Transient light-activated gene expression in Chinese hamster ovary cells

Abstract

Background: Chinese hamster ovary (CHO) cells are widely used for industrial production of biopharmaceuticals. Many genetic, chemical, and environmental approaches have been developed to modulate cellular pathways to improve titers. However, these methods are often irreversible or have off-target effects. Development of techniques which are precise, tunable, and reversible will facilitate temporal regulation of target pathways to maximize titers. In this study, we investigate the use of optogenetics in CHO cells. The light-activated CRISPR-dCas9 effector (LACE) system was first transiently transfected to express eGFP in a light-inducible manner. Then, a stable system was tested using lentiviral transduction. Results: Transient transfections resulted in increasing eGFP expression as a function of LED intensity, and activation for 48 hours yielded up to 4-fold increased eGFP expression compared to cells kept in the dark. Fluorescence decreased once the LACE system was deactivated, and a half-life of 14.9 hours was calculated, which is in agreement with values reported in the literature. In cells stably expressing the LACE system, eGFP expression was confirmed, but there was no significant increase in expression following light activation. Conclusions: Taken together, these results suggest that optogenetics can regulate CHO cell cultures, but development of stable cell lines requires optimized expression levels of the LACE components to maintain high dynamic range.

Enhancing the capability of Klebsiella pneumoniae to produce 1, 3‐propanediol by overexpression and regulation through CRISPR‐dCas9

“…The T7 promoter in pET28a was replaced by P32, hence reducing the cost of the whole process. At the same time, according to literature, P32 promoter is conducive to improving production (Minami and Shah, 2021 ). The fermentation experiment showed that K.P .…”

“…Thus, a new gene regulatory tool was developed (Prykhozhij et al ., 2017 ; Saifaldeen et al ., 2021 ). CRISPR‐dCas9 has advantages over common genome regulatory techniques, such as species restrictions, multiple gene regulatory methods, high gene modification rate and simple simultaneous regulation of multiple genes (Deaner et al ., 2018 ; Schwartz et al ., 2018 ; Velegzhaninov et al ., 2020 ; Minami and Shah, 2021 ).…”