Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration
- Iowa State University, United States
- Mayo Clinic, United States
Abstract
The ability to regulate gene activity spatially and temporally is essential to investigate cell type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression, >99% reduction of endogenous gene expression, and recapitulated known indel loss of function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss of function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Table 2, Figure 4, Figure 4 - figure supplement 1, Figure 5, Figure 5 - figure supplement 2, Figure 6, Figure 6 - figure supplement 2, Figure 7, Figure 7 - figure supplement 1, Figure 7 - figure supplement 2, Figure 8, Figure 9
Article and author information
Author details
Funding
NIH Office of the Director (R24 OD 020166)
- Stephen C Ekker
- Karl J Clark
- Jeffrey J Essner
- Maura McGrail
CNPq Brazilian National Council for Scientific and Technological Development
- Maira P Almeida
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Use of zebrafish for research in this study was performed according to the Guidelines for Ethical Conduct in the Care and Use of Animals (APA, 1986), and carried out in accordance with Iowa State University Animal Care and Use Committee IACUC-18-279 and IACUC-20-058 approved protocols. All methods involving zebrafish were in compliance with the American Veterinary Medical Association (2020), ARRIVE (Percie du Sert et al., 2020) and NIH guidelines for the humane use of animals in research.
Copyright
© 2022, Liu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,785
- views
-
- 882
- downloads
-
- 19
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration
eLife 11:e71478.
https://doi.org/10.7554/eLife.71478
Further reading
-
- Developmental Biology
Wing dimorphism is a common phenomenon that plays key roles in the environmental adaptation of aphid; however, the signal transduction in response to environmental cues and the regulation mechanism related to this event remain unknown. Adenosine (A) to inosine (I) RNA editing is a post-transcriptional modification that extends transcriptome variety without altering the genome, playing essential roles in numerous biological and physiological processes. Here, we present a chromosome-level genome assembly of the rose-grain aphid Metopolophium dirhodum by using PacBio long HiFi reads and Hi-C technology. The final genome assembly for M. dirhodum is 447.8 Mb, with 98.50% of the assembled sequences anchored to nine chromosomes. The contig and scaffold N50 values are 7.82 and 37.54 Mb, respectively. A total of 18,003 protein-coding genes were predicted, of which 92.05% were functionally annotated. In addition, 11,678 A-to-I RNA-editing sites were systematically identified based on this assembled M. dirhodum genome, and two synonymous A-to-I RNA-editing sites on CYP18A1 were closely associated with transgenerational wing dimorphism induced by crowding. One of these A-to-I RNA-editing sites may prevent the binding of miR-3036-5p to CYP18A1, thus elevating CYP18A1 expression, decreasing 20E titer, and finally regulating the wing dimorphism of offspring. Meanwhile, crowding can also inhibit miR-3036-5p expression and further increase CYP18A1 abundance, resulting in winged offspring. These findings support that A-to-I RNA editing is a dynamic mechanism in the regulation of transgenerational wing dimorphism in aphids and would advance our understanding of the roles of RNA editing in environmental adaptability and phenotypic plasticity.
-
- Developmental Biology
The evolutionarily conserved Hippo (Hpo) pathway has been shown to impact early development and tumorigenesis by governing cell proliferation and apoptosis. However, its post-developmental roles are relatively unexplored. Here, we demonstrate its roles in post-mitotic cells by showing that defective Hpo signaling accelerates age-associated structural and functional decline of neurons in Caenorhabditis elegans. Loss of wts-1/LATS, the core kinase of the Hpo pathway, resulted in premature deformation of touch neurons and impaired touch responses in a yap-1/YAP-dependent manner, the downstream transcriptional co-activator of LATS. Decreased movement as well as microtubule destabilization by treatment with colchicine or disruption of microtubule-stabilizing genes alleviated the neuronal deformation of wts-1 mutants. Colchicine exerted neuroprotective effects even during normal aging. In addition, the deficiency of a microtubule-severing enzyme spas-1 also led to precocious structural deformation. These results consistently suggest that hyper-stabilized microtubules in both wts-1-deficient neurons and normally aged neurons are detrimental to the maintenance of neuronal structural integrity. In summary, Hpo pathway governs the structural and functional maintenance of differentiated neurons by modulating microtubule stability, raising the possibility that the microtubule stability of fully developed neurons could be a promising target to delay neuronal aging. Our study provides potential therapeutic approaches to combat age- or disease-related neurodegeneration.
