Team:NTU-Singapore/Basic Part

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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Best Basic Part Special Prize

Similar to part BBa_K2818001, dCasRx-ADAR2DD(D338) (BBa_K3250012) is a fusion protein of ADAR2 deaminase domain (with the existing E488Q hypermutation) and a Type VI CRISPR-associated RNA-guided ribonuclease, Cas13d. The Cas13d is mutated to be catalytically inactive but retains the ability of binding to the RNA target with a guide RNA (gRNA) sequence. As this Cas13d was derived from Ruminococcus flavefaciens XPD3002, we refer to this variant as CasRx (or dCasRx for the catalyically inactive ribonuclease). The ADAR2 is inserted within the coding region (loop) of dCasRx at D338. It can be used to selectively edit adenosine to inosine (A-to-I editing) in RNA molecules using a gRNA. A nuclear localization signal was also added to facilitate localisation of constructs in the nucleus for editing of RNA transcripts.

Figure 1. On-target RNA editing of ADAR2_DD inserted into different dCasRx internal sites. The higher the relative luminescence unit (RLU), the higher the RNA editing activity.

Figure 2. Off-target RNA editing of ADAR2_DD inserted into different dCasRx internal sites. The higher the relative luminescence unit (RLU), the lower the RNA editing specificity.

Figure 3. Scatter plot of average editing rate of the different dCasRx-ADAR2_DD mutants from amplicon sequencing data. Average editing rate calculated for all 4 on-target genes (GAPDH, KRAS, PPIB, RAB7A) and 3 off-target genes (F11R, APOOL, XIAP). Values normalised to dCasRx V1.

Our improvements stem from shifting the ADAR2_DD to internal sites within the dCasRx enzyme. From there, we generated 7 internal site mutants. Based on on-target (Figure 1) and off-target (Figure 2) luciferase assay, we found that D338 resulted in the best combination of high on-target and low off-target editing. Further, we performed amplicon sequencing (Illumina) on 4 on-target genes and 3 off-target genes. Based on the average score across the different on- and off- target genes (Figure 3), we found that D338 had similar levels of efficiency while being more specific. Thus, we nominate our D338 mutant as the new best basic part for its improved specificity in A-to-I editing.