摘要
目的针对人源的CCL17和CCL22基因进行CRISPR/Cas9-gRNA设计,并验证设计的sgRNA的有效敲除效率。方法利用CRISPR网站提供的工具对CCL17和CCL22基因的靶向编辑位点进行筛选设计,然后构建CRISPR-Cas9-CCL17和CRISPR-Cas9-CCL22敲除质粒,并将构建好的质粒转入293 T细胞系,然后通过基因组PCR产物的T-A克隆测序方法分别验证设计的sgRNA敲除质粒的编辑效率。结果成功构建针对于人源CCL17和CCL22基因的sgRNA敲除质粒,并有着良好的编辑效率,均达70%以上。结论针对CCL17和CCL22基因设计的CRISPR敲除质粒对靶基因有着很好的编辑效果,为后续基于CCL17和CCL22的相关性研究奠定了基础。
Objective To design CRISPR/Cas9-gRNA targeting human CCL17 and CCL22 gene,and to evaluate the editing effectiveness of the sgRNA.Methods The targeted editing sites of CCL17 and CCL22 genes were screened using tools provided by CRISPR website,then the knockout plasmid of CRISPR-Cas9-CCL17 and CRISPR-Cas9-CCL22 were constructed and separately transfected into 293 T cells.Finally,the T-A editing and sequencing methods of genomic PCR products were used to verify the editing efficiency of the designed sgRNA knockout plasmid.Results The knockout plasmid of CRISPR-sgRNA targeting human CCL17 and CCL22 gene was successfully constructed with high editing efficiency,reached more than 70%.Conclusion The CRISPR-Cas9-CCL17 and CRISPR-Cas9-CCL22 show good editing efficiency,which provide foundation for the deep research of the function on CCL17 and CCL22.
作者
高淑慧
瞿创
吴凤麟
沈晗
GAO Shu-hui;QU Chuang;WU Feng-lin;SHEN Han(School of Biosciences and Biopharmaceutics,Guangdong Pharmaceutical University,Guangdong Province Key Laboratoryfor Biotechnology Drug Candidate,Guangzhou 510006,China)
出处
《中国当代医药》
2018年第12期4-8,共5页
China Modern Medicine
基金
国家自然科学基金项目(31300737)
广东省自然科学基金项目(2015A030310310)
广东省科技计划项目(2014A020212311)
关键词
CRISPR
引导RNA
趋化因子
基因编辑
Clustered regularly interspaced short palindromic reapeat
sgRNA
Chemokines
Genome editing
