Multiplex Grna Expression
Multiplex Grna Expression - Related Videos
10:32
A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
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Cited by 2 •
2017
Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.
08:40
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
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Cited by 15 •
2016
Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.
10:37
Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform
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Cited by 7 •
2016
We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.
13:14
Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications
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Cited by 4 •
2015
Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.
10:00
A Customizable Protocol for String Assembly gRNA Cloning (STAgR)
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Cited by 2 •
2018
Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.
FAQs
These processing signals are placed within a polycistronic guide-RNA array and help separate the encoded guides inside the cell. Processing produces individual guide RNAs that can function with the Cas protein rather than remaining as one continuous transcript. The design therefore connects compact genetic encoding with production of multiple usable targeting molecules.
A single construct must encode several guide sequences while preserving the processing signals that separate them into functional units. Each resulting guide directs the Cas protein toward its own complementary DNA target. This organization allows multiple genomic sites to be addressed from one genetic design, supporting coordinated changes across different loci.
Sequential editing addresses targets one after another, whereas a multiplex arrangement coordinates several guide RNAs from one construct in the same cell. This can make simultaneous manipulation of related loci more efficient and is especially useful when the biological question depends on gene interactions or pathway-level effects rather than an isolated genomic change.
Researchers first encode multiple guide sequences in one polycistronic construct, incorporating tRNA sequences or self-cleaving ribozymes as processing signals. After the construct enters a cell, these signals help generate separate guide RNAs. The guides then associate with a Cas protein and direct it to their corresponding complementary DNA targets for the intended genomic intervention.
Depending on the CRISPR system and experimental design, multiple guides can support gene knockout, gene activation, gene repression, or precise genome editing at several sites. Combining these capabilities allows investigators to alter more than one genetic element in a coordinated experiment, which can reveal how distinct loci contribute to a shared biological outcome.
Simultaneous targeting lets researchers perturb several genes or genomic sites within the same cellular context. That design can expose interactions among genes and help investigators examine coordinated effects across a biological pathway. It also supports higher-throughput genetic screens by enabling broader sets of targets to be tested more efficiently than sequential editing.