Figure 2. Engineering dCas13b-ADAR fusions for RNA editing.

A) Schematic of RNA editing by dCas13b-ADAR_DD fusion proteins. Catalytically dead Cas13b (dCas13b) is fused to the deaminase domain of human ADAR (ADAR_DD), which naturally deaminates adenosines to insosines in dsRNA. The crRNA specifies the target site by hybridizing to the bases surrounding the target adenosine, creating a dsRNA structure for editing, and recruiting the dCas13b-ADAR_DD fusion. A mismatched cytidine in the crRNA opposite the target adenosine enhances the editing reaction, promoting target adenosine deamination to inosine, a base that functionally mimics guanosine in many cellular reactions.

B) Schematic of Cypridina luciferase W85X target and targeting guide design. Deamination of the target adenosine restores the stop codon to the wildtype tryptophan. Spacer length is the region of the guide that contains homology to the target sequence. Mismatch distance is the number of bases between the 3’ end of the spacer and the mismatched cytidine. The cytidine mismatched base is included as part of the mismatch distance calculation.

C) Quantification of luciferase activity restoration for dCas13b-ADAR1_DD(E1008Q) (left) and dCas13b-ADAR2_DD(E488Q) (right) with tiling guides of length 30, 50, 70, or 84 nt. All guides with even mismatch distances are tested for each guide length. Values are background subtracted relative to a 30nt non-targeting guide that is randomized with no sequence homology to the human transcriptome.

D) Schematic of the sequencing window in which A to I edits were assessed for Cypridinia luciferase W85X.

E) Sequencing quantification of A to I editing for 50-nt guides targeting Cypridinia luciferase W85X. Blue triangle indicates the targeted adenosine. For each guide, the region of duplex RNA is outlined in red. Values represent mean +/− S.E.M. Non-targeting guide is the same as in Fig. 2C.