Shortening the Half-Life of Cas9 Maintains Its Gene Editing Ability and Reduces Neuronal Toxicity

Virus-mediated expression of CRISPR/Cas9 is commonly used for genome editing in animal brains to model or treat neurological diseases, but the potential neurotoxicity of overexpressing bacterial Cas9 in the mammalian brain remains unknown. Through RNA sequencing (RNA-seq) analysis, we find that virus-mediated expression of Cas9 influences the expression of genes involved in neuronal functions. Reducing the half-life of Cas9 by tagging with geminin, whose expression is regulated by the cell cycle, maintains the genome editing capacity of Cas9 but significantly alleviates neurotoxicity. Thus, modification of Cas9 by shortening its half-life can help develop CRISPR/Cas9-based therapeutic approaches for treating neurological disorders. [Display omitted] •Linking N-terminal Geminin promotes the degradation of Cas9 in G1/G0 cell phase•Geminin-tagged Cas9 has a shorter half-life in mouse brain than wild-type Cas9•AAV transduction of wild-type Cas9 affects genes involved in neuronal functions•Geminin-tagged Cas9 has less impact on genes involved in neuronal functions Yang et al. use Geminin-tagged Cas9 to show that reducing the half-life of Cas9 diminishes neurotoxicity.