A model organism for tubulinopathy causing cortical developmental malformation

Abstract

The invention relates to the modelling of a disorder called tubulinopathy, which refers to a wide range of cortical developmental malformations (CDM) resulting from defects in genes encoding tubulin proteins that regulate neuronal migration during brain development, by knocking out the tubulin gamma 1 (Tubg1) gene in zebrafish (Danio rerio) embryos using CRISPR / Cas9 technology. The transgenic animal in question allows the investigation of the phenotype and molecular pathological mechanisms of CDMs resulting from the deficiency of the Tubg1 protein during the developmental process. In the invention, the region located in the third exon of the Tubg1 gene in zebrafish is targeted and knocked out with a guide RNA having the nucleotide sequence SEQ ID NO: 1.

Description

[0001] A MODEL ORGANISM FOR TUBULINOPATHY CAUSING CORTICAL DEVELOPMENTAL MALFORMATION

[0002] Technical Area

[0003] The invention relates to the modeling of a disorder called tubulinopathy, which refers to a wide range of cortical developmental malformations (CDM) resulting from defects in genes encoding tubulin proteins that regulate neuronal migration during brain development, by knocking out the tubulin gamma 1 (Tubgl) gene in zebrafish (Danio rerio) embryos using CRISPR / Cas9 technology. The transgenic animal allows the phenotype and molecular pathological mechanisms of CDMs resulting from the deficiency of the Tubgl protein to be investigated during the developmental process.

[0004] State of the Art

[0005] Tubulin gamma 1 (Tubgl is the gene that encodes the y-Tubulin protein, one of the most basic microtubule elements that form the cytoskeleton and play an important role in many basic cellular processes such as cell division, cell movement, intracellular transport, and cell shape

[0001] . Microtubules are microscopic hollow tubes made of beta and alpha tubulin proteins, a network of protein filaments that are part of a cell's cytoskeleton, giving the cell its shape, extending throughout the cell, and holding its organelles in place. y-Tubulin is a tubulin superfamily protein that is structurally conserved among eukaryotes and is involved in the nucleation and polymerization of microtubules. y-Tubulin, unlike a- and [3-tubulins that polymerize to form microtubules, is not directly involved in microtubule structure, but interacts with several proteins in the cytoplasm and participates in the structure of protein complexes such as y-Tubulin small complex (y-Tusc) and y-Tubulin ring complex (y-Turc) formed for the polymerization of a- and [3-tubulin dimers, ensuring the stability of these complexes [2], In eukaryotes, y-Tubulin is encoded by the genes Tubgl and Tubg2. Tubgl, which plays a more critical role in microtubule nucleation during and after organism development, affects microtubule-dependent cellular events such as mitosis and cell migration [3].

[0006] The organization and nucleation of microtubules are particularly necessary for the proper differentiation and migration of neurons [4], Mutations in the Tubgl gene prevent microtubules from forming and functioning properly. This disrupts the ability of neurons to migrate to their correct locations, leading to neurodevelopmental disorders. Therefore, mutations in the Tubgl gene can lead to a series of cortical developmental malformations (CDMs), such as lissencephaly, microcephaly, and pachygyria. Cortical developmental malformations (CDMs) are rare central nervous system anomalies that occur due to disorders in the development, migration, and settlement of neuronal cells. During normal brain development, neurons migrate from one brain layer to specific brain regions in the correct order. While a healthy brain has numerous folds (sulci and gyri), in lissencephaly, where abnormalities are observed in the development of the folds in the brain, the brain is almost flat, and the brain folds are much fewer and less defined than in a healthy brain [5]. This occurs because microtubules cannot polymerize completely in the absence of y-Tubulin and neurons cannot migrate to the correct regions [6].

