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Inhibiting alternative end-joining(Alt-EJ) pathway enhances CRISPR/Cas9 mediated site-specific knock-in efficiency in CHO-K1 cells

Chuanjie Wang  Ming Wang  Zhaolin Sun  Hongxu Cao  Kai Tu  Jingqing Liu  Zeliang Wei  Beifeng Shen  Jiannan Feng  Jing Wang  
【摘要】:Chinese hamster ovary(CHO) cells are the preferred host to produce a wide array of biopharmaceuticals,especially in antibody-drug production.However,development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration.Although CRISPR/Cas9 with the homology-directed repair(HDR) pathway enables precise integration of transgenes into target genomic sites.However,inherent recalcitrance to HDR-mediated targeted integration of transgenes in CHO cells results in low targeting efficiency.In mammalian cells,DSBs are predominantly repaired by non-homologous endjoining(NHEJ) and HDR.Repair of DSBs via NHEJ encompasses two major sub-pathways:canonical/classical NHEJ(cNHEJ),and non-canonical,alternative end-joining(alt-EJ).Recent reports have shown that inactivation of a-family DNA polymerase θ(Polθ) encoded by the POLQ gene in mouse ES cells and Nalm6 cells could substantially improve the efficiency of gene targeting through blocking alternative end-joining(Alt-EJ) pathway.Whereas,whether this consequence suits for CHO cells has not been demonstrated.Based on above discovery,we establish deficiency of Polθ in CHO-K1 cells by CRISPR-Cas9 and test the efficiency of gene targeting in ROSA26 safe locus using different reporter vectors including fluorescence and puromycin-resistance system.In flow cytometry analysis,we found the in-targeting insertion efficiency was increased nearly 20-fold compared with wild type.Further experiments showed that about 100% integration efficiency of stable cells screened by puromycin had been identified by junction PCR.To our astonishment,there was no obvious impairment of growth to POLQ~(-/-) CHO-K1 cells in contrast to ES cells in previous studies.The establishment of POLQ~(-/-) cell line not only improve the exogenous gene targeting integration efficiency,which accelerating the selection of high-producing and stable cell clones,but also may diminish the side-effect to the host cell by the application of ROSA26 safe locus,which benefits for the continuous production.In short our work provides a simple and efficiency strategy for targeted generation of stable CHO-K1 production cell lines for industrial application in future.

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