CRISPR-Cas9-induced gene knockout in zebrafish

Medishetti, Raghavender; Balamurugan, Keerthana; Yadavalli, Krishnaveni; Rani, Rita; Sevilimedu, Aarti; Challa, Anil K.; Parsa, Kishore V. L.; Chatti, Kiranam

doi:10.1016/j.xpro.2022.101779

Cited by 10 publications

(9 citation statements)

References 8 publications

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“…We used the standard CRISPR workflow to generate fmr1 knockout zebrafish lines (Medishetti et al, 2022). Single guide RNAs were designed to target early exons (3 and 4) of zebrafish fmr1 , in order to maximise the likelihood of complete loss of function (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Impaired ovarian development in a zebrafishfmr1knockout model

Rani,

Sri,

Medishetti

et al. 2024

Preprint

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Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder and the leading genetic cause of autism spectrum disorders. FXS is caused by loss of function mutations in Fragile X mental retardation protein (FMRP), an RNA binding protein that is known to regulate translation of its target mRNAs, predominantly in the brain and gonads. The molecular mechanisms connecting FMRP function to neurodevelopmental phenotypes are well understood. However, neither the full extent of reproductive phenotypes, nor the underlying molecular mechanisms have been as yet determined. Here, we developed newfmr1knockout zebrafish lines and show that they mimic key aspects of FXS neuronal phenotypes across both larval and adult stages. Results from thefmr1knockout females also showed that altered gene expression in the brain, via the neuroendocrine pathway contribute to distinct abnormal phenotypes during ovarian development and oocyte maturation. We identified at least three mechanisms underpinning these defects, including altered neuroendocrine signaling in sexually mature females resulting in accelerated ovarian development, altered expression of germ cell and meiosis promoting genes at various stages during oocyte maturation, and finally a strong mitochondrial impairment in late stage oocytes from knockout females. Our findings have implications beyond FXS in the study of reproductive function and female infertility. Dissection of the translation control pathways during ovarian development using models like the knockout lines reported here may reveal novel approaches and targets for fertility treatments.Abstract FigureGraphical Abstract

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Section: Resultsmentioning

confidence: 99%

“…CRISPR/Cas9 mediated knockout generation was performed as described previously (Medishetti et al, 2022). Briefly, one cell stage zebrafish embryos were injected with 5nl of Cas9-sgRNA mix (400ng of Cas9 protein and 0.2-0.5µg of sgRNA in 5µl of 10mM Tris pH 8.0).…”

Section: Microinjection and Generation Of Fmr Knockout Linesmentioning

confidence: 99%

Impaired ovarian development in a zebrafishfmr1knockout model

Rani,

Sri,

Medishetti

et al. 2024

Preprint

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“…As such, we expect this protocol to work for generating genetically modified fish lines using CRISPR (Jao et al, 2013, Medishetti et al, 2022, Mi and Andersson, 2023, or integrase-based methods (Lalonde et al, 2023). In the near future we plan to update our preprint to demonstrate some of these use cases.…”

Section: Discussionmentioning

confidence: 99%

“…Gene delivery methods in zebrafish can generate transient products in cells or lead to alterations in genomes that persist through generations. Common applications of gene delivery include the expression of transient payloads, gene knock-down, -out or -in, and transposon or integrase-mediated genome integration of transgenic constructs (Auer et al, 2014, Jao et al, 2013, Kwan et al, 2007, Medishetti et al, 2022, Mi and Andersson, 2023, Lalonde et al, 2023.…”

Section: Introductionmentioning

confidence: 99%

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery in Zebrafish

Terzi,

Lao,

Jacobo

2024

Preprint

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Zebrafish, a widely used model organism in developmental and biomedical research, offers several advantages such as external fertilization, embryonic transparency, and genetic similarity to humans. However, traditional methods for introducing exogenous genetic material into zebrafish embryos, particularly microinjection, pose significant technical challenges and limit throughput. To address this, we developed a novel approach utilizing Lipofectamine ® LTX for the efficient delivery of nucleic acids into zebrafish embryos by lipid-based transfection. Our protocol bypasses the need for microinjection, offering a cost-effective, high-throughput, and user-friendly alternative. This protocol outlines new strategies for gene delivery in zebrafish to enhance the efficiency and scope of genetic studies in this model system.

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“…sgRNAs were synthesized, as described in the studies by Medishetti et al and Sorlien. 18,19 Briefly, templates for sgRNA3 and sgRNA4 were obtained by carrying out overlap PCR using T7 promoter forward primer and tail oligo reverse primer. The product was ethanol precipitated and in vitro transcribed using HiScribe™ T7 RNA synthesis kit (E2040S, New England Biolabs, Massachusetts, USA) to get the individual sgRNAs.…”

Section: Methodsmentioning

confidence: 99%

Biallelic Variants inCCN2Underlie an Autosomal Recessive Kyphomelic Dysplasia

Singh,

Danda,

Sharma

et al. 2024

Preprint

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Kyphomelic dysplasia, characterized by bowing of the limbs severely affecting femora, has eluded a clear molecular etiology for nearly four decades. We ascertained two unrelated consanguineous families with kyphomelic dysplasia. We performed clinical and radiographic evaluation followed by exome sequencing in three probands. Impact of CCN2 knockout was studied in zebrafish models via CRISPR-Cas9 gene editing. All the probands had short stature, cleft palate, micro-retrognathia. Kyphomelic femora, bowing of long bones, radial head dislocations and spondyloepimetaphyseal dysplasia. We noted two novel biallelic (homozygous) variants in CCN2 as possible candidates: a missense variant c.443G>A; p.(Cys148Tyr) in exon 31 and a frameshift variant, c.779_786del; p.(Pro260LeufsTer7) in exon 5. CCN2 is crucial for proliferation and differentiation of chondrocytes. Earlier studies have shown that CCN2-deficient mice exhibit twisted limbs, short and kinked sterna, broad vertebrae, domed cranial vault, shorter mandibles, cleft palate and impaired osteoclastogenesis. We generated F0 knockouts of ccn2a in zebrafish, which showed altered body curvature, impaired cartilage formation in craniofacial region and either bent or missing tails recapitulating the human phenotype. Our observations confirm biallelic loss of function variants in CCN2 result in an autosomal recessive kyphomelic dysplasia, for the first time.

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CRISPR-Cas9-induced gene knockout in zebrafish

Cited by 10 publications

References 8 publications

Impaired ovarian development in a zebrafishfmr1knockout model

Impaired ovarian development in a zebrafishfmr1knockout model

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery in Zebrafish

Biallelic Variants inCCN2Underlie an Autosomal Recessive Kyphomelic Dysplasia

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