Supplementary Figure 1: Exploration of an efficient RNA editing platform.
From: Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs
(a) Schematic of dLbuCas13a-ADAR1DD (E1008Q) fusion protein and the corresponding crRNA. The catalytic inactive LbuCas13a was fused to the deaminase domain of ADAR1 (hyperactive E1008Q variant) using 3× GGGGS linker. The crRNA (crRNACas13a) consisted of Lbu-crRNA scaffold and a spacer which was complementary to the targeting RNA with an A-C mismatch as indicated. (b) Schematic of dual fluorescent reporter system and the Lbu-crRNA with various lengths of spacers as indicated. (c) Quantification of the EGFP positive (EGFP+) cells. HEK293T cells stably expressing the Repoter-1 were transfected with indicated lengths of crRNACas13a, with or without co-expression of the dLbuCas13a-ADAR1DD (E1008Q), followed by FACS analysis. (d) Representative FACS result from the experiment performed with the Ctrl crRNACas13a (70-nt random spacer) or crRNACas13a (70-nt targeting spacer). (e) Representative FACS result from the experiment performed with REPAIR. Left, the Ctrl crRNACas13b (70-nt random spacer) or crRNACas13b (70-nt targeting spacer) was co-transfected with or without dPspCas13b-ADAR2DD-E488Q into HEK293T cells, which stably express Reporter-1. Right, the Ctrl crRNACas13b (70-nt random spacer) or crRNACas13b (70-nt targeting spacer) was co-transfected with Reporter-1 into HEK293T ADAR1−/− cells, in the presence or absence of dPspCas13b-ADAR2DD-E488Q. The 70-nt random or targeting spacer was fused to the 3′-end of PspCas13b-crRNA scaffold. The RNA editing effects were quantified by the percentages of EGFP+ cells. Data are presented as the mean ± s.e.m. (n = 3, n represents the number of independent experiments performed in parallel).
