Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells
Year: 2026
Type: article
Abstract: Itaconate mimetics inadequately represent endogenous itaconate, a negative regulator of innate immune-driven pro-inflammatory cytokines. We present a CRISPR-Cas9 protocol to delete a 4-nucleotide region in the immunoresponsive gene 1 (IRG1), ablating ACOD1 (the itaconate-producing enzyme) in THP-1 cells. We describe the functional validation of ACOD1 deletion using immunoblotting, ELISA, and liquid chromatography-mass spectrometry (LC-MS) quantification of itaconate, enabling the study of endogenous itaconate in THP-1 macrophages. For complete details on the use and execution of this protocol, please refer to Bourner et al. 1 • Instructions for CRISPR-Cas9-mediated IRG1 knockout (KO) in THP-1 macrophages • Cross-validation of KO by immunoblotting for IRG1 and LC-MS quantitation of itaconate • Steps for measuring IFN-β in stimulated wild-type and IRG1 KO THP-1 cells by ELISA Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Itaconate mimetics inadequately represent endogenous itaconate, a negative regulator of innate immune-driven pro-inflammatory cytokines. We present a CRISPR-Cas9 protocol to delete a 4-nucleotide region in the immunoresponsive gene 1 (IRG1), ablating ACOD1 (the itaconate-producing enzyme) in THP-1 cells. We describe the functional validation of ACOD1 deletion using immunoblotting, ELISA, and liquid chromatography-mass spectrometry (LC-MS) quantification of itaconate, enabling the study of endogenous itaconate in THP-1 macrophages.
Source: STAR Protocols
Institution: Eli Lilly (United States)
Language: English
Topic: Immune cells in cancer
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Funder: Eli Lilly and Company
OpenAlex ID: W7125404689