In Vivo Silencing of Regulatory Elements Using a Single AAV-CRISPRi Vector

CRISPR-Cas9 based transcriptional repressors (CRISPRi) have emerged as robust tools for functional epigenetic silencing of regulatory elements. Adeno-associated viruses (AAV) are promising delivery vectors for cardiovascular research and therapy. However, compact vectors enabling codelivery of all CRISPRi components to cardiac cells 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 AAVi 6 and cardiotropic AAVi 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 more than 90% reduction of gene expression. This effect was orchestrated by loss of chromatin accessibility and establishment of heterochromatin specifically at the target promoter region. We confirmed the broad applicability of AAVi to different 2D and 3D cardiomyocyte cell culture models by silencing several genes in primary neonatal rat ventricular cardiomyocytes (NRVM), human iPSC-derived cardioids and iPSC-CM. To demonstrate the efficacy of AAVi in vivo in the heart 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 cardiac hypertrophy and Nppa transcription. AAViNppa efficiently silenced Nppa transcription as shown 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. ### Competing Interest Statement The authors have declared no competing interest.