Cas9 Nuclease
Cas9 Nuclease - Related Videos
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.
12:04
Mouse Genome Engineering Using Designer Nucleases
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Cited by 14 •
2014
Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.
08:13
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
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2025
This article describes a methodology for overexpressing recombinant Nsp15, a toxic nuclease, in a C41(DE3) expression system, followed by purification of the tagged protein utilizing affinity and size exclusion chromatography. These protocols can be adapted for other challenging toxic proteins.
05:12
All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System
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2025
In this video, we demonstrate all-in-one CRISPR-Cas9 based genome editing in cultured cells where Cas9 and sgRNA are provided as a single plasmid construct to the cells. The CRISPR-Cas9 system and desired gene to be inserted was introduced in cells through electroporation technique to facilitate successful gene editing.
FAQs
Cas9 nuclease cleaves only when the guide-directed DNA match lies next to a compatible protospacer adjacent motif, or PAM. This requirement adds a second layer of sequence recognition beyond guide complementarity and determines which genomic sites are accessible for editing. In infection studies, PAM availability therefore influences which host or microbial genes can be selected for functional analysis.
The single-guide RNA provides the sequence-recognition component of the system by pairing with a complementary DNA target. Cas9 uses this RNA-DNA match together with PAM recognition to position its cutting activity at the intended site. Changing the guide sequence can redirect cleavage to different genes, allowing researchers to examine distinct host factors, immune pathways, or microbial functions.
Cas9 cuts both DNA strands at a selected locus, producing a targeted double-strand break rather than a nonspecific alteration across the genome. This defined lesion underlies gene knockout and targeted genome-editing experiments. In immunology and infection research, the resulting genetic disruption can reveal whether a particular host or pathogen gene contributes to entry, replication, virulence, or immune responses.
A typical study selects a host or microbial gene of interest, designs a single-guide RNA complementary to a target sequence with a compatible PAM, and uses Cas9 to create a break at that locus. Researchers then examine the biological consequences of the targeted gene disruption, such as altered pathogen entry, replication, virulence, or host immune behavior.
Targeted disruption of host genes allows investigators to test whether those genes influence pathogen entry, replication, or the immune response. Comparing the effects of different gene knockouts connects a specific host factor with an infection-related phenotype. This approach supports functional rather than purely observational analysis, helping clarify which cellular components contribute to host-pathogen interactions.
In microbial studies, targeted cutting can support functional investigation of virulence genes, linking specific genetic elements to traits that influence infection. The same programmable activity provides a basis for exploring potential antimicrobial strategies and therapeutic approaches. These applications extend Cas9 research beyond host-gene analysis to questions about pathogen behavior and ways to modify infection outcomes.