Heart-specific gene silencing via single-AAV-delivered base editing [Amplicon-Seq]

We used an adenine base editor to target the translation start site and mRNA splicing site of Camk2d in order to knock out CaMKIIδ. We found that editing the 5' splice site of intron 7 can lead to premature translation termination, effectively knocking out CaMKIIδ. We first designed 10 sgRNAs, transfected them into Neuro2a cells using plasmids. After 48 hours, we extracted DNA and performed amplicon sequencing to detect the editing efficiency of each sgRNA. Then, we delivered the top 3 sgRNAs with the highest editing efficiency using a single AAV vector into mice to evaluate the editing efficiency in the heart and the knockout effect of CaMKIIδ. Finally, we split the single AAV vector into dual AAV vectors and compared the editing efficiency in the mouse heart between single AAV and dual AAV under the same total AAV dosage.