| 邓倩云 常雪莹 王斯佳 肖君华 李凯 周宇荀.利用CRISPR 系统介导人类mir-505基因位点的编辑[J].,2016,16(2):257-260 |
| 利用CRISPR 系统介导人类mir-505基因位点的编辑 |
| Human mir-505 Genomic Loci Editing by CRISPR Systems |
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| DOI： |
| 中文关键词: CRISPR 系统 Mir-505 SgRNA T7E1 assay 剪切效率 |
| 英文关键词: CRISPR system Mir-505 SgRNA T7E1 assay Cleavage Efficiency |
| 基金项目:中央高校基本科研业务专项资金（13D110521） |
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| 中文摘要: |
| 目的：通过针对人源mir-505 基因，设计不同sgRNA，从而利用CRIPSR 系统对mir-505 基因位点进行编辑，并探讨CRISPR
系统对靶点的剪切效率的影响因素。方法：针对人源mir-505 基因设计两条具有不同GC%的sgRNA，随后分别将其构建入
41824-sgRNA 表达载体和42230-Cas9 蛋白/sgRNA共表达载体，并将表达载体利用脂质体转染法转染入人类细胞，通过T7E1
assay检测不同CRISPR系统对靶点剪切效率的影响。结果：对靶点的剪切与Cas9 蛋白和sgRNA 的剂量成正比；当sgRNA与
Cas9 蛋白共传递时，也能够明显提高靶点的剪切效率；而较低的sgRNA的GC%会降低其对靶点的剪切效率。结论：本研究利用
CRISPR 系统靶向人源细胞的mir-505 基因，使得mir-505 基因发生突变。CRISPR 系统对靶点的剪切效率和sgRNA和Cas9 蛋白
的剂量、sgRNA 是否和Cas9蛋白共传递以及sgRNA的GC%有关。 |
| 英文摘要: |
| Objective:To design different sgRNAs targeted to human mir-505 genomic loci, and investigate the influencing factors
of cleavage efficiency in CRISPR system.Methods:Two different sgRNA with different GC% were designed using the online software,
and were constructed into 41824 vector and 42230 vector. Then the vectors were transfected into human cells, and the cleavage efficiency
were detected by T7E1 assay.Results:Higher concentration of CRISPR vector could increase the cleavage efficiency; sgRNAs
codelivered with Cas9 nuclease could also increase the cleavage efficiency; however, targeted to the same mir-505 genomic loci by two
sgRNAs with different GC%, the lower one decreased the cleavage efficiency.Conclusion:Using CRISPR system could target to
mir-505 genomic loci in human cells, and induced the mutagenesis of mir-505. The cleavage efficiency of CRISPR systemwas associated
with the dose of sgRNAs and Cas9 nuclease, codelivery of sgRNAs and Cas9 nuclease, and the GC%of sgRNAs. |
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