bims-cateng 2023-09-17 papers

Issue of 2023–09–17
five papers selected by
Chance Bowman, Dartmouth College

bioRxiv. 2023 Aug 29. pii: 2023.08.29.555404. [Epub ahead of print]

Repair of CRISPR-guided RNA breaks enables site-specific RNA editing in human cells.

Anna Nemudraia, Artem Nemudryi, Blake Wiedenheft.

Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. Here, we show that site-specific RNA breaks generated with RNA-targeting CRISPR complexes are repaired in human cells, and this repair can be used for programmable deletions in human transcripts that restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications.
One-Sentence Summary: CRISPR-guided RNA breaks are repaired in human cells, and this RNA repair can be used for programmable editing of human transcriptomes.

DOI: https://doi.org/10.1101/2023.08.29.555404

Opt Express. 2023 Aug 28. 31(18): 29174-29186

Fabrication and mechanical characterization of hydrogel-based 3D cell-like structures.

Randhir Kumar, Dustin Dzikonski, Elena Bekker, Robert Vornhusen, Valerio Vitali, Jörg Imbrock, Cornelia Denz.

In this article, we demonstrate the fabrication of 3D cell-like structures using a femtosecond laser-based two-photon polymerization technique. By employing poly(ethylene glycol) diacrylate monomers as a precursor solution, we fabricate 3D hemispheres that resemble morphological and biomechanical characteristics of natural cells. We employ an optical tweezers-based microrheology technique to measure the viscoelastic properties of the precursor solutions inside and outside the structures. In addition, we demonstrate the interchangeability of the precursor solution within fabricated structures without impairing the microstructures. The combination of two-photon polymerization and microrheological measurements by optical tweezers demonstrated here represents a powerful toolbox for future investigations into cell mimic and artificial cell studies.

DOI: https://doi.org/10.1364/OE.496888

Biosens Bioelectron. 2023 Aug 25. pii: S0956-5663(23)00576-6. [Epub ahead of print]241 115634

Optimizing mRNA transfection on a high-definition electroporation microelectrode array results in 98% efficiency and multiplexed gene delivery.

Bastien Duckert, Dennis Lambrechts, Dries Braeken, Liesbet Lagae, Maarten Fauvart.

Spatially resolved transfection, intracellular delivery of proteins and nucleic acids, has the potential to drastically speed up the discovery of biologically active cargos, for instance for the development of cell therapies or new genome engineering tools. We recently demonstrated the use of a high-density microelectrode array for the targeted electrotransfection of cells grown on its surface, a process called High-Definition Electroporation (HD-EP). We also developed a framework based on Design of Experiments to quickly establish optimized electroporation conditions across five different electrical pulse parameters. Here, we used this framework to optimize the transfection efficiency of primary fibroblasts with a mCherry-encoding mRNA, resulting in 98% of the cells expressing the desired fluorescent protein without any sign of cell death. That transfection yield is the highest reported so far for electroporation. Moreover, varying the pulse number was shown to modulate the fluorescence intensity of cells, indicating the dosage-controlled delivery of mRNA and protein expression. Finally, exploiting the single-electrode addressability of the microelectrode array, we demonstrated spatially resolved, high efficiency, sequential transfection of cells with three distinct mRNAs. Since the chip can be easily redesigned to feature a much large number of electrodes, we anticipate that this methodology will enable the development of dedicated screening platforms for analysis of mRNA variants at scale.

Keywords: Adherent cells; CMOS microelectrode arrays; Design of experiments; RNA delivery; Spatially resolved electroporation; Transfection

DOI: https://doi.org/10.1016/j.bios.2023.115634

Analyst. 2023 Sep 12.

Implantable microfluidics: methods and applications.

Tao Luo, Lican Zheng, Dongyang Chen, Chen Zhang, Sirui Liu, Chongjie Jiang, Yu Xie, Dan Du, Wei Zhou.

Implantable microfluidics involves integrating microfluidic functionalities into implantable devices, such as medical implants or bioelectronic devices, revolutionizing healthcare by enabling personalized and precise diagnostics, targeted drug delivery, and regeneration of targeted tissues or organs. The impact of implantable microfluidics depends heavily on advancements in both methods and applications. Despite significant progress in the past two decades, continuous advancements are still required in fluidic control and manipulation, device miniaturization and integration, biosafety considerations, as well as the development of various application scenarios to address a wide range of healthcare issues. In this review, we discuss advancements in implantable microfluidics, focusing on methods and applications. Regarding methods, we discuss progress made in fluid manipulation, device fabrication, and biosafety considerations in implantable microfluidics. In terms of applications, we review advancements in using implantable microfluidics for drug delivery, diagnostics, tissue engineering, and energy harvesting. The purpose of this review is to expand research ideas for the development of novel implantable microfluidic devices for various healthcare applications.

DOI: https://doi.org/10.1039/d3an00981e

Cell Syst. 2023 Sep 08. pii: S2405-4712(23)00216-8. [Epub ahead of print]

Synthetic symmetry breaking and programmable multicellular structure formation.

Noreen Wauford, Akshay Patel, Jesse Tordoff, Casper Enghuus, Andrew Jin, Jack Toppen, Melissa L Kemp, Ron Weiss.

During development, cells undergo symmetry breaking into differentiated subpopulations that self-organize into complex structures.1,2,3,4,5 However, few tools exist to recapitulate these behaviors in a controllable and coupled manner.6,7,8,9 Here, we engineer a stochastic recombinase genetic switch tunable by small molecules to induce programmable symmetry breaking, commitment to downstream cell fates, and morphological self-organization. Inducers determine commitment probabilities, generating tunable subpopulations as a function of inducer dosage. We use this switch to control the cell-cell adhesion properties of cells committed to each fate.10,11 We generate a wide variety of 3D morphologies from a monoclonal population and develop a computational model showing high concordance with experimental results, yielding new quantitative insights into the relationship between cell-cell adhesion strengths and downstream morphologies. We expect that programmable symmetry breaking, generating precise and tunable subpopulation ratios and coupled to structure formation, will serve as an integral component of the toolbox for complex tissue and organoid engineering.

Keywords: adhesion; agent-based model; biophysics; morphogenesis; simulations; symmetry breaking; synthetic biology

DOI: https://doi.org/10.1016/j.cels.2023.08.001