Figure 3. Characterizing the split-ADAR2 deaminase domains.

(a) The components of the split-ADAR2 system based on pair 12 were tested for their ability to edit the RAB7A transcript. Editing was observed only when every component was delivered. Values represent mean ± SEM (n = 3). (b) 2D histograms comparing the transcriptome-wide A-to-G editing yields observed with each construct (y-axis) to the yields observed with the control sample (x-axis). Each histogram represents the same set of reference sites, where read coverage was at least 10 and at least one putative editing event was detected in at least one sample. Bins highlighted in red contain sites with significant changes in A-to-G editing yields when comparing treatment to control sample. Red crosses in each plot indicate the 100 sites with the smallest adjusted p-values. Blue circles indicate the intended target A-site within the RAB7A transcript. (c) The split-ADAR2 system was assayed for editing the KRAS and CKB transcripts. Values represent mean ± SEM (n = 3). All experiments were carried out in HEK293FT cells.

Figure 3—figure supplement 1. Specificity profiles of the ADAR2 deaminase expression constructs.

(a) Heatmap depicting hyper-editing observed with the split-ADAR2 system corresponding to the plot in Figure 3a. The red arrow indicates the target adenosine. (b) 2D histograms comparing the transcriptome-wide A-to-G editing yields (fraction of edited transcripts) observed with constructs from Figure 3a (y-axis) to the yields observed with the control sample (x-axis). Each histogram represents the same set of 27,587 reference sites, where read coverage was at least 10 and at least one putative editing event was detected in at least one sample. Bins highlighted in red contain sites with significant changes in A-to-G editing yields when comparing treatment to control sample. Red crosses in each plot indicate the 100 sites with the smallest adjusted p-values. Blue circles indicate the intended target A-site within the RAB7A transcript. Large counts in bins near the lower-left corner likely correspond not only to low editing yields in both test and control samples, but also to sequencing errors and alignment errors. Large counts in bins near the upper-right corner of each plot likely correspond to homozygous single nucleotide polymorphisms (SNPs), as well as other differences between the reference genome and the genome of the HEK293FT cell line used in the experiments.

Figure 3—figure supplement 2. Specificity profiles of the ADAR2 deaminase expression constructs.

2D histograms comparing the transcriptome-wide A-to-G editing yields observed with each split-ADAR2 construct (y-axis) to the yields observed with the control sample (x-axis). Blue circles indicate the intended target A-site within the RAB7A transcript.

Figure 3—figure supplement 3. Specificity profiles of the split-ADAR2 deaminase expression constructs.

2D histograms comparing the transcriptome-wide A-to-G editing yields observed with each split-ADAR2 construct (y-axis) to the yields observed with the control sample (x-axis). Blue circles indicate the intended target A-site within the KRAS transcript.