Overcoming the eIF2α Brake in Human Cell-Derived Translation Systems

Cell-free translation from human cells is a powerful platform for studying mammalian gene expression and building synthetic biology tools, but productivity is often curtailed by inhibitory phosphorylation of eIF2 on residue Ser52. Here we systematically explored complementary strategies to bypass this initiation block across editable and hard-to-edit human cell types. In Expi293F suspension cells, precise genome editing of EIF2S1 to block Ser52 phosphorylation (eIF2-S52A) produced high-activity extracts. Genetic knockout of EIF2AK2 (PKR)-the principal eIF2 kinase engaged in eIF2 phosphorylation in Expi293F lysates-also improved translation, further establishing eIF2 phosphorylation as the dominant bottleneck in Expi293F translation extracts. Because genome editing is impractical in many contexts including primary human cells, we also implemented expression-based rescue of eIF2 phosphorylation: stable piggyBac integration of truncated GADD34 (PPP1R15A) and K3L, a viral eIF2 decoy, under control of a Tet-inducible promotor in induced pluripotent stem cells (iPSCs) and primary human fibroblasts. After differentiating engineered KOLF2.1J iPSCs into cardiomyocytes, we found that stable GADD34/K3L expression increased translation output in cardiomyocyte translation extracts. Using the piggyBac expression system in primary fibroblasts also resulted in improved translational output. Together these data pinpoint eIF2 phosphorylation as the key barrier to robust translation in human cell translation extracts. They also show that editing eIF2 or removing PKR is optimal where genome editing is feasible, while providing a portable GADD34/K3L expression cassette enables production of translationally active lysates from systems where genome editing is challenging or not possible.