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800 μg/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?10 μmol/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-lactoglobulin (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 cell 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.
国家转基因新品种培育重大专项 (No. 2014ZX08008-004) 资助。