RS-1提高CRISPR-Cas9系统介导的人乳铁蛋白基因敲入效率

发布时间：2018-07-11 13:14

本文选题：CRISPR-Cas + RS-　；参考：《生物工程学报》2017年08期

【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(h LF)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sg RNA和Cas9共表达载体p Cas9-sg BLG,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p BHA-h LF-NIE(包含NEO/EGFP);将该打靶载体与p Cas9-sg BLG载体共转染至山羊耳成纤维细胞,分别用0、5、10和20μmol/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/m L 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h LF定点敲入的阳性细胞克隆。结果显示:设计的sg RNA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,20μmol/L RS-1处理组的效率是对照组的3.5倍;利用G418筛选h LF敲入阳性细胞克隆后,当RS-1浓度为0-10μmol/L时,h LF敲入效率随着RS-1浓度增加而升高,在10μmol/L时阳性克隆率最高为32.61%,然而在20μmol/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h LF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。
[Abstract]:Using CRISPR-Cas9 system to knockout 尾 -lactoglobulin (BLG) gene from goat genome, the human lactoferrin (hLF) gene was knocked into the BLG locus, and the effects of different concentrations of RAD51 protein activator (RS-1) on the efficiency of homologous recombination were further investigated. Firstly, the co-expression vector of sg RNA and Cas9 was designed and constructed for the first exon of goat BLG. The vector was transfected into goat ear fibroblasts. The genomic editing activity was verified by PCR and T7EN1. Then, the BLG gene targeting vector pBHA-h LF-NIE (including neo / EGFP) was further constructed and co-transfected with pCas9-sg BLG vector into goat ear fibroblasts. The expression efficiency of green fluorescent protein was analyzed after treated with 0 渭 mol / L and 20 渭 mol / L RS-1, respectively. At the same time, 800 渭 g / mL G418 was used to screen the cells treated with different concentrations of RS-1, and the EGFP positive cell clones were selected and further identified by PCR and sequencing. The results showed that the efficiency of the designed sg RNA editing goat BLG locus was 25-31.The expression efficiency of the reporter gene suggested that RS-1 could promote the increase of gene knockin efficiency, and the efficiency was 3.5 times higher in the 20 渭 mol / L RS-1 treatment group than in the control group. When the concentration of RS-1 was 0-10 渭 mol / L, the knockout efficiency of hLF was increased with the increase of RS-1 concentration. The highest positive clone rate was 32.61 at 10 渭 mol / L, but the knockout positive clone rate decreased to 22.22 at 20 渭 mol / L, and the number of senescent cell clones increased. These results indicate that the CRISPR-Cas9 system can be used to knockout BLG gene and hLF gene in goat ear fibroblasts, and the appropriate concentration of RS-1 can significantly improve the efficiency of gene knockout. This study provides a reference for the efficient use of CRISPR-Cas9 system to obtain knockout cells.
【作者单位】：南京农业大学江苏省家畜胚胎工程实验室;
【基金】：国家转基因新品种培育重大专项(No.2014ZX08008-004)资助~~
【分类号】：Q78

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