Fig. 1 RNA and DNA editing activity of each TadA monomer in ABEmax.

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Fig. 1 RNA and DNA editing activity of each TadA monomer in ABEmax. RNA and DNA editing activity of each TadA monomer in ABEmax. (A) ABEmax (shown as a schematic model) comprises three proteins fused in a single chain: TadA-TadA*-Cas9(D10A). (B) The two TadA monomers (shown as a schematic model) in ABEmax. The schematic models in (A) and (B) are generated from independently solved Cas9 [Protein Data Bank (PDB) id: 4un3] and E. coli TadA (PDB id: 1z3a) structures, as the structure of ABE has not yet been solved. (C) Average A-to-I conversion frequency in three mRNA transcripts from each treatment analyzed by high-throughput sequencing (HTS). (D) The number of adenosines within a 220- to 240-nt region of the indicated mRNA that are converted to inosine [read as a G after cDNA synthesis and DNA sequencing] at a detectable level (≥0.1%). Cas9(D10A) controls show the number of adenosines that are edited by endogenous cellular adenosine deaminases. The amplified regions of RSL1D1, CTNNB1, and IP90 mRNA have 46, 59, and 77 sequenced adenosines, respectively. (E) DNA base editing at seven genomic loci from ABEmax or by ABEmax with mutations at catalytic Glu59 in TadA or TadA*. The protospacer position of the target A and the sequence context of the A are shown. (F) RNA editing frequencies at various adenosines within the RSL1D1 amplicon after treatment with the indicated base editors. The adenosine homologous to TadA’s native substrate is at position 152 within the amplicon. (G) On-target DNA base editing with the low-density lipoprotein receptor (LDLR) sgRNA leads to a U-to-C (red to blue) edit in the LDLR mRNA in the transcriptome-wide RNA sequencing (RNA-seq) data. Alignments were visualized in the Integrated Genomics Viewer (IGV) and aligned to hg38. (H) Transcriptome-wide RNA-seq analysis showing the number of high-confidence (Phred quality score, ≥20; see Materials and Methods) A-to-I variant calls after treatment with the indicated base editors. The line represents the number of A-to-I conversions in the transcriptome from endogenous deaminase activity as measured in the Cas9(D10A) control samples. (I) The average frequency (%) of A-to-I RNA editing across all transcripts. For (A) to (F), data are shown as individual data points and means ± SD for n = 3 independent biological replicates performed on different days. For (H) and (I), data are shown as means ± SEM. The alignment was generated by combining reads from three independent biological replicates performed on different days. Holly A. Rees et al. Sci Adv 2019;5:eaax5717 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).