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High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins. Here, we performed a cross-species approach by applying a mouse whole-genome siRNA library to CHO cells, optimized the protocol for suspension cultured cells, as this is the industrial practice for CHO cells, and developed an  in silico  method to identify functioning siRNAs, which also revealed the limitations of using cross-species libraries. With this method, we were able to identify several genes that, upon knockdown, enhanced the total productivity in the primary screen. A second screen validated two of these genes,  Rad21  and  Chd4 , whose knockdown was tested in additional CHO cell lines, confirming the induced high productivity phenotype, but also demonstrating the cell line/clone specificity of engineering effects.

A cross-species whole genome siRNA screen in suspension-cultured Chinese hamster ovary cells identifies novel engineering targets