Here, we introduce \'TICIT\', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish\'s genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.

The most current in vitro genetic methods, including gene preservation, gene editing and developmental modelling, require a significant number of healthy cells. In poultry species, primordial germ cells (PGCs) are great candidates for all the above-mentioned purposes, given their easy culturing and well-established freezing method for chicken. However, the constant monitoring of cultures can be financially challenging and consumes large amounts of solutions and accessories. This study aimed to introduce the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) complex into the chicken PGCs. FUCCI is a powerful transgenic tool based on the periodic protein expression changes during the cell cycle. It includes chromatin licensing and DNA replication factor 1 attached monomeric Kusabira-Orange and Geminin-attached monomeric Azami-Green fluorescent proteins, that cause the cells to express a red signal in the G1 phase and a green signal in S and G2 phases. Modification of the chicken PGCs was done via electroporation and deemed to be successful according to confocal microscopy, DNA sequencing and timelapse video analysis. Stable clone cell lines were established, cryopreserved, and injected into recipient embryos to prove the integrational competency. The cell health monitoring was tested with medium change experiments, that proved the intended reactions of the FUCCI transgene. These results established the future for FUCCI experiments in chicken, including heat treatment and toxin treatment.

Delivering molecular tools into oocytes is essential for developmental and reproductive biology. Microinjection, the conventional method, is equipment-intensive, often technically challenging, and low-yield, and is impractical in species with delicate oocytes or restricted spawning seasons. To overcome these limitations, we developed VitelloTag, a cost-effective, high-throughput system using vitellogenin-derived fusion proteins to enable efficient cargo delivery via receptor-mediated endocytosis. We demonstrate its utility by delivering Cas9/sgRNA complexes in two distantly related species for gene knockout.

Cnidarians have become valuable models for understanding many aspects of developmental biology including the evolution of body plan diversity, novel cell type specification, and regeneration. Most of our understanding of gene function during early development in cnidarians comes from a small number of experimental systems including the sea anemone, Nematostella vectensis. Few molecular tools have been developed for use in hard corals, limiting our understanding of this diverse and ecologically important clade. Here, we report the development of a suite of tools for manipulating and analyzing gene expression during early development in the northern star coral, Astrangia poculata. We present methods for gene knockdown using short hairpin RNAs, gene overexpression using exogenous mRNAs, and endogenous gene tagging using CRISPR-mediated gene knock-in. Combined with our ability to control spawning in the laboratory, these tools make A. poculata a tractable experimental system for investigative studies of coral development. Further application of these tools will enable functional analyses of embryonic patterning and morphogenesis across Anthozoa and open new frontiers in coral biology research.

BACKGROUND: The Tol2 transposable element is the most widely used transgenesis tool in zebrafish. However, its high activity almost always leads to multiple unlinked integrations of the transgenic cassette in F1 fish. Each of these transgenes is susceptible to positional effects from the surrounding regulatory landscape, which can lead to altered expression and, consequently, activity. Scientists therefore must strike a balance between the need to maximize reproducibility by establishing single-insertion transgenic lines and the need to complete experiments within a reasonable timeframe.
RESULTS: In this article, we introduce a simple competitive dilution strategy for rapid generation of single-insertion transgenics. By using cry:BFP reporter plasmid as a competitor, we achieved a nearly fourfold reduction in the number of the transgene of interest integrations while simultaneously increasing the proportion of single-insertion F1 generation transgenics to over 50%. We also observed variations in transgene of interest expression among independent single-insertion transgenics, highlighting that the commonly used ubiquitous ubb promoter is susceptible to position effects.
CONCLUSIONS: Wide application of our competitive dilution strategy will save time, reduce animal usage, and improve reproducibility of zebrafish research.

The skin-penetrating gastrointestinal parasitic nematode Strongyloides stercoralis causes strongyloidiasis, which is a neglected tropical disease that is associated with severe chronic illness and fatalities. Unlike other human-infective nematodes, S. stercoralis cycles through a single free-living generation and thus serves as a genetically tractable model organism for understanding the mechanisms that enable parasitism. Techniques such as CRISPR/Cas9-mediated mutagenesis and transgenesis are now routinely performed in S. stercoralis by introducing exogenous DNA into free-living adults and then screening their F1 progeny for transgenic or mutant larvae. However, transgenesis in S. stercoralis has been severely hindered by the inability to establish stable transgenic lines that can be propagated for multiple generations through a host; to date, studies of transgenic S. stercoralis have been limited to heterogeneous populations of transgenic F1 larvae. Here, we develop an efficient pipeline for the generation of stable transgenic lines in S. stercoralis. We also show that this approach can be used to efficiently generate stable transgenic lines in the rat-infective nematode Strongyloides ratti. The ability to generate stable transgenic lines circumvents the limitations of working with heterogeneous F1 populations, such as variable transgene expression and the inability to generate transgenics of all life stages. Our transgenesis approach will enable novel lines of inquiry into parasite biology, such as transgene-based comparisons between free-living and parasitic generations.

