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. 2022 Jan 19;11:e75555. doi: 10.7554/eLife.75555

Figure 2. Engineering split-ADAR2 deaminase domains (ADAR2-DD).

(a) Schematic of the split-ADAR2 engineering approach. (b) Sequence of the ADAR2-DD. The protein was split between residues labelled in red, and a total of 18 pairs were evaluated. (c) The ability of each split pair from (b) to correct a premature stop codon when transfected with a chimeric BoxB-MS2 ADAR-recruiting RNA (adRNA) was assayed via a luciferase assay. The pairs 1–18 correspond to the residues in red in (b) in the order in which they appear. The residues in (b) in bold red correspond to pairs 9–12. Values represent mean (n = 2). All experiments were carried out in HEK293FT cells.

Figure 2—source data 1. Engineering split-ADAR2 deaminase domains.

Figure 2.

Figure 2—figure supplement 1. Optimization of the split-ADAR2 deaminase constructs.

Figure 2—figure supplement 1.

(a) All components of the split-ADAR2 system were tested for their ability to edit RNA via the luciferase assay. Restoration of luciferase activity is observed only when every component is delivered. Values represent mean (n = 2). (b) The importance of orientation of the N- and C-terminal fragments in forming a functional ADAR2 deaminase domain (ADAR2-DD) is assayed via the luciferase assay. Chimeric and non-chimeric ADAR-recruiting RNA (adRNA) are used to recruit the split-ADAR2 pairs. Values represent mean (n = 2). (c) Engineering of humanized split-ADAR2 variant based on pair 12 and assayed of its ability to correct a stop codon in the cluc transcript. Values represent mean (n = 2). All experiments were carried out in HEK293FT cells.