Therapeutic in vivo gene editing with highly specific nucleases has the potential to revolutionize treatment for a wide range of human diseases, including genetic disorders and latent viral infections like herpes simplex virus (HSV). However, challenges regarding specificity, efficiency, delivery, and safety must be addressed before its clinical application. A key concern is the risk of off-target effects, which can cause unintended and potentially harmful genetic changes. We previously developed a curative in vivo gene editing approach to eliminate latent HSV using HSV-specific meganuclease delivered by an AAV vector. In this study, we investigate off-target effects of meganuclease by identifying potential off-target sites through GUIDE-tag analysis and assessing genetic alterations using amplicon deep sequencing in tissues from meganuclease treated mice. Our results show that meganuclease expression driven by a ubiquitous promoter leads to high off-target gene editing in the mouse liver, a non-relevant target tissue. However, restricting the meganuclease expression with a neuron-specific promoter and/or a liver-specific miRNA target sequence efficiently reduces off-target effects in both liver and trigeminal ganglia. These findings suggest that incorporation of regulatory DNA elements for tissue-specific expression in viral vectors can reduce off-target effects and improve the safety of therapeutic in vivo gene editing.