Crispr/cas9 Knockout
Crispr/cas9 Knockout - Related Videos
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.
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.
08:25
Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
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Cited by 6 •
2017
The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.
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.
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.
FAQs
The key outcome is often a disruption of the gene’s reading frame. After Cas9 cuts both DNA strands, non-homologous end joining repairs the break without precisely restoring the original sequence. Insertions or deletions created during this repair can shift how the gene is read, preventing production of a functional protein and making the resulting cells useful for studying gene loss.
Guide design determines whether Cas9 is directed toward the intended gene sequence and whether the resulting edit can support a clear interpretation. A suitable guide should promote disruption of the target while limiting unintended matching sites elsewhere in the genome. Careful design therefore improves confidence that an observed phenotype reflects the intended gene knockout rather than an off-target edit.
A disruption becomes informative when the loss of functional protein can be connected to a measurable biological consequence. Comparing the altered system with an appropriate unedited context helps researchers evaluate how the gene contributes to a cellular pathway or trait. This approach links a specific genetic change to gene function, while validation helps separate the intended effect from other experimental influences.
A typical workflow begins by selecting a target sequence within the gene and designing a matching guide RNA. Cas9 is then directed to that sequence to create the DNA break, after which cellular non-homologous end joining can generate disruptive insertions or deletions. Researchers subsequently validate the edited material to determine whether the intended gene disruption occurred and to assess possible off-target effects.
Validation should determine whether the targeted gene carries a disruptive change and whether the result is consistent with loss of functional protein production. It should also examine the possibility of off-target edits and other experimental effects that could explain the outcome. This verification is essential before attributing a cellular or organismal phenotype specifically to the intended knockout.
Researchers apply the approach to investigate gene function, model the effects of gene loss, and examine cellular pathways. It can be used in cultured cells as well as organisms, allowing questions to be studied in different biological settings. The method also supports disease modeling, where disrupting a selected gene helps researchers examine consequences relevant to genetic disease mechanisms.