Yeast Gene Knockout
Yeast Gene Knockout - Related Videos
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.
08:48
Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
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Cited by 5 •
2017
A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.
02:29
In-vitro Mutagenesis (Gene Knockouts)
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2026
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes. The Process Genes can be randomly knocked out, or specific genes can be targeted. To knock out a particular gene, an engineered piece of DNA called a targeting vector is used to replace the...
11:53
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
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Cited by 17 •
2017
This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.
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.
FAQs
These matching flanking sequences guide homologous recombination, allowing the engineered DNA to align with the corresponding chromosome regions. That alignment supports replacement of the selected gene rather than an unrelated genomic segment. The design therefore determines whether the intended locus is disrupted, which is essential for connecting the observed phenotype to loss of that gene.
A selectable marker provides a way to identify cells that carry the engineered gene replacement. After the DNA modification step, researchers use the marker to distinguish candidate cells from those that did not acquire the replacement. Isolating marker-containing cells enriches for strains suitable for subsequent comparisons of gene-loss effects.
The normal strain provides a reference for assessing changes associated with losing the target gene. Differences in growth, metabolism, stress responses, or cell division can then be evaluated against the unmodified cellular background. This comparison helps separate effects linked to the gene disruption from characteristics that are part of ordinary yeast biology.
Researchers first select the gene whose function they want to examine, then prepare engineered DNA containing a selectable marker and sequences matching the gene’s flanking regions. Following homologous recombination, cells carrying the replacement are identified through the marker and isolated. The resulting strain can be compared with normal yeast in relevant cellular assays.
The technique is useful when researchers need to connect an individual gene with a broader cellular process. Examining knockout effects can reveal roles in metabolism, growth, stress responses, or cell division, while comparisons across related genes can support pathway analysis. These results help clarify how genetic components contribute to coordinated biological functions.
Loss-of-function yeast strains can provide experimentally tractable systems for examining genes or pathways relevant to disease biology. Their phenotypes may help researchers analyze pathway behavior and identify cellular functions that warrant further investigation. The same approach can support evaluation of potential drug targets by showing how disrupting a gene affects important cellular outcomes.