Crispr Cas9 Knock-in
Crispr Cas9 Knock-in - Related Videos
05:12
All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System
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2025
In this video, we demonstrate all-in-one CRISPR-Cas9 based genome editing in cultured cells where Cas9 and sgRNA are provided as a single plasmid construct to the cells. The CRISPR-Cas9 system and desired gene to be inserted was introduced in cells through electroporation technique to facilitate successful gene editing.
07:56
Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells
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Cited by 34 •
2019
Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.
02:55
Genetically Modifying CAR T Cells Using a CRISPR-Cas9 System
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2025
The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.
11:32
Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
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Cited by 7 •
2021
This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.
06:52
CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts
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Cited by 2 •
2022
This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.
FAQs
Specificity begins with guide RNA recognition of a matching DNA sequence. When Cas9 reaches this target, it creates a double-strand break at the selected genomic site. That break provides the entry point for donor-directed repair. Because the donor carries the intended sequence, repair can connect the engineered DNA to a defined location, supporting controlled changes in cellular function.
The donor DNA template carries the sequence intended for insertion, while homology-directed repair, or HDR, uses that template after Cas9 creates the break. The donor therefore determines whether the engineered cell receives a reporter gene, mutation, or tagged-protein sequence. Choosing the donor is central to linking the editing event to the planned functional question.
Selecting a genomic location determines where the engineered sequence is placed and therefore which cellular function can be examined. A knock-in at a relevant gene or regulatory site can connect the introduced reporter, mutation, or tag with the biology under study. This positional logic is especially useful when interpreting changes in signaling or gene regulation.
The workflow links three elements: a guide RNA that matches the target, Cas9-mediated creation of a double-strand break, and a donor DNA template. First, the guide RNA directs Cas9 to the chosen site. After cleavage, the donor supplies the sequence that homology-directed repair incorporates. This order enables a planned genetic change in an immune or host cell.
Reporter-gene knock-ins can connect a defined genomic change with studies of cellular activity. In immunology research, this strategy supports investigation of gene regulation and signaling by introducing a reporter at a selected location. The resulting engineered model helps researchers examine how a targeted genetic alteration relates to cellular function and immune responses.
A tagged-protein knock-in can add a defined sequence to a protein for focused investigation. In immunology and infection studies, this design can help examine signaling, gene regulation, or interactions between host cells and pathogens. Its value comes from connecting the engineered protein to a specific cellular question rather than making an undefined genomic alteration.
Host-cell knock-ins can be used to study pathogen interactions alongside immune signaling and gene regulation. By introducing a reporter, mutation, or tagged protein into a host cell, researchers can examine how a defined genetic change relates to the cell's response to infection. These models may improve understanding of disease mechanisms and immune responses.