A CRISPR-Based Toolbox for Studying T Cell Signal Transduction.

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• The CRISPR/Cas9 system offers powerful genome editing capabilities across various cell types.
• Understanding T cell signaling pathways is crucial for immunology research.
• Existing tools for large-scale genetic screens in T cells are limited.

Purpose of the Study:

• To create a versatile CRISPR/Cas9 toolbox for genetic screens in human Jurkat T cells.
• To enable both loss-of-function and gain-of-function genetic screens for T cell signaling pathway analysis.

Main Methods:

• Generation of three Jurkat T cell lines expressing distinct Cas9 variants: wild-type Cas9, dCas9-KRAB, and sunCas9.
• Utilizing the toolbox with genome-wide sgRNA libraries for genetic screening.
• Demonstrating gene expression disruption at the DNA level and modulation at the transcriptional level.

Main Results:

• The toolbox facilitates rapid disruption of endogenous gene expression.
• The system enables efficient repression or activation of gene expression.
• Successful application in human Jurkat T cells for genetic screening.

Conclusions:

• The developed CRISPR/Cas9 toolbox is effective for large-scale genetic screens in T cells.
• This resource will accelerate the systematic investigation of T cell signal transduction pathways.
• The toolbox supports both loss-of-function and gain-of-function studies in T cell biology.