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Updated: Jan 11, 2026

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Published on: November 13, 2021

Normal macrophage signaling and gene expression in Rosa26 Cas9-expressing mice.

Monika D Hermann¹, Nuria Fernandez Perez¹, Assa Yeroslaviz¹

¹Max Planck Institute of Biochemistry, Martinsried, Germany.

|November 10, 2025

Summary

Cas9-FZ mice, widely used for gene editing, show no significant macrophage signaling defects. This study clarifies potential differences in macrophage culture techniques for Crispr screening applications.

Keywords:

Crispr/Cas9 Rosa26 macrophage macrophage polarization signaling

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Last Updated: Jan 11, 2026

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Published on: November 13, 2021

Depletion and Reconstitution of Macrophages in Mice

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Area of Science:

Immunology
Genetics
Molecular Biology

Background:

Rosa26 locus Cas9 expression is common for gene manipulation and CRISPR screening.
Prior studies suggested potential TRIF signaling differences in macrophages from Rosa26-Cas9 mice.

Purpose of the Study:

To investigate macrophage signaling pathways in Cas9-Feng Zhang (Cas9-FZ) mice.
To clarify potential discrepancies in macrophage signaling reported in literature.
To provide guidance for using Cas9-expressing macrophages in research.

Main Methods:

Macrophage isolation and culture under various polarization and signaling conditions.
RNA sequencing to analyze gene expression.
Immunoblotting to measure Toll-like receptor (TLR) signaling.

Main Results:

Cas9-FZ mice macrophages exhibited no detectable defects in common signaling pathways.
Differences in macrophage culture techniques were identified compared to previous studies.
No significant TRIF signaling alterations were observed under tested conditions.

Conclusions:

Cas9-FZ mice are suitable for macrophage-based studies without inherent signaling defects.
Understanding macrophage culture nuances is critical for reproducible CRISPR screening.
This work aids researchers in the application of Cas9-expressing macrophages.