Mehravar Maryam, Shirazi Abolfazl, Mehrazar Mohammad Mehdi, Nazari Mahboobeh, Banan Mehdi, Salimi Maryam
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.
Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.
Iran J Biotechnol. 2019 Jan 11;17(1):e2205. doi: 10.21859/ijb.2205. eCollection 2019 Jan.
Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences.
The main purposes of this study were to achieve complete knock-out of RAG1 gene by investigating the nature of mutations in mutant mESC and to generate RAG1 knock-out mESCs containing homozygous indels with the aim of creating desired and specific RAG-1 -/- mutant mouse in a shorter period of time.
Here, we first utilized CRISPR/Cas9 system to target RAG1/RAG2 genes in NIH3T3 cells to test the activity and efficiency of our CRISPR system. Then we used the system for targeting RAG1 gene in mouse embryonic stem cell (mESCs) to generate knock-out embryonic stem cells. This method combined with highly active single guide RNA (sgRNA) is an efficient way to produce new RAG1-knockout mESCs in the selected regions of early coding DNA sequence, approximately between nucleotide c. 512-c. 513 and nucleotide c. 725-c. 726 of RAG1 coding sequence that had not been targeted previously.
CRISPR gene editing resulted in a multitude of engineered homozygous and compound heterozygous mutations, including both in-frame and out-of-frame indels in 92% of mES cell clones. Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-frame homozygous indel mutations. The RAG1-knockout mESC clones retained normal morphology and pluripotent gene expression.
Our study demonstrated that CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell clones carried out-of-frame homozygous indel mutations.
重组激活基因（RAG）突变的胚胎干细胞是无法进行V（D）J重组的胚胎干细胞（ES细胞）。这些细胞可用于生成免疫缺陷小鼠。通过CRISPR/Cas9基因组编辑创建双等位基因突变已成为在不同序列中产生位点特异性突变的强大技术。
本研究的主要目的是通过研究突变型小鼠胚胎干细胞（mESC）中的突变性质来实现RAG1基因的完全敲除，并生成含有纯合插入缺失的RAG1敲除mESC，以便在更短的时间内创建所需的特定RAG-1-/-突变小鼠。
在此，我们首先利用CRISPR/Cas9系统靶向NIH3T3细胞中的RAG1/RAG2基因，以测试我们的CRISPR系统的活性和效率。然后我们使用该系统靶向小鼠胚胎干细胞（mESC）中的RAG1基因，以生成敲除胚胎干细胞。该方法与高活性单向导RNA（sgRNA）相结合，是在早期编码DNA序列的选定区域（大约在RAG1编码序列的核苷酸c.512-c.513和核苷酸c.725-c.726之间，这些区域以前未被靶向）产生新的RAG1敲除mESC的有效方法。
CRISPR基因编辑导致大量工程化的纯合和复合杂合突变，包括92%的mES细胞克隆中的框内和框外插入缺失。CRISPR/Cas9系统产生的大多数突变是框外的，导致基因完全敲除。此外，59%的突变ES细胞克隆携带框外纯合插入缺失突变。RAG1敲除mESC克隆保持正常形态和多能基因表达。
我们的研究表明，CRISPR/Cas9系统可以在约92%的突变mESC克隆中有效地创建包含纯合和复合杂合RAG1突变的双等位基因插入缺失。59%的突变ES细胞克隆携带框外纯合插入缺失突变。
