Browsing by Author "Cabrera, Alan"

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    Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control
    (Springer Nature, 2023) Mahata, Barun; Cabrera, Alan; Brenner, Daniel A.; Guerra-Resendez, Rosa Selenia; Li, Jing; Goell, Jacob; Wang, Kaiyuan; Guo, Yannie; Escobar, Mario; Parthasarathy, Abinand Krishna; Szadowski, Hailey; Bedford, Guy; Reed, Daniel R.; Kim, Sunghwan; Hilton, Isaac B.
    Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR–Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR–dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.
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    The sound of silence: Transgene silencing in mammalian cell engineering
    (Cell Press, 2022) Cabrera, 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 W.; Collins, James J.; Rosser, Susan J.; Polizzi, Karen; Elowitz, Michael B.; Fussenegger, Martin; Hilton, Isaac B.; Leonard, Joshua N.; Bintu, Lacramioara; Galloway, Kate E.; Deans, Tara L.; Bioengineering
    To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.
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    Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription
    (Elsevier, 2022) Reed, Kathleen S. M.; Davis, Eric S.; Bond, Marielle L.; Cabrera, Alan; Thulson, Eliza; Quiroga, Ivana Yoseli; Cassel, Shannon; Woolery, Kamisha T.; Hilton, Isaac; Won, Hyejung; Love, Michael I.; Phanstiel, Douglas H.; Bioengineering; Biosciences
    To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly spaced time points of macrophage activation. Enhancers and genes connected by loops exhibit stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. At these looped enhancer-promoter pairs, changes in acetylation at distal enhancers precede changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, and lost loops are often accompanied by high levels of transcription within the loop boundaries themselves. These results are consistent with a reciprocal relationship where loops can facilitate increased transcription by connecting promoters to distal enhancers, whereas high levels of transcription can impede loop formation.