A precise and sustainable doxycycline-inducible cell line development platform for reliable mammalian cell engineering with gain-of-function mutations

Shin, Sung Wook; Min, Honggi; Kim, Jiwon; Lee, Jae Seong

doi:10.1016/j.ymben.2024.09.004

Metabolic Engineering

2024

DOI: 10.1016/j.ymben.2024.09.004

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A precise and sustainable doxycycline-inducible cell line development platform for reliable mammalian cell engineering with gain-of-function mutations

Sung Wook Shin,

Honggi Min,

Jiwon Kim

et al.

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2024

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STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells

Blanch-Asensio,

Ploessl,

Wang

et al. 2024

Preprint

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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 tetracyclineinducible 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.

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STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells

Blanch-Asensio,

Ploessl,

Wang

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

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A precise and sustainable doxycycline-inducible cell line development platform for reliable mammalian cell engineering with gain-of-function mutations

Cited by 1 publication

References 72 publications

STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells

STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells

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