Related Experiment Video
Updated: Sep 27, 2025
Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays
Published on: January 7, 2019
[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML]
Ling-Yan Wang1, Pei-Fang Jiang1, Jia-Zheng Li1
1Department of Hematology, Fujian Medical University Union Hospital, Fujian Provincial Key Laboratory of Hematology, Fujian Institute of Hematology, Fuzhou 350001, Fujian Province, China.
Objective:
Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.
Methods:
The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.
Results:
In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.
Conclusion:
The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.
Insights
Dual sgRNA CRISPR/Cas9 effectively knocks out the miR-155 gene in FLT3-ITD+ AML cells, enhancing drug sensitivity and apoptosis. This dual sgRNA approach shows superior gene editing efficiency compared to single sgRNA.
Area of Science:
- Molecular Biology
- Gene Editing
- Cancer Research
Background:
- FLT3-ITD+ AML is a high-risk leukemia.
- MicroRNA-155 (miR-155) plays a role in AML pathogenesis.
- CRISPR/Cas9 technology offers precise gene editing capabilities.
Purpose of the Study:
- To compare the efficiency of single versus dual sgRNA transfection for miR-155 gene knockout in FLT3-ITD+ AML cells.
- To evaluate the impact of miR-155 knockout on cell proliferation, drug sensitivity, and apoptosis.
Main Methods:
- Generation of lentiviral vectors with single or dual sgRNAs for CRISPR/Cas9.
- Transfection of MV411 (FLT3-ITD+ AML) cells.
- Assessment of gene editing efficiency via PCR.
- Quantification of miR-155 expression using qPCR.
- Evaluation of cell proliferation (CCK-8 assay) and apoptosis (Annexin V/7-AAD staining).
- Determination of drug sensitivity to adriamycin and quizartinib.
Main Results:
- Dual sgRNA transfection successfully achieved gene editing, indicated by a cleavage band.
- Significantly lower miR-155-5p expression was observed in dual sgRNA transfected cells compared to single sgRNA.
- Dual sgRNA transfected cells exhibited increased sensitivity to adriamycin and quizartinib (lower IC50) and a higher apoptosis rate.
Conclusions:
- Dual sgRNA transfection is more effective in inhibiting miR-155 gene expression than single sgRNA.
- Dual sgRNA mediated miR-155 knockout significantly inhibits proliferation, reverses drug resistance, and induces apoptosis in FLT3-ITD+ AML cells.
- Dual sgRNA transfection represents a highly efficient gene editing strategy for targeting miR-155 in AML.
