Cut and paste: Using CRISPR/Cas9 to model RAG1 deficiency

Abstract

In order to recognize and eliminate the many different pathogens that the immune system can encounter, it generates an enormous repertoire of different unique T-cell receptors and B-cell immunoglobulins. The main contributor to this diversity is the process of V(D)J recombination, in which first different V, (D) and J genes are combined, then more diversity is created at the junction sites between the V, (D) and J genes. Recombination-activating gene 1 (RAG1) and 2 (RAG2) are crucial for this process. Mutations in RAG1 and RAG2 are associated with a broad spectrum of distinct clinical and immunological phenotypes, including T- B- severe combined immune deficiency (SCID), Omenn syndrome (OS), atypical/leaky SCID (LS), γδ SCID and combined immunodeficiency with granuloma and/or autoimmunity (CID-G/AI). Previous to this work, in vitro assays and structural modeling of the RAG complex have given some insights into why some mutations cause a more severe phenotype than others. In particular, mutations that allow for more residual RAG activity result in phenotypes such as leaky SCID or CID-G/AI. However, phenotypic variability remains even between patients with mutations that affect the same region of RAG1, showing the same residual RAG1 activity using in vitro models. How these mutations cause these different phenotypes remains an open question. The main aim of this thesis was to generate novel in vitro and in vivo models of RAG1 deficiency to study the mechanisms underlying the different RAG phenotypes with a focus on the CID-G/AI phenotype. These models will also be useful to test novel treatment options for patients with mutations in RAG1. A promising novel treatment is gene correction, so improving the genome-editing strategy used to generate models of RAG1 deficiency should also contribute to the development of a gene therapy that corrects patient stem cells.