Crispr/cas9 Mediated Deletion
Crispr/cas9 Mediated Deletion - Related Videos
11:31
Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
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Cited by 23 •
2016
Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).
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.
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...
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 guide RNA directs Cas9 to a complementary genomic target, but the biological result depends on how the cell repairs the resulting double-strand break. Error-prone non-homologous end joining can introduce disruptive mutations or remove the DNA sequence between targeted sites. Consequently, researchers may obtain either a sequence deletion or a mutation that interferes with gene function, making repair outcome important when interpreting phenotypes.
The guide RNA supplies the sequence-recognition component of the editing system. Its complementary sequence directs the Cas9 nuclease toward a chosen genomic location, where Cas9 creates the double-strand break. This pairing connects the intended target, such as a developmental gene or regulatory region, with the cellular repair event that produces the genetic change examined in the experiment.
Deleting a developmental gene directly tests how loss or disruption of that gene affects development, whereas removing an enhancer or other regulatory region examines how controlling DNA influences gene activity and developmental processes. This distinction helps researchers separate the contribution of a gene itself from the contribution of genomic elements that regulate developmental programs or signaling pathways.
Researchers first select a gene, enhancer, or other DNA segment whose developmental role they want to examine. A guide RNA directs Cas9 to the relevant genomic target, and the resulting double-strand break is repaired by the cell. The edited cells, embryos, or model organisms are then examined for phenotypes that indicate effects on differentiation, tissue formation, or overall development.
This approach is useful when researchers need a functional test of a specific developmental gene, enhancer, regulatory region, or signaling pathway. By altering the selected DNA directly in cells, embryos, or model organisms, they can connect the resulting genetic change with developmental consequences. It therefore supports experiments that move beyond association toward testing whether a component contributes to development.
Observed phenotypes can show whether the targeted sequence contributes to cell differentiation, tissue formation, or organismal development. In developmental biology, these outcomes provide functional evidence about the roles of genes, enhancers, and signaling pathways. Interpreting the phenotype alongside the edited target helps identify which developmental process is affected by disrupting or removing that genomic element.