Protocol for one-step design of a triple sgRNA-based CRISPR/Cas9 construct for neuronal gene knockout in the mouse brain

Abstract

Summary CRISPR-Cas9 enables genome editing through the expression of Cas9 and single guide RNAs (sgRNAs). Here, we present a protocol for designing and constructing the vector expressing three sgRNAs targeting a single gene in the mouse brain. We describe steps for CRISPR knockout sgRNA design, plasmid construction and verification, animal preparation, and neonatal adeno-associated viral (AAV) vector delivery. We then detail procedures for brain preparation and immunofluorescence-based validation of gene disruption. This protocol enables rapid, one-step assembly of triple-sgRNA expression cassettes. For complete details on the use and execution of this protocol, please refer to Ogawa et al.1