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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Mráz, Marek (referee)
Markéta Sypecká CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, because of complications from CLL, but most patients live 5-10 years. However, every stage of this disease has significantly higher level of miR-155, which is known as oncomiR. Micro RNAs represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets. We hypothesized that by gene editing of CLL B - cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Method we use for gene editing is CRISPR/Cas9, which enables to delete sequence of mature miR-155 in genome of leukemic B-cells. Methods: CRISPR/Cas9, nucleofection, qRT-PCR, FACS Results:We achieved to isolate clone that bears one allelic deletion (miR-155-/+) in sequence for mature...
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