Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9

Area of Science:

• * Molecular Biology
• * Genetics
• * Immunology

Background:

• * Recombination Activating Genes (RAG) mutated embryonic stem cells (ESCs) are crucial for generating immunodeficient mice.
• * V(D)J recombination is essential for adaptive immunity.
• * CRISPR/Cas9 genome editing enables precise gene modifications.

Purpose of the Study:

• * To achieve complete knockout of the RAG1 gene in mouse ESCs.
• * To investigate the nature of mutations induced by CRISPR/Cas9 in mESCs.
• * To generate RAG1-knockout mESCs for rapid production of RAG-1 -/- mutant mice.

Main Methods:

• * CRISPR/Cas9 system was optimized using NIH3T3 cells.
• * Targeted RAG1 gene in mouse ESCs using CRISPR/Cas9 and single guide RNA (sgRNA).
• * Focused on specific early coding regions of the RAG1 gene (nucleotides c. 512-c. 513 and c. 725-c. 726).

Main Results:

• * CRISPR/Cas9 induced diverse homozygous and compound heterozygous mutations in 92% of mESC clones.
• * Out-of-frame indels, leading to gene knockout, were the predominant mutation type.
• * 59% of mutant ESC clones exhibited homozygous out-of-frame indels; cells maintained normal morphology and pluripotency.

Conclusions:

• * CRISPR/Cas9 system is highly efficient for creating biallelic RAG1 mutations in mESCs.
• * Achieved significant rates of homozygous out-of-frame indels, confirming RAG1 gene knockout.
• * This approach accelerates the generation of RAG-1 -/- mutant mice.