山东理工大学学报（自然科学版）

利用CRISPR/Cas9基因编辑技术和同源重组技术在PD-L1基因的特定位置敲入绿色荧光蛋白(GFP)序列，构建含有PD-L1-GFP报告基因的结肠癌细胞(HT29)稳定细胞株。根据CRISPR-Cas9靶点设计原则，针对PD-L1基因终止密码子设计两对sgRNA,退火形成双链后连接至Lenti-V2空载质粒中，转化培养后提取质粒并测序验证。对于正确的Lenti-V2-sgRNA重组质粒，T7E1酶切验证其基因编辑效率。根据靶点位置设计左同源臂+GFP+右同源臂序列合成Donor片段，双酶切后连接至pUC19,重组载体转化扩增后同样进行质粒提取和测序验证。将验证成功的Lenti-V2-sgRNA和pUC19-donor-GFP共转染HT29细胞，荧光显微镜检测GFP表达情况，菌落PCR及基因测序验证GFP报告基因的靶向插入效果。经酶切和测序鉴定，靶向编辑PD-L1的Cas9载体Lenti-V2-gRNA和含GFP基因的Donor质粒pUC19-donor-GFP构建成功。两个重组质粒转染HT29细胞后，显微观察、多克隆验证、单克隆筛选及鉴定结果显示，GFP成功转入HT29细胞并进行了表达，经有限稀释法筛选出的单克隆细胞荧光均一，且与对照组相比，阳性细胞克隆的基因组PCR均检测到特异条带，表明在PD-L1终止密码子前成功插入了GFP片段，细胞株构建成功。通过基因编辑技术成功构建了稳定表达PD-L1-GFP报告基因的HT29结肠癌细胞株，建立了一个可以直观在细胞上观察PD-L1是否表达以及表达的强弱程度的系统，为后期体内外筛选调控PD-L1的上游新靶点及药物奠定了基础。

Using CRISPR/Cas9 and homologous recombination technology to tap green fluorescent protein(GFP) sequence at specific position of PD-L1 gene, we constructed human colon cancer cell(HT29) stable cell line containing PD-L1-GFP reporter gene. According to the design principle of CRISPR-Cas9 target, two pairs of sgRNAs were designed for the stop codon of PD-L1 gene. After annealing, the sgRNAs were connected to the Lenti-V2 plasmid. The plasmid was extracted and verified by sequencing after amplification of the receptor cells. For the correct Lenti-V2-gRNA recombinant plasmid, the efficiency of gene editing was verified by T7E1 digestion. According to the target location, Donor fragment was synthesized from left homologous arm +GFP+ right homologous arm sequence, which was ligated to pUC19 after double enzyme digestion. Plasmid extraction and sequencing were also performed after recombinant vector transformation and amplification. HT29 cells were co-transfected with Lenti-V2-gRNA and pUC19-donor-GFP, and the expression of GFP protein was detected by fluorescence microscopy. Finally, the targeted insertion effect of GFP reporter gene was verified by colony PCR and gene sequencing. The Cas9 vector Lenti-V2-gRNA and Donor plasmid pUC19-donor-GFP containing PD-L1 were successfully constructed by enzyme digestion and sequencing. After the two recombinant plasmids were transfected into HT29 cells, the results of microscopic observation, polyclonal verification, monoclonal screening and identification showed that GFP was successfully transferred into HT29 cells and expressed, and the monoclonal cells screened by limited dilution method had uniform fluorescence. Moreover, compared with the control group, specific bands were detected in the genomic PCR of the clones of positive cells, indicating that the GFP fragment was successfully inserted before the PD-L1 stop codon, and the cell line was successfully constructed. HT29 colon cancer cell line with stable expression of PD-L1-GFP reporter gene was successfully constructed by gene editing technology, and a system was established to directly observe whether PD-L1 is expressed and the degree of expression, which laid a foundation for subsequent in vitro and in vivo screening of upstream new targets and drugs regulating PD-L1.