Abstract
Mutations in the Recombination Activating Gene 1 (RAG1) can cause a wide variety of clinical and immunological phenotypes in humans, ranging from absence of T and B lymphocytes to occurrence of autoimmune manifestations associated with expansion of oligoclonal T cells and production of autoantibodies. Although the mechanisms underlying this phenotypic heterogeneity remain poorly understood, some genotype-phenotype correlations can be made. Currently, mouse models of Rag deficiency are restricted to RAG1−/− mice and to knock-in models carrying severe missense mutations. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system is a novel and powerful gene-editing strategy that permits targeted introduction of DNA double strand breaks with high efficiency through simultaneous delivery of the Cas9 endonuclease and a guide RNA (gRNA). Here, we report on CRISPR-based, single-step generation and characterization of mutant mouse models in which gene editing was attempted around residue 838 of RAG1, a region whose functional role had not been studied previously.
Highlights
The Recombination Activating Gene 1 (RAG1) and RAG2 are critical for T and B cell development
In order to compare the ability of guide RNA (gRNA)-A and gRNA-B to induce CRISPR associated 9 (Cas9)-mediated introduction of on-site double strand breaks (DSBs) at the Rag1 locus, we analyzed the frequency of indels as detected by high-throughput sequencing (HTS) in nucleofected embryonic stem cells (ESCs)
We showed that Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 can be efficiently used to generate several unique Rag1 murine models in six weeks counting from the start of zygote injection to an F0 generation of weaning age, circumventing www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget months of breeding as required for the traditional ES cell blastocyst injection
Summary
The Recombination Activating Gene 1 (RAG1) and RAG2 are critical for T and B cell development. Mutations in RAG1 or RAG2 can lead to a wide variety of clinical and immunological phenotypes in humans, including complete absence of T and B cells (T- B- Severe Combined Immunodeficiency (SCID)) [2]; Omenn syndrome (OS) with lymphadenopathy, increased serum IgE, eosinophilia, erythroderma and detectable autologous, oligoclonal and activated T lymphocytes [3,4,5,6,7,8,9]; RAG deficiency with expansion of TCRγδ+ T cells[9]; atypical/leaky SCID (LS) with some T and B cells but no typical OS features[10, 11]; Combined Immunodeficiency with granuloma and/or autoimmunity (CID/G/A)[12,13,14], and CD4 lymphopenia[15]. The fourth class involves the RAG1/RAG2 interface [18]
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