Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

iSort enables automated complex microfluidic droplet sorting in an effort to democratize technology.

Fluorescence-activated droplet sorting (FADS) is a widely used microfluidic technique for high-throughput screening. However, it requires highly trained specialists to determine optimal sorting parameters, and this results in a large combinatorial space that is challenging to optimize systematically. Additionally, it is currently challenging to track every single droplet within a screen, leading to compromised sorting and "hidden" false-positive events. To overcome these limitations, we have developed a setup in which the droplet frequency, spacing, and trajectory at the sorting junction are monitored in real time using impedance analysis. The resulting data are used to continuously optimize all parameters automatically and to counteract perturbations, resulting in higher throughput, higher reproducibility, increased robustness, and a beginner-friendly character. We believe this provides a missing piece for the spreading of phenotypic single-cell analysis methods, similar to what we have seen for single-cell genomics platforms.

Fluorescence crosstalk reduction by modulated excitation-synchronous acquisition for multispectral analysis in high-throughput droplet microfluidics.

Crosstalk between fluorescent biomarkers significantly limits the resolution of multispectral fluorescence analysis in real-time droplet-microfluidics applications. The crosstalk is a result of overlapping emission and excitation spectra of different fluorophores in multiplexed analyses. To mitigate this crosstalk, we present a method that modulates multiple laser beams to selectively and sequentially excite the fluorophores by a single beam of a particular wavelength using acousto-optic modulators at a frequency of 0.1 MHz. An FPGA based data acquisition algorithm synchronized with the modulation signal then acquires the emission signals only from the fluorescence channel that corresponds to the excitation wavelength provided in that particular time window. We applied our method for fluorescence-based droplet analysis in microfluidics and demonstrate that the method is able to reduce crosstalk contribution between channels by >97% and can resolve fluorescence populations that are indistinguishable with conventional droplet analysis methods.

Design and construction of a microfluidics workstation for high-throughput multi-wavelength fluorescence and transmittance activated droplet analysis and sorting.

Droplet microfluidics has revolutionized quantitative high-throughput bioassays and screening, especially in the field of single-cell analysis where applications include cell characterization, antibody discovery and directed evolution. However, droplet microfluidic platforms capable of phenotypic, fluorescence-based readouts and sorting are still mostly found in specialized labs, because their setup is complex. Complementary to conventional FACS, microfluidic droplet sorters allow the screening of cell libraries for secreted factors, or even for the effects of secreted or surface-displayed factors on a second cell type. Furthermore, they also enable PCR-activated droplet sorting for the isolation of genetic material harboring specific markers. In this protocol, we provide a detailed step-by-step guide for the construction of a high-throughput droplet analyzer and sorter, which can be accomplished in ~45 working hours by nonspecialists. The resulting instrument is equipped with three lasers to excite the fluorophores in droplets and photosensors that acquire fluorescence signals in the blue (425-465 nm), green (505-545 nm) and red (580-630 nm) spectrum. This instrument also allows transmittance-activated droplet sorting by analyzing the brightfield light intensity transmitting through the droplets. The setup is validated by sorting droplets containing fluorescent beads at 200 Hz with 99.4% accuracy. We show results from an experiment where droplets hosting single cells were sorted on the basis of increased matrix metalloprotease activity as an application of our workstation in single-cell molecular biology, e.g., to analyze molecular determinants of cancer metastasis.

A microfluidic Braille valve platform for on-demand production, combinatorial screening and sorting of chemically distinct droplets.

Droplet microfluidics is a powerful tool for a variety of biological applications including single-cell genetics, antibody discovery and directed evolution. All these applications make use of genetic libraries, illustrating the difficulty of generating chemically distinct droplets for screening applications. This protocol describes our Braille Display valving platform for on-demand generation of droplets with different chemical contents (16 different reagents and combinations thereof), as well as sorting droplets with different chemical properties, on the basis of fluorescence signals. The Braille Display platform is compact, versatile and cost efficient (only ~US$1,000 on top of a standard droplet microfluidics setup). The procedure includes manufacturing of microfluidic chips, assembly of custom hardware, co-encapsulation of cells and drugs into droplets, fluorescence detection of readout signals and data analysis using shared, freely available LabVIEW and Python packages. As a first application, we demonstrate the complete workflow for screening cancer cell drug sensitivities toward 74 conditions. Furthermore, we describe here an assay enabling the normalization of the observed drug sensitivity to the number of cancer cells per droplet, which additionally increases the robustness of the system. As a second application, we also demonstrate the sorting of droplets according to enzymatic activity. The drug screening application can be completed within 2 d; droplet sorting takes ~1 d; and all preparatory steps for manufacturing molds, chips and setting up the Braille controller can be accomplished within 1 week.

Differential contribution of nuclear size scaling mechanisms between Xenopus species.

