Engineering of AAV-Mediated in Vivo Targeted DNA Methylation Editing System via Staphylococcus Aureus Derived Cas9-SunTag

DNA methylation aberrations have been implicated in the pathogenesis of various psychiatric and developmental disease states. However, basic research and therapeutic in vivo applications targeting aberrant DNA methylation are still limited due to the lack of targeted DNA methylation editing systems that work on capacity-limited adeno-associated virus (AAV), a highly versatile in vivo gene delivery vector. Here, we report the engineering of AAV-based vector system for in vivo targeted DNA methylation/demethylation by integrating small Cas9 orthologues (SaCas9) and a dCas9-SunTag methodology. In this system, dSaCas9-SunTag targeting protein and anti-SunTag-mini-antibody (scFv) fused effector protein were packaged into different vectors for efficient in vivo delivery by AAV. We demonstrated that co-introduction of AAV vectors carrying dSaCas9-SunTag and scFv-fused Dnmt3A, Dnmt3L, or Dnmt3A-Dnmt3L fusion proteins induced robust DNA methylation on the Bdnf gene locus in a guide RNA dependent manner in vitro . The scFv-fused TET1 and TET2 successfully induced robust targeted DNA demethylation in the H19 ICR. We also constructed a doxycycline (Dox)-inducible target DNA methylation system utilizing dSaCas9-SunTag and scFv-Dnmt3AL. Finally, we showed that AAV-mediated delivery of dSaCas9-SunTag system induced DNA methylation on the Bdnf gene locus and suppressed activity-dependent but not basal Bdnf expression after contextual fear learning in the Hippocampus of adult mouse. Our in vivo targeted DNA methylation editing system can be a promising platform for understating the causal relationship between psychiatric disease and aberrant DNA modifications.