CN-122004173-A - Targeting agent for preparing fragile X syndrome animals and application thereof

Abstract

The invention provides a targeting agent for preparing fragile X syndrome animals and application thereof. The invention discloses a target which is matched with a non-human primate and is suitable for genetic modification, namely a third exon and/or a fourth exon of an FMR1 gene, a targeting reagent is prepared by targeting the target, and an animal model of fragile X syndrome with typical disease symptoms can be obtained by performing down regulation.

Inventors

• XIONG ZHIQI
• SUN QIANG
• ZHUANG LING
• ZHANG RUNZE
• LI CHUNYANG
• Bian Xinyan
• ZHANG XIAOLEI
• KONG DELUN

Assignees

• 中国科学院脑科学与智能技术卓越创新中心

Dates

Publication Date

20260512

Application Date

20241112

Claims (11)

1. Use of an sgRNA that down-regulates an FMR1 gene of an animal, said down-regulation targeting a third exon and/or a fourth exon of an FMR1 gene, in the preparation of an animal with fragile X syndrome, said animal being a non-human primate.
2. 2. The use of claim 1, wherein the sgRNA comprises: sgRNA of target sequence SEQ ID NO:3 targeting the fourth exon of the FMR1 gene, and/or And targeting the sgRNA of SEQ ID NO 2 and/or SEQ ID NO 1 in the third exon of the FMR1 gene.
3. 3. The use according to claim 2, wherein the sgRNA comprises a sgRNA targeting the target sequence SEQ ID NO. 3 in the fourth exon of the FMR1 gene and a sgRNA targeting the target sequence SEQ ID NO. 2 in the third exon of the FMR1 gene.
4. 4. The use of claim 2, wherein the sgrnas comprise a sequence selected from the group consisting of: the nucleotide sequence of the sgRNA targeting the target sequence SEQ ID NO. 3 is shown as SEQ ID NO. 6; The sgRNA targeting the target sequence SEQ ID NO. 2 has a nucleotide sequence shown as SEQ ID NO. 5; the sgRNA targeting the target sequence SEQ ID NO. 1 has the nucleotide sequence shown as SEQ ID NO. 4.
5. 5. The method of claim 4, wherein the sgRNA and the Cas mRNA or the construct capable of forming the sgRNA and the Cas9 mRNA are introduced into fertilized eggs of an animal, and preferably further comprising developing the fertilized eggs to obtain the fragile X syndrome animal.
6. 6. The use according to claim 1, wherein the fragile X syndrome animal has a phenotype selected from the group consisting of increased total activity and repetitive notch activity, increased anxiety, presentation of language development disorder, reduced social willingness, memory disorder, reduced cognitive flexibility, attention deficit.
7. 7. The sgRNA for preparing fragile X syndrome animals, which targets the target sequence SEQ ID NO 3 in the fourth exon, SEQ ID NO 2 or SEQ ID NO 1 in the FMR1 gene of the animal.
8. 8. The sgRNA for use in the preparation of an animal with fragile X syndrome of claim 7, wherein the sgRNA is a combination of sgRNAs comprising the target sequence SEQ ID NO 3 targeted to the fourth exon in the FMR1 gene of the animal and the target sequence SEQ ID NO 2 targeted to the third exon in the FMR1 gene of the animal.
9. 9. The sgrnas for use in the preparation of fragile X syndrome animals of claim 8, wherein the target sequence is SEQ ID No. 3, the nucleotide sequence is SEQ ID No. 6, the target sequence is SEQ ID No. 2, the nucleotide sequence is SEQ ID No. 5, or the target sequence is SEQ ID No. 1, the nucleotide sequence is SEQ ID No. 4.
10. 10. Kit for preparing an animal with fragile X syndrome, comprising the sgRNA for preparing an animal with fragile X syndrome according to any one of claims 7 to 10.
11. 11. The kit of claim 10, further comprising a Cas mRNA or a construct capable of forming a Cas mRNA.

Description

Targeting agent for preparing fragile X syndrome animals and application thereof Technical Field The invention belongs to the fields of medicine and biology, and more particularly relates to a fragile X syndrome animal model, and construction and application thereof. Background Fragile X syndrome (Fragile X Syndrome, FXS) is a hereditary intellectual disorder disease with a incidence next to down syndrome, and is also the most common type of autism spectrum disorder, with a male incidence of 1/2500-1/5000 and a female incidence of 1/4000-1/6000.FXS patients have various clinical manifestations, mainly representing moderate to severe mental disorder, and often have IQ values of 20-60, and FXS patients also have abnormal appearance characteristics, such as forehead protrusion, large and protruding mandibular, large ears, high palate arch, thick lips, protruding lower lips, etc., and male males have large testes in puberty. Some patients exhibit behavioral disorders such as hyperactivity, autism (e.g., aggressive, social and pragmatic behaviors, etc.), and 20% have sporadic seizures. FXS is caused by mutation of FMR1 gene, the gene is located on an X chromosome q27.3, CGG repeated sequences exist in a 5 '-untranslated region (5' -UTR) of FMR1 gene, the number of CGG trinucleotide repeated sequences of normal FMR1 gene is generally between 6 and 54, the number of CGG trinucleotide repeated sequences of individuals with pre-mutation is 55-200, the copy number of CGG of women carrying the pre-mutation is unstable, and the CGG is easy to be converted into full mutation in the process of transferring to offspring. When the number of CGG trinucleotide repeats exceeds 200, referred to as total mutation, an epigenetic change occurs in the FMR1 gene, and the CGG repeats and the promoter of FMR1 are methylated, resulting in silencing of FMR1 gene expression. Over 99% of patients are caused by additional expansion (> 200 copies) of CGG trinucleotide repeats in the 5' -UTR of FMR1 gene, the remaining 1% being due to FMR1 gene point mutations and gene duplication/deletion. The FMRP protein coded by the FMR1 gene is expressed in various tissues of mammals, is particularly rich in brain and testis, and is expressed in brain neurons and various glial cells. FMRP is an RNA-binding protein whose expression can be regulated by binding to mRNA, and whose selectivity for RNA targets is largely dependent on two central KH domains and a C-terminal arginine-rich glycine domain. It is currently believed that FMRP is primarily responsible for the negative regulation of neuronal local protein expression, most of which are associated with dendritic growth and receptor signaling pathways, and that these regulated proteins are increased in expression following the absence of FMRP. At present, the specific mechanism of FXS caused by FMRP deficiency is not clear, and no specific medicine for treating FXS is clinically available, mainly for symptomatic treatment and supportive treatment. Animal models are an important basis for human disease mechanism research and new drug development. The current FXS animal models are drosophila model, zebra fish model, mouse model and rat model. But these animals also show great limitations in mimicking FXS disease. The drosophila homology gene of the FMR1 gene is Dfmr.sup.1. DFMRP-deleted Drosophila showed several FXS patients Associated phenotypes, including sleep problems, memory deficits, social interaction deficits, and neurodevelopment deficits. But they did not exhibit increased activity and anxiety phenotypes nor typical repetitive plating. The zebra fish model also exhibited some phenotypes in some FXS patients, including behavioral characteristics such as anxiety and hyperactivity, which were exhibited when Fmr knockout zebra fish were transferred to new fish tanks. Craniofacial dysplasia was also observed in Fmr knocked-out zebra fish larvae. