Cas9 Mrna Preparation
Cas9 Mrna Preparation - Related Videos
06:57
Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing
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Cited by 1 •
2023
This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.
08:33
Cell-Free In Vitro mRNA Translation In Drosophila Lysates
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2026
Here, we provide details on how to prepare lysates and perform in vitro translation using Drosophila embryos.
02:22
Regulated mRNA Transport
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2020
In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing specific...
02:31
Nuclear Export of mRNA
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2020
Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
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.
FAQs
These processing steps convert the transcript into the capped and polyadenylated form used for cellular translation. They are performed after or during in vitro transcription, depending on the preparation strategy, and precede purification. The resulting RNA can then support transient production of Cas9 after delivery into cells with a compatible guide RNA.
Cas9 mRNA provides the temporary instructions for producing the Cas9 enzyme, but the guide RNA directs where the resulting enzyme acts. Together, they enable a targeted DNA break rather than an untargeted editing event. Cellular repair of that break can then produce gene disruption or sequence modification at the selected genomic location.
Because the delivered material is messenger RNA, Cas9 production is transient rather than sustained by continued expression from a genetic template. This limits the duration of enzyme availability in cells while still allowing a targeted DNA break and subsequent repair. The feature is useful when experiments aim to reduce prolonged Cas9 activity during genome editing.
A typical workflow begins with a DNA template containing the Cas9 coding sequence and uses in vitro transcription to generate the RNA. The transcript then receives or incorporates a 5′ cap and poly(A) tail, followed by purification. Purification removes the DNA template, enzymes, and abnormal RNA products before the material is used for cell delivery.
Purification is intended to remove residual DNA template, transcription enzymes, and abnormal RNA products generated during synthesis. Removing these components produces a cleaner preparation for delivery into cells and helps ensure that the material used in a CRISPR experiment is primarily the intended Cas9 transcript rather than a mixture of reaction components.
The prepared transcript supports CRISPR experiments in which transient Cas9 production creates a targeted DNA break for cellular repair. Its applications include gene disruption, sequence modification, functional genomics, disease modeling, and potential therapeutic development. These uses connect RNA preparation quality directly to experiments that investigate gene function or model genetically relevant changes.