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Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis

Summary

This article examines the use of CRISPR-Cas9 gene editing tools to inactivate a non-coding regulatory DNA element found upstream of the mouse tyrosinase (Tyr) gene. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression, as inferred from the decrease of coat pigmentation, thus supporting the functionality of the boundary sequence in vivo. Several potential off-targets were analyzed and found to be largely intact. This study illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.

Q&As

What is the purpose of the CRISPR-Cas9 system?
The purpose of the CRISPR-Cas9 system is to allow simple and rapid genetic modification in most model organisms and human cell lines.

What effects did the inactivation of the genomic insulator have in the mouse?
The inactivation of the genomic insulator resulted in a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation.

What evidence was found to support the functionality of the boundary sequence in vivo?
Evidence was found to support the functionality of the boundary sequence in vivo by the production and analysis of mice carrying the inactivation via deletion of the genomic insulator.

How can the regulatory elements described by the ENCODE and EPIGENOME projects be validated?
The regulatory elements described by the ENCODE and EPIGENOME projects can be systematically validated by the CRISPR-Cas9 system.

What other methods of DNA manipulation have been described?
Other methods of DNA manipulation that have been described include DNA microinjection, somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections, and the G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.

AI Comments

👍 This article provides an in-depth analysis of the functionality of mouse tyrosinase non-coding regulatory DNA elements and the effect of CRISPR-cas9 mediated mutagenesis on them.

👎 The article lacks concrete evidence to support its claims about the functionality of mouse tyrosinase non-coding regulatory DNA elements.

AI Discussion

Me: It discusses the implications of using CRISPR-Cas9-mediated mutagenesis to functionally validate non-coding regulatory DNA elements found 5' upstream of the mouse tyrosinase (Tyr) gene. They found that the deletion of this element resulted in a decrease in coat pigmentation, thus providing evidence of its functionality in vivo.

Friend: Interesting. So this technology has a wide range of potential applications then?

Me: Absolutely. It can be used to validate non-coding DNA elements, and can even be used to study and treat diseases. It can also be used to generate knockout mice, which can be used to study gene function. Furthermore, it can be used to study conserved cis-regulatory elements, which could provide insight into gene regulation.

Action items

Technical terms

CRISPR-Cas9
A gene-editing tool that uses a combination of a guide RNA and a Cas9 enzyme to target and modify specific sequences of DNA.
Non-coding regulatory DNA elements
DNA sequences that do not code for proteins, but instead regulate gene expression.
Genome-editing
The process of making specific changes to the DNA of an organism.
Inactivation
The process of making a gene or gene product non-functional.
Off-targets
Genes or gene products that are affected by a gene-editing tool, but are not the intended target.
Inversion
A type of genetic mutation in which a segment of DNA is reversed.
RNA guides
Short pieces of RNA that are used to target a specific sequence of DNA for editing.
ENCODE and EPIGENOME projects
Projects that aim to identify and map all of the functional elements in the human and mouse genomes.

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