基于Crispr/Cas9技术构建真核细胞激酶敲除文库质粒  

Construction of human kinase knock-out library by using CRISPR/Cas9 technique

作　　者：肖斌[1] 全静雯[1] 陈丽丹[1] 杭建峰[1] 张卫云[1] 张蓉[1] 廖扬[1] 陈建芸[1] 孙朝晖[1] 李林海[1] 

机构地区：[1]广州军区广州总医院检验科,广州市510010

出　　处：《实用医学杂志》2017年第24期4038-4042,共5页The Journal of Practical Medicine

基　　金：2015年广州市科创委项目(编号:201604040003)

摘　　要：目的拟靶向细胞内一群重要的信号转导因子—激酶,利用CRISPR/Cas9技术构建真核细胞敲除文库质粒,并探讨其构建方案。方法拟靶向编码人源激酶蛋白的507个基因开放阅读框,针对每个基因片段设计10个作用靶点,通过芯片法合成引物库(oligo pools)后,将引物从芯片上洗脱下来;合成的引物模板序列经PCR扩增、割胶回收后,与Cas9慢病毒载体连接,电转化至感受态细胞并提取质粒;质粒转化至Stbl3感受态细胞后,随机挑取单克隆进行摇菌,并送测序。结果 (1)对选取的507个激酶基因进行GO分析,发现细胞激酶广泛参与各种重要的细胞信号通路;(2)以oligo pool为模板,利用合成的通用引物进行PCR扩增,得到大小约为140 bp的条带,符合预期;(3)在鉴定的40个文库质粒克隆中,共有34个克隆样品测序成功,其中25个样品与设计的目标序列完全一致,9个样品存在一定引物合成突变。部分克隆存在摇菌失败、测序无信号或双峰等因素。结论构建好的CRISPR/Cas9激酶敲除文库可广泛用于筛选介导细胞增殖、转移、耐药、自噬等表型的重要激酶,为阐明激酶在疾病发生发展中的作用奠定基础。Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results（1） GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.（2）The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.（3）In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.

关 键 词：基因敲除 CRISPR/Cas9文库 激酶 高通量 

分 类 号：R346[医药卫生—基础医学]