Hemocyte Nuclei Isolation from Adult Drosophila melanogaster for snRNA-seq.

In adult Drosophila, most of the hemocytes are macrophage-like cells (so called plasmatocytes), which serve various functions in organ homeostasis and immune defense. Ontogeny and functions are largely conserved between vertebrate and invertebrate macrophages. Hence, Drosophila offers a powerful genetic toolbox to study macrophage function and genetically modulate these cells. Technological advances in high-throughput sequencing approaches allowed to give an in-depth characterization of vertebrate macrophage populations and their heterogenous composition within different organs as well as changes in disease. Embryonic and larval hemocytes in Drosophila have been recently analyzed in single-cell RNA-sequencing (scRNA-seq) approaches during infection and steady state. These analyses revealed anatomical and functional Drosophila hemocyte subtypes dedicated to specific tasks. Only recently, the Fly Cell Atlas provided a whole transcriptomic single-cell atlas via single-nuclei RNA-sequencing (snRNA-seq) of adult Drosophila including many different tissues and cell types where hemocytes were also included. Yet, a specific protocol to isolate nuclei from adult hemocytes for snRNA-seq and study these cells in different experimental conditions was not available. In this chapter, we give a detailed protocol to purify hemocyte nuclei from adult Drosophila, which can be used in subsequent analyses such as snRNA-seq.

Combined Analysis of mRNA Expression and Open Chromatin in Microglia.

The advance of single-cell RNA-sequencing technologies in the past years has enabled unprecedented insights into the complexity and heterogeneity of microglial cell states in the homeostatic and diseased brain. This includes rather complex proteomic, metabolomic, morphological, transcriptomic, and epigenetic adaptations to external stimuli and challenges resulting in a novel concept of core microglia properties and functions. To uncover the regulatory programs facilitating the rapid transcriptomic adaptation in response to changes in the local microenvironment, the accessibility of gene bodies and gene regulatory elements can be assessed. Here, we describe the application of a previously published method for simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq) on microglia nuclei isolated from frozen mouse brain tissue.

Visual and rapid detection of escolar (Lepidocybium flavobrunneum) using loop mediated isothermal amplification in conjunction with a specific molecular beacon probe.

Species adulteration has become a main reason for the unexpected exposure to escolar, which is often related with the gastrointestinal disease called keriorrhea. Sensitive and accurate identification of escolar is required to protect consumers from commercial and health frauds. The present study established a visual and rapid method for escolar detection using LAMP (loop-mediated isothermal amplification) in conjunction with a MB (molecular beacon) probe. The visual MB-LAMP assay demonstrated high specificity and superb sensitivity (1 pg DNA) for escolar and low to 0.1 % (w/w) simulated adulteration could be detected within 25 min. Additionally, method validation on commercial products highlighted the umbrella term of white tuna for escolar on Chinese market. All these results indicated that the MB-LAMP method is a useful tool for rapid, sensitive and convenient detection of escolar and can also be used as a point-of-care molecular diagnostic technique since it does not require the expensive equipment.

Functional Genomics of Novel Rhabdomyosarcoma Fusion-Oncogenes Using Zebrafish.

Clinical sequencing efforts continue to identify novel putative oncogenes with limited strategies to perform functional validation in vivo and study their role in tumorigenesis. Here, we present a pipeline for fusion-driven rhabdomyosarcoma (RMS) in vivo modeling using transgenic zebrafish systems. This strategy originates with novel fusion-oncogenes identified from patient samples that require functional validation in vertebrate systems, integrating these genes into the zebrafish genome, and then characterizing that they indeed drive rhabdomyosarcoma tumor formation. In this scenario, the human form of the fusion-oncogene is inserted into the zebrafish genome to understand if it is an oncogene, and if so, the underlying mechanisms of tumorigenesis. This approach has been successful in our models of infantile rhabdomyosarcoma and alveolar rhabdomyosarcoma, both driven by respective fusion-oncogenes, VGLL2-NCOA2 and PAX3-FOXO1. Our described zebrafish platform is a rapid method to understand the impact of fusion-oncogene activity, divergent and shared fusion-oncogene biology, and whether any analyzed pathways converge for potential clinically actionable targets.

Delivering Traumatic Brain Injury to Larval Zebrafish.

