CRISPR–Cas9 Genome Editing in<i>Nothobranchius furzeri</i>for Gene Knockout and Knock-In

Rozenberg, Itai; Moses, Eitan; Harel, Itamar

doi:10.1101/pdb.prot107742

Cited by 8 publications

(6 citation statements)

References 13 publications

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“…The copyright holder for this preprint this version posted May 1, 2023. ; https://doi.org/10.1101/2023.05.01.538839 doi: bioRxiv preprint heterogeneity of cancer and metastasis. Together, we add to the continently expanding toolkit for the killifish model system [64][65][66][67] , by providing a transformative resource for advanced colorbased anatomical and lineage analyses during vertebrate aging and disease.…”

Section: Discussionmentioning

confidence: 99%

“…To enable transplantation into other organs, aside from the testis, we generated an immunocompromised genetic model by mutating the rag2 gene [35][36][37] (Figure 3). Using our recently updated CRISPR protocol 20,52 we generated a ~250 base-pair deletion allele (Δ250) in the single exon of the killifish rag2 coding sequence (Figure 3a, left). We outcrossed this line for several generations and generated a fertile homozygous fish.…”

Section: Generation and Characterization Of A Rag2 Genetic Modelmentioning

confidence: 99%

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A genetic toolbox for the turquoise killifish identifies sporadic age-related cancer

Rozenberg

Atlan

Franěk

et al. 2023

Preprint

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Using lineage tracing and fate mapping strategies to study vertebrate aging has lagged behind developmental studies, primarily due to of the relatively long lifespans of classical models. Here, we introduce theKillibow, an inducible transgenic model forin-vivomulticolor lineage tracing in the naturally short-lived turquoise killifish (N. furzeri). We demonstrate that Cre-mediated recombination in transgenic fish can generate robust and stochastic labeling that remains stable during aging and regeneration. In addition, to achieve inducible control of recombination, we either utilizein-vivoCre electroporation or use the tamoxifen system inKillibow-derived cells. To further enable transplantation assays, we establish the first immunocompromised killifish model by mutatingrag2. RNA sequencing reveals thatrag2mutants exhibit severely compromised expression of V(D)J recombination products, including immunoglobulins. Accordingly, we demonstrate that clearance of transplantedKillibow-derived cells is delayed inrag2recipients, and present a proof-of-principle for a KRASG12Dcancer model that is compatible with lineage tracing. Our platform provides the opportunity to examine tissue homeostasis, stem cell function, cancer dynamics, and tissue regeneration at unprecedented temporal resolution during vertebrate aging and disease.

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Section: Discussionmentioning

confidence: 99%

Section: Generation and Characterization Of A Rag2 Genetic Modelmentioning

confidence: 99%

A genetic toolbox for the turquoise killifish identifies sporadic age-related cancer

Rozenberg

Atlan

Franěk

et al. 2023

Preprint

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“…Tissues samples were processed as described previously ( Harel et al, 2015 ; Astre et al, 2022b ; Harel and Brunet, 2015 ; Astre et al, 2021 ; Harel et al, 2022 ; Nathan et al, 2008 ; Theis et al, 2010 ; Harel and Tzahor, 2012 ; Harel et al, 2012 ; Benayoun et al, 2019 ; Rozenberg et al, 2023b ; Astre et al, 2022a ; Harel et al, 2016 ; Rozenberg et al, 2023a ; Van Keymeulen et al, 2009 ; Moses and Harel, 2023 ; Valenzano et al, 2015 ; Harel et al, 2009 ; Gruenbaum-Cohen et al, 2012 ). Briefly, fish were euthanized with 500 mg/l tricaine (MS222, #A5040, Sigma).…”

Section: Methodsmentioning

confidence: 99%

A scalable and tunable platform for functional interrogation of peptide hormones in fish

Moses,

Franek,

Harel

2023

eLife

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Pituitary hormones play a central role in shaping vertebrate life history events, including growth, reproduction, metabolism, and aging. The regulation of these traits often requires precise control of hormone levels across diverse timescales. However, fine tuning circulating hormones in-vivo has traditionally been experimentally challenging. Here, using the naturally short-lived turquoise killifish (N. furzeri), we describe a high-throughput platform that combines loss- and gain-of-function of peptide hormones. Mutation of three primary pituitary hormones, growth hormone (gh1), follicle stimulating hormone (fshb), and thyroid stimulating hormone (tshb), alters somatic growth and reproduction. Thus, suggesting that while the killifish undergoes extremely rapid growth and maturity, it still relies on vertebrate-conserved genetic networks. As the next stage, we developed a gain-of-function vector system in which a hormone is tagged using a self-cleavable fluorescent reporter, and ectopically expressed in-vivo through intramuscular electroporation. Following a single electroporation, phenotypes, such as reproduction, are stably rescued for several months. Notably, we demonstrate the versatility of this approach by using multiplexing, dose-dependent, and doxycycline-inducible systems to achieve tunable and reversible expression. In summary, this method is relatively high-throughput, and facilitates large-scale interrogation of life-history strategies in fish. Ultimately, this approach could be adapted for modifying aquaculture species and exploring pro-longevity interventions.

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“…Tissue samples were processed as described previously 29,30,32,33,35,36,111,138–145 . Briefly, paraffin sections from one-month old fish of the indicated sex and genotype were used.…”

Section: Methodsmentioning

confidence: 99%

The killifish germline regulates longevity and somatic repair in a sex-specific manner

Moses,

Atlan,

Sun

et al. 2023

Preprint

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Classical evolutionary theories propose tradeoffs between reproduction, damage repair, and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. Here, we use the turquoise killifish (N. furzeri) to genetically arrest germline differentiation at discrete stages, and examine how different ‘flavors’ of infertility impact life-history. We first constructed a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. Next, investigating our genetic models revealed that only germline depletion enhanced female damage repair, while arresting germline differentiation did not. Conversely, germline-depleted males were significantly long-lived, indicating that the mere presence of the germline can negatively affect lifespan. Transcriptomic analysis highlighted enrichment of pro-longevity pathways and genes, with functional conservation in germline-depletedC. elegans. Finally, germline depletion extended male healthspan through rejuvenated metabolic functions. Our results suggest that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative mechanisms to classical evolutionary tradeoffs.

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CRISPR–Cas9 Genome Editing inNothobranchius furzerifor Gene Knockout and Knock-In

Cited by 8 publications

References 13 publications

A genetic toolbox for the turquoise killifish identifies sporadic age-related cancer

A genetic toolbox for the turquoise killifish identifies sporadic age-related cancer

A scalable and tunable platform for functional interrogation of peptide hormones in fish

The killifish germline regulates longevity and somatic repair in a sex-specific manner

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