Programmable promoter editing for precise control of transgene expression

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 20, 2024

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL building editable promoters that allow fine-scale, heritable transgene Using DIAL, increase by recombinase-mediated excision of spacers between the binding sites synthetic zinc finger transcription factor core promoter. By nesting varying numbers lengths spacers, generates tunable range unimodal setpoints from single Through small-molecule control factors recombinases, supports temporally defined, user-guided is additional factors. Lentiviral delivery multiple primary iPSCs. As promoter editing stable states, are heritable, facilitating mapping levels phenotypes. The opens new opportunities tailoring improving predictability performance circuits across diverse applications.

Language: Английский

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Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 25, 2024

Abstract To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within window efficacy. Differences copy number and other sources extrinsic noise generate variance transgene expression limit performance synthetic gene circuits. In a context, supraphysiological can compromise phenotypes lead to toxicity. ensure narrow range expression, we design characterize Co mpact m icroRNA- M ediated A ttenuator N oise D osage ( ComMAND ), single-transcript, microRNA-based incoherent feedforward loop. We experimentally tune output profile, model system explore additional tuning strategies. By comparing two-gene implementations, highlight precise control afforded by single-transcript architecture, particularly at relatively low numbers. show that tightly regulates from lentiviruses precisely controls primary human T cells, rat neurons, mouse embryonic fibroblasts, induced pluripotent stem cells. Finally, effectively sets levels clinically relevant FMRP1 FXN window. Together, is compact tool well-suited specify cargoes.

Language: Английский

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Cells transit through a quiescent-like state to convert to neurons at high rates

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 25, 2024

Abstract While transcription factors (TFs) provide essential cues for directing and redirecting cell fate, TFs alone are insufficient to drive cells adopt alternative fates. Rather, rely on receptive states induce novel identities. Cell state emerges from is shaped by cellular history the activity of diverse processes. Here, we define molecular properties a highly amenable factor-mediated direct conversion fibroblasts induced motor neurons. Using well-defined model post-mitotic identify proliferative, that transiently during conversion. Through examining chromatin accessibility, histone marks, nuclear features, find reprogram characterized global reductions in size transcriptional activity. Supported globally increased levels H3K27me3, enter quiescent-like reduced RNA metabolism elevated expression REST p27, markers quiescent neural stem cells. From this transient state, convert neurons at high rates. Inhibition Ezh2, catalytic subunit PRC2 deposits abolishes Our work offers roadmap changes processes with different potentials may generalize other cell-fate transitions. Highlights Proliferation drives compact TF-mediated Increased receptivity corresponds volumes. Reprogrammable display global, genome-wide increases H3K27me3. High H3K27me3 support cells’ transits through altered metabolism. Ezh2 size, reduces quiescence marker p27. Acute inhibition neuron One Sentence Summary Cells transit Graphical

Language: Английский

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Rewinding the Tape to Identify Intrinsic Determinants of Reprogramming Potential

Cellular Reprogramming, Journal Year: 2024, Volume and Issue: 26(4), P. 117 - 119

Published: Aug. 1, 2024

Via retrospective isolation of clones using Rewind, Jain et al. identified primed states cells that reprogram to induced pluripotent stem cells. Examining clones, they find retain memory over several rounds cell division. Moreover, show extrinsic factors change the number cells, suggesting there exist diverse paths reprogramming and priming.

Language: Английский

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