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CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

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Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9.

Jamie L Everman¹, Cydney Rios², Max A Seibold^2,3

¹Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, USA. evermanj@njhealth.org.

Methods in Molecular Biology (Clifton, N.J.)

|February 10, 2018

Summary

This study details a new protocol for gene editing in human airway epithelial cells (AECs) using CRISPR-Cas9 technology delivered via lentivirus. The method efficiently generates gene-knockout AECs for studying airway diseases.

Keywords:

Airway epithelial cells CRISPR Gene editing Gene knockout Lentivirus Primary cells

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Area of Science:

Molecular Biology
Gene Editing
Cell Biology

Background:

CRISPR-Cas9 is a versatile gene editing tool.
Standard transfection is inefficient for primary human airway epithelial cells (AECs).
Lentiviral delivery offers efficient gene editing in AECs.

Purpose of the Study:

To develop and present a protocol for generating gene-knockout AECs.
To enable the study of gene function in airway diseases.
To establish a robust primary cell model system.

Main Methods:

Design and generation of lentivirus for CRISPR-Cas9 gene knockout.
Efficient lentiviral transduction of primary human basal AECs.
Culture, selection, screening, expansion, and differentiation of edited AECs into mucociliary cultures.

Main Results:

Successful generation of bulk edited AEC populations.
Molecular screening confirmed Cas9 cutting and specific gene edits.
Established protocol for creating gene-knockout mucociliary epithelial cultures.

Conclusions:

The developed protocol provides an efficient method for gene knockout in primary human AECs.
This model system is valuable for investigating gene function in airway dysfunction and disease.
Lentiviral delivery combined with advanced cell culture techniques enhances gene editing efficiency in AECs.