RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9 KRAB ) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9 KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9 , a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9 KRAB and a Pcsk9 -targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9 KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9 KRAB . In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.