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A CRISPR interference strategy for gene expression silencing in multiple myeloma cell lines
Josep Escrivá-Fernández1, Cristina Cueto-Ureña1,2, Amalia Solana-Orts1
1Department of Biomedical Sciences, School of Health Sciences, Universidad Cardenal Herrera-CEU, CEU Universities, Alfara del Patriarca, E-46115, Valencia, Spain.
Journal of Biological Engineering
|May 4, 2023
Summary
We developed a CRISPR interference strategy to reduce Rnd3 gene expression in multiple myeloma (MM) cell lines. This gene silencing approach successfully altered the cells' transcriptomic profile, offering a new tool for MM research.
Area of Science:
- Hematologic Malignancies
- Gene Editing Technologies
- Cancer Cell Biology
Background:
- Multiple myeloma (MM) is a prevalent hematologic neoplasm characterized by bone marrow plasma cell infiltration and resistance to treatment.
- CRISPR/Cas9 technology offers a powerful platform for investigating genetic alterations in hematologic malignancies.
- The application of CRISPR/Cas9 for gene editing in MM remains underexplored.
Purpose of the Study:
- To establish a CRISPR interference (CRISPRi) strategy for repressing Rnd3 expression in MM cell lines.
- To investigate the functional consequences of Rnd3 knockdown in MM.
Main Methods:
- Designed and cloned guide RNAs into a lentiviral plasmid for CRISPRi.
- Produced lentiviral particles by co-transfecting HEK 293T cells with packaging plasmids.
- Transduced RPMI 8226 and JJN3 MM cell lines to achieve Rnd3 knockdown.
- Performed 3' UTR RNA sequencing for transcriptomic analysis.
Main Results:
- Successfully generated lentiviral particles for CRISPRi-mediated Rnd3 knockdown.
- Demonstrated Rnd3 gene silencing at both transcriptional and protein levels in MM cell lines.
- Transcriptomic analysis revealed distinct profiles in Rnd3 knockdown cells compared to controls.
Conclusions:
- Developed and validated a CRISPRi strategy for stable Rnd3 knockdown in MM cell lines via lentiviral transduction.
- Proposed CRISPRi as a viable tool for gene silencing in MM research.
- Confirmed that Rnd3 silencing significantly alters the cellular transcriptomic landscape in MM.
