| Autor: |
Blanch-Asensio A; Department of Anatomy and Embryology, Leiden University Medical Center, 2300RC Leiden, The Netherlands.; The Novo Nordisk Foundation Center for Stem Cell Medicine, reNEW, Leiden University Medical Center., Ploessl DS; Department of Chemical Engineering, Massachusetts Institute of Technology, MA 02139 Cambridge, USA., Wang NB; Department of Chemical Engineering, Massachusetts Institute of Technology, MA 02139 Cambridge, USA., Mummery CL; Department of Anatomy and Embryology, Leiden University Medical Center, 2300RC Leiden, The Netherlands.; The Novo Nordisk Foundation Center for Stem Cell Medicine, reNEW, Leiden University Medical Center., Galloway KE; Department of Chemical Engineering, Massachusetts Institute of Technology, MA 02139 Cambridge, USA.; Senior author., Davis RP; Department of Anatomy and Embryology, Leiden University Medical Center, 2300RC Leiden, The Netherlands.; The Novo Nordisk Foundation Center for Stem Cell Medicine, reNEW, Leiden University Medical Center.; Senior author. |
| Abstrakt: |
Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influences transgene silencing, and demonstrate the impact of these design features on forward programming of hiPSCs into neurons. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely-used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of both transcription factors and functional reporters. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering. |
