In vivo silencing of regulatory elements using a single AAV-CRISPRi vector
CRISPR-Cas9 based transcriptional repressors (CRISPRi) have emerged as specific and robust tools for functional epigenetic silencing of regulatory elements. Adeno-associated viruses (AAVs) are promising CRISPRi delivery vectors for cardiovascular research and therapy. However, compact vectors enabling codelivery of all CRISPRi components by a single AAV are needed for an enhanced and consistent performance. We engineered a 4.7kb all-in-one CRISPRi construct compatible with AAV-mediated delivery and produced cardiotropic AAVi 6 and 9 particles for in vitro and in vivo application, respectively. AAVi vectors targeting the Nppa promoter (AAViNppa) reduced gene expression in cultivated cardiomyocytes (HL-1 cells) in a dose-dependent manner. The maximum effect was a >95% reduction as measured by qPCR and RNA-seq. This effect was orchestrated by loss of chromatin accessibility (ATAC-seq) and establishment of heterochromatin (H3K9me3 ChIP-seq) specifically at the target promoter region. We confirmed the broad applicability of AAVi to different cardiomyocyte cell culture systems by silencing several genes in primary neonatal rat ventricular cardiomyocytes (NRVMs), human iPSC-derived cardioids and iPSC-CMs. To demonstrate the efficacy of AAVi in vivo we injected 8-week-old C57Bl/6 WT mice with a single dose of AAViNppa and implanted osmotic minipumps releasing Phenylephrine (50 mg/kg/d) and Angiotensin II (0.45 mg/kg/d) to induce Nppa transcription. AAViNppa silenced Nppa transcription as revealed by qPCR and single nuclei RNA-seq even under stress conditions. On the epigenome layer AAViNppa induced closed chromatin at the Nppa promoter site comparable to the in vitro effect. Here, we present an efficient AAV-based method for CRISPRi-mediated epigenetic silencing of gene expression in cardiac myocytes in vivo and in vitro. This functional epigenetic approach provides an efficient way to modulate gene expression in the heart and could become a standard method for cardiovascular disease modelling and translational research.