Efficient gene deletion of Integrin alpha 4 in primary mouse CD4 T cells using CRISPR RNA pair-mediated fragmentation.

The functional specialization of CD4 T lymphocytes into various subtypes, including T _H 1 and T _FH cells, is crucial for effective immune responses. T _FH cells facilitate B cell differentiation within germinal centers, while T _H 1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of Integrin α4 in regulating T _FH and T _H 1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating deletions in the gene. By analyzing the effects of Itga4 deficiency on T _FH and T _H 1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the T _H 1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of Integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2024 Wi, Choi, Kim, Choi, Pipkin and Choi.)