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.

Age-dependent expression pattern in the mammalian brain of a novel, small peptide encoded in the 22q11.2 deletion syndrome region.

22q11.2 deletion syndrome (22q11.2DS) carries increased risk for both physical and psychiatric symptoms, including a high risk for schizophrenia. Understanding the genetic elements within the deletion region therefore has the potential to unlock the mysteries of both diseases. While most of the protein-coding genes in this region have been characterized, novel elements, such as non-coding RNAs and small Open Reading Frames (sORFs) remain unstudied. We have identified a novel, highly-conserved mouse sORF in a region of the mouse genome that is orthologous to a portion of the 22q11.2 deletion. This region was previously associated with age-dependent synaptic plasticity abnormalities. We refer to it as the Plasticity Associated Neural Transcript Short, or Pants. In developing and aging mouse brain, Pants expression is strongest in hippocampus, especially in areas CA3 and CA2, throughout the dorsoventral axis. The Pants peptide is expressed throughout the hippocampus, with an age-dependent increase in stratum lucidum at 16 weeks of age. This expression pattern suggests a potential role for Pants in many hippocampal behaviors, as well as a potential role in the age-dependent neurologic deficits displayed by 22q11.2DS model mice and patients.