Engineering RNA export for measurement and manipulation of living cells

Horns, Felix; Martinez, Joe A.; Fan, Chengcheng; Haque, Md. Intesaful; Linton, Jamie; Tobin, Victoria; Santat, Leah A.; Maggiolo, Ailiena O.; Björkman, Pamela J.; Lois, Carlos; Elowitz, Michael B.

doi:10.1016/j.cell.2023.06.013

Cited by 28 publications

(22 citation statements)

References 85 publications

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“…Unlike mRNA delivery systems that require chemical modification of an sgRNA to achieve gene editing, a Cas protein can protect an sgRNA from cellular RNAses when bound to it and delivered to cells as an RNP 19 . While a number of different VLP systems have been developed that can deliver mRNA to cells, these VLPs have struggled to achieve gene editing with CRISPR-Cas systems unless an sgRNA is supplied to cells exogenously 12,20 . Here we circumvented this challenge to achieve cell-cell genetic engineering with a CRISPR-Cas9 adenine base editor, by SPIT, via its packaging and delivery to recipient cells as an RNP.…”

Section: Discussionmentioning

confidence: 99%

Secreted Particle Information Transfer (SPIT) – A Cellular Platform forIn VivoGenetic Engineering

Charlesworth,

Homma,

Suchy

et al. 2024

Preprint

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A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

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Section: Discussionmentioning

confidence: 99%

Secreted Particle Information Transfer (SPIT) – A Cellular Platform forIn VivoGenetic Engineering

Charlesworth,

Homma,

Suchy

et al. 2024

Preprint

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“…Other potential extensions of HyperSeq include chromatin accessibility 12,58 , whole-genome sequencing 59 at the sequencing level, and analysis of subcellular organelles 60,61 at imaging level. Hyperseq could also be used on engineered cells that export samples of the transcriptome nondestructively 62 , potentially achieving data sets similar to Live-seq with a simpler workflow.…”

Section: Discussionmentioning

confidence: 99%

Integration of hyperspectral imaging and transcriptomics from individual cells with HyperSeq

Xie,

Habibalahi,

Anwer

et al. 2024

Preprint

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Microscopy and omics are complementary approaches to probe the molecular state of cells in health and disease, combining granularity with scalability. While important advances have been achieved over the last decade in each area, integrating both imaging- and sequencing-based assays on the same cell has proven challenging. In this study, a new approach called HyperSeq that combines hyperspectral autofluorescence imaging with transcriptomics on the same cell is demonstrated. HyperSeq was applied to Michigan Cancer Foundation 7 (MCF-7) breast cancer cells and identified a subpopulation of cells exhibiting bright autofluorescence rings at the plasma membrane in optical channel 13 (ex = 431 nm, em = 594 nm). Correlating the presence of a ring with the gene expression in the same cell indicated that ringed cells are more likely to express hallmark genes of apoptosis and less likely to express genes associated with ATP production. Further, correlation of cell morphology with gene expression suggested that multiple members of the spliceosome were downregulated in larger MCF-7 cells. Multiple genes were evenly expressed across cell sizes but also exhibited higher usage of specific exons in larger or smaller cells. Finally, correlation between gene expression and fluorescence within the spectral range of Nicotinamide adenine dinucleotide hydrogen (NADH) provided insight into the metabolic states of MCF-7 cells. These observations provided a link between the cell's optical spectrum and its internal molecular state, demonstrating the utility of HyperSeq to study cell biology at single cell resolution by integrating spectral, morphological and transcriptomic analyses into a single, streamlined workflow.

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“…Clathrin assembles on the cytoplasmic side of intracellular vesicles, forming a coat that facilitates vesicle formation and transport. Designed proteins that form adaptable clathrin-like assemblies with defined size ranges could potentially be used as bio-orthogonal systems for encapsulating membrane-bounded vesicles, or could conceivably encapsulate other classes of biomolecules such as nucleic acids or ribonucleoprotein complexes 9,24,75 . It is intriguing to consider that interactions between such cargos and oligomorphic protein coats may shift the distribution or even type of architectures formed by the coats, allowing them to adapt to cargos of various kinds and sizes.…”

Section: Discussionmentioning

confidence: 99%

Local structural flexibility drives oligomorphism in computationally designed protein assemblies

Khmelinskaia,

Bethel,

Fatehi

et al. 2023

Preprint

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Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

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Engineering RNA export for measurement and manipulation of living cells

Cited by 28 publications

References 85 publications

Secreted Particle Information Transfer (SPIT) – A Cellular Platform forIn VivoGenetic Engineering

Secreted Particle Information Transfer (SPIT) – A Cellular Platform forIn VivoGenetic Engineering

Integration of hyperspectral imaging and transcriptomics from individual cells with HyperSeq

Local structural flexibility drives oligomorphism in computationally designed protein assemblies

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