We describe a straightforward, scalable method for administering traumatic brain injury (TBI) to zebrafish larvae. The pathological outcomes appear generalizable for all TBI types, but perhaps most closely model closed-skull, diffuse lesion (blast injury) neurotrauma. The injury is delivered by dropping a weight onto the plunger of a fluid-filled syringe containing zebrafish larvae. This model is easy to implement, cost-effective, and provides a high-throughput system that induces brain injury in many larvae at once. Unique to vertebrate TBI models, this method can be used to deliver TBI without anesthetic or other metabolic agents. The methods simulate the main aspects of traumatic brain injury in humans, providing a preclinical model to study the consequences of this prevalent injury type and a way to explore early interventions that may ameliorate subsequent neurodegeneration. We also describe a convenient method for executing pressure measurements to calibrate and validate this method. When used in concert with the genetic tools readily available in zebrafish, this model of traumatic brain injury offers opportunities to examine many mechanisms and outcomes induced by traumatic brain injury. For example, genetically encoded fluorescent reporters have been implemented with this system to measure protein misfolding and neural activity via optogenetics.

Cancer Modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).

Transgenic expression of genes is a mainstay of cancer modeling in zebrafish. Traditional transgenic techniques rely upon injection into one-cell embryos, but ideally these transgenes would be expressed only in adult somatic tissues. We provide a method to model cancer in adult zebrafish in which transgenes can be expressed via electroporation. Using melanoma as an example, we demonstrate the feasibility of expressing oncogenes such as BRAFV600E as well as CRISPR/Cas9 inactivation of tumor suppressors such as PTEN. These approaches can be performed in any genetic background such as existing fluorophore reporter lines or the casper line. These methods can readily be extended to other cell types allowing for rapid adult modeling of cancer in zebrafish.

Genetic Identification of Neural Circuits Essential for Active Avoidance Fear Conditioning in Adult Zebrafish.

Inhibition or ablation of neuronal activity combined with behavioral assessment is crucial in identifying neural circuits or populations essential for specific behaviors and to understand brain function. In the model vertebrate zebrafish, the development of genetic methods has allowed not only visualization but also targeted manipulation of neuronal activity, and quantitative behavioral assays allow precise measurement of animal behavior. Here, we describe a method to inhibit a specific neuronal population in adult zebrafish brain and assess their role in a learning behavior. We employed the Gal4-UAS system, gene trap and enhancer trap methods, and isolated transgenic zebrafish lines expressing Gal4FF transactivator in specific populations of neurons in the adult zebrafish brain. In these lines, a genetically engineered neurotoxin, botulinum toxin B light chain, was expressed and the fish were assessed in the active avoidance fear conditioning paradigm. The transgenic lines that showed impaired avoidance response were isolated and, in these fish, the Gal4-expressing neurons were analyzed to identify the neuronal circuits involved in avoidance learning.

Selective Cell Ablation Using an Improved Prodrug-Converting Nitroreductase.

Selective cell ablation is an invaluable tool to investigate the function of cell types, the regeneration of cells, and the modeling of diseases associated with cell loss. The nitroreductase (NTR)-mediated cell ablation system is a simple method enabling the elimination of targeted cells through the expression of a nitroreductase enzyme and the application of a prodrug (such as metronidazole). The prodrug is reduced to a cytotoxic product by nitroreductase, thereby leading to DNA damage-induced cell death. In species with elevated regenerative capacity such as zebrafish, removing the prodrug allows endogenous tissue to replace the lost cells. Herein, we describe a method for the use of a markedly improved nitroreductase enzyme for spatially and temporally controlled targeted cell ablation in the zebrafish. Recently, we identified an NTR variant (NTR 2.0) that achieves effective targeted cell ablation at concentrations of metronidazole well below those causing toxic side effects. NTR 2.0 thereby enables the ablation of "resistant" cell types and novel cell ablation paradigms. These advances simplify investigations of cell function, enable interrogations of the effects of chronic inflammation on regenerative processes and facilitate modeling of degenerative diseases associated with chronic cell loss. Techniques for transgenic nitroreductase expression and prodrug application are discussed.

Generation of Conditional Knockout Zebrafish Using an Invertible Gene-Trap Cassette.

Conditional knockout (cKO) is a genetic technique to inactivate gene expression in specific tissues or cell types in a temporally regulated manner. cKO analysis is essential to investigate gene function while avoiding the confounding effects of global gene deletion. Genetic techniques enabling cKO analysis were developed in mice based on culturable embryonic stem cells that were not generally available in zebrafish, which hampered precise analysis of genetic mechanisms of organ development and regeneration. However, recent advances in genome editing technologies have resolved this limitation, providing a platform for the generation of cKO models in any organism. Here we describe a detailed protocol for the generation of cKO zebrafish using a Cre-dependent genetic switch.

Holographic Optogenetic Activation of Neurons Eliciting Locomotion in Head-Embedded Larval Zebrafish.

