Extended Data Fig. 3: G·U wobbles significantly outperform previous solutions in bystander suppression at nearest neighbor (NN) sites.
From: Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA
(a) Design of the cis-acting RNA editing reporter constructs used in e) and f). (b) Design of the linear trans-acting CLUSTER guide RNAs used in g). (c) Illustration of the base-pairing motifs applied to the constructs in a) (ocher) and b) (grey) that were used to suppress bystander editing at the 5´-U[AAG] sequence motif in e) and g). (d) As c) but for the 5´-[UAA]U sequence motif and the results in f). (e) Suppression of bystander editing at the 5´-NN A and concurrent effect on the target A editing yield in a 5’-U[AAG] sequence motif. (f) Similar to e) but targeting the A in a 5’-[UAA]U sequence motif and suppressing bystander editing at a 3´-NN A. Editing in e) and f) was performed in ADAR1 p110 Flp-In T-REx cells using plasmid-borne reporters as displayed in a). (g) Suppression of bystander editing at a 5´-NN A when editing a 5’-U[AAG] site (K984) of the human BMPR2 transcript using trans-acting CLUSTER guide RNAs. (h) Meta-analysis of bystander suppression at 5´- and 3´-NN sites over three different target transcripts (AHI1, BMPR2 and COL3A1), three different A-rich target triplets flanked either 5’ and/or 3’ by uridines (5’-[UAA]U, 5’-U[AAG], 5’-U[AAA]U), and applying two trans-acting site-directed RNA editing systems (CLUSTER/endogenous ADAR, boxB/λN-ADAR2Q). The full data set of the meta-analysis is shown in Supplementary Fig. S5. Editing in g) and h) was performed in HeLa cells using plasmid-borne editase (λN-ADAR2Q) and/or guide RNA (2x boxB, CLUSTER). Data in e) – g) are shown as the mean editing percentage ± s.d. of N = 3 biological replicates. Data in h) are shown as the mean editing percentage ± s.d. of three 5’-NN off-target sites, seven on-target sites, and five 3’-NN off-target sites containing N = 3 biological replicates per site. The center line represents the median, the plus-sign the mean. The whiskers extend to the minimum and maximum values. The box limits the 25th and 75th percentile. For statistical analysis, a student’s t-test (two-tailed, parametric) was applied.
