Mehravar Maryam, Shirazi Abolfazl, Mehrazar Mohammad Mehdi, Nazari Mahboobeh, Banan Mehdi
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
Avicenna J Med Biotechnol. 2019 Apr-Jun;11(2):149-155.
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 genes to produce mutant mouse NIH/3T3 cell line. The 1 and 2 genes are essential for generation of mature B and T lymphocytes. Disruption of 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.
Four single guide RNAs were designed to target sequences in the coding region of the 1 and 2 genes. Four sgRNA-CAS9 plasmids were tested to target and .
Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in 1 resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in 2 gene resulted in indel mutations at both sites. In this report, fragment deletion in 1 gene was detected in about 50% of transfected cells.
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.
成簇规律间隔短回文重复序列（CRISPR）及其相关蛋白（Cas）系统已成为基因组工程的有力工具。在本研究中，报道了该系统用于靶向基因以产生突变小鼠NIH/3T3细胞系的应用。1号和2号基因对于成熟B淋巴细胞和T淋巴细胞的产生至关重要。这些基因的破坏会导致如重症联合免疫缺陷综合征（SCID）等疾病。在此，用高活性的单向导RNA（sgRNA）测试了CRISPR系统在NIH/3T3小鼠细胞系中产生新突变的效率和特异性。
设计了4条单向导RNA靶向1号和2号基因编码区的序列。测试了4种sgRNA-CAS9质粒靶向1号和2号基因。
基于T7核酸内切酶检测和测序分析，靶向1号基因两个位点的sgRNA的表达导致中间DNA片段的缺失。靶向2号基因两个位点的sgRNA与Cas9共同表达导致两个位点均出现插入缺失突变。在本报告中，在约50%的转染细胞中检测到了1号基因的片段缺失。
因此，当合理设计向导RNA（gRNA）时，CRISPR/Cas9系统可具有高效性和特异性，并为细胞和模式动物的基因工程提供了一种强大的方法。