Understanding how motor circuits are organized and recruited in order to perform complex behavior is an essential question of neuroscience. Here we present an optogenetic protocol on larval zebrafish that allows spatial selective control of neuronal activity within a genetically defined population. We combine holographic illumination with the use of effective opsin transgenic lines, alongside high-speed behavioral monitoring to dissect the motor circuits of the larval zebrafish.

Scalable CRISPR Screens in Zebrafish Using MIC-Drop.

CRISPR-Cas9 is a powerful tool to interrogate gene function in a targeted and systematic manner. Although the technology has been scaled up for large-scale genetic screens in cell culture, similar scale screens in vivo have been extremely challenging due to the cost, labor, and time required to generate and keep track of thousands of mutant animals. We reported the development of Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform that makes large-scale reverse genetic screens possible in zebrafish. In this chapter, we provide a detailed protocol to conduct large-scale genetic screens using this novel platform.

The Goldfish Genome and Its Utility for Understanding Gene Regulation and Vertebrate Body Morphology.

Goldfish, widely viewed as an ornamental fish, is a member of Cyprinidae family and has a very long history in research for both genetics and physiology studies. Among Cyprinidae, the chromosomal locations of orthologs and the amino acid sequences are usually highly conserved. Adult goldfish are 1000 times larger than adult zebrafish (who are in the same family of fishes), which can make it easier to perform several types of experiments compared to their zebrafish cousins. Comparing mutant phenotypes in orthologous genes between goldfish and zebrafish can often be very informative and provide a deeper insight into the gene function than studying the gene in either species alone. Comparative genomics and phenotypic comparisons between goldfish and zebrafish will provide new opportunities for understanding the development and evolution of body forms in the vertebrate lineage.

Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression.

The controlled expression of Cas9 and/or sgRNA in transgenic zebrafish made it possible to knock out a gene in a spatially and/or temporally controlled manner. This transgenic approach can be more useful if multiple sgRNAs are efficiently expressed since we can improve the biallelic frame-shift mutation rate and circumvent the functional redundancy of genes and genetic compensation. We developed the tRNA-based system to express multiple functional sgRNAs from a single transcript in zebrafish and found that it is applicable to the transgenic expression of multiple sgRNAs. In this chapter, we describe a procedure for the generation of plasmids containing multiple sgRNAs flanked by tRNAs and a method to induce multiple CRISPR/Cas9-mediated genome modifications in transgenic zebrafish.

Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish.

Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines.

In Vivo Optogenetic Phase Transition of an Intrinsically Disordered Protein.

Proteins containing intrinsically disordered regions (IDRs) control a wide variety of cellular processes by assembly of membrane-less organelles via IDR-mediated liquid-liquid phase separation. Dysregulated IDR-mediated phase transition has been implicated in the pathogenesis of diseases characterized by deposition of abnormal protein aggregates. Here, we describe a method to enhance interactions between the IDRs of the RNA/DNA-binding protein and TAR DNA-binding protein 43 (TDP-43) by light to drive its phase transition in the motor neurons of zebrafish. The optically controlled TDP-43 phase transition in motor neurons, in vivo, provides a unique opportunity to evaluate the impact of dysregulated TDP-43 phase transition on the physiology of motor neurons. This will help to address the etiology of neurodegenerative diseases associated with abnormal TDP-43 phase transition and aggregation, including amyotrophic lateral sclerosis (ALS).

Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells.

Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces - one composed of the atypical cadherin Fat2 (also known as Kugelei) and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptor Plexin A. Here, we show that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at leading edges of cells, but Lar and Semaphorin5c play little role in the localization of Fat2. Fat2 is also more stable at interfaces than Lar or Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. We propose that Fat2 serves as the organizer of this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells.

Engineering of an adaptive tandem CRISPR/Cas12a molecular amplifier permits robust analysis of Vibrio parahaemolyticus.

Seeking new molecular diagnostic method for pathogenic bacteria detection is of utmost importance for ensuring food safety and protecting human health. Herein, we have engineered an adaptive tandem CRISPR/Cas12a molecular amplifier specifically designed for robust analysis of vibrio parahaemolyticus (V. parahaemolyticus), one of the most harmful pathogens. Our strategy involves the integration of three crucial processes: recombinase polymerase amplification (RPA) for copy number amplification, terminal deoxynucleotidyl transferase (TdT) for template-free strand elongation, and CRISPR/Cas12a-mediated trans-cleavage of a reporter molecule. By combining these processes, the target genomic DNA extracted from V. parahaemolyticus is able to activate many CRISPR/Cas12a units (CRISPR/Cas12an) simultaneously, resulting in a greatly amplified target signal to indicate the presence and concentration of V. parahaemolyticus. This unique model offers more advantages compared to traditional amplification models that use one RPA amplicon to activate one CRISPR/Cas12a unit. Under optimized conditions, our method enables the detection of target V. parahaemolyticus within a linear range of 1 × 102-1 × 107 CFU/mL, with an impressive limit of detection as low as 12.4 CFU/mL. It is conceivable that the adaptive tandem CRISPR/Cas12a molecular amplifier could be adapted as routine diagnostic kits in future for in-field detection of pathogens.

