山羊CSN2基因内含子7靶向的CRISPR-Cas9系统高效切割位点筛选

发布时间：2018-05-12 22:43

本文选题：山羊 + CSN基因　；参考：《甘肃农业大学学报》2017年01期

【摘要】：【目的】使用CRISPR-Cas9系统在山羊CSN2基因座内含子7上筛选出高效的切割位点.【方法】根据"20N+NGG"原则在CSN2内含子7序列中设计了5个sgRNA靶向位点.分别连接具有sgRNA到和Cas9表达框的PX330-P2A-eGFP载体上,通过脂质体转染到实验室保存山羊胎儿成纤维细胞系中,PCR扩增转染阳性的细胞基因组CSN2内含子7序列,连接到Pmd19-T载体中,测序比较不同的sgRNA介导切割产生的突变.【结果】使用5个sgRNA均可以在山羊CSN2基因内含子7上造成有效切割,产生碱基删除或者插入突变.实验设计的5个sgRNA介导的CRISPR-Cas9系统效率均在30.7%以上,效率最高的2号位点(sgRNA2)介导的切割效率达到了61.5%.【结论】研究确定了CRISPR-Cas9系统在山羊CSN2基因内含子7内进行高效切割的sgRNA,为之后在该基因位点进行高效的非同源介导基因敲入奠定了基础.
[Abstract]:[objective] to screen efficient cleavage sites on intron 7 of goat CSN2 locus using CRISPR-Cas9 system. [methods] five sgRNA targeting sites were designed in CSN2 intron 7 sequence according to the "20N NGG" principle. SgRNA was ligated into the PX330-P2A-eGFP vector with sgRNA and Cas9 expression frame respectively. The positive genomic CSN2 intron 7 sequence was amplified by liposome transfection into goat fetal fibroblast cell line and ligated to Pmd19-T vector. [results] all 5 sgRNA could effectively cleavage the goat CSN2 gene intron 7, resulting in base deletion or insertion mutation. The efficiency of the five sgRNA mediated CRISPR-Cas9 systems was over 30.7%. The cleavage efficiency mediated by the most efficient site 2 was 61.5. [conclusion] the CRISPR-Cas9 system was identified as highly efficient cleavage in intron 7 of the goat CSN2 gene, which was used as a highly efficient non-homologous medium for the gene locus. [conclusion] the results indicate that the CRISPR-Cas9 system is highly effective in the cleavage of goat CSN2 gene intron 7. The conduction gene knockin lays the foundation.
【作者单位】：甘肃农业大学生命科学技术学院;甘肃农业大学动物医学院;
【基金】：兰州市科技局人才创新创业项目(2015-RC-7) 甘肃省高校科研业务基本费(2011ZX08008-003)
【分类号】：Q78;S827

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