Vector-borne diseases (VBDs) impose devastating effects on human health and a heavy financial burden. Malaria, Lyme disease, and dengue fever are just a few examples of VBDs that cause severe illnesses. The current strategies to control VBDs consist mainly of environmental modification and chemical use, and to a small extent, genetic approaches. The genetic approaches, including transgenesis/genome modification and gene-drive technologies, provide the basis for developing new tools for VBD prevention by suppressing vector populations or reducing their capacity to transmit pathogens. The regulatory elements such as promoters are required for a robust sex-, tissue-, and stage-specific transgene expression. As discussed in this review, information on the regulatory elements is available for mosquito vectors but is scant for other vectors.

Malaria remains a persistent global public health challenge because of the limitations of current prevention tools. The use of transgenic mosquitoes incapable of transmitting malaria, in conjunction with existing methods, holds promise for achieving elimination of malaria and preventing its reintroduction. In this context, population modification involves the spread of engineered genetic elements through mosquito populations that render them incapable of malaria transmission. Significant progress has been made in this field over the past decade in revealing promising targets, optimizing genetic tools, and facilitating the transition from the laboratory to successful field deployments, which are subject to regulatory scrutiny. This review summarizes recent advances and ongoing challenges in \'curing\' Anopheles vectors of the malaria parasite.

Functional gene and protein characterizations in parasitic protists are often limited by their genetic tractability. Despite the development of CRISPR-Cas9-derived or inspired approaches for a handful of protist parasites, the overall genetic tractability of these organisms remains limited. The intestinal parasite Giardia lamblia is one such species, with the added challenge of a paucity of reliable selection markers. To address this limitation, we tested the feasibility of using Nourseothricin as an effective selection agent in Giardia. Here, we report that axenically-grown WB Giardia cells are sensitive to Nourseothricin and that engineering expression of the streptothricin acetyltransferase (SAT-1) gene from Streptomyces rochei in transgenic parasites confers resistance to this antibiotic. Furthermore, we determine that SAT-1-expressing parasites are cross-resistant neither to Neomycin nor Puromycin, which are widely used to select for transgenic parasites. Consequently, we show that Nourseothricin can be used in sequential combination with both Neomycin and Puromycin to select for dual transfection events. This work increases the number of reliable selection agents and markers for Giardia genetic manipulation, expanding the limited molecular toolbox for this species of global medical importance.

The zebrafish (Danio rerio) has emerged as an appreciated and versatile model organism for studying cardiovascular and metabolic diseases, offering unique advantages for both basic research and drug discovery. The genetic conservation between zebrafish and humans and their high fecundity and transparent embryos allow for efficient large-scale genetic and drug-oriented screening studies. Zebrafish possess a simplified cardiovascular system that shares similarities with mammals, making them particularly suitable for modeling various aspects of heart development, function, and disease. The transparency of zebrafish embryos enables the real-time visualization of cardiovascular dynamics, offering insights into early embryonic events and facilitating the study of heart-related anomalies. In metabolic research, zebrafish provide a cost-effective platform for modeling obesity, type 2 diabetes, hyperlipidemia, and other metabolic disorders. Their high reproductive rate allows for the generation of large cohorts for robust statistical analyses, while advanced genetic tools, such as CRISPR/Cas9, enable precise gene editing with which to model specific genetic mutations associated with human diseases. Zebrafish metabolic models have been instrumental in elucidating the molecular mechanisms underlying metabolic diseases, studying the effects of environmental factors, and identifying potential therapeutic targets. Additionally, the permeability of zebrafish embryos to small molecules facilitates drug discovery and screening, offering a rapid and economical approach to identifying compounds with therapeutic potential. In conclusion, zebrafish cardiovascular and metabolic disease models continue to contribute significantly to our perception of disease pathogenesis, providing a platform for translational research and developing novel therapeutic interventions. The versatility, scalability, and genetic manipulability of zebrafish position them as an invaluable asset in unraveling the complexities of cardiovascular and metabolic diseases. This review presents an overview of the zebrafish model\'s key features and contributions to investigating cardiovascular and metabolic disorders. We discuss the benefits and drawbacks of using zebrafish models to study human disease and the critical findings revealed by the progress in this endeavor to date.