Crispr/cas9 Gene Knockout
Crispr/cas9 Gene Knockout - Related Videos
11:35
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Cited by 6 •
2017
Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.
04:13
CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells
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2025
This video describes a gene knockout technique using a CRISPR-concatemer to simultaneously knock out multiple genes in cultured mouse intestinal organoid cells. This method is used to knock out a diseased gene and to elucidate the function of a gene and its paralogues.
12:25
Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells
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2026
Here, we present a protocol for generating chemoresistant cells using a dose-escalation approach, followed by a CRISPR/Cas9-based screen using a focused sgRNA library targeting epigenetic modifiers to identify regulators of acquired chemoresistance. The protocol also provides multiple optimization points tailored to chemoresistant cell models, offering a robust framework for researchers investigating resistance mechanisms.
10:42
A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
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Cited by 21 •
2017
We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.
04:22
A Technique for Gene Editing in Natural Killer Cells Using CRISPR Cas9
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2025
This video demonstrates a technique for Cas9 ribonucleoprotein-mediated genetic modification of primary natural killer (NK) cells. A Cas9 ribonucleoprotein, consisting of a Cas9 endonuclease bound to a guide RNA (gRNA) formed by base pairing a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), is introduced into primary natural killer cells via electroporation. The ribonucleoprotein targets and cleaves the host DNA at the target site, leading to gene knockout via modification of the...
FAQs
The guide RNA determines where Cas9 acts by pairing with a complementary DNA sequence. Cas9 then cuts that selected location, linking sequence recognition to a physical break in the genome. This division of labor gives the method its adaptability: changing the guide RNA can redirect the editing system toward a different gene or genomic target for functional studies.
Gene disruption depends on what happens after the cut. Error-prone repair, often through nonhomologous end joining, can insert or delete DNA bases at the break. If these indels shift the gene’s reading frame, the resulting sequence can prevent normal gene expression. Thus, the knockout outcome is tied not only to targeting, but also to repair.
The double-strand break creates the critical opportunity for gene disruption because repair can alter the original DNA sequence. In CRISPR/Cas9 gene knockout, error-prone nonhomologous end joining may generate insertions or deletions rather than restoring the sequence exactly. Those changes can produce a frameshift, connecting the molecular repair event to loss of gene expression and observable biological effects.
A basic workflow begins by selecting a gene of interest and using a guide RNA designed to recognize a complementary DNA sequence. Cas9 is directed to that site and creates a double-strand break. The cell’s repair process can introduce disruptive insertions or deletions, after which researchers study how loss of the gene affects the cell, organism, or biological pathway.
Researchers use this approach when they need to connect a gene with its biological function or examine the consequences of losing its activity. It can support studies of inherited diseases, biological pathways, and drug-target validation. Because the target can be selected through the guide RNA, the method can be adapted to many functional genomics investigations.
In genetics, gene knockout can help model inherited diseases by examining the effects associated with disruption of a relevant gene. It also allows researchers to test whether a gene is a plausible drug target by studying the consequences of its loss. These findings contribute to broader functional genomics and may inform potential therapeutic strategies.