A CRISPR interference strategy for gene expression silencing in multiple myeloma cell lines

Abstract

Background: Multiple myeloma is the second most common hematologic neoplasm which is characterized by proliferation and infiltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to treatment resistance. The CRISPR/Cas9 gene editing system has been recently used in several cell lines and mouse models to understand the role of various genetic alterations in pathogenesis of hematologic malignancies. Despite the recent advances of gene edition tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy. Results: We have designed different guide RNAs and cloning them into a lentiviral plasmid, which contain all the machinery necessary for develop the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids for the production of lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression.Conclusions: We have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines.