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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
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Protocol for Using CRISPR-Cas9 to Generate a Monocyte Cell Line Harboring a Single-Nucleotide Polymorphism.
Amar Chandra Mahatha1, Octavio Ramos-Espinosa1, Dhemerson Souza de Lima1
1Division of Infectious Diseases, Allergy and Immunology, Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, MO, USA.
Bio-Protocol
|April 27, 2026
Summary
We developed a CRISPR-Cas9 method to precisely edit single-nucleotide polymorphisms in human immune cells. This enables the study of genetic variants in inflammatory gene regulation.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- Investigating the functional impact of genetic variations in immune regulatory genes is crucial for understanding inflammatory diseases.
- Human monocyte cell lines, such as THP-1, are valuable models for studying immune responses.
- Precise genome editing tools are needed to create specific genetic modifications in these cell lines.
Purpose of the Study:
- To establish a robust protocol for generating single-nucleotide mutations in the promoter region of immune regulatory genes in human monocyte THP-1 cells.
- To create a precise cellular model for investigating the role of specific single-nucleotide polymorphisms (SNPs) in regulating inflammatory gene expression.
- To demonstrate the applicability of the CRISPR-Cas9 system for functional studies of noncoding and coding variants in innate immune genes.
Main Methods:
- Utilized a plasmid-based CRISPR-Cas9 system with a homology-directed repair (HDR) template.
- Designed and cloned a single-guide RNA (sgRNA) into a Cas9-expressing plasmid.
- Delivered plasmid constructs and single-stranded oligonucleotide repair templates (ssODNs) into THP-1 cells via transfection.
- Employed single-cell cloning, PCR-based genotyping, and Sanger sequencing for validation.
Main Results:
- Successfully generated a specific single-nucleotide mutation (rs1024611 SNP) in the endogenous CCL2 promoter of THP-1 cells.
- Established THP-1 monocytic cell lines that differ solely at the rs1024611 locus, enabling allele-specific effect comparisons.
- Validated the precision and efficiency of the CRISPR/Cas9-mediated genome editing protocol.
Conclusions:
- The developed CRISPR-Cas9 strategy provides a precise and relevant model for studying the functional impact of SNPs on inflammatory gene expression in human monocytes.
- This protocol is broadly applicable for functional characterization of both noncoding and coding variants in innate immune genes.
- The methodology serves as a framework for future investigations into other regulatory SNPs and noncoding variants in immune-related genes.
