Abstract:

Single nucleotide variations (SNVs) represent the most common form of pathogenic mutations in humans, while base editing technology offers an ideal solution for treating such pathogenic variants. RNA editing has become a hotspot in current gene therapy due to its reversible action, which avoids long-term risks by not permanently altering the genome, and the compact size of the editors. Among these, the mini-dCas13X.1-mediated RNA adenine base editing (mxABE) system demonstrates highly efficient RNA base editing; however, it still suffers from non-target nucleotide editing caused by the bystander editing effect, which constitutes a major off-target risk. In this study, by deleting the nucleotide opposite the non-target adenosine in the sgRNA sequence, we effectively reduced the bystander editing effect of the mxABE system. In vitro results showed that this strategy successfully controlled the bystander editing rate below 5% while maintaining highly efficient on-target editing of approximately 70%. In a murine model of DMD (Duchenne muscular dystrophy), a single administration of AAV (adeno-associated virus)-delivered mxABE system demonstrated significant therapeutic efficacy in the tibialis anterior muscle, with successful elimination of off-target adenosine bystander editing. This study provides a novel precision-targeting strategy for treating monogenic genetic diseases using the mxABE RNA editing system.

Key words: bystander editing, mxABE, Duchenne muscular dystrophy (DMD), single-base editing