The sound of silence: Transgene silencing in mammalian cell engineering

Cabera, Alan; Edelstein, Hailey I.; Glykofrydis, Fokion; Love, Kasey S.; Palacios, Sebastian; Tycko, Josh; Zhang, Meng; Lensch, Sarah; Shields, Cara E.; Livingston, Mark; Weiss, Ron; Zhao, Huimin; Haynes, Karmella A.; Morsut, Leonardo; Chen, Yvonne Y.; Khalil, Ahmad S.; Wong, Wilson; Collins, James J.; Rosser, Susan J.; Polizzi, Karen M.; Elowitz, Michael B.; Fussenegger, Martin; Hilton, Isaac B.; Leonard, Joshua N.; Bintu, Lacramioara; Galloway, Kate E.; Deans, Tara L.

doi:10.1016/j.cels.2022.11.005

Cited by 67 publications

(43 citation statements)

References 259 publications

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“…Loss of CAR expression can mitigate this and continued expression of the SJR allows these cells to spatially reactivate where they are again exposed to the tumor. If undesirable, this limitation could be overcome by increasing transcription factor levels before division 32, 64 or reactivating silenced genes through another synthetic circuit 67, 68 .…”

Section: Discussionmentioning

confidence: 99%

Design and Mathematical Analysis of Activating Amplifiers that Enable Modular Temporal Control in Synthetic Circuits

Lam

2023

Preprint

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The ability to control mammalian cells such that they self-organize or enact therapeutic effects as desired has incredible implications. Not only would it further our understanding of native processes such as development and the immune response, but it would also have powerful applications in medical fields such as regenerative medicine and immunotherapy. This control is typically obtained by synthetic circuits that use synthetic receptors, but control remains incomplete. For example, the synthetic juxtacrine receptors (SJRs) are widely used as they are fully modular and enable spatial control, but they have limited gene expression amplification and temporal control. I therefore designed transcription factor based amplifiers that amplify gene expression and enable unidirectional temporal control by prolonging duration of target gene expression. Using an in silico framework for SJR signaling, I combined these amplifiers with SJRs and show that these SJR amplifier circuits can improve the quality of self-organization and direct different spatiotemporal patterning. I then show that these circuits can improve chimeric antigen receptor (CAR) T cell tumor killing against two different types of tumors. These amplifiers are flexible tools that improve control over SJR based circuits and have both basic and therapeutic applications.

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Section: Discussionmentioning

confidence: 99%

Design and Mathematical Analysis of Activating Amplifiers that Enable Modular Temporal Control in Synthetic Circuits

Lam

2023

Preprint

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“…The use of hPSC lines that express an inducible Cas9 gene has helped overcome such challenges by controlling Cas9 expression during or after differentiation ( Tian et al, 2019 ; Esk et al, 2020 ; Dräger et al, 2022 ; Li et al, 2022 ). Additional challenges associated with hPSCs need to be taken in consideration, such as possible transgene silencing in the pluripotent and/or differentiated cell states ( Ordovás et al, 2015 ; Cabrera et al, 2022 ), and hPSC population bottlenecking that might arise due to significant cell loss from Cas9-induced double-strand breaks ( Ihry et al, 2018 ).…”

Section: Experimental Considerations Limitations and Future Directionsmentioning

confidence: 99%

Understanding neural development and diseases using CRISPR screens in human pluripotent stem cell-derived cultures

Ahmed

Muffat

2023

Front. Cell Dev. Biol.

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The brain is arguably the most complex part of the human body in form and function. Much remains unclear about the molecular mechanisms that regulate its normal and pathological physiology. This lack of knowledge largely stems from the inaccessible nature of the human brain, and the limitation of animal models. As a result, brain disorders are difficult to understand and even more difficult to treat. Recent advances in generating human pluripotent stem cells (hPSCs)-derived 2-dimensional (2D) and 3-dimensional (3D) neural cultures have provided an accessible system to model the human brain. Breakthroughs in gene editing technologies such as CRISPR/Cas9 further elevate the hPSCs into a genetically tractable experimental system. Powerful genetic screens, previously reserved for model organisms and transformed cell lines, can now be performed in human neural cells. Combined with the rapidly expanding single-cell genomics toolkit, these technological advances culminate to create an unprecedented opportunity to study the human brain using functional genomics. This review will summarize the current progress of applying CRISPR-based genetic screens in hPSCs-derived 2D neural cultures and 3D brain organoids. We will also evaluate the key technologies involved and discuss their related experimental considerations and future applications.

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“…Using the protocols outlined here will enable the growth and expansion of mESCs, the differentiation of mESCs into MKs in vitro , and studies for inserting transgenes and synthetic gene circuits into the Hipp11 chromosome of mESCs. These methods will facilitate studying the molecular rules and dynamic processes governing cell fate transitions into MKs, in addition to the mechanisms of transgene silencing, the challenge of losing expression of the inserted transgene(s) over time [17, 18].…”

Section: Introductionmentioning

confidence: 99%

In vitrogeneration of megakaryocytes from engineered mouse embryonic stem cells

Lewis

Deans

2023

Preprint

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Thein vitrodifferentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

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The sound of silence: Transgene silencing in mammalian cell engineering

Cited by 67 publications

References 259 publications

Design and Mathematical Analysis of Activating Amplifiers that Enable Modular Temporal Control in Synthetic Circuits

Design and Mathematical Analysis of Activating Amplifiers that Enable Modular Temporal Control in Synthetic Circuits

Understanding neural development and diseases using CRISPR screens in human pluripotent stem cell-derived cultures

In vitrogeneration of megakaryocytes from engineered mouse embryonic stem cells

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