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

Overview

1. Department 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

Reproduced under the paper's license (CC BY), from the paper cited above.

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Data and code availability

All data reported in this paper will be shared by the lead contact upon request. All original code is available in this paper’s supplemental information (File S1). Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 2 authors, 3 keywords, 1 funder, 10 references, 5 RRIDs.

Cite

This paper

Dowd, G., & Ogawa, Y. (2026). Protocol for one-step design of a triple sgRNA-based CRISPR/Cas9 construct for neuronal gene knockout in the mouse brain. STAR protocols, 7(3), 104715. https://doi.org/10.1016/j.xpro.2026.104715

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