Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Discovery

Background:

• Phenotypic assays with human primary cells are crucial for identifying and validating drug targets.
• Efficient protein knockdown (KD) methods are needed to study target roles in disease models.
• CRISPR/Cas9 is an effective gene inactivation tool, primarily demonstrated in immortalized cell lines.

Purpose of the Study:

• To evaluate adenoviral (AdV) CRISPR/Cas9 for efficient gene inactivation in human primary cells.
• To assess the utility of AdV CRISPR/Cas9 in phenotypic assays relevant to fibrosis.
• To compare different AdV CRISPR/Cas9 delivery methods for gene editing efficiency.

Main Methods:

• Utilized adenoviral vectors expressing CRISPR/Cas9 and SMAD3-targeting guide RNAs (gRNAs).
• Applied the system to normal human lung fibroblasts and human bronchial epithelial cells.
• Investigated gene inactivation effects in TGF-β-induced fibroblast to myofibroblast transition (FMT) and epithelial to mesenchymal transition (EMT) assays.
• Compared co-transduction of separate AdV Cas9 and gRNA with a single all-in-one AdV Cas9/gRNA vector.

Main Results:

• Achieved efficient genome editing (insertion/deletion formation) and significant SMAD3 protein reduction and nuclear translocation inhibition.
• Observed downstream phenotypic changes, including decreased alpha smooth muscle actin and fibronectin 1 expression, key markers for FMT and EMT.
• Demonstrated comparable indel formation rates between separate and all-in-one AdV CRISPR/Cas9 vector systems.

Conclusions:

• Adenoviral CRISPR/Cas9 is a powerful and efficient tool for protein knockdown in human primary cell-based phenotypic assays.
• This technology offers advantages over existing methods, potentially accelerating target discovery and validation in drug development.
• AdV CRISPR/Cas9 facilitates the study of gene function in disease-relevant primary cell models.