficiency given different input based on changes to the spacer region of the gRNA.

The RESCUE Reporter system was tested in HEK293T mammalian cell lines transfected with base editor 3 and 4 made up of rAPOBEC1 and dCas9. It could successfully report DNA editing events under realistic conditions. Take a look at our results for more information on the demonstration.

We have submitted three parts for this category, namely The EGFP-GLB1 WT (BBa_K2807014), EGFP-GLB1 CCG mutant, (BBa_K2807015), and dPspCas13b (BBa_K2807001). All these constructs can successfully express proteins of the expected size when transfected in HEK293T cells.

We hosted the very first Singapore iGEM team meeting to discuss potential collaborations. We have also collaborated with Team Washington, Team IIT-Madras, and Team OUC-China in translations of outreach materials and survey and lastly, Team EPFL for human practices. Visit our collaborations page to see more details!

NUS Singapore-Sci have sought to clarify the public’s myths and misconceptions with scientists and bioethics committee about genetic editing. Have a look at our human practices to find out what myths and misconceptions were busted!

Take a look at our attribution page for details on our project breakdown.

Take a look at our Interlab Study for details.