Gene expression editing in myeloma cell lines using CRISPR/Cas9 technique

Sammanfattning: Multiple myeloma, or myeloma, is a bone marrow cancer which characterizes by uncontrolled proliferation of mutant plasma cells. It is a disease that claims many lives every year, mostly due to the absence of curative treatment. Finding a suitable treatment is therefor of great importance. One way to study different diseases is to use a gene editing method for knockdown or knockout of specific genes. The main aim of this project was to design guide RNAs, to be able to use CRISPR/Cas9 for knockout of the two genes BMPR1A and BMPR2 in different myeloma cell lines (KJON, INA-6 and IH-1). This, to be able to study the expression and function of these genes. Further aim of the project was to investigate potential SMAD activation by treatment with different bone morphogenetic proteins (BMPs). However, due to limited time this could not be carried through. Six guide RNAs were designed and ligated into pLentiCRISPRv2. Plasmid amplification was done by transformation of Escherichia coli. To check the quality of the plasmids, PCR, gel electrophoresis and Sanger sequencing was performed. The results from the gel electrophoresis showed that nine of the twelve samples for BMPR1A and seven of the thirteen samples for BMPR2, that were tested, were positive. The results from the Sanger sequencing confirmed that all guides that were tested (BMPR1A 3.2.3, BMPR1A 4.2.2, BMPR2 1.1.4 and BMPR2 2.1.2), were properly ligated into the plasmids. The main aim of the project was successfully accomplished, but additional work is needed for any further conclusions.