Adenovirus vector-mediated delivery of the CRISPR-CasRx system enables gene knockdown with suppressed collateral activity.

The CRISPR-Cas13 system is a programmable gene knockdown tool widely used in basic and clinical research. Meanwhile, RNA-targeting by Cas13 exhibits collateral activity, i.e., cleavage of non-target RNAs, raising serious concerns. Accumulating evidence suggests the collateral activity comes from high expression levels of the Cas13 system. Therefore, controlled expression of the Cas13 system using viral vectors could solve the problem. Adenovirus vectors are efficient gene delivery vehicles and commonly used for clinical therapy. Here, we generated adenovirus vectors carrying a CRISPR-Cas13 system using CasRx (Ad-CasRx). Ad-CasRx efficiently knocked down the on-target reporter with suppressed collateral activity in cell lines, while plasmid transfection-mediated delivery exhibited strong collateral activity. Moreover, Ad-CasRx efficiently knocked down Pcsk9 in cultured murine hepatic cells without suppressing abundant endogenous RNAs, suggesting that collateral activity was largely suppressed when targeting endogenous genes with Ad-CasRx. Finally, we targeted hepatic Pcsk9 in mice by intravenous administration of Ad-CasRx. Pcsk9 mRNA and serum total cholesterol levels were reduced without affecting the expression of major genes related to cholesterol metabolism and hepatic function. Serum ALT levels were not elevated. Overall, controlled delivery of Ad-CasRx enabled in vivo gene knockdown without evident side effects from collateral activity or Cas13 expression itself in liver.