Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR.

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• Long noncoding RNAs (lncRNAs) are increasingly implicated in various diseases.
• Studying lncRNA function is challenging due to their unique molecular characteristics and species-specific expression.
• Investigating lncRNA roles in disease requires robust methods for manipulating their expression levels.

Purpose of the Study:

• To present a detailed protocol for in vitro overexpression of long noncoding RNAs (lncRNAs).
• To demonstrate the utility of activating CRISPR technology for endogenous lncRNA manipulation.
• To establish a method for studying lncRNA function in disease contexts.

Main Methods:

• Development and visualization of a protocol for lncRNA overexpression.
• Utilizing activating CRISPR technology to target transcriptional start sites for gene activation.
• Employing a Jurkat T-cell model to overexpress the inflammatory bowel disease-associated lncRNA, IFNG-AS1.

Main Results:

• Successful implementation of a three-step protocol: gRNA design/vector construction, virus generation/transduction, and colony screening.
• Achieved over 20-fold enhancement of IFNG-AS1 expression in Jurkat T cells.
• Demonstrated the efficacy of activating CRISPR for robust overexpression of multiple lncRNA splice forms.

Conclusions:

• The presented protocol provides a reliable method for endogenous lncRNA overexpression in vitro.
• Activating CRISPR technology offers a powerful tool for dissecting lncRNA functions, particularly in disease-related studies.
• This method facilitates the investigation of lncRNAs like IFNG-AS1 in cellular models.