Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.

(m) The overlapping ratio for RNA A-to-I edits calculated by using HaplotypeCaller (as denominator) and MuTect2 tools was shown from the results shown in b-d.
(n) 9 MuTect2-specific RNA edits (ABE-MU-OFT-1 to ABE-MU-OFT-8; also shown as S1-S8) were selected for PCR validation (from cDNA) and Sanger sequencing. The A-to-G conversion efficiency was also presented. The primers for amplifying the fragments containing these edits are listed in Supplementary Table 3. (c) HEK293T cells were transfected with ABEmax and an sgRNA targeting ABE site 8. The DNA on-target A-to-G editing efficiency with two independent replicates was shown from Sanger sequencing data.
(d) Two replicates (ABEmax rep1 and rep3) in c were collected for RNA-seq analysis.
(e) Sequence logos centered by the edited adenine (A) from pooled RNA-seq data in d for edited adenines with indicated editing efficiencies (> 50%, > 40%, > 30%, > 20%, and all). The nucleotides around the edited A were shown (21 nucleotides in total). (a) Protein sequence alignment of Staphylococcus aureus TadA (SaTadA) and E. coli TadA. The conserved essential amino acids were highlighted in red. The secondary structure of TadA was also labelled. , -helix; , -sheet.
(b) Sanger sequencing for DNA A-to-G editing and RNA A-to-I editing of ecHokb induced by wild-type ABEmax and eight variants containing indicated mutations. The diagram for on-target DNA editing of HEK site 3 was also presented.
(c-d) Deep sequencing analysis of the fraction of by-products, including A-to-nonG editing (c) and indels (d), induced by wildtype ABEmax and engineered variants. Three independent replicates are presented as mean values ± s.d..
(e) The predicted crystal structure presenting the interaction between SaTadA and tRNA.
The contact sites between N46, E59, and R153 of ecTadA and tRNA were highlighted.    (a) The number of RNA A-to-I edits by using HaplotypeCaller in Fig. 2a was presented. (b) The RNA A-to-I edits induced by ABEmax or its variants (del153/dele153* and mini del153) expressed in HEK293T cells co-transfected with an sgRNA targeting ABE site 8 were calculated by using HaplotypeCaller and MuTect2 tools separately. Then, the RNA A-to-I edits generated by the two tools were overlapped for each sample, and the number of overlapped edits, HaplotypeCaller-/MuTect2-specific edits, and merged edits was shown. ABEmax, rep 3 and rep4; del153/dele153*, rep1 and rep2; mini del153: rep1 and rep2. Two independent replicates are presented separately.

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(c) Histograms showing numbers of off-target edited RNA adenines (y-axis) and corresponding RNA A-to-I editing frequencies (x-axis) for two replicates shown in Fig.   2b (using MuTect2). Solid red line represents the mean frequency, and dashed red line represents the median frequency.  Figure S7. Comparison of engineered ABE variants.

A A C A C A A A
DNA A-to-G editing (%)  as mean values ± s.d..