Optimized protocol for CRISPR knockout of human iPSC-derived macrophages

Abstract

Summary: Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression.For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• High Throughput Screening
• Microbiology
• Molecular Biology
• CRISPR