Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR
March 1st, 2019
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Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.
Overview
This article discusses a protocol utilizing CRISPR technology to overexpress multiple splice variants of long noncoding RNAs. Traditional cDNA-based methods are limited in their ability to handle the complexity of these isoforms.
Key Study Components
Area of Science
- Neuroscience
- Genetics
- Molecular Biology
Background
- Noncoding RNAs can have multiple isoforms.
- Studying all expressed isoforms in vitro is challenging.
- CRISPR technology allows for precise gene expression.
- This method utilizes the endogenous gene-splicing machinery.
Purpose of Study
- To develop a reliable method for overexpressing long noncoding RNA isoforms.
- To enhance the understanding of the functionality of these isoforms.
- To provide a protocol that can be replicated in various research settings.
Methods Used
- CRISPR technology for gene editing.
- Designing guide RNAs specific to target genes.
- In vitro expression studies of noncoding RNAs.
- Demonstration of the procedure by a post-doc researcher.
Main Results
- The protocol successfully allows for the expression of multiple isoforms.
- Endogenous splicing machinery is utilized effectively.
- Demonstrated technique enhances the study of noncoding RNA functionality.
- Provides a framework for future research on long noncoding RNAs.
Conclusions
- CRISPR technology is a powerful tool for studying noncoding RNAs.
- The method improves the understanding of RNA isoform functionality.
- This approach can be adapted for various noncoding RNA studies.
Frequently Asked Questions
What are long noncoding RNAs?
Long noncoding RNAs are RNA molecules that do not encode proteins but play crucial roles in regulating gene expression.
How does CRISPR technology work?
CRISPR technology allows for precise editing of DNA by using guide RNAs to target specific genes for modification.
What is the significance of isoforms?
Isoforms can have different functions and regulatory roles, making their study important for understanding gene expression dynamics.
Can this method be used for other types of RNA?
While this protocol focuses on long noncoding RNAs, similar CRISPR techniques can be adapted for other RNA types.
Who demonstrated the procedure?
The procedure was demonstrated by Robert Rankin, a post-doctoral researcher in the laboratory.
Executive Industry Relevance
Overexpressing long noncoding RNAs (lncRNAs) using CRISPR activation enables functional interrogation of disease-associated transcripts without cDNA limitations, supporting target validation in discovery pipelines. This approach addresses challenges posed by low expression, tissue specificity, and isoform complexity, providing a mechanism to assess lncRNA involvement in inflammatory pathways. By leveraging endogenous splicing, the method enhances predictive confidence in preclinical models and supports mechanistic de-risking of lncRNA targets.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of lncRNA function in disease-relevant contexts such as inflammatory bowel disease using IFNG-AS1 as a model.
- Operational Value: Supports overexpression of multiple endogenous splice forms, increasing confidence in target engagement and pathway modulation.
- Scientific Value: Facilitates biological de-risking by linking lncRNA overexpression to downstream phenotypic readouts like cytokine production.
Screening & Assay Development
- Scientific Value: Generates standardized, inducible overexpression systems suitable for high-content screening of lncRNA-modulating compounds.
- Operational Value: Produces quantifiable outputs via RT-qPCR with housekeeping gene normalization, enabling dose-response and temporal expression profiling.
- Scientific Value: Ensures expression of native isoforms, improving assay relevance compared to ectopic cDNA approaches.
Translational & Preclinical Research
- Scientific Value: Uses human Jurkat T-cells, enhancing translational relevance for immune-mediated diseases.
- Operational Value: Enables clonal expansion and selection, supporting consistent preclinical model generation.
- Scientific Value: Links lncRNA overexpression to functional immune outputs, supporting biomarker-aligned mechanism of action studies.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target hypothesis testing to lead identification, particularly for noncoding RNA targets where traditional overexpression fails.
- Discovery Biology: Supports hypothesis testing by enabling controlled overexpression of lncRNAs to assess phenotypic consequences in immune cells.
- Screening: Produces reproducible, inducible expression systems amenable to compound library screening for modulators of lncRNA activity.
- Analytics: Delivers quantitative, normalized gene expression data via RT-qPCR, facilitating comparison across conditions and clones.
- Translational Research: Uses human T-cell model, enabling direct relevance to inflammatory disease pathways and preclinical validation.
- Enterprise Reuse: Establishes a modular platform for lncRNA overexpression applicable across multiple targets and cell types.
Operational & Enterprise Impact
- Scientific Value: Increases target validation confidence by enabling physiologic overexpression of multiple lncRNA isoforms.
- Operational Value: Provides a standardized, lentiviral-based system for consistent, scalable overexpression across experiments.
- Strategic Value: Reduces false negatives in target assessment by overcoming limitations of low-abundance lncRNA detection.
- Portfolio Impact: Supports go/no-go decisions in lncRNA target programs by providing mechanistic insight prior to investment.
Implementation Considerations
- Requires expertise in molecular cloning, lentiviral production, and CRISPR-based transcriptional activation.
- Necessitates biosafety level 2 (BSL-2) facilities for handling lentiviral vectors and human cell lines.
- Depends on accurate gRNA design within 100 bp of transcriptional start site for effective activation.
- Requires validation of overexpression via isoform-specific or pan-transcript RT-qPCR with appropriate controls.
- Involves multi-day workflow including virus production, transduction, selection, and clonal expansion.
Biopharma FAQs
Why does gRNA design near the transcriptional start site matter for lncRNA overexpression?
Positioning the gRNA within 10 to 100 base pairs of the transcriptional start site ensures effective recruitment of the CRISPR activation complex to the endogenous locus, enabling robust overexpression of IFNG-AS1 and its splice variants in Jurkat T-cells.
How does using a two-plasmid system support the overexpression workflow?
The two-plasmid system allows separate transduction of dCas9 and gRNA-MS2 enhancer components, enabling selection of double-positive cells and enhancing transcriptional activation of the target lncRNA through synergistic effector recruitment.
What quantitative measurement confirms successful lncRNA overexpression in this protocol?
RT-qPCR using primers against all known IFNG-AS1 transcripts demonstrated a 20-fold increase in expression relative to controls, with housekeeping gene HPRT1 normalization confirming specific upregulation.
Why are replication requirements important for validating lncRNA overexpression results?
Replication across clonal lines and experimental runs ensures that observed overexpression is not due to insertional effects or clonal variability, supporting reliable phenotype-genotype links in functional follow-up studies.
What statistical analysis is recommended before implementing this overexpression method in a discovery pipeline?
Normalization to housekeeping genes, comparison across multiple clones, and assessment of splice variant expression are recommended to distinguish specific overexpression from nonspecific effects and ensure data robustness prior to downstream application.
Chapters in this video
0:04
Title
0:40
Vector Design and Generation
1:52
Virus Generation
4:24
Particle Count using p24 Enzyme-linked Immunosorbent Assay Kit
7:07
dCas9 and gRNA Transduction, Selection, and Clone Creation
10:18
Results: Using CRISPR Technology to Overexpress Multiple Splice Variants of a lncRNA
12:08
Conclusion
