Fishing for understanding: Unlocking the zebrafish gene editor's toolbox.

The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing. As the zebrafish gene editing toolkit continues to grow, it is becoming increasingly important to understand when and how to utilize which of these technologies for maximum efficacy in a particular project. While CRISPR-Cas9 has brought broad attention to the field of genome engineering in recent years, designer endonucleases have been utilized in genome engineering for more than two decades. This chapter provides a brief overview of designer endonuclease and other gene editing technologies in zebrafish as well as some of their known functional benefits and limitations depending on specific project goals. Finally, selected prospects for additional gene editing tools are presented, promising additional options for directed genomic programming of this versatile animal model system.

Tryptophan-Rich Sensory Protein/Translocator Protein (TSPO) from Cyanobacterium Fremyella diplosiphon Binds a Broad Range of Functionally Relevant Tetrapyrroles.

Tryptophan-rich sensory protein/translocator protein (TSPO) is a membrane protein involved in stress adaptation in the cyanobacterium Fremyella diplosiphon. Characterized mammalian and proteobacterial TSPO homologues bind tetrapyrroles and cholesterol ligands. We investigated the ligand binding properties of TSPO from F. diplosiphon (FdTSPO1), which was functionally characterized in prior genetic studies. Two additional TSPO proteins (FdTSPO2 and FdTSPO3) are present in F. diplosiphon; they are similar in size to reported bacterial TSPOs and smaller than FdTSPO1. The longer cyanobacterial TSPO1 is found almost exclusively in filamentous cyanobacteria and has a relatively low degree of homology to bacterial and mammalian TSPO homologues with confirmed tetrapyrrole binding. To probe distinctions of long-form TSPOs, we tested the binding of porphyrin and bilin to FdTSPO1 and measured binding affinities in the low micromolar range, with the highest binding affinity detected for heme. Although tetrapyrrole ligands bound FdTSPO1 with affinities similar to those previously reported for proteobacterial TSPO, binding of cholesterol to FdTSPO1 was particularly poor and was not improved by introducing an amino acid motif known to enhance cholesterol binding in other bacterial TSPO homologues. Additionally, we detected limited binding of bacterial hopanoids to FdTSPO1. Cyanobacterial TSPO1 from the oxygenic photosynthetic F. diplosiphon, thus, binds a range of tetrapyrroles of functional relevance with efficiencies similar to those of mammalian and proteobacterial homologues, but the level of cholesterol binding is greatly reduced compared to that of mammalian TSPO. Furthermore, the ΔFdTSPO1 mutant exhibits altered growth in the presence of biliverdin compared to that of wild-type cells under green light. Together, these results suggest that TSPO molecules may play roles in bilin homeostasis or trafficking in cyanobacteria.

A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish.

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington's disease and Parkinson's disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

The FusX TALE Base Editor (FusXTBE) for Rapid Mitochondrial DNA Programming of Human Cells In Vitro and Zebrafish Disease Models In Vivo.

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo. Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

Rapid Adaptation and Remote Delivery of Undergraduate Research Training during the COVID-19 Pandemic.

When COVID-19 caused worldwide cancellations of summer research immersion programs in 2020, Mayo Clinic rallied to create an alternate virtual experience called Summer Foundations in Research (SFIR). SFIR was designed not only to ensure the continuance of science pathways training for undergraduate scientists but also to support undergraduate mental wellbeing, given the known pandemic stressors. A total of 170 participants took part in the program and were surveyed pre-post for outcomes in biomedical research career knowledge, biomedical research career interest, research skills confidence, and three dimensions of mental wellbeing. Knowledge of and interest in careers involving biomedical research rose significantly following participation in SFIR. The participants' mean research skills confidence also rose between 0.08 and 1.32 points on a 7-point scale across 12 items from the Clinical Research Appraisal Inventory. Success in science pathways support was accompanied by positive shifts in participant mental wellbeing. Measurable decreases in stress (Perceived Stress Scale, p < 0.0001) accompanied gains in resilience (Brief Resilience Scale, p < 0.0001) and life satisfaction (Satisfaction with Life Scale, p = 0.0005). Collectively, the data suggest that core objectives of traditional in-person summer research programming can be accomplished virtually and that these programs can simultaneously impact student wellbeing. This theoretical framework is particularly salient during COVID-19, but the increased accessibility of virtual programs such as SFIR can continue to bolster science education pathways long after the pandemic is gone.

Rapid adaptation and remote delivery of undergraduate research training during the COVID 19 Pandemic.

COVID-19 continues to alter daily life around the globe. Education is particularly affected by shifts to distance learning. This change has poignant effects on all aspects of academic life, including the consequence of increased mental stress reported specifically for students. COVID-19 cancellations of many summer fellowships and internships for undergraduates across the country increased students' uncertainty about their educational opportunities and careers. When the pandemic necessitated elimination of on-campus programming at Mayo Clinic, a new program was developed for remote delivery. Summer Foundations in Research (SFIR) was drafted around 4 aims: 1) support the academic trajectory gap in research science created by COVID-19; 2) build sustainable scientific relationships with mentors, peers, and the community; 3) create opportunities for participants to share and address concerns with their own experiences in the pandemic; and 4) provide support for individual wellbeing. SFIR included research training, but also training in communication through generative Dialogue and resilience through Amit Sood's SMART program. 170 participants were followed for outcomes in these spaces. Knowledge of and interest in careers involving biomedical research rose significantly following SFIR. Participants' mean confidence levels in 12 Key areas of research rose between 0.08 to 1.32 points on a 7-point scale. The strongest gains in mean confidence levels were seen in designing a study and collaborating with others. SFIR participants demonstrated gains in perceived happiness, and measured resilience and a reduction in stress. Participants' qualitative responses indicated exceptionally positive mentor relationships and specific benefit of both the SMART program and Dialogue.

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

Precision gene editing technology and applications in nephrology.

The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR-Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision. Progress has also been made in harnessing endogenous DNA repair machineries, such as non-homologous end joining, homology-directed repair and microhomology-mediated end joining, to functionally manipulate genetic sequences. As understanding of how DNA damage results in deletions, insertions and modifications increases, the genome becomes more predictably mutable. DNA-binding platforms such as TALEs and CRISPR can also be used to make locus-specific epigenetic changes and to transcriptionally enhance or suppress genes. Although many challenges remain, the application of precision gene editing technology in the field of nephrology has enabled the generation of new animal models of disease as well as advances in the development of novel therapeutic approaches such as gene therapy and xenotransplantation.