Luciferase Reporter Assay

As described in our REPAIR project, both the dCas13b-ADAR2DD and dCas13d-ADAR2DD have demonstrated A-to-I base modification activities on exogeneous mRNAs in the reporter assay under appropriate RNA guides. With a guanosine in the codon of Rluc luciferase, luminescence can only be observed if the constructs successfully perform the base modification. The graph below shows the results for luminescence intensity after transfection of the necessary constructs. On all five editing sites, all constructs showed some, if not significant level of base editing activities, proving the functionality of our constructs.

Figure 1. Editing rate of different RNA editors at different target positions on Rluc (n = 2)

Besides just proving that our constructs’ functionality, we further characterized important design parameters for optimal RNA base editing using dCas13d-ADAR2DD, like spacer length and the location of coverage of the target sequence. Below shows the different luminescence levels when the dCas13d construct is guided with guides of different parameters. More detailed analysis can be found on this page.

Figure 2. Editing rate of different RNA editors with different spacer lengths and different guide mismatch distances. (n = 2)

where dash line shows the non-targeting control

 Endogenous RNA Editing

After the proof-of-concept with the successful A-to-I editing in our luciferase assay, we further demonstrated the functionality of our constructs when targeting endogenous mRNA transcripts of the PPIB and KRAS genes. Sanger sequencing showed that a significant portion of the targeted adenosine were called as guanosine for both the dCas13b-ADAR2DD and the dCas13d-ADAR2DD, which is the functional equivalence of inosine in base pairing. Figure below illustrates such an observation, suggesting that our constructs indeed perform as expected in mammalian cell lines.

Figure 5. Editing rate of different RNA editors with different spacer lengths and different guide mismatch distances
on mRNA transcripts of the endogenous PPIB and KRAS gene. (n = 2)