Optimized protocol for CRISPR knockout of human iPSC-derived macrophages

Area of Science:

• Stem Cell Biology
• Gene Editing
• Immunology

Background:

• Macrophages play critical roles in immune responses, inflammation, neurodegenerative diseases, and cancer.
• Efficient genetic manipulation of human induced pluripotent stem cell (iPSC)-derived macrophages is crucial for studying these roles.
• Existing protocols for gene editing in iPSC-derived macrophages can be inefficient or complex.

Purpose of the Study:

• To present a detailed protocol for lentiviral delivery of CRISPR-Cas9 genome editing technology to iPSC-derived macrophages.
• To establish an efficient method for generating gene knockouts in iPSC-derived macrophages for functional studies.
• To facilitate research into the involvement of macrophages in various disease processes.

Main Methods:

• Co-incubation of iPSC-derived macrophages with VPX virus-like particles (VPX-VLPs) for lentiviral delivery.
• Production of polybrene and puromycin kill curves for optimizing transduction efficiency.
• VPX viral production and titration using western blotting.
• Lentiviral transduction of iPSC macrophage precursors and subsequent CRISPR-Cas9-based knockout.

Main Results:

• Successful lentiviral delivery of CRISPR-Cas9 to iPSC-derived macrophages was achieved using the VPX-VLP co-incubation method.
• The protocol includes detailed steps for optimizing transduction and performing genome editing.
• Demonstrated efficiency of CRISPR-Cas9-based knockout in iPSC-derived macrophages.

Conclusions:

• This protocol provides an efficient and reproducible method for genome editing in iPSC-derived macrophages.
• The developed technique enables robust investigation of macrophage functions in various physiological and pathological contexts.
• Facilitates advanced research in immunology, neurobiology, and oncology by enabling precise genetic manipulation of macrophages.