Modeling thoracic aortic genetic variants in the zebrafish: useful for predicting clinical pathogenicity?

Frontiers in Cardiovascular Medicine, Journal Year: 2025, Volume and Issue: 12

Published: Feb. 19, 2025

Thoracic aortic aneurysm and dissection (TAAD) significantly impact cardiovascular morbidity mortality. A large subset of TAAD cases, particularly those with an earlier onset, is linked to heritable genetic defects. Despite progress in characterizing genes associated both syndromic non-syndromic TAAD, the causative gene remains unknown most cases. Another important bottleneck correct timely diagnosis proportion variants significance (VUS) that are routinely encountered upon medical testing. Reliable functional modeling data required accurately identify new causal determine pathogenicity VUS. To address this gap, our collaborative effort-comprising teams from Yale University, University Kentucky, Ghent University-explores a novel approach: zebrafish. Leveraging unique advantages animal model promises allow for accelerated variant assessment, ultimately enhancing patient care. In review, we critically explore currently available zebrafish-based approaches can be used testing related offer outlook on implementation these strategies clinical applications.

Language: Английский

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Evaluation of Cas13d as a tool for genetic interaction mapping

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Feb. 14, 2025

Abstract Mapping genetic interactions (GIs) is crucial for understanding network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, GI mapping and compare it to Cas9 Cas12a, two DNA nucleases commonly used mapping. We find that Cas13d induces faster target gene perturbation generates more uniform cell populations with double perturbations than or Cas12a. then encounter gRNA-gRNA interference when concatenating gRNAs different genes into one gRNA array, which overcome by dual promoter expression strategy. Moreover, three same are able maximize Cas13d-mediated knockdown effects. Combining these strategies enhances proliferation phenotypes while reducing library size facilitates reproducible quantification GIs in oncogenic signaling pathways. Our study highlights potential mapping, promising advancements therapeutically relevant drug response

Language: Английский

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Reproductive Adaptation of Astyanax mexicanus Under Nutrient Limitation

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 17, 2025

Abstract Reproduction is a fundamental biological process for the survival and continuity of species. Examining changes in reproductive strategies offers valuable insights into how animals have adapted their life histories to different environments. Since reproduction one most energy-intensive processes female animals, nutrient scarcity expected interfere with ability invest gametes. Lately, new model study adaptation limitation has emerged; Mexican tetra Astyanax mexicanus . This fish species exists as two morphs, surface river morph cave-dwelling morph. The dark, biodiversity, nutrient-limited cave environment consequently evolved an impressive starvation resistance. However, limitations this remains poorly understood. Here, we compared breeding activities maternal contributions between laboratory-raised cavefish. We found that cavefish produce clutch sizes eggs larger yolk fish, indicating greater deposition embryos. To systematically characterize compositions, used untargeted proteomics lipidomics approaches analyze protein lipid profiles 2-cell stage embryos increased proportion sphingolipids fish. Additionally, generated transcriptomic ovaries using combination single cell bulk RNA sequencing examine differences contribution. genes essential hormone regulation were upregulated follicular somatic cells evaluate whether these contribute abilities under natural-occurring stress, induced starved Remarkably, maintained breed starvation, whereas largely lost ability. identified insulin-like growth factor 1a receptor ( igf1ra ) potential candidate gene mediating downregulation ovarian development genes, potentially contributing starvation-resistant fertility Taken together, investigated , which will provide adaptations environments extreme deficit.

Language: Английский

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Reproductive Adaptation of Astyanax mexicanus Under Nutrient Limitation

Developmental Biology, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

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Transposon insertion causes ctnnb2 transcript instability that results in the maternal effect zebrafish ichabod (ich) mutation

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: March 4, 2025

Abstract The maternal-effect mutation ichabod ( ich ) results in ventralized zebrafish embryos due to impaired induction of the dorsal canonical Wnt-signaling pathway. While previous studies linked phenotype reduced ctnnb2 transcript levels, causative remained unidentified. Using long-read sequencing, we discovered that stems from insertion a non-autonomous CMC-Enhancer/Suppressor-mutator (CMC-EnSpm) transposon 3’UTR gene. Through reporter assays, demonstrate while wild type mRNAs exhibit remarkably high stability throughout early stages development, dramatically reduces stability. Genome-wide mapping CMC-EnSpm transposons across multiple strains also indicated ongoing transposition activity genome. Our findings not only resolve molecular basis but highlight continuing mutagenic potential endogenous and reveal unexpected aspects maternal regulation during development.

Language: Английский

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Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: March 16, 2025

CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity mammalian cells mouse models. Moreover, competence could be improved vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations enhance the CRISPR-RfxCas13d system zebrafish. We i) use chemically modified gRNAs allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, iii) compare different computational models determine most accurate predict gRNA Furthermore, demonstrate that can effectively deplete endogenous mRNAs zebrafish embryos without inducing effects, except when targeting extremely abundant ectopic RNAs. Finally, implement alternative CRISPR-Cas such CRISPR-Cas7-11 CRISPR-DjCas13d. Altogether, these findings contribute technology optimization for through approaches assist progression of vivo applications. comprise an invaluable set tools fields Moreno-Sánchez, Hernández-Huertas, Nahón-Cano et al. enhanced targeted depletion mRNAs.

Language: Английский

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Critical role of Spatio-Temporally Regulated Maternal RNAs in Zebrafish Embryogenesis

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 8, 2024

Summary The maternal-to-zygotic transition shifts regulatory control from maternal to zygotic messenger RNAs (mRNA) through mRNA degradation. While temporal aspects of decay are known, spatial mechanisms remain underexplored. Using CRISPR-Cas9 and CRISPR-Cas13d systems, we functionally dissected the contribution versus fractions overcame challenges studying embryonic lethal genes. We identified differentially distributed mRNAs in specific cells evidenced critical role five mRNAs, cth1, arl4d, abi1b, foxa lhx1a, embryogenesis. Further, focused on uncharacterized cth1 gene, revealing its essential gametogenesis Cth1 acts as a spatio-temporal RNA factor regulating stability accumulation targets manner 3’UTR recognition during early development. Furthermore, drives localization. Our findings provide new insights into highlight dual CRISPR-Cas strategies Highlights Differentially marginal have Cas13d complements Cas9 limitation study functions is for required regulation. dynamics.

Language: Английский

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