Single-point mutations can give rise to a wide spectrum of genetic diseases. Recent therapeutic approaches aim at editing the mutated mRNA, providing a safe, reversible and tunable treatment. However, these methods are currently not efficient due to knowledge gaps in the understanding of the rules determining RNA editing. Here we provide a powerful method to enhance the efficiency of RNA editing for treating genetic diseases associated with single-point mutations.
Single-point nonsense or missense mutations are responsible for several diseases, including neurofibromatosis, sickle-cell anemia, cystic fibrosis etc. Since therapeutic genome editing may give rise to severe side-effects, targeted RNA editing has become a major interest in recent years, offering a potentially safer alternative to correct single-point mutations. One of the most prevalent forms of RNA editing is Adenosine (A) to Inosine (I) RNA editing, in which adenosines are deaminated into inosines by adenosine deaminase acting on RNA (ADAR) enzymes. Inosine is read as guanine by the translational machinery of the cell, instead of the original adenosine that was encoded in the genome. Using different strategies, several groups have sought to guide either the endogenous or engineered exogenous editing machinery towards pre-designed targets. One such strategy is by delivering antisense guide RNA oligos that create editable structures around a target adenosine that will be recognized by ADAR. However, all these endeavors resulted in low editing efficiency, since the fundamental rules determining which sites within the RNA will be edited and to what extent remain poorly understood.
The group of Prof. Schraga Schwartz conducted a large-scale systematic screening to uncover the rules that govern A-to-I RNA editing. The rules they discovered enable increased targeting efficiency.
The group conducted a large-scale systematic screening to explore the structure and sequence context determining editability. They have generated two highly edited hairpin shaped RNA reporters, and designed an oligo library with systematically disrupted sequences and structures. These constructs were then transfected into several human and mouse cell lines. Single-molecule level editing transcriptome analysis of the of the transfected cell lines revealed a set of structural principles independent of sequence that can significantly improve A-to-I editing efficiency. For instance, they show that editing is robustly induced at fixed intervals from structural disruption. The group went on to show that an RNA targeting oligo designed based on these rules increased editing by 3-fold.
- Safe, reversible and tunable relative to genome editing approaches
- A more effective RNA editing method compared to other available RNA editing platforms.
The team uncovered the rules for efficient site-directed RNA editing and demonstrated their applicability by designing a targeting mRNA for SMAD4 to recruit endogenous ADAR1.
Additional information will be available under a CDA.