LAUSR

Chinese Hamster Ovary (CHO) cells are widely used for the high‐level production of recombinant proteins. We created a multiauxotrophic mutant of CHO‐K1 cells, CHO8A, that is deficient in eight enzymatic steps in the purine/pyrimidine biosynthetic pathways. Prototrophy was restored by transfections with complementary DNA‐based genes for the eight missing activities. CHO8A cells permit: (1) selection of transfectant clones that have incorporated genes for eight or more different polypeptides, suitable for engineering complex proteins, or pathways; and (2) the single‐step selection of high producers of a particular protein. The latter is achieved by simultaneous use of eight vectors, each bearing one of the eight rescue genes and a cargo protein gene. Screening as few as 10 surviving colonies yielded high producers secreting mAbs at 84 picograms per cell per day or more. CHO8A was isolated by CRISPR‐Cas9 knockout of 10 genes in the pathways to pyrimidines (Dhodh, Umps, Ctps1, Ctps2, and Tyms) and purines (Paics, Atic, Impdh1, Impdh2, and Gmps).