Novel CRISPR/Cas9-mediated knockout of LIG4 increases efficiency of site-specific integration in Chinese hamster ovary cell line

Autor: Chuanjie Wang, Zhaolin Sun, Ming Wang, Zhiyang Jiang, Mengmeng Zhang, Hongxu Cao, Longlong Luo, Chunxia Qiao, He Xiao, Guojiang Chen, Xinying Li, Jinqing Liu, Zeliang Wei, Beifen Shen, Jing Wang, Jiannan Feng
Rok vydání: 2022
Předmět:
Zdroj: Biotechnology Letters. 44:1063-1072
ISSN: 1573-6776
0141-5492
DOI: 10.1007/s10529-022-03282-7
Popis: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells.CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.
Databáze: OpenAIRE
Externí odkaz: