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Establishing a Primary Epithelial Cell Model to Investigate the Role of MicroRNAs in IL-13-Mediated Mucus Regulation Using Non-Lentiviral Delivery of CRISPR/Cas9
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A7185 - Establishing a Primary Epithelial Cell Model to Investigate the Role of MicroRNAs in IL-13-Mediated Mucus Regulation Using Non-Lentiviral Delivery of CRISPR/Cas9
Author Block: S. Siddiqui, L. R. Bonser, O. Le Tonqueze, T. L. Roth, K. Koh, W. Zhao, L. Zlock, A. Marson, W. E. Finkbeiner, P. G. Woodruff, D. J. Erle; University of California, San Francisco, San Francisco, CA, United States.
Rationale: IL-13-induced goblet cell metaplasia contributes to mucus plugging and airway remodeling in asthma. IL-13 stimulation of cultured primary human bronchial epithelial cells (HBE) induces large changes in miRNA expression, many of which are observed in people with asthma. The role of miRNAs in goblet cell differentiation, however, is largely unexplored.
We hypothesize that airway epithelial miRNAs play a role in IL-13-induced goblet cell differentiation and/or mucus regulation. We aim to identify miRNAs with roles in mucous metaplasia and establish methods for using the CRISPR/Cas9 gene editing system to inactivate candidate miRNAs.
Methods: HBE cells were cultured at air-liquid interface (ALI) in the presence or absence of IL-13. Fixed and permeabilized cells were stained with an antibody for the goblet cell marker, MUC5AC, and sorted by flow cytometry prior to RNA isolation. Select miRNAs were measured via qPCR (n=4 donors) in the IL13-treated, sorted MUC5AC+ and MUC5AC- populations. As a proof-of-principle experiment, we targeted miR-21 by introducing a RNP (ribonucleoprotein) complex (recombinant Cas9 and a combination of crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA)) into HBE cells using nucleofection. Four crRNAs were designed to target miR-21. We assessed efficiency of miR-21 editing >5 days later by Sanger sequencing followed by TIDE (Tracking of Indels by Decomposition) analysis.
Results: A zinc-based fixative preserved RNA integrity over formalin prior to cell staining and sorting. We selected candidate miRNAs from the qPCR results performed on the MUC5AC+ and MUC5AC- cell populations. We will target these miRNAs using CRISPR/Cas9 to determine their roles in mucus regulation. We were able to detect ~85% efficiency of CRISPR/Cas9 gene editing in miR-21 in primary HBE cells.
Significance: We have established methods to assess differences in miRNAs in mucous cells and efficiently edit miRNA genes, together, which will allow for further investigation of the roles of miRNAs in mucous cell differentiation and function.
Author Block: S. Siddiqui, L. R. Bonser, O. Le Tonqueze, T. L. Roth, K. Koh, W. Zhao, L. Zlock, A. Marson, W. E. Finkbeiner, P. G. Woodruff, D. J. Erle; University of California, San Francisco, San Francisco, CA, United States.
Rationale: IL-13-induced goblet cell metaplasia contributes to mucus plugging and airway remodeling in asthma. IL-13 stimulation of cultured primary human bronchial epithelial cells (HBE) induces large changes in miRNA expression, many of which are observed in people with asthma. The role of miRNAs in goblet cell differentiation, however, is largely unexplored.
We hypothesize that airway epithelial miRNAs play a role in IL-13-induced goblet cell differentiation and/or mucus regulation. We aim to identify miRNAs with roles in mucous metaplasia and establish methods for using the CRISPR/Cas9 gene editing system to inactivate candidate miRNAs.
Methods: HBE cells were cultured at air-liquid interface (ALI) in the presence or absence of IL-13. Fixed and permeabilized cells were stained with an antibody for the goblet cell marker, MUC5AC, and sorted by flow cytometry prior to RNA isolation. Select miRNAs were measured via qPCR (n=4 donors) in the IL13-treated, sorted MUC5AC+ and MUC5AC- populations. As a proof-of-principle experiment, we targeted miR-21 by introducing a RNP (ribonucleoprotein) complex (recombinant Cas9 and a combination of crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA)) into HBE cells using nucleofection. Four crRNAs were designed to target miR-21. We assessed efficiency of miR-21 editing >5 days later by Sanger sequencing followed by TIDE (Tracking of Indels by Decomposition) analysis.
Results: A zinc-based fixative preserved RNA integrity over formalin prior to cell staining and sorting. We selected candidate miRNAs from the qPCR results performed on the MUC5AC+ and MUC5AC- cell populations. We will target these miRNAs using CRISPR/Cas9 to determine their roles in mucus regulation. We were able to detect ~85% efficiency of CRISPR/Cas9 gene editing in miR-21 in primary HBE cells.
Significance: We have established methods to assess differences in miRNAs in mucous cells and efficiently edit miRNA genes, together, which will allow for further investigation of the roles of miRNAs in mucous cell differentiation and function.
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