AUTHOR=Chege Kuria Timothy , Schneider Andrea , Baraka Favoured , Wanzek Jana , Vogg Lisa , Brey Stefanie , Habenicht Katharina M. , Winkler Thomas H. 

TITLE=In vivo analysis of CRISPR-edited germinal center murine B cells

JOURNAL=Frontiers in Immunology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1473760

DOI=10.3389/fimmu.2024.1473760

ISSN=1664-3224

ABSTRACT=<p>The germinal center (GC) reaction is crucial for somatic hypermutation, affinity maturation, and the selection of high-affinity B cells, all of which are hallmarks of the humoral immune response. Understanding the distinct roles of various B cell genes is essential for elucidating the selection mechanisms within the GC reaction. Traditionally, studying B cell gene function in the GC reaction involved generating knock-out mice, a highly time-consuming method that necessitates complex vectors. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has simplified the creation of knock-out mice. However, even with CRISPR, the generation of knock-out mice still faces challenges, including being time-consuming, costly, having low knock-out efficiency, and raising ethical concerns regarding animal use. To address these challenges, we developed an alternative method to traditional knock-out mouse generation. Our approach entails the <italic>ex vivo</italic> CRISPR editing of B cells from transgenic donor mice with different B cell receptor affinities followed by their adoptive transfer into recipient mice. We present a cost-effective, rapid, versatile, and adaptable CRISPR-Cas9 method for <italic>in vivo</italic> loss-of-function studies of individual murine B cell genes within the context of the GC reaction. This method provides a valuable tool for investigating the complex roles of different B cell genes in the GC selection process. As proof of concept, we validated our approach by examining the role of the pro-apoptotic gene Fas in the GC selection process. We adoptively transferred a mix of Fas knock-out (Fas<sup>KO</sup>) low-affinity B cells, Fas wild-type (Fas<sup>WT</sup>) low-affinity B cells, and Fas<sup>WT</sup> high-affinity B cells into recipient mice. From our results, Fas<sup>KO</sup> low-affinity B cells were still outcompeted by the Fas<sup>WT</sup> high-affinity B cells for selection in the GC. An important observation was the accumulation of Fas<sup>KO</sup> low-affinity GC B cells when compared to the Fas<sup>WT</sup> low-affinity B cells, which suggested a role of Fas in the GC selection process.</p>