The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive approach to efficiently identify suitable clones in the cell line generation process for biologics manufacture, for which GS-knockout (GS-KO) CHO cell lines are commonly used. Since genome analysis indicated that there are two GS genes in CHO cells, deleting only 1 GS gene could potentially result in the activation of other GS genes, consequently reducing the selection efficiency. Therefore, in this study, both GS genes identified on chromosome 5 (GS5) and 1 (GS1) of CHO-S and CHO-K1, were deleted using CRISPR/Cpf1. Both single and double GS-KO CHO-S and K1 showed robust glutamine-dependent growth. Next, the engineered CHO cells were tested for their efficiency of selection of stable producers of two therapeutic antibodies. Analysis of pool cultures and subclones after a single round of 25 µM methionine sulfoxinime (MSX) selection indicated that for CHO-K1 the double GS5,1-KO was more efficient as in the case of a single GS5-KO the GS1 gene was upregulated. In CHO-S, on the other hand, with an autologously lower level of expression of both variants of GS, a single GS5-KO was more robust and already enabled selection of high producers. In conclusion, CRISPR/Cpf1 can be efficiently used to knock out GS genes from CHO cells. The study also indicates that for the generation of host cell lines for efficient selection, the initial characterisation of expression levels of the target gene as well as the identification of potential escape mechanisms is important.