CRISPR/Cas9 screen of B cell differentiation pathways

Abstract To provide immunity to pathogens, naïve B cells undergo differentiation to several states such as plasma cells and memory B cells. The transcription factors and epigenetic modifiers that program a naïve B cell to adopt a cell fate have not been fully characterized. During differentiation, B cells undergo extensive changes in transcription factor networks and gene expression that are underpinned by changes in the epigenome. One such example is a transition from expression of glycolysis related genes to increased expression of genes related to oxidative phosphorylation metabolism pathways. We previously demonstrated that in vivo, naïve B cells take 8 cell divisions to reach the plasma cell terminal differentiation and that at each division, the B cells also go through a hierarchy of reprogramming before becoming plasma cells after exposure to T independent antigens. Our data also showed that at division 3–4 a second branch emerged that expressed genes associated with the memory B cell fate. In order to find genes that play a key role in committing a naïve B cell to the plasma cell or memory B cell fate, cells committed to the two branches after T independent antigen exposure were sorted and analyzed with RNA sequencing. Analysis of this data revealed 67 genes involved in transcriptional and epigenetic regulation that were significantly differentially expressed between the two branches. In order to determine the role these genes play in B cell lineage path commitment, retrovirus libraries containing gRNAs for each of the 67 genes were constructed and tested in a targeted CRISPR/Cas9 knock out screen. This study identifies mechanisms of early memory B cell fate commitment. This work was supported by NIH R01 AI123733, P01 AI125180 and T32 GM008490 to JMB