Dataset Information

Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.

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.

SUBMITTER: Ott de Bruin L

PROVIDER: S-EPMC4914335 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.

Ott de Bruin Lisa L Yang Wei W Capuder Kelly K Lee Yu Nee YN Antolini Maddalena M Meyers Robin R Gellert Martin M Musunuru Kiran K Manis John J Notarangelo Luigi L

Oncotarget 20160301 11

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 ...[more]

PMID: 26887046

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Project description:While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Dataset Information

Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.

Publications

Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.

Similar Datasets

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