Construction of human kinase knock-out library by using CRISPR/Cas9 technique
10.3969/j.issn.1006-5725.2017.24.005
- VernacularTitle:基于Crispr/Cas9技术构建真核细胞激酶敲除文库质粒
- Author:
Bin XIAO
1
;
Jingwen QUAN
;
Lidan CHEN
;
Jianfeng HANG
;
Weiyun ZHANG
;
Rong ZHANG
;
Yang LIAO
;
Jianyun CHEN
;
ohui Zha SUN
;
Linhai LI
Author Information
1. 广州军区广州总医院检验科
- Keywords:
gene knock-out;
CRISPR/Cas9 library;
kinase;
high throughput
- From:
The Journal of Practical Medicine
2017;33(24):4038-4042
- CountryChina
- Language:Chinese
-
Abstract:
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.
