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1. Biolistic approach for transient gene expression studies in plants

   Lacroix, B; Citovsky, V. (April 2020, Methods in molecular biology)

   Since its inception in the late 1980s, the delivery of exogenous nucleic acids into living cells via high-velocity micro-projectiles (biolistic, or micro-particle bombardment) has been an invaluable tool for both agricultural and fundamental plant research. Here, we review the technical aspects and the major applications of the biolistic method for studies involving transient gene expression in plant cells. These studies cover multiple areas of plant research, including gene expression, protein subcellular localization and cell-to-cell movement, plant virology, silencing and the more recently developed targeted genome editing via transient expression of customized endonuclease. 

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2. A synthetic circuit for buffering gene dosage variation between individual mammalian cells

   https://doi.org/10.1038/s41467-021-23889-0  

   Yang, Jin; Lee, Jihwan; Land, Michelle A.; Lai, Shujuan; Igoshin, Oleg A.; St-Pierre, François (December 2021, Nature Communications)

   null (Ed.)

   Abstract Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits – Equalizers – that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell. 

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3. Exploring plant cis‐regulatory elements at single‐cell resolution: overcoming biological and computational challenges to advance plant research

   https://doi.org/10.1111/tpj.16351  

   Mendieta, John Pablo; Sangra, Ankush; Yan, Haidong; Minow, Mark A. A.; Schmitz, Robert J. (June 2023, The Plant Journal)

   SUMMARY Cis‐regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with the inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall has limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Advances in single‐cell epigenomics have revolutionized the field of identifying cell‐type‐specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single‐cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single‐cell research, challenges, and common pitfalls in the analysis of plant single‐cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single‐cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes. 

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4. Ionizable Lipid Nanoparticles with Integrated Immune Checkpoint Inhibition for mRNA CAR T Cell Engineering

   https://doi.org/10.1002/adhm.202301515  

   Hamilton, Alex G; Swingle, Kelsey L; Joseph, Ryann A; Mai, David; Gong, Ningqiang; Billingsley, Margaret M; Alameh, Mohamad‐Gabriel; Weissman, Drew; Sheppard, Neil C; June, Carl H; et al (December 2023, Advanced Healthcare Materials)

   Abstract The programmed cell death protein 1 (PD‐1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment. While clinical approaches to inhibiting the PD‐1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD‐1 suppression, increasing the risk of autoimmune reactions. This study reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic gene expression and RNA interference (RNAi)‐mediated transient gene knockdown in T cells. In developing this platform, interesting interactions are observed between the two RNA cargoes when co‐encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co‐delivery platform is adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD‐1 to primary human T cells ex vivo and strong CAR expression and PD‐1 knockdown are observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including the development of improved cancer immunotherapies. 

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5. Altering metabolite distribution at Xenopus cleavage stages affects left–right gene expression asymmetries

   https://doi.org/10.1002/dvg.23418  

   Onjiko, Rosemary M.; Nemes, Peter; Moody, Sally A. (April 2021, genesis)

   Summary The left–right (L–R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L–R patterning. Using single‐cell high‐resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal‐animal blastomeres of the eight‐cellXenopusembryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L–R patterning. Using lineage tracing, we found that injecting right‐enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left‐enriched metabolites were injected into the right cell. At later stages, injecting left‐enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetricalnodalexpression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L–R gene expression that then can be compensated for during organogenesis. 

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