Abstract
Laboratory Automation and High-Throughput Biology
Integrated, Automated Maintenance, Expansion and Differentiation of 2D and 3D Patient-Derived Cellular Models for High-Throughput Drug Screening
Patient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way toward high-throughput screenings (HTS), including the implementation of laboratory advanced automation. However, the maintenance and expansion of cells for HTS remains largely manual work. Here, Boussaad et al. describe an integrated, complex automated platform for HTS in a translational research setting also designed for the maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for the development of methods for variable cell types. The authors demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSCs), and neural progenitor cells (NPCs) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, the authors provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine. (Boussaad, I., et al. Sci. Rep.
Automated Screening for Oxidative or Methylation-Induced DNA Damage in Human Cells
The assessment of genotoxicity upon exposure to chemical and environmental agents plays an important role in basic research as well as in the pharmaceutical, chemical, cosmetic, and food industries. Low sensitivity and lack of interlaboratory comparability are considered problematic issues in genotoxicity testing. Moreover, commonly used mutagenicity assays lack information about early and specific genotoxic events. Previously, the authors developed an automated version of the Fluorimetric Detection of Alkaline DNA Unwinding (FADU) assay as a high-throughput screening method for the detection of DNA strand breaks in living cells. Here, Mack et al. report an enzyme-modified version of the cell-based FADU assay (emFADU) for the determination of oxidative and methylation lesions in cellular DNA. The authors’ method is based on the use of formamidopyrimidine DNA glycosylase or human alkyladenine DNA glycosylase for the detection of chemically induced nucleobase modifications in lysates of immortalized cell lines, growing in suspension or as adherent cells, and in peripheral blood mononuclear cells. The authors could show that upon treatment with subcytotoxic doses of known genotoxins, oxidative and methylation lesions are readily detectable. This fast, inexpensive, and convenient method could be useful as a high-content screening approach for the sensitive and specific assessment of genotoxicity in human cells. Thus, when implemented in the early compound development in an industrial setting, the emFADU assay could help reduce the number of animals used for toxicity testing. Furthermore, as Mack et al. established the method for different cell types, this new assay may provide an opportunity for population studies and/or mechanistic research into DNA repair pathways. (Mack, M., et al. ALTEX
Accelerating Strain Engineering in Biofuel Research via Build and Test Automation of Synthetic Biology
Biofuels are a type of sustainable and renewable energy. However, for the economical production of bulk-volume biofuels, biosystems design is particularly challenging to achieve sufficient yield, titer, and productivity. Because of the lack of predictive modeling, high-throughput screening remains essential. Recently established biofoundries provide an emerging infrastructure to accelerate biological design-build-test-learn (DBTL) cycles through the integration of robotics, synthetic biology, and informatics. In this review, Zhang et al. first introduce the technical advances of build and test automation in synthetic biology, focusing on the use of industry-standard microplates for DNA assembly, chassis engineering, and enzyme and strain screening. Proof-of-concept studies on prototypes of automated foundries are then discussed, for improving biomass deconstruction, metabolic conversion, and host robustness. Zhang et al. conclude with future challenges and opportunities in creating a flexible, versatile, and data-driven framework to support biofuel research and development in biofoundries. (Zhang, J., et al. Curr. Opin. Biotechnol.
Microfluidics
Microfluidics for Microalgal Biotechnology
Microalgae have expanded their roles as renewable and sustainable feedstocks for biofuel, smart nutrition, biopharmaceutical, cosmeceutical, biosensing, and space technologies. They accumulate valuable biochemical compounds from protein, carbohydrate, and lipid groups, including pigments and carotenoids. Microalgal biomass, which can be adopted for multivalorization under biorefinery settings, allows the production of not only various biofuels but also other value-added biotechnological products. However, state-of-the-art technologies are required to optimize yield, quality, and the economical aspects of both upstream and downstream processes. As such, the need to use microfluidic-based devices for both fundamental research and industrial applications of microalgae arises due to their microscale sizes and dilute cultures. Microfluidics-based devices are superior to their competitors through their ability to perform multiple functions such as sorting and analyzing small amounts of samples (nanoliter to picoliter) with higher sensitivities. Here, the authors review emerging applications of microfluidic technologies on microalgal processes in cell sorting, cultivation, harvesting, and applications in biofuels, biosensing, drug delivery, and nutrition. (Ozdalgic, B., et al. Biotechnol. Bioeng.
