Background
The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line.
Conclusion
Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals.
Methods
Four single guide RNAs were designed to target sequences in the coding region of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2.
Results
Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was detected in about 50% of transfected cells.