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Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins
- Publication Year :
- 2020
- Publisher :
- Cold Spring Harbor Laboratory, 2020.
-
Abstract
- SummaryGenome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.
- Subjects :
- 0303 health sciences
Myeloid
CD14
Inflammation
Nucleofection
Biology
3. Good health
Cell biology
03 medical and health sciences
0302 clinical medicine
medicine.anatomical_structure
030220 oncology & carcinogenesis
medicine
CRISPR
medicine.symptom
Gene knockout
030304 developmental biology
Cancer immunology
SAMHD1
Subjects
Details
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....8290ee4feeb36c7da93a729b5cf03042