Size of the nucleus, a membrane-bound organelle for DNA replication and transcription in eukaryotic cells, varies to adapt nuclear functions to the surrounding environment. Nuclear size strongly correlates with cytoplasmic size and genomic content. Previous studies using Xenopus laevis have unraveled two modes, cytoplasmic and chromatin-based mechanisms, for controlling nuclear size. However, owing to limited comparative analyses of the mechanisms among eukaryotic species, the contribution of each mechanism in controlling nuclear size has not been comprehensively elucidated. Here, we compared the relative contribution utilizing a cell-free reconstruction system from the cytoplasmic extract of unfertilized eggs of Xenopus tropicalis to that of the sister species X. laevis. In this system, interphase nuclei were reconstructed in vitro from sperm chromatin and increased in size throughout the incubation period. Using extracts from X. tropicalis, growth rate of the reconstructed nuclei was decreased by obstructing the effective cytoplasmic space, decreasing DNA quantity, or inhibiting molecules involved in various cytoplasmic mechanisms. Although these features are qualitatively identical to that shown by the extract of X. laevis, the sensitivities of experimental manipulation for each cellular parameter were different between the extracts from two Xenopus species. These quantitative differences implied that the contribution of each mode to expansion of the nuclear envelope is coordinated in a species-specific manner, which sets the species-specific nuclear size for in vivo physiological function.

Technological and computational advances driving high-throughput oncology.

Engineering and computational advances have opened many new avenues in cancer research, particularly when being exploited in interdisciplinary approaches. For example, the combination of microfluidics, novel sequencing technologies, and computational analyses has been crucial to enable single-cell assays, giving a detailed picture of tumor heterogeneity for the very first time. In a similar way, these 'tech' disciplines have been elementary for generating large data sets in multidimensional cancer 'omics' approaches, cell-cell interaction screens, 3D tumor models, and tissue level analyses. In this review we summarize the most important technology and computational developments that have been or will be instrumental for transitioning classical cancer research to a large data-driven, high-throughput, high-content discipline across all biological scales.

Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics.

We report a droplet microfluidic method to target and sort individual cells directly from complex microbiome samples and to prepare these cells for bulk whole-genome sequencing without cultivation. We characterize this approach by recovering bacteria spiked into human stool samples at a ratio as low as 1:250 and by successfully enriching endogenous Bacteroides vulgatus to the level required for de novo assembly of high-quality genomes. Although microbiome strains are increasingly demanded for biomedical applications, a vast majority of species and strains are uncultivated and without reference genomes. We address this shortcoming by encapsulating complex microbiome samples directly into microfluidic droplets and amplifying a target-specific genomic fragment using a custom molecular TaqMan probe. We separate those positive droplets by droplet sorting, selectively enriching single target strain cells. Finally, we present a protocol to purify the genomic DNA while specifically removing amplicons and cell debris for high-quality genome sequencing.

Targeted Perturb-seq enables genome-scale genetic screens in single cells.

The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer-target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer-target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.

Patient-specific logic models of signaling pathways from screenings on cancer biopsies to prioritize personalized combination therapies.

Mechanistic modeling of signaling pathways mediating patient-specific response to therapy can help to unveil resistance mechanisms and improve therapeutic strategies. Yet, creating such models for patients, in particular for solid malignancies, is challenging. A major hurdle to build these models is the limited material available that precludes the generation of large-scale perturbation data. Here, we present an approach that couples ex vivo high-throughput screenings of cancer biopsies using microfluidics with logic-based modeling to generate patient-specific dynamic models of extrinsic and intrinsic apoptosis signaling pathways. We used the resulting models to investigate heterogeneity in pancreatic cancer patients, showing dissimilarities especially in the PI3K-Akt pathway. Variation in model parameters reflected well the different tumor stages. Finally, we used our dynamic models to efficaciously predict new personalized combinatorial treatments. Our results suggest that our combination of microfluidic experiments and mathematical model can be a novel tool toward cancer precision medicine.

Microfluidics as an Enabling Technology for Personalized Cancer Therapy.

Tailoring patient-specific treatments for cancer is necessary in order to achieve optimal results but requires new diagnostic approaches at affordable prices. Microfluidics has immense potential to provide solutions for this, as it enables the processing of samples that are not available in large quantities (e.g., cells from patient biopsies), is cost efficient, provides a high level of automation, and allows the set-up of complex models for cancer studies. In this review, individual solutions in the fields of genetics, circulating tumor cell monitoring, biomarker analysis, phenotypic drug sensitivity tests, and systems providing controlled environments for disease modeling are discussed. An overview on how these early stage achievements can be combined or developed further is showcased, and the required translational steps before microfluidics becomes a routine tool for clinical applications are critically discussed.

Microfluidics as an Emerging Precision Tool in Developmental Biology.

Microfluidics has become a precision tool in modern biology. It enables omics data to be obtained from individual cells, as compared to averaged signals from cell populations, and it allows manipulation of biological specimens in entirely new ways. Cells and organisms can be perturbed at extraordinary spatiotemporal resolution, revealing mechanistic insights that would otherwise remain hidden. In this perspective article, we discuss the current and future impact of microfluidic technology in the field of developmental biology. In addition, we provide detailed information on how to start using this technology even without prior experience.

Antibiotics-induced monodominance of a novel gut bacterial order.

OBJECTIVE: The composition of the healthy human adult gut microbiome is relatively stable over prolonged periods, and representatives of the most highly abundant and prevalent species have been cultured and described. However, microbial abundances can change on perturbations, such as antibiotics intake, enabling the identification and characterisation of otherwise low abundant species. DESIGN: Analysing gut microbial time-series data, we used shotgun metagenomics to create strain level taxonomic and functional profiles. Community dynamics were modelled postintervention with a focus on conditionally rare taxa and previously unknown bacteria. RESULTS: In response to a commonly prescribed cephalosporin (ceftriaxone), we observe a strong compositional shift in one subject, in which a previously unknown species, UBorkfalki ceftriaxensis, was identified, blooming to 92% relative abundance. The genome assembly reveals that this species (1) belongs to a so far undescribed order of Firmicutes, (2) is ubiquitously present at low abundances in at least one third of adults, (3) is opportunistically growing, being ecologically similar to typical probiotic species and (4) is stably associated to healthy hosts as determined by single nucleotide variation analysis. It was the first coloniser after the antibiotic intervention that led to a long-lasting microbial community shift and likely permanent loss of nine commensals. CONCLUSION: The bloom of UB. ceftriaxensis and a subsequent one of Parabacteroides distasonis demonstrate the existence of monodominance community states in the gut. Our study points to an undiscovered wealth of low abundant but common taxa in the human gut and calls for more highly resolved longitudinal studies, in particular on ecosystem perturbations.

A microfluidics platform for combinatorial drug screening on cancer biopsies.

Screening drugs on patient biopsies from solid tumours has immense potential, but is challenging due to the small amount of available material. To address this, we present here a plug-based microfluidics platform for functional screening of drug combinations. Integrated Braille valves allow changing the plug composition on demand and enable collecting >1200 data points (56 different conditions with at least 20 replicates each) per biopsy. After deriving and validating efficient and specific drug combinations for two genetically different pancreatic cancer cell lines and xenograft mouse models, we additionally screen live cells from human solid tumours with no need for ex vivo culturing steps, and obtain highly specific sensitivity profiles. The entire workflow can be completed within 48 h at assay costs of less than US$ 150 per patient. We believe this can pave the way for rapid determination of optimal personalized cancer therapies.

A Versatile, Low-Cost, Multiway Microfluidic Sorter for Droplets, Cells, and Embryos.

Partitioning and sorting particles, including molecules, cells and organisms, is an essential prerequisite for a diverse range of applications. Here, we describe a very economical microfluidic platform (built from parts costing about U.S. $6800 for a stand-alone system or U.S. $3700, when mounted on an existing fluorescence microscope connected to a computer) to sort droplets, cells and embryos, based on imaging data. Valves operated by a Braille display are used to open and close microfluidic channels, enabling sorting at rates of >2 Hz. Furthermore, we show microfluidic 8-way sorting for the first time, facilitating the simultaneous separation and collection of objects with diverse characteristics/phenotypes. Due to the high flexibility in the size of objects that can be sorted, the low cost, and the many possibilities enabled by imaging technology, we believe that our approach nicely complements existing FACS and µFACS technology.

Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells.

Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells.

Modulation of Phase Shift between Wnt and Notch Signaling Oscillations Controls Mesoderm Segmentation.

How signaling dynamics encode information is a central question in biology. During vertebrate development, dynamic Notch signaling oscillations control segmentation of the presomitic mesoderm (PSM). In mouse embryos, this molecular clock comprises signaling oscillations of several pathways, i.e., Notch, Wnt, and FGF signaling. Here, we directly address the role of the relative timing between Wnt and Notch signaling oscillations during PSM patterning. To this end, we developed a new experimental strategy using microfluidics-based entrainment that enables specific control of the rhythm of segmentation clock oscillations. Using this approach, we find that Wnt and Notch signaling are coupled at the level of their oscillation dynamics. Furthermore, we provide functional evidence that the oscillation phase shift between Wnt and Notch signaling is critical for PSM segmentation. Our work hence reveals that dynamic signaling, i.e., the relative timing between oscillatory signals, encodes essential information during multicellular development.

Microfluidic single-cell technology in immunology and antibody screening.

Single-cell technology has a major impact on the field of immunology. It enables the kinetics and logic of immune signaling and immune cell migration to be elucidated, facilitates antibody screening and allows massively parallelized analysis of B- and T-cell repertoires. Impressive progress has been made over the last decade, strongly boosted by microfluidic approaches. In this review, we summarize the most powerful microfluidic systems based on continuous flow, nanowells, valves and droplets and we analyze their benefits for phenotypic characterization, drug discovery and next generation sequencing experiments. We describe current limitations and provide an outlook on important future applications.