The R229Q mutation of Rag2 does not characterize severe immunodeficiency in mice

Jin, Yoonhee; Lee, Ara; Oh, Ja Hyun; Lee, Han Woong; Ha, Sang Jun

doi:10.1038/s41598-019-39496-5

Cited by 4 publications

(3 citation statements)

References 34 publications

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“…The chimeric embryo represents features of both origins, and feather crossings are required to obtain the animal with desired phenotype. The second method, microinjection into mouse zygotes, allows us to modify genomes in different ways, generating knockouts [11], point mutations [12], knock-ins [13][14][15], allele humanization [13], and large chromosome rearrangements [6,[16][17][18], as well as producing genetically modified animals, in one step. Both methods have advantages and disadvantages, but microinjection into zygotes is a less-costly and less-time-consuming technique, with a high-efficiency outcome.…”

Section: Introductionmentioning

confidence: 99%

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

Korablev

Lukyanchikova

Serova

et al. 2020

IJMS

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Genome engineering has been tremendously affected by the appearance of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based approach. Initially discovered as an adaptive immune system for prokaryotes, the method has rapidly evolved over the last decade, overtaking multiple technical challenges and scientific tasks and becoming one of the most effective, reliable, and easy-to-use technologies for precise genomic manipulations. Despite its undoubtable advantages, CRISPR/Cas9 technology cannot ensure absolute accuracy and predictability of genomic editing results. One of the major concerns, especially for clinical applications, is mutations resulting from error-prone repairs of CRISPR/Cas9-induced double-strand DNA breaks. In some cases, such error-prone repairs can cause unpredicted and unplanned large genomic modifications within the CRISPR/Cas9 on-target site. Here we describe the largest, to the best of our knowledge, undesigned on-target deletion with a size of ~293 kb that occurred after the cytoplasmic injection of CRISPR/Cas9 system components into mouse zygotes and speculate about its origin. We suppose that deletion occurred as a result of the truncation of one of the ends of a double-strand break during the repair.

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Section: Introductionmentioning

confidence: 99%

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

Korablev

Lukyanchikova

Serova

et al. 2020

IJMS

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“…Of note, the C57BL/6 strain of mice that our RAG1 NX mutation is present in is notoriously resistant to autoimmunity in the absence of additional environmental stimuli (26). Similarly, the R229Q RAG2 mutation on the C57BL/6 background does not present with overt autoimmunity (27).…”

Section: Discussionmentioning

confidence: 88%

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms

et al. 2020

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The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 59 region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 59 RAG1 frameshift mutations. We show that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1 mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1 and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of 59 nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

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“…Within a setting of less efficient negative selection as with homozygous Rag1 P326G mutation, an increased predisposition to αβ T cell mediated autoimmunity would be expected. However, we did not observe overt autoimmune symptoms within PG mice, possibly because mice of the C57BL/6 background are resistant to spontaneous autoimmune diseases (45,46). Thus, to investigate whether the Rag1 P326G mutation can predispose to autoimmunity, we utilized an induced model of autoimmune colitis in C57BL/6 mice and a spontaneous model of autoimmune diabetes in NOD mice.…”

Section: Mature αβ T Cells Of Homozygous Rag1 P326g Mice Exhibit Increased Predisposition To Autoimmunitymentioning

confidence: 94%

The RAG1 Ubiquitin Ligase Domain Enhances the Assembly and Selection of T Cell Receptor Genes to Restrain the Autoimmune Hazard of Generating T Cell Receptor Diversity

Burn

Miot

Gordon

et al. 2021

Preprint

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RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte antigen receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. However, this process creates highly self-reactive cells that must be negatively selected to suppress autoimmunity. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity. We report here that mice lacking RAG1 ubiquitin ligase activity exhibit diminished recombination of T cell receptor (TCR) b and a loci, and impaired thymocyte developmental transitions that require the assembly of these genes and signaling by their proteins. The mice also have reduced expression of TCR signaling proteins within thymocytes, less efficient negative selection of highly self-reactive thymocytes, and mature ab T cells of elevated autoimmune potential. Thus, we propose that the RAG1 ubiquitin ligase domain provides ab T cell developmental stage-specific means to augment TCR signaling and thereby enhance selection for beneficial TCR genes and against ab TCRs possessing high autoimmune potential.

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The R229Q mutation of Rag2 does not characterize severe immunodeficiency in mice

Cited by 4 publications

References 34 publications

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

On-Target CRISPR/Cas9 Activity Can Cause Undesigned Large Deletion in Mouse Zygotes

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms

The RAG1 Ubiquitin Ligase Domain Enhances the Assembly and Selection of T Cell Receptor Genes to Restrain the Autoimmune Hazard of Generating T Cell Receptor Diversity

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