摘要：
尝试利用CRISPR-Cas9系统敲除山羊基因组中β-乳球蛋白(BLG)基因,以实现在BLG基因座敲入人乳铁蛋白(hLF)基因,并进一步探讨了不同浓度RAD51蛋白激活剂(RS-1)对同源重组效率的影响.首先针对山羊BLG的第一外显子设计并构建了sgRNA和Cas9共表达载体pCas9-sgBLG,将该载体转染至山羊耳成纤维细胞,利用PCR和T7EN1法验证了其基因组编辑活性;然后进一步构建了BLG基因打靶载体pBHA-hLF-NIE(包含NEO/EGFP);将该打靶载体与pCas9-sgBLG载体共转染至山羊耳成纤维细胞,分别用0、5、10和20 μmol/L RS-1处理细胞,分析了绿色荧光蛋白的表达效率;同时用800μg/mL G418对不同浓度RS-1处理后的细胞进行筛选,挑取EGFP阳性细胞克隆,进一步通过PCR和测序鉴定hLF定点敲入的阳性细胞克隆.结果显示:设计的sgRNA编辑山羊BLG位点的效率为25％-31％;报告基因的表达效率提示RS-1可以促进基因敲入效率的提高,其效率与RS-1浓度呈正相关,20 μmol/L RS-1处理组的效率是对照组的3.5倍;利用G418筛选hLF敲入阳性细胞克隆后,当RS-1浓度为0-10μmol/L时,hLF敲入效率随着RS-1浓度增加而升高,在10 μmol/L时阳性克隆率最高为32.61％,然而在20 μmol/L时敲入阳性克隆率下降至22.22％,且衰老细胞克隆增多.以上结果表明,利用CRISPR-Cas9系统可以实现在山羊耳成纤维细胞中敲除BLG基因和敲入hLF基因,且适宜浓度的RS-1可以显著提升基因敲入效率,本试验为高效利用CRISPR-Cas9系统获得基因敲入的细胞提供了参考依据.%This study aims to knock out the goat β-1actoglobulin (BLG) gene using CRISPR-Cas9 system and knock in human lactoferrin (hLF) at the BLG locus,and further study the effect of RAD51 stimulatory compound (RS-1) on homologous recombination efficiency.First,we designed an sgRNA targeting the first exon of goat BLG gene and constructed a co-expression vector pCas9-sgBLG.This sgRNA vector was then transfected into goat ear fibroblasts (GEFs),and the target region was examined by T7EN1 assay and sequencing.Second,we constructed a targeting vector pBHA-hLF-NIE including NEO and EGFP genes based on BLG gene locus.This targeting vector together with pCas9-sgBLG expression vector was co-transfected into GEFs.Transfected cells were then treated with 0,5,10 and 20 μmol/L RS-1 for 72 h to analyse the EGFP expression efficiency.Next,we used 800 μg/mL G418 to screen G418-resistent cell clones,and studied hLF site-specific knock-in cell clones by PCR and sequencing.The editing efficiency of sgBLG was between 25％ and 31％.The EGFP expression efficiency indicated that the gene knock-in efficiency was improved by RS-1 in a dose-dependent manner,which could reach 3.5-fold compared to the control group.The percentage of positive cells with hLF knock-in was increased to 32.61％ when 10 μmol/L RS-1 was used.However,when the concentration of RS-1 increased to 20 μmol/L,the percentage of positive cells decreased to 22.22％ and resulted in an increase of senescent celt clone number.These results suggested that hLF knock-in and BLG knock-out in GEFs were achieved by using CRISPR/Cas9 system,and optimum concentration of RS-1 could improve knock-in efficiency,which provides a reference for efficiently obtaining gene knock-in cells using CRISPR/Cas9 in the future.