[0007] The complex structure of the multilayered cerebral cortex is determined in three important steps: the development of neurons from stem cells, the migration of new neurons to appropriate positions, the differentiation of neurons, and the establishment of axonal connections between neurons [7], Considering that microtubules are critical in these steps, mutations in genes encoding members of the tubulin superfamily have been associated with various CDMs [8]. These CDMs are defined as tubulinopathies encompassing a variety of clinical and phenotypes resulting from mutations in tubulin superfamily genes such as Tubala, Tubb2a, Tubb2b, Tubb3, Tubgcp2, and Tubgl. Variants of these genes, such as microcephaly, lissencephaly, and pachygyria, have been associated with variants of these genes [9]. Variations in the Tubgl gene have been associated with CDMs in patients

[0010] . In vitro and in vivo studies modeling Tubgl variations reveal that Tubgl mutations impair neuronal cell migration and microtubule dynamics

[0011] .

[0008] Zebrafish (Danio rerio) is a powerful vertebrate model for studying the functional roles of genes in development due to its many advantages such as external fertilization, giving birth to many offspring at a time, transparent embryonic development, being suitable for forward and reverse genetic applications, showing high genomic similarity to humans, and having a CNS similar to the mammalian central nervous system (CNS)

[0012] , The zebrafish Tubgl gene, which is the ortholog of the human TUBG1 gene, encodes the Tubgl protein. In the current state of the art, in vitro models are not suitable for investigating the molecular mechanisms of the disease, cell-cell and cell-tissue interactions, because they contain a single cell type. In vivo models, on the other hand, contain missense mutations of the Tubgl gene and do not represent the knockout situation in which the protein, the gene product, is largely or completely eliminated. On the other hand, homozygous Tubgl- / - knockout mice die during early embryonic development, while heterozygous Tubgl + / - mice develop normally and show no histological defects

[0013] . Therefore, it is not possible to examine tubulinopathy-related CDMs resulting from Tubgl gene knockout during neurodevelopment and / or to test therapeutic approaches with current animal models.

[0009] In another known case of the technique, the developmental period of the current animals used as models in the study of mutations in the Tubgl gene is long and the maintenance costs are high. In this case, the time loss and high costs experienced during the research process during neurodevelopment of tubulinopathy-related CDMs resulting from the knockout of the Tubgl gene do not prevent.

[0010] Due to the limitations and inadequacies of the current technical solutions, the impossibility of examining tubulinopathy-associated CDMs resulting from Tubgl gene knockout during neurodevelopment and / or testing therapeutic approaches with current animal models, the time loss and high costs experienced during the research process, it has become necessary to develop the model organisms used to investigate CDMs.

[0011] Brief Description and Objectives of the Invention

[0012] The invention relates to the modelling of a disorder called tubulinopathy, which refers to a broad spectrum of cortical developmental malformations (CDM) resulting from defects in genes encoding tubulin proteins that regulate neuronal migration during brain development, by knocking out the tubulin gamma 1 (tubgl) gene in zebrafish (Danio rerio) embryos using CRISPR / Cas9 technology.

[0013] The aim of the invention is to create a transgenic animal model that allows detailed examination of the molecular mechanisms and pathophysiology of tubulinopathy- associated CDMs and neurodevelopmental disorders. In the modeling subject to the invention, the animal model in question is created by knocking out the tubulin gamma 1 (tubgl gene in zebrafish (Danio rerio) embryos using CRISPR / Cas9 technology. Another aim of the invention is to allow the characterization of CDM phenotypes at cellular, molecular and behavioral levels. The invention is characterized based on the zebrafish embryo / larva model, which allows obtaining mosaic knockouts in the FO generation. Thanks to this method, knockout embryos and larvae can be monitored for 5 days and characterized in detail.

[0014] Explanation of Figures

[0015] Figure 1. Microscopic image of zebrafish Tubgl GO KO larvae in which the Tubgl gene was knocked out with the CRISPR / Cas9 system, on day 5 after fertilization.

