Reference : PhD Bernardo Reina San Martin
During the course of immune responses B cells diversify their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR). These reactions establish highly specific and adapted humoral responses by increasing antibody affinity and by allowing the expression of a different antibody isotype with unique immunological functions. SHM diversifies the variable region Immunoglobulin heavy (IgH) and light (IgL) chain genes, producing families of related clones bearing mutated receptors that are positively selected on the basis of antigen binding affinity. CSR diversifies the B cell repertoire by combining a single heavy chain variable region with a different constant region, switching the antibody isotype expressed (from IgM to IgG, IgE or IgA) while retaining the antigen specificity of the receptor. Both of these reactions are initiated by Activation Induced Cytidine Deaminase (AID), an enzyme that deaminates cytosines in DNA. AID-induced lesions are recognized by the DNA repair machinery and are processed in different ways to trigger mutations or double stranded DNA break intermediates during CSR. AID has the potential to induce significant collateral DNA damage and has been implicated in the generation of B cell malignancies. At present in is not understood how AID is specifically targeted to immunoglobulin genes or how collateral genomic damage is restricted. We have recently conducted a genome-wide CRISPR/Cas9 knockout screen using CH12 cells (a cell line that undergoes CSR very efficiently in vitro). In this screen we have identified a number of candidate genes potentially regulating CSR and/or AID's activity.
The project's goal is to generate individual knockouts in CH12 cells for the most interesting candidates to verify the phenotype and conduct initial mechanistic studies. Based on these results, conditional knockout mouse models will be generated to study SHM and CSR in vivo.
Application Deadline : Nov. 1, 2018