Sep 2026· STAR Protocols· Vol 7, pp. 104837· 0 citations· 17 references
Medicine
TL;DR
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.
Abstract
Summary CRISPR-Cas9 is a powerful tool for editing genomic loci, however achieving high knockout efficiency at certain targets remains challenging. Here, we present a protocol for gene knockout using an all-in-one, quad-guide RNA-expressing vector. We describe steps for plasmid construction, virus preparation, transduction, and subsequent gene editing and functional validation within DLD-1 colorectal adenocarcinoma cells. This strategy provides an efficient workflow for gene knockout that is rapidly confirmed through PCR amplification of mRNA derived from the targeted gene loci.
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
Adeno associated viral (AAV) delivery of CRISPR-Cas9 enables efficient, targeted hepatic gene disruption composed of both small insertions and deletions (INDELs) and larger DNA structural variants (DNA-SVs), which are difficult to quantify. Here, we present a protocol for quantifying AAV-CRISPR-mediated gene editing in...
K. Jarrett, Rodrigo Baltazar-Nunez, Chloe Anne Borja et al.· STAR Protocols· 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
The knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development and can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses.
Vasileios Morfos, M. Frie, Daniel Peschkov et al.· bioRxiv· 0 citations
Functional validation of the gRNAs designed to target exon 2 of the COL1A1 gene is confirmed through in vitro cleavage activity and support its candidate selection for subsequent cellular studies targeting COL1A1, representing an initial step toward future knockout-style cellular models of Osteogenesis Imperfecta.
D. M. Melo, Sabrina Rodrigues Sarquis, Paolla Manzoli Luz et al.· Methods· 0 citations
Recent advances in genome editing technologies have enabled transformative therapeutic strategies for hematological disorders. Efficient implementation of these approaches requires reliable delivery of genome editing components into primary hematopoietic stem and progenitor cells (HSPCs), which are particularly sensiti...
Nikoletta Y. Papaioannou, P. Papasavva, P. Patsali et al.· Methods in molecular biology· 0 citations
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