[0016] Figure 2. Expressions of genes whose mutations were associated with lissencephaly and microcephaly in zebrafish Tubgl GO larvae in which the Tubgl gene was knocked out with the CRISPR / Cas9 system, at 5 dpf: (A: Tubgl gene, B: Tubala gene, C: Lamb 1b gene, D: Kif5c gene, ***: p< 0.001 )

[0017] Figure 3. Expressions of neuronal differentiation markers in Tubgl GO-KO zebrafish larvae in which the Tubgl gene was knocked out by the CRISPR / Cas9 system at 5 dpf: E1 : Atohla gene, E2: NeuroDI gene, E3: Elavl3 gene, ns: nonsignificant, **: p<0 01 ***■ p<0.001 ).

[0018] Figure 4. Expressions of pro-apoptotic genes in Tubgl KO zebrafish larvae in which the Tubgl gene was knocked out by the CRISPR / Cas9 system at 5 dpf (F: baxa gene, G: bcl2like11 gene, H: bcl2a gene, I: mcl1a gene, ns: nonsignificant, *:p<0.05, **: p<0.01 , ***:p<0.001 ).

[0019] Detailed Description of the Invention

[0020] The invention concerns the modeling of a disorder called tubulopathy, which expresses a wide range of cortical developmental malformations (CDM) caused by defects in genes encoding tubulin proteins that regulate neuronal migration during brain development, by knocking out the tubulin gamma 1 (Tubgl) gene in zebrafish (Danio rerio) embryos using CRISPR / Cas9 technology. The transgenic animal in question allows the investigation of the phenotype and molecular pathological mechanisms of CDMs caused by the deficiency of the Tubgl protein in the development process. The invention concerns the knockout of the Tubgl gene, one of the genes encoding tubulin proteins, whose mutations are associated with CDMs. The knockout animal model is created in zebrafish embryos using CRISPR / Cas9 technology. This model accommodates neurodevelopmental disorders caused by the deterioration of microtubule structures. These include reduced head and eyes, alteration in the expression of genes associated with lissencephaly, microcephaly, and pachygiria, decreased return of genes associated with neuronal differentiation, and increased apoptotic cell death.

[0021] In the invention, by using zebrafish, the Tubgl gene is knocked out faster, more cost- effectively and with a high success rate compared to rodent models.

[0022] In the invention, the region located in the third exon of the Tubgl gene in zebrafish is targeted and knocked out with the guide RNA having the nucleotide sequence of SEQ ID NO: 1

[0014] , In order to target the zebrafish Tubgl gene, the target sequence having the nucleotide sequence of SEQ ID NO: 2 of the Tubgl gene is selected using the cloud-based computing platform. The GRCzl 1 genome data set is selected for target determination and guanine nucleotide is added to the 5' part of the target sequence to increase in vitro transcription efficiency. The template is amplified by PCR using high- fidelity DNA polymerase with the forward primer having the nucleotide sequence of SEQ ID NO: 3 containing the T7 promoter and the targeted guide RNA sequence and the reverse primer having the nucleotide sequence of SEQ ID NO: 4 encoding the standard chimeric guide RNA scaffold. Scrambled guide RNA is used as a control at this stage. After purification of the DNA template, guide RNAs are synthesized in vitro using the HiScribe™ T7 Quick High Yield RNA synthesis kit. After purification of transcription products, RNA quantity is determined with NanoDrop.

[0023] For the ribonucleoprotein (RNP) complex, guide RNA (1000 ng / pL) and recombinant Guide-it Cas9 protein (500 ng / pL) are mixed and incubated at room temperature for 5 min. 0.05% phenol red is added to the RNP complex mixture for easy monitoring of the injection, and 1 -1.5 nL of the mixture is microinjected directly into the cytoplasm of zebrafish embryos. In Figure 1 , it is observed that the head and eyes are smaller compared to the control in zebrafish Tubgl GO KO larvae in which the Tubgl gene was knocked out with the CRISPR / Cas9 system on the 5th day after fertilization (5 dpf).

[0024] In Figure 2, in zebrafish Tubgl GO larvae in which the Tubgl gene was knocked out with the CRISPR / Cas9 system, the expressions of genes whose mutations are associated with lissencephaly and microcephaly are reduced compared to the control at 5 dpf.

