Team:NTU-Singapore/Improve

Home Team Members Collaboration Attributions Description Mutation-internal Open / Structured Demonstrate Characterisation Improve Basic Parts Part Collection Human Practices Outreach Medal Criteria Judging Form

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Improve

Structure of dCasRx, with 7 internal sites mapped on the structure.

Average on-target and off-target editing rate of ADAR2_DD inserted into D338 (In-D338) as compared to CasRx v1, based on amplicon sequencing. Values were normalised to CasRx V1.

This year, our project focused on improving the specificity (i.e. reduce off-targets) of the dCas13-directed A-to-I editing platform. Team NTU-Singapore 2018 previously characterised REPAIRv1 and CasRx v1 (BBa_K2818001). However, both had high off-target activities, possibly due to the E488Q hyperactivating mutation in ADAR2_DD. This year, we improved on CasRx v1 by reducing its off-target activity. dCasRx was chosen as the base for improvement due to its smaller size, which facilitates in vivo therapeutic delivery. Through protein modelling, we inserted ADAR2_DD into D338 (In-D338) within the dCasRx helical loop region to create a new part, BBa_K3250012.

Figure 1. Comparison of APOOL trans off-target editing between dCasRx V1 and dCasRx D338.

Figure 2. Comparison of F11R trans off-target editing between dCasRx V1 and dCasRx D338.

Figure 3. Comparison of KRAS cis off-target editing between dCasRx V1 and dCasRx D338.

We performed Amplicon sequencing on APOOL and F11R, which were used to analyse for trans off-target effects in our project. From Figure 1 and 2, it is observed that D338 has less off-target sites compared to dCasRx V1. This is indicated by the number of A>G mutations at each position (higher the G, higher the off-target activity). Similarly, cis off-target editing on the KRAS on-target mRNA was also reduced when we transfected D338 with the KRAS gRNA, compared to CasRx V1 with the KRAS gRNA.

In conclusion, In-D338 is an improvement over CasRx v1 as it has a lower off-target editing rate while still retaining its relatively high on-target editing rate. This means In-D338 is more specific while being almost equally efficient as CasRx v1.