A Microfluidics-Based Stem Cell Model of Early Postimplantation Human Development
Early postimplantation human embryonic development has been challenging to study due to both technical limitations and ethical restrictions. Proper modeling of the process is important for infertility and toxicology research. Here the authors provide details of the design and implementation of a microfluidic device that can be used to model human embryo development. The microfluidic human embryo model is established from human pluripotent stem cells (hPSCs), and the resulting structures exhibit molecular and cellular features resembling the progressive development of the early postimplantation human embryo. The compartmentalized configuration of the microfluidic device allows the formation of spherical hPSC clusters in prescribed locations in the device, enabling the two opposite regions of each hPSC cluster to be exposed to two different exogenous chemical environments. Under such asymmetrical chemical conditions, several early postimplantation human embryo developmental landmarks, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and gastrulating cells (or mesendoderm cells), can be robustly recapitulated using the microfluidic device. The microfluidic human embryo model is compatible with high-throughput studies, live imaging, immunofluorescence staining, fluorescent in situ hybridization, and single-cell sequencing. This protocol takes ~5 days to complete, including microfluidic device fabrication (2 days), cell seeding (1 day), and progressive development of the microfluidic model until gastrulation-like events occur (1–2 days). (Zheng, Y., et al. Nat. Protoc.
A High-Throughput Multiplexed Microfluidic Device for COVID-19 Serology Assays
The applications of serology tests to the virus SARS-CoV-2 are diverse, ranging from diagnosing COVID-19, understanding the humoral response to this disease, and estimating its prevalence in a population, to modeling the course of the pandemic. COVID-19 serology assays will significantly benefit from sensitive and reliable technologies that can process dozens of samples in parallel, thus reducing costs and time; however, they will also benefit from biosensors that can assess antibody reactivities to multiple SARS-CoV-2 antigens. Here, the authors report a high-throughput microfluidic device that can assess antibody reactivities against four SARS-CoV-2 antigens from up to 50 serum samples in parallel. This semiautomatic platform measures IgG and IgM levels against four SARS-CoV-2 proteins: the spike protein (S), the S1 subunit (S1), the receptor-binding domain (RBD), and the nucleocapsid (N). After assay optimization, the authors evaluated sera from infected individuals with COVID-19 and a cohort of archival samples from 2018. The assay achieved a sensitivity of 95% and a specificity of 91%. Nonetheless, both parameters increased to 100% when evaluating sera from individuals in the third week after symptom onset. To further assess their platform’s utility, the authors monitored the antibody titers from five COVID-19 patients over a time course of several weeks. Rodriguez-Moncayo et al.’s platform can aid in global efforts to control and understand COVID-19. (Rodriguez-Moncayo, R., et al. Lab Chip
High-Throughput Monitoring of Bacterial Cell Density in Nanoliter Droplets: Label-Free Detection of Unmodified Gram-Positive and Gram-Negative Bacteria
Droplet microfluidics disrupted analytical biology with the introduction of digital PCR and single-cell sequencing. The same technology may also bring important innovation in the analysis of bacteria, including antibiotic susceptibility testing at the single-cell level. Still, despite promising demonstrations, the lack of a high-throughput label-free method of detecting bacteria in nanoliter droplets prohibits analysis of the most interesting strains and widespread use of droplet technologies in analytical microbiology. Pacocha et al. use a sensitive and fast measurement of scattered light from nanoliter droplets to demonstrate reliable detection of the proliferation of encapsulated bacteria. The authors verify the sensitivity of the method by simultaneous readout of fluorescent signals from bacteria expressing fluorescent proteins and demonstrate label-free readout on unlabeled Gram-negative and Gram-positive species. The authors’ approach requires neither genetic modification of the cells nor the addition of chemical markers of metabolism. It is compatible with a wide range of bacterial species of clinical, research, and industrial interest, opening the microfluidic droplet technologies for adaptation in these fields. (Pacocha, N., et al. Anal. Chem.
