Structure-guided chemical modification of guide RNA enables potent non-viral Cas9-mediated in vivo genome editing

CRISPR/Cas9 has emerged as a versatile and transformative genome editing technology. However, to date, efficient in vivo genome editing has required, at least in part, the use of viral delivery systems. In vivo Cas9-mediated gene inactivation, or indel formation, requires nuclear delivery of the Cas9 protein and a short guide RNA (sgRNA). Translational efforts to develop RNA therapeutics have shown that chemical modification of RNA can dramatically improve in vivo delivery, but that modification must be done judiciously, as non-natural nucleotides can inhibit activity as well. In this study, we develop a framework for chemical modification of the sgRNA that preserves, and in some cases improves, Cas9 function in vitro and in vivo. Guided by the structure of the Cas9-guide RNA complex, we develop “enhanced” sgRNA (e-sgRNA). We identify regions of sgRNA that tolerate chemical modification without inhibiting Cas9 and sgRNA interaction, while maintaining or enhancing genome editing activity. Formulations of e-sgRNA and mRNA encoding Cas9 were developed that are capable of inducing nearly complete gene editing of hepatocytes in the liver following a single intravenous dose. As proof of principle, a single injection of this formulation was able to induce >80% editing of Pcsk9 in the liver, resulting in complete disappearance of serum PCSK9 and significantly lower cholesterol levels. We believe the information provided here paths a generalized strategy for enabling non-viral, Cas9-based genome editing in vivo, and may enable a range of both rapid animal model generation and therapeutic applications.