Cas9 Activation
Cas9 Activation - 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.
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...
11:37
Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Cited by 21 •
2019
Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.
02:36
Establishing a Murine Tumor Cell Line In Vitro Using a Virus-Cas9 System
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2026
Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System. J. Vis. Exp. (2020)This video demonstrates the in vitro transformation of murine epithelial cells through viral delivery of Cre recombinase and mutation-inducing components. It outlines the steps for Cas9 activation, cancer-gene modification, and the establishment of a tumorigenic murine cell line.
FAQs
Guide RNA binding places Cas9 in a configuration capable of locating a complementary DNA sequence. The RNA provides the sequence-specific information, while the resulting protein-RNA complex recognizes DNA containing a matching target next to a protospacer adjacent motif, or PAM. This pairing requirement makes activation dependent on both guide complementarity and the appropriate neighboring DNA sequence.
The PAM supplies a required sequence context next to the DNA protospacer. Cas9 does not rely on guide complementarity alone; target recognition also depends on finding this adjacent motif. Consequently, the PAM helps determine whether a potential DNA site can support productive activation and cleavage, making it an important variable when selecting targets for genome engineering.
When the guide RNA pairs with its complementary DNA sequence, the interaction forms an R-loop, a structure in which the RNA-DNA pairing displaces the opposite DNA strand. This event is not merely a recognition step. It triggers conformational changes in Cas9 that align its nuclease regions, converting target binding into a cleavage-competent state.
Activation positions the HNH and RuvC nuclease domains so they can cut the two DNA strands. Their coordinated activity produces a targeted double-strand break, typically at the recognized site. This division of catalytic roles is central to Cas9 function because sequence recognition by the guide RNA must be coupled to synchronized cleavage of both strands.
A productive target must satisfy several linked conditions: Cas9 must bind the guide RNA, the guide must pair with a complementary DNA sequence, and that sequence must lie next to a suitable PAM. Pairing then has to form an R-loop and induce the conformational changes that align the HNH and RuvC domains. These steps connect recognition with cleavage.
In genome engineering, activation creates a targeted DNA double-strand break at a selected sequence. That break can support gene disruption and precise genome editing, while the programmable guide sequence determines the intended target. The same mechanism also underlies functional genomics studies, where researchers use targeted cleavage to investigate gene function.
Cas9 activation is a central mechanism in bacterial immunity as well as a foundation for engineered technologies. Its programmable DNA recognition and cleavage have enabled applications in functional genomics and contributed to the development of diagnostic and therapeutic technologies. These uses arise from linking guide-directed targeting to controlled activation of the nuclease domains.