Genomic Screening of B cell differentiation by using CRIPSR/Cas9

Abstract The differentiation of naïve B cells to antibody secreting plasma cells plays a crucial role in humoral immunity. This process is regulated by multiple factors including, transcription factors and epigenetic modifiers. Type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems can be used to identify genes that influence a phenotype of interest. Herein, we established this system to identify novel regulators of plasma cell differentiation. To accomplish this, we transduced hematopoietic stem cells derived from mice expressing Cas9 specifically in B cells (Rosa26-Cas9, Cd19-Cre) with lentiviral based gRNAs targeting Ptprc (CD45), Irf8 and Bach2 and transplanted the stem cells into congenically disparate hosts. Hematopoietic stem cells are enriched with lineage depletion kit and then sorted by C-kit and Sca-1, followed by culture for 24 hours and spin-infection with corresponding lentivirus. After another 24-hour recovery, these cells are transferred to radioactively immune system destroyed hosts. Following immune constitution, B cells were isolated and the deletion of CD45, IRF8 and BACH2 was confirmed. Consistent with previous findings, deletion of IRF8 and BACH2 augmented plasma cell formation in ex vivo differentiation assays. Additionally, we used a genome-wide CRISPR gRNA lentiviral library targeting 18,424 genes to screen for factors that influenced B cell differentiation to LPS, IL-2 and IL-5. In conclusion, the CRISPR/Cas9 system allows us to rapidly generate genetic deletions to explore B cell differentiation in vivo and to discover novel factors that regulate plasma cell formation.