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

Abstract

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.

Abstract

Repression of gene transcription using CRISPR-Cas9 has been achieved in vitro but not for delivery into adult animal models. Here, the authors use AAV8 to deliver the transcriptional repressor dSaCas9 ^KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.