Advances in Gene Editing
In Situ Generation of Large Numbers of Genetic Combinations for Metabolic Reprogramming via CRISPR-Guided Base Editing
Reprogramming complex cellular metabolism requires simultaneous regulation of multigene expression. Ex situ cloning-based methods are commonly used, but the target gene number and combinatorial library size are severely limited by cloning and transformation efficiencies. In situ methods such as multiplex automated genome engineering (MAGE) depend on high-efficiency transformation and incorporation of heterologous DNA donors, which are limited to few microorganisms. Here, Wang et al. describe a Base Editor-Targeted and Template-free Expression Regulation (BETTER) method for simultaneously diversifying multigene expression. BETTER repurposes CRISPR-guided base editors and in situ generates large numbers of genetic combinations of diverse ribosome binding sites, 5′ untranslated regions, or promoters, without library construction, transformation, and incorporation of DNA donors. The authors apply BETTER to simultaneously regulate expression of up to 10 genes in industrial and model microorganisms Corynebacterium glutamicum and Bacillus subtilis. Variants with improved xylose catabolism, glycerol catabolism, or lycopene biosynthesis are respectively obtained. This technology will be useful for large-scale fine-tuning of multigene expression in both genetically tractable and intractable microorganisms. (Wang, Y., et al. Nat. Commun.
The Establishment of a Novel High-Throughput Screening System Using RNA-Guided Genome Editing to Identify Chemicals That Suppress Aldosterone Synthase Expression
Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, Ito et al. attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. The authors generated a cell line by inserting Nanoluc gene as a reporter into the CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. The authors then identified nine compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. The authors’ screening system may therefore be a useful tool for the development of novel medicines against PA. (Ito, R., et al. Biochem. Biophys. Res. Commun.
Precise and Broad Scope Genome Editing Based on High-Specificity Cas9 Nickases
RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, Wang et al. report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by “tiptoeing” over preexisting single-base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, the authors demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multitier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount. (Wang, Q., et al. Nucleic Acids Res.
Application of Genome Editing in Tomato Breeding: Mechanisms, Advances, and Prospects
Plants regularly face the changing climatic conditions that cause biotic and abiotic stress responses. The abiotic stresses are the primary constraints affecting crop yield and nutritional quality in many crop plants. The advances in genome sequencing and high-throughput approaches have enabled the researchers to use genome editing tools for the functional characterization of many genes useful for crop improvement. The present review focuses on the genome editing tools for improving many traits, such as disease resistance, abiotic stress tolerance, yield, quality, and nutritional aspects of tomato. Many candidate genes conferring tolerance to abiotic stresses such as heat, cold, drought, and salinity stress have been successfully manipulated by gene modification and editing techniques such as RNA interference, insertional mutagenesis, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9). In this regard, the genome editing tools such as CRISPR/Cas9, which is a fast and efficient technology, can be exploited to explore the genetic resources for the improvement of tomato and other crop plants in terms of stress tolerance and nutritional quality. The review presents examples of gene editing responsible for conferring both biotic and abiotic stresses in tomato simultaneously. The literature on using this powerful technology to improve fruit quality, yield, and nutritional aspects in tomato is highlighted. Finally, the prospects and challenges of genome editing and public and political acceptance in tomato are discussed. (Salava, H., et al. Int. J. Mol. Sci.
Gut-Licensed IFNγ+ NK Cells Drive LAMP1+TRAIL+ Anti-Inflammatory Astrocytes
Astrocytes are glial cells that are abundant in the central nervous system (CNS) and that have important homeostatic and disease-promoting functions. However, little is known about the homeostatic anti-inflammatory activities of astrocytes and their regulation. Here, using high-throughput flow cytometry screening, single-cell RNA sequencing, and CRISPR-Cas9-based cell-specific in vivo genetic perturbations in mice, the authors identify a subset of astrocytes that expresses the lysosomal protein LAMP1 and the death receptor ligand TRAIL. LAMP1+TRAIL+ astrocytes limit inflammation in the CNS by inducing T-cell apoptosis through TRAIL-DR5 signaling. In homeostatic conditions, the expression of TRAIL in astrocytes is driven by interferon-γ (IFNγ) produced by meningeal natural killer (NK) cells, in which IFNγ expression is modulated by the gut microbiome. TRAIL expression in astrocytes is repressed by molecules produced by T cells and microglia in the context of inflammation. Altogether, Sanmarco et al. show that LAMP1+TRAIL+ astrocytes limit CNS inflammation by inducing T-cell apoptosis, and that this astrocyte subset is maintained by meningeal IFNγ+ NK cells that are licensed by the microbiome. (Sanmarco, L. M., et al. Nature
Footnotes
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.