Crispr Shuttle Cloning
Crispr Shuttle Cloning - Related Videos
04:25
CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
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2025
We describe a protocol for a high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which allows the transfer of DNA fragments of interest between vectors without the need for PCR amplification of the DNA fragments.
06:48
CRISPR Guide RNA Cloning for Mammalian Systems
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Cited by 16 •
2018
Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.
01:59
CRISPR
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2019
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
09:55
Molecular Cloning
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2023
Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called...
08:30
Yeast Transformation and Cloning
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2023
S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures. This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast...
FAQs
Compatibility allows the assembled CRISPR components to move between plasmid contexts while preserving their ability to be propagated in one host and used in another. This separation of maintenance and application supports reagent construction in bacterial cells before delivery to the target system, helping researchers manage guide RNA and Cas nuclease expression cassettes across different experimental settings.
Researchers insert guide RNA sequences and Cas nuclease expression cassettes into the shuttle plasmid through restriction enzyme ligation or sequence-directed assembly. These approaches provide alternative routes for placing the required CRISPR elements into a compatible vector. The resulting construct can then be maintained in bacterial cells before transfer to the intended target system.
Controlled expression determines how the CRISPR machinery is made available in the target system after the construct is transferred. By organizing guide RNA and Cas nuclease sequences within expression cassettes, the strategy supports use of the editing components in diverse organisms. This is particularly relevant when studying gene function or biochemical pathways in different biological contexts.
A typical workflow begins by assembling the guide RNA sequence and Cas nuclease expression cassette in a compatible shuttle plasmid. Researchers then propagate the construct in bacterial cells, where the plasmid is maintained, before delivering it to the target system. This sequence separates reagent construction and plasmid propagation from the later gene-editing experiment.
The strategy is useful when researchers need to construct gene-editing reagents for experiments involving gene knockout, sequence modification, or functional screening. In biochemistry, these applications can support analysis of protein pathways and metabolic pathways. Moving the assembled components through a shuttle plasmid helps connect molecular reagent preparation with downstream functional studies.
By enabling CRISPR components to reach a target system, the approach supports targeted gene disruption, sequence alteration, and screening for functional effects. Those outcomes can be used to investigate how genes influence protein or metabolic pathways. The specific result depends on whether the experiment is designed for knockout, modification, or broader functional analysis.