| 高晓彤 付伟 陈娟娟 胡彬 李丹慧
黄斯勇 李国辉 张阳萍 刘利 梁英民.应用Surrogate 报告载体提高RISPR/Cas9 对TMEM215基因的打靶效率*[J].,2016,16(4):652-656 |
| 应用Surrogate 报告载体提高RISPR/Cas9 对TMEM215基因的打靶效率* |
| Improving Gene Targeting Efficiency on TMEM215 Mediated byCRISPR/Cas9 by Using Surrogate Reporter |
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| DOI： |
| 中文关键词: CRISPR/Cas9 Surrogate 报告系统 |
| 英文关键词: CRISPR/Cas9 Surrogate reporter |
| 基金项目:国家自然科学基金项目青年科学基金项目(81300397) |
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| 中文摘要: |
| 目的：构建Surrogate 报告载体，并利用Surrogate 报告载体提高CRISPR/Cas9 对HEK293T 细胞tmem215基因打靶效率。
方法：构建针对人TMEM的CRISPR/Cas9表达载体及相应Surrogate 报告载体，两者共转HEK293T 细胞，通过流式分析、
T7EI检测、TA 克隆测序等明确Surrogate 报告载体对不同sgRNA打靶效率的检测及对基因修饰细胞的筛选富集作用。结果：流
式分析结果表明, Surrogate 报告载体成功检测出不同sgRNA的打靶效率，并筛选出高效率sgRNA；T7EI检测及TA 克隆测序显
示，外加嘌呤霉素抗性筛选时，Surrogate 报告载体可有效富集基因修饰细胞。结论：成功构建Surrogate 报告载体，并利用
Surrogate 报告载体提高CRISPR/Cas9 对HEK293T 细胞TMEM215基因的打靶效率。 |
| 英文摘要: |
| Objective:This work aims to construct the surrogate reporter and use it to improve gene targeting efficiency on human TMEM215
in HEK293T cells.Methods:The CRISPR/Cas9 expressing vectors targeting human TMEM215 and the related surrogate
reporter vectors were constructed and co-transfected into HEK293T cell line. The gene targeting efficiency was determined by
Fluorescence-activated cell sorting (FACS), T7EI assay and sequencing analysis.Results:FACS results showed that the surrogate
reporters can be used as an indicator of the gene targeting efficiency of each sgRNA. T7EI assay and sequencing analysis further
confirmed the results and showed that puromycin selected cell populations are highly enriched with cells containing mutations induced byCas9/sgRNA complex.Conclusion:Surrogate reporter can improve gene targeting efficiency mediated by CRISPR/Cas9 on
in HEK293T cell line. |
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