Abridged solid-phase extraction with alkaline Poly(ethylene) glycol lysis (ASAP) for direct DNA amplification.

Complexity of sample preparation decelerate the development of sample-in-answer-out devices for point-of-need nucleic acid amplification testing. Here, we present the consolidation of alkaline poly(ethylene) glycol-based lysis and solid-phase extraction for rapid and simple sample preparation compatible with direct on-bead amplification. Simultaneous cell lysis and binding of DNA were achieved using an optimised reagent comprising 15% PEG8000, 0.5 M NaCl, and 3.5 mM KOH. This was combined with direct, on-bead amplification using 1.5 µg beads per 20 µL PCR reaction mix. The novel single reagent, 5-min method improved the detection limit by 10 and 100-fold compared with commercial DNA extraction kits and the original alkaline PEG lysis method, respectively. The sensitivity can be further enhanced by one amplification cycle with an ethanol wash or by extending the incubation to 10 min before collecting the magnetic particles. Both methods successfully detected a single copy of Escherichia coli DNA. In biological fluids (saliva, sweat, and urine), the 5-min method was delayed by about one cycle compared to the 15-min method. The proposed methods are attractive for incorporation in the workflow for point-of-need testing of biological samples by providing a practical and chemical method for simple alternative DNA sample preparation.

Target-triggered stochastic DNAzyme motors on spherical nucleic acids for simultaneous fluorescence assay of double miRNAs.

Simultaneous quantifications of multiple miRNAs in the single-sampling system would be conducive to the accurate diagnosis of diseases in contrast with single miRNA analysis. In this work, a stochastic DNAzyme motor on spherical nucleic acids (SNAs) for simultaneous fluorescence assay of double miRNAs was established. Hairpin 1 (H1)-FAM-7a and H1-TAMRA-133a-functionalized magnetic beads (MBs) as SNAs were mixed. Targets (let-7a and miRNA-133a) reacted with two different S1 and S2, triggering the formation of two types of metal DNAzymes. The DNAzymes can further react with H1 stem-loop DNA on SNAs to release the two fluorescent DNA-FAM and DNA-TAMRA fragments in the presence of Mg2+. Meanwhile, the DNAzyme as DNA motors were separated from the previous H1 probe to participate the next cycling operations, resulting in the signal amplification toward the simultaneous and sensitive detection of let-7a and miRNA-133a. SNAs with three dimensional nanostructures provided enough space for the operation of DNAzyme walker, promoting the sensitivity of this proposed analytical system. The two mixed SNAs enable one-step and specific quantification of miRNA let-7a and miRNA-133a with lower detection limits of 90.5 fM and 74.9 fM, respectively. Finally, this proposed strategy was employed to simultaneously detect double miRNAs in practical applicability.

High-sensitive and rapid electrochemical detection of miRNA-31 in saliva using Cas12a-based 3D nano-harvester with improved trans-cleavage efficiency.

Salivary miRNA-31 is a reliable diagnostic marker for early-stage oral squamous cell carcinoma (OSCC), but accurate detection of miRNA-31 in saliva samples is a challenge because of its low level and high sequence homology. The CRISPR/Cas12a system has the exceptional potential to enable simple nucleic acid analysis but suffers from low speed and sensitivity. To achieve rapid and high-sensitive detection of miRNA-31 using the CRISPR/Cas12a system, a Cas12a-based nano-harvester activated by a polymerase-driven DNA walker, named as dual 3D nanorobots, was developed. The target walked rapidly on the surface of DNA hairpin-modified magnetic nanoparticles driven by DNA polymerase, generating numerous double-strand DNA (dsDNA). Then, the Cas12a bound to the generated dsDNA for activating its trans-cleavage activity, forming 3D nano-harvester. Subsequently, the harvester cut and released methylene blue-labeled DNA hairpins immobilized on the sensing interface, leading to the change in electrochemical signal. We found that the trans-cleavage activity of the harvester was higher than the conventional CRISPR/Cas12a system. The developed dual 3D nanorobots could enable rapid (detection time within 60 min), high-sensitive (detection limit of femtomolar), and specific analysis of miRNA-31 in saliva samples. Thus, our established electrochemical biosensing strategy has great potential for early diagnosis of OSCC.