Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Cystic Fibrosis (CF) is characterized by widespread organ inflammation.
• It remains unclear if this inflammation stems from inherent cellular properties or the inflammatory environment.
• The role of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in regulating inflammation is not fully understood.

Purpose of the Study:

• To investigate the direct role of CFTR in inflammatory gene regulation.
• To determine if CFTR absence inherently causes inflammation or primes cells for an inflammatory response.

Main Methods:

• CRISPR/Cas9 gene editing was used to create CFTR-deficient (CFTR-) and CFTR-functional (CFTR+) Caco-2 cell lines.
• Functional assessment included forskolin response and measurement of tight junction formation.
• Gene expression analysis, including transcriptomics, was performed before and after inflammatory stimulation (TNFα, IL1β).

Main Results:

• CFTR- cell lines lacked forskolin-stimulated currents, confirming CFTR deficiency.
• Baseline cytokine (IL6, CXCL8) expression was similar, but CFTR- lines showed higher CXCL8 induction after TNFα stimulation.
• Transcriptomic analysis revealed CFTR absence altered gene expression, with some differences becoming apparent only after inflammatory stimulation.

Conclusions:

• CFTR absence modulates cellular transcriptional networks, particularly in response to inflammatory stimuli.
• These findings support a model where CFTR deficiency creates a poised, pro-inflammatory cellular state.
• This state is unmasked by environmental triggers, highlighting CFTR's role in maintaining cellular homeostasis during inflammation.