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Updated: Aug 6, 2026
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Published on: May 23, 2016
Protocol for one-step design of a triple sgRNA-based CRISPR/Cas9 construct for neuronal gene knockout in the mouse
1Department of Biological Sciences, McCausland College of Arts and Sciences, University of South Carolina, Columbia, SC 29208, USA.
Abstract:
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.
