<p>Background: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) 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 <em>Rag</em> genes to produce mutant mouse NIH/3T3 cell line. The <em>Rag1</em> and <em>Rag2</em> genes are essential for generation of mature B and T lymphocytes. Disruption of <em>Rag</em> 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.<br /> Methods: Four single guide RNAs were designed to target sequences in the coding region of the <em>Rag1</em> and <em>Rag2</em> genes. Four sgRNA-CAS9 plasmids were tested to target <em>Rag1</em> and <em>Rag2</em>.&nbsp;<br /> Results: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in <em>Rag1</em> resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in <em>Rag2 </em>gene resulted in indel mutations at both sites. In this report, fragment deletion in <em>Rag1</em>&nbsp;gene was detected in about 50% of transfected cells.<br /> 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.</p>