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.
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.
Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder associated with deficits in social communication and stereotypical behaviors. Numerous ASD-related genetic mutations have been identified and genome editing methods have been developed but successful genome editing in the whole-brain scale to alleviate autistic-like behaviors in animal models has not been achieved. Here we report the development of a new CRISPR-mediated cytidine base editor (CBE) system, which converts C/G base pairs to T/A. We demonstrate the effectiveness of this system by targeting an ASD-associated de novo mutation in the MEF2C gene (c.104T>C, p.L35P). We constructed a Mef2c L35P knock-in mouse and observed that Mef2c L35P heterozygous mice displayed autistic-like behaviors, including deficits in social behaviors and repetitive behaviors. We programmed the CBE to edit the C/G base pairs of the mutated Mef2c gene (c.104T>C, p.L35P) to T/A base pairs and delivered it via a single dose intravenous injection of blood brain barrier (BBB)-crossing AAV-PHPeB vector into the mouse brain. This treatment restored MEF2C protein levels and reversed impairments in social interactions and repetitive behaviors in Mef2c L35P heterozygous mice. Together, this work presents an in vivo gene editing strategy in which correcting a single nucleotide mutation in the whole-brain scale could be successfully achieved, further providing a new therapeutic framework for neurodevelopmental disorders.
Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder with deficits in social communication and stereotypical behaviors. Whole-brain genome editing to correct single-base mutations and alleviate autistic-like behaviors in animal models has not been achieved. Here we developed an APOBEC-embedded cytosine base editor (AeCBE) system, for converting C·G to T·A base pairs. We demonstrate the effectiveness by targeting AeCBE to an ASD-associated mutation of the MEF2C gene (c.104T>C, p.L35P) in vivo. We constructed a Mef2c L35P heterozygous mouse, which exhibited autistic-like behavioral deficits. We programmed AeCBE to edit the mutated C·G base pairs of Mef2cin the mouse brain, via the intravenous injection of blood brain barrier (BBB)-crossing AAV. This treatment restored MEF2C protein levels and reversed impairments in social interactions and repetitive behaviors in Mef2c mutant mice. This work presents an in vivo base editing paradigm in which a single-base mutation in the brain could be successfully corrected. One-Sentence Summary Base editing in vivo in the mouse brain corrects autistic-like behaviors.
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