猪HBB基因中先导编辑效率研究  

Study on efficiency of prime editing in pig HBB gene

作　　者：柒依琦 陆小丹 伍钊涛 梁佳华 黄丽柔 秦可 王晓丽[1] 唐小川 QI Yiqi;LU Xiaodan;WU Zhaotao;LIANG Jiahua;HUANG Lirou;QIN Ke;WANG Xiaoli;TANG Xiaochuan(College of Animal Science and Technology,Guangxi University,Nanning 530004,China)

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《黑龙江畜牧兽医》2024年第17期1-7,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(32002240)。

摘　　要：为了验证先导编辑(prime editing,PE)在猪细胞HBB基因中的编辑效率,并比较其在人细胞与猪细胞中的编辑效率,试验通过生物信息学分析,选择β珠蛋白生成障碍性贫血致病位点-28(A>G)、CD17(A>T)、CD41/42(-TCTT)设计并合成基因编辑质粒,通过脂质体Lipofectamine 2000转染人肾上皮细胞系(HEK293T)和猪肾上皮细胞系(PK-13)并进行药物筛选,提取细胞基因组进行PCR扩增,对PCR扩增产物进行TA克隆后测序,统计各位点的编辑效率;然后通过改进向导RNA骨架序列、添加小分子药物等方法对PK-13中的先导编辑进行优化。结果表明:先导编辑系统PEmax在PK-13中具有编辑作用,猪HBB基因-28(A>G)、CD17(A>T)、CD41/42(-TCTT)位点先导编辑的正确编辑效率分别为9.44%、8.89%和4.76%,较人HBB基因相应位点的正确编辑效率低33.89%、42.22%和26.11%,差异显著(P<0.05)。而经过优化pegRNA序列后猪细胞中正确编辑效率达到了12.78%[CD17(A>T)]、11.67%[CD41/42(-TCTT)],均显著高于pegRNA骨架序列优化前的7.22%和5.33%,是未优化前的1.8~2.2倍,差异显著(P<0.05)。说明先导编辑在猪细胞中具有编辑效率,且可以通过改进pegRNA的策略进一步提高正确编辑效率。To verify the editing efficiency of prime editing(PE)in HBB gene in pig cells and compare its editing efficiency in human cells and pig cells,the pathogenic sites-28(A>G),CD17(A>T),CD41/42(-TCTT)ofβ-thalassemia were selected through bioinformatics analysis,gene editing plasmids were designed and synthesized.Human and pig renal epithelial cell lines(HEK293T,PK-13)were transfected with Lipofectamine 2000 and drug screening was performed.The cell genome was extracted for PCR amplification,and its products were cloned and sequenced after TA cloning,and the editing efficiency of each site was calculated.Then,the lead editing in PK-13 was optimized by improving the guide RNA skeleton sequence and adding small molecule drugs.The results showed that PEmax had editing effect in PK-13,and the correct editing efficiency of pig HBB gene-28(A>G),CD17(A>T)and CD41/42(-TCTT)were 9.44%,8.89%and 4.76%,respectively.The editing efficiencies of pig HBB gene were 33.89%,42.22%and 26.11%lower that those of human HBB.The exact editing efficiency in pig cells was still significantly different from that in human cells(P<0.05).After optimizing the pegRNA sequence,the correct editing efficiencies of CD17(A>T)and CD41/42(-TCTT)in pig cells were 12.78%and 11.67%,which were significantly higher than those before pegRNA skeleton sequence optimization which were 7.22%and 5.33%,and 1.8~2.2 times than before optimization(P<0.05).These results indicated that prime editing had editing efficiency in porcine cells,and the correct editing efficiency could be further improved by improving the pegRNA strategy.

关 键 词：先导编辑 猪 肾上皮细胞系 HBB基因 Β珠蛋白生成障碍性贫血 

分 类 号：S828[农业科学—畜牧学] Q78[农业科学—畜牧兽医]