Jul 2026· STAR Protocols· Vol 7, pp. 104715· 1 citation· 23 references
Medicine
TL;DR
This protocol enables rapid, one-step assembly of triple-sgRNA expression cassettes and describes steps for CRISPR knockout sgRNA design, plasmid construction and verification, animal preparation, and neonatal adeno-associated viral vector delivery.
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
This protocol enables quantification of INDELs and DNA-SVs, allowing for validation of gene disruption, and describes guide RNA (gRNA) design, plasmid cloning, delivery of AAV-CRISPR to the liver, and execution of liver DNA-editing analysis.
K. Jarrett, Rodrigo Baltazar-Nunez, Chloe Anne Borja et al.· STAR Protocols· 0 citations
This strategy provides an efficient workflow for gene knockout that is rapidly confirmed through PCR amplification of mRNA derived from the targeted gene loci that is rapidly confirmed through PCR amplification of mRNA derived from the targeted gene loci.
Michael Tobin, Jie Dai, Li-Chuan Chan et al.· STAR Protocols· 0 citations
Summary Jurkat cells are a versatile cell-line model utilized across multiple areas of biology. Here, we present a protocol for inserting DNA sequences (knockins) at an endogenous locus using CRISPR-Cas9 technology via homology-directed repair (HDR). We describe steps for designing, constructing, and validating endogen...
Joana R. Costa, Yang Li, S. Ahrabi et al.· STAR Protocols· 0 citations
This chapter detail a methodology using a rAAV vector carrying Staphylococcus aureus Cas9 (SaCas9) to target the murine Hao1 gene in liver parenchymal cells as a curative treatment for primary hyperoxaluria type 1 (PH1).
Andrea Llanos-Ardaiz, N. Zabaleta, Laura Torella et al.· Methods in molecular biology· 0 citations
A detailed protocol for the production of high-titer, third-generation LVs with a dual-expression cassette for Cas13d and a customizable guide RNA is described, which can be used for stable transduction and efficient RNA knockdown in a broad range of mammalian cell types.
R. Stilhano, L. Martin· Methods in molecular biology· 0 citations
A systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing, which provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translation...
Giacomo Roman, K. Lauritzen, B. Smolková et al.· Stem Cell Reviews and Report...· 0 citations
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