Programmable RNA base editing with a single gRNA-free enzyme

Abstract

Programmable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60–80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.

RNA Editing Therapeutics: Advances, Challenges and Perspectives on Combating Heart Disease

“…Most of the currently available SDRE strategies use ADARs for A-to-I or C-to-U editing, which is why ADARs are at the centre of this review. To our knowledge, there are only two studies that engineered an SDRE construct using an APOBEC deaminase domain [94,95], and for one other we are aware of, the authors have expressed the possibility of APOBECs being used as effectors [96].…”

“…1A). Therefore, to our knowledge, all ADAR SDRE methods developed so far, except for one [95], use guide RNAs (gRNAs). Guide RNAs are single-stranded deoxyribonucleotides, which interact with a target mRNA via Watson-Crick base-pairing, thereby forming a dsRNA structure.…”

“…Therefore, the use of gRNAs might reduce editing efficiency. SDRE systems that use programmable RNA-binding motifs for target recognition instead of gRNAs might be the solution to this issue [95,116]. Specifically, the programmable RNA-binding motif of the human Pumilio and FBF homology (PUF) proteins has been recently engineered into a novel SDRE system called REWIRE [95,117].…”

“…SDRE systems that use programmable RNA-binding motifs for target recognition instead of gRNAs might be the solution to this issue [95,116]. Specifically, the programmable RNA-binding motif of the human Pumilio and FBF homology (PUF) proteins has been recently engineered into a novel SDRE system called REWIRE [95,117]. Fused to an ADAR1/2dd or A3Add, REWIRE achieved high-efficiency A-to-I and C-to-U editing, respectively.…”

Improvement of C‐to‐U RNA editing using an artificial MS2‐APOBEC system

“…[13] In addition, Wang's group developed the CU-REWIRE system to perform the C-to-U RNA editing, which uses human APOBEC3A fused to PUF domain. [25] MS2 tagging is a technique based on the natural interaction between the MS2 bacteriophage coat protein and a stem-loop structure from the phage genome. [26] This technique is utilized for the biochemical purification of RNA-protein complexes and is combined with green fluorescent protein (GFP) expression for the detection of RNA in living cells.…”