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

Nat Commun. 2021 Apr 16;12(1):2287. doi: 10.1038/s41467-021-22519-z.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / metabolism
  • Adenosine Deaminase / genetics*
  • Adenosine Deaminase / metabolism
  • Bacterial Toxins / genetics
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • Cell Line, Tumor
  • Cytosine / metabolism
  • DNA / genetics*
  • DNA / metabolism
  • Escherichia coli Proteins / genetics*
  • Escherichia coli Proteins / metabolism
  • Gene Editing / methods*
  • Genes, Reporter
  • HEK293 Cells
  • Humans
  • Inosine / genetics
  • Inosine / metabolism
  • Protein Engineering
  • RNA Editing / genetics
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • RNA-Seq
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Bacterial Toxins
  • Escherichia coli Proteins
  • HokB protein, E coli
  • Recombinant Fusion Proteins
  • Inosine
  • Cytosine
  • DNA
  • RNA, Transfer
  • CRISPR-Associated Protein 9
  • Adenosine Deaminase
  • TadA protein, E coli
  • Adenine