Julia Boese
Julia Boese
Julia Boese
Helios Scholar
School: Arizona State University
Hometown: Gilbert, Arizona
Mentor: Jonathan Keats, Ph.D.

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CRISPR knock-down of long-non-coding RNA NEAT1 in multiple myeloma

Multiple myeloma (MM) is a currently incurable hematological cancer of plasma cells that affects 2% of all cancer patients.  Our laboratory has shown that NEAT1, the long non-coding RNA structural backbone of the nuclear paraspeckles complex, is highly expressed in MM patients.  Of particular interest, patients in the top quartile of NEAT1 expression exhibit poor survival outcomes over the course of their disease and treatment, regardless of molecular classification.  A separate study recently demonstrated that NEAT1 repression sensitizes melanoma cancer cells to chemotherapy and PARP inhibitors, making NEAT1 repression a potential clinical target in melanoma. Based on these data, we hypothesized that NEAT1, or its regulated pathways, may be therapeutically-actionable targets in MM.  To test the effects of NEAT1 knock-down in MM, we employed a repressive dCAS9-KRAB and sgRNA two-component CRISPR system in KMS-34, a myeloma cell line with high NEAT1 expression.  We designed and cloned a 23 base-pair guide sequence targeting the NEAT1 transcription start site into pLX-sgRNA, which can be used to produce NEAT1-sgRNA lentiviral particles in HEK-293T cells.  Additionally, we generated a stable, puromycin-selected KMS-34-dCAS9-KRAB cell line using dCAS9-KRAB lentivirus, also generated in HEK-293T cells.  After dosing the transfected KMS-34 line with the guide lentivirus, we will confirm NEAT1 repression through qRT-PCR.  Future work will include studying the effects of NEAT1 knock-down on the morphology, viability, and growth changes of KMS-34.  In addition, qRT-PCR will be used to investigate dysregulation of NEAT1-related genes such as PSPC1, p53, and NEAT2. The results of this study can be used to further investigate the clinical treatment potential of NEAT1 knock-down in treating myeloma patients.