[0025] In Figure 3, in Tubgl GO KO zebrafish larvae in which the Tubgl gene was knocked out with the CRISPR / Cas9 system, the expressions of neuronal differentiation markers are reduced compared to the control at 5 dpf.

[0026] In Figure 4, in Tubgl KO zebrafish larvae in which the Tubgl gene was knocked out with the CRISPR / Cas9 system, the expression of pro-apoptotic genes at 5 dpf is increased compared to the control.

[0027] Following microinjection of the RNP complex, embryos were monitored daily under a stereomicroscope, and at 5 dpf, most embryos showed a significant phenotypic difference compared to the control. Genomic DNA was then isolated from individual larvae at 5 dpf, and Sanger sequencing was performed to identify insertions and deletions (indels). According to Sanger sequencing, indels in the Tubgl gene were confirmed with approximately 85% efficiency. Further characterization was performed using stereomicroscope observation, quantitative real-time PCR (qPCR), and whole tissue in situ hybridization to detect mRNA expression of brain marker genes in the Tubgl gene knockout zebrafish to examine brain development.

[0028] The invention is based on a zebrafish embryo / larva model that allows obtaining mosaic knockout in the FO generation, allowing the resulting CDM phenotypes to be characterized at cellular, molecular, and behavioral levels. The phenotypic effect appears from day 3 of development and reaches its most effective state on day 5. From that day on, the larvae slowly lose their lives.

[0029] These results reveal a model that allows the monitoring of knockout embryos and larvae for 5 days, their detailed characterization, and studies on the diagnosis and treatment of CDM.

[0030] In Tubgl knockout zebrafish larvae, where the Tubgl gene was knocked out with the CRISPR / Cas9 system, it was found that the head and eyes were much smaller at 5 dpf compared to the control group, the expressions of genes whose mutations were associated with lissencephaly and microcephaly were reduced, the expressions of neuronal differentiation markers were reduced, and apoptotic cell death was increased.

[0031] The method of producing a zebrafish with a Tubulin gamma 1 (Tubgl) gene knockout, which is the subject of the invention, includes the following steps: i. determining the target sequence of the Tubgl gene in zebrafish, ii. forming the ribonucleoprotein (RNP) complex with guide RNA and Cas9, iii. microinjection of the ribonucleoprotein (RNP) complex into the cytoplasm of zebrafish embryos.

[0032] Transgenic zebrafish produced in this way can be used as an in vivo model organism to investigate the molecular mechanisms of CDMs such as lissencephaly and pachygyria, cell-cell and cell-tissue interactions, and to test potential therapeutic approaches and candidate chemicals. In particular, the fact that it can be used in drug discovery studies creates significant commercial value for biotechnology companies using pharmaceutical technologies. REFERENCES

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[0011] Poirier, K., Lebrun, N., Broix, L., Tian, G., Saillour, Y., Boscheron, C., Chelly, J. 2013. "Mutations in TUBG1 , DYNC1 H1 , KIF5C and KIF2A cause malformations of cortical development and microcephaly". Nature genetics, 45(6), 639-647.

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Claims

CLAIMS1. A transgenic zebrafish (Danio rerio , characterized by the knockout of the Tubulin gamma 1 (TubgT) gene.

2. A transgenic zebrafish (Danio rerio) according to claim 1 , to be used as a model organism for tubulinopathy disease causing cortical developmental malformation (CDM).

3. A production method of a transgenic zebrafish according to claim 1 or 2, characterized by the following steps: i. determining the target sequence of the Tubgl gene in zebrafish, ii. forming the ribonucleoprotein (RNP) complex with guide RNA and Cas9, iii. microinjection of the ribonucleoprotein (RNP) complex into the cytoplasm of zebrafish embryos,4. A method according to claim 3, characterized by the following steps: said target sequence has the nucleotide sequence of SEQ ID NO:2.

5. A method according to claim 3, characterized in that the said guide RNA has the nucleotide sequence SEQ ID NO: 1 .

6. A method according to claim 4, characterized in that the said target sequence with the nucleotide sequence SEQ ID NO: 2 is located in exon 3.

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