<em>HOX</em> Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Huacheng Luo; Amin Sobh; Christopher  D. Vulpe; Edmond Brewer; Sinisa Dovat; Yi Qiu; Suming Huang

doi:10.3791/59382

HOX Loci Focused CRISPR/sgRNA biblioteca Screening identificazione critico CTCF confini

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Genetics

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JoVE Journal Genetics

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

HOX Loci Focused CRISPR/sgRNA biblioteca Screening identificazione critico CTCF confini

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10:10 min

March 31, 2019

DOI: 10.3791/59382-v

Huacheng Luo¹, Amin Sobh², Christopher D. Vulpe², Edmond Brewer¹, Sinisa Dovat¹, Yi Qiu³, Suming Huang¹

¹Department of Pediatrics,Pennsylvania State University College of Medicine, ²Department of Physiological Sciences,University of Florida, ³Department of Anatomy and Cell Biology,University of Florida

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Overview

This study explores the use of a HOX loci specific sgRNA library to investigate the role of CTCF boundaries in gene regulation. The approach aims to enhance understanding of noncoding elements and their impact on HOX gene expression.

Key Study Components

Area of Science

Gene regulation
CRISPR technology
HOX gene function

Background

CTCF is a key regulator of chromatin structure and gene expression.
Understanding CTCF boundaries can provide insights into gene regulation mechanisms.
HOX genes play critical roles in development and disease.
Previous studies have not fully explored the impact of CTCF on HOX loci.

Purpose of Study

To elucidate the function of CTCF boundaries in HOX gene regulation.
To assess the feasibility of using an sgRNA library for this purpose.
To investigate the implications for early embryonic development and leukemias.

Methods Used

Design of sgRNA targeting CTCF binding sites.
Cloning of the sgRNA library into cell preparations.
Packaging of lentivirus using HEK293 T cells.
Screening for effects on gene expression.

Main Results

Successful design and implementation of a HOX loci specific sgRNA library.
Identification of the role of CTCF in regulating HOX gene expression.
Insights into the function of noncoding elements in gene regulation.
Potential applications in understanding leukemias with aberrant HOX signatures.

Conclusions

The study demonstrates the utility of sgRNA libraries in gene regulation research.
CTCF boundaries significantly influence HOX gene expression.
Findings may have broader implications for developmental biology and cancer research.

Frequently Asked Questions

What is the significance of CTCF in gene regulation?

CTCF is crucial for maintaining chromatin structure and regulating gene expression by forming boundaries that influence enhancer-promoter interactions.

How does the sgRNA library work?

The sgRNA library allows for targeted disruption of specific genomic sites to study their functional roles in gene regulation.

What are HOX genes?

HOX genes are a group of related genes that control the body plan of an embryo along the head-tail axis and are essential for proper development.

What potential applications do the findings have?

The findings can help in understanding developmental processes and the mechanisms underlying certain cancers associated with HOX gene dysregulation.

What methods were used to package the lentivirus?

Lentivirus was packaged by cotransfecting HEK293 T cells with specific plasmid vectors for the sgRNA library and envelope proteins.

Can this approach be applied to other genes?

Yes, the sgRNA library approach can be adapted to study other genes and regulatory elements beyond HOX loci.

Una libreria CRISPR/sgRNA è stata applicata a interrogare i geni di proteina-codificazione. Tuttavia, la fattibilità di una libreria di sgRNA per scoprire la funzione di un confine CTCF nella regolazione genica rimane inesplorata. Qui, descriviamo una raccolta di specifiche sgRNA loci HOX per delucidare la funzione dei confini CTCF in loci HOX .

Il nostro sgRNA in pool chiamato screening della biblioteca, è un potente approccio genetico per identificare e valutare la funzione biologica degli elementi non di rivestimento in tutto il genoma. Questa tecnica ci consente di esaminare l'effetto di interrompere i siti di legame associati, i confini cromatici o altri elementi regolatori sull'espressione dei geni mirati. Il nostro approccio può essere utilizzato per identificare il ruolo del CTCF, gli RNA e gli esaltatori lunghi non codificanti, nella regolazione genica HOX nello sviluppo embrionale precoce, così come alcune leucemie con una firma genica HOX aberrante.

Iniziare questa procedura con la progettazione di uno sgRNA destinato ai siti di associazione CTCF. Clonazione della libreria sgRNA nella preparazione delle celle come descritto nel protocollo di testo. Per confezionare il lentivirus, cotrasfetto cellule HEK293 T con 20 microgrammi di vettori di libreria purificata, 15 microgrammi del plasmide confezionato e 10 microgrammi del plasmide dell'involucro, per 48 ore prima di raccogliere i virus.

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