Targeted RNA editing in brainstem alleviates respiratory dysfunction in a mouse model of Rett syndrome.

Rett syndrome is a neurological disease due to loss-of-function mutations in the transcription factor, Methyl CpG binding protein 2 (MECP2). Because overexpression of endogenous MECP2 also causes disease, we have exploited a targeted RNA-editing approach to repair patient mutations where levels of MECP2 protein will never exceed endogenous levels. Here, we have constructed adeno-associated viruses coexpressing a bioengineered wild-type ADAR2 catalytic domain (Editasewt) and either Mecp2-targeting or nontargeting gfp RNA guides. The viruses are introduced systemically into male mice containing a guanosine to adenosine mutation that eliminates MeCP2 protein and causes classic Rett syndrome in humans. We find that in the mutant mice injected with the Mecp2-targeting virus, the brainstem exhibits the highest RNA-editing frequency compared to other brain regions. The efficiency is sufficient to rescue MeCP2 expression and function in the brainstem of mice expressing the Mecp2-targeting virus. Correspondingly, we find that abnormal Rett-like respiratory patterns are alleviated, and survival is prolonged, compared to mice injected with the control gfp guide virus. The levels of RNA editing among most brain regions corresponds to the distribution of guide RNA rather than Editasewt. Our results provide evidence that a targeted RNA-editing approach can alleviate a hallmark symptom in a mouse model of human disease.

Precise in vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA.

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5'-UAN triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.

Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites.

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5' and 3' nearest neighbor nucleotides (e.g., 5' U, 3' G). Current rational design approaches are well-suited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA-protein interactions.