Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

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 rCas9 (recombinant Cas9) 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 SPDEF, which enco

SUBMITTER: Koh KD 

PROVIDER: S-EPMC7055692 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature