Efficient RNP-Directed Human Gene Targeting Reveals SPDEF is Required for IL-13-Induced Mucostasis.
Efficient RNP-Directed Human Gene Targeting Reveals SPDEF is Required for IL-13-Induced Mucostasis.
Am J Respir Cell Mol Biol. 2019 Oct 09;:
Authors: Koh KD, Siddiqui S, Cheng D, Bonser LR, Sun DI, Zlock LT, Finkbeiner WE, Woodruff PG, Erle DJ
Abstract
Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here, we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and recombinant Cas9 (rCas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near complete disruption of gene expression. To demonstrate the value of this system, we targeted <i>SPDEF</i>, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting <i>SPDEF</i> led to proportional decreases in <i>MUC5AC</i> expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of <i>SPDEF</i> abolished IL-13-ind...
Source: Am J Respir Cell Mol... - Category: Respiratory Medicine Authors: Koh KD, Siddiqui S, Cheng D, Bonser LR, Sun DI, Zlock LT, Finkbeiner WE, Woodruff PG, Erle DJ Tags: Am J Respir Cell Mol Biol Source Type: research