中文摘要：

CRISPR/Cas9技术作为一项新起的基因工程技术,在细胞及动物模型基因的编辑修饰中起着越来越重要的角色。CRISPR相关Cas9核酸酶可在特殊设定的单一导向RNA（single guide RNA,sg RNA）的引导下造成基因组DNA双链断裂（DSBs）,从而引起同源重组修复（HDR）。利用这种特性,我们构建出了带有CD34-GFP报告基因的293AD细胞系。根据CD34基因组序列,我们设计出了针对CD34基因特异性的单向导向RNA（sg RNA）和带有CD34-GFP报告基因的外源性同源序列。在与Cas9共转染之后,利用嘌呤霉素筛选出基因组DNA中插入有CD34-GFP报告基因的293AD细胞。之后我们对筛选出的293AD细胞进行单细胞分选,并利用PCR扩增对CD34绿色荧光报告基因的插入进行了验证。最后,我们将PCR扩增结果阳性的单克隆293AD细胞系的基因组进行DNA测序。DNA测序结果显示,分选出来的细胞基因组里确实包含有CD34-GFP报告基因。最终,我们成功利用CRISPR/Cas9系统构建出了绿色荧光标记的CD34报告基因293AD细胞系,为后续人体细胞诱导去分化成造血干细胞提供了有利的工具。

英文摘要：

CRISPR/Cas 9 pla ys a more and more important role in gene editing of cell and animal models as a newly developed gene engineering technology. CRISPR associated Cas9 nucleases can cause the double strand breaks（DSBs） of genome DNA through binding to target site under the guidance of the specific designed single guide RNA（sg RNA） which leads to the homology directed repair（HDR）. According to the character,we reported the establishment of a 293 AD cell line carrying CD34-GFP report gene. CD34 specific sg RNA and foreign homologous sequences of CD34 gene carrying CD34-GFP report gene were designed according to the sequence of CD34 gene. After co-transfection with Cas9,cells contain with the CD34-GFP reported gene in the genomic DNA can be selected by puromycin. Then we performed single cell sorting of the puromycin selected cells,and did PCR amplification verification of CD34 GFP report gene insertion. Finally,the genomic DNA of the monoclonal cells with positive PCR results were sent for DNA sequencing. Sequencing results of genomic DNA indicated that the sorted cells contained CD34-GFP report gene in their genomic DNA. In summary,we have successfully used CRISPR/Cas9 system to construct a CD34 GFP report cell line,and it will be a helpful tool for studying reprogramming of human somatic cells into hematopoietic stem cells.