Cas9 Endonuclease
Cas9 Endonuclease - Related Videos
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Cited by 4 •
2012
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
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...
04:40
Modeling Prostate Cancer in Genetically-engineered Mouse Models: A CRISPR/Cas9-mediated Localized Gene Editing Technique in Mouse Anterior Prostate Lobe Cells
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2023
This video describes a method for editing specific genes in prostate gland cells using an adenovirus-based delivery system. The approach allows for orthotopic and localized alteration of gene expression utilizing CRISPR technology to develop novel mouse models for prostate cancer.
02:30
A CRISPR-Cas9 Technique for Gene Editing in T Cells
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2025
This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell's non-homologous end-joining mechanism, leading to gene knockout.
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 protospacer adjacent motif, or PAM, provides a required DNA feature next to the sequence recognized by the guide RNA. Cas9 therefore does not respond solely to guide-RNA complementarity; the target must also be positioned beside an appropriate PAM. This requirement helps determine which DNA sites can be selected for genome editing and influences targeting options in biological experiments.
The guide RNA supplies the sequence-matching information that brings the Cas9 protein complex to a complementary DNA region. Because this recognition information can be programmed, investigators can direct cleavage toward different genomic sequences by changing the guide RNA. In practice, the selected target sequence and its neighboring PAM jointly determine whether the intended site can be recognized and cut.
A double-strand break creates a repair problem that the cell must resolve. The resulting repair can modify the cut site or restore it, producing outcomes that support gene disruption or, when sequence replacement is intended, alteration of the targeted region. Thus, the biological effect depends not only on Cas9 cleavage but also on how cellular repair processes handle the break.
A typical workflow begins by selecting a DNA sequence that is complementary to a guide RNA and lies next to a suitable PAM. The guide RNA is then associated with Cas9, allowing the complex to recognize the target and cut both DNA strands. Researchers subsequently examine how cellular repair modified or restored the site to assess the editing outcome.
Cas9-based methods are useful when researchers need to examine what a gene does or change a selected DNA sequence. They can support gene disruption, sequence replacement, and functional analysis, linking a targeted genetic alteration to a biological outcome. These applications make the system relevant to studies of gene function, disease mechanisms, biotechnology, and potential therapeutic development.
Cas9 also provides a model for understanding bacterial adaptive immunity, rather than serving only as a genome-editing tool. Its RNA-guided recognition and DNA-cleavage activity illustrate how genetic information can direct a protein toward a specific nucleic-acid target. Studying this system connects molecular mechanisms of immunity with biological methods for manipulating and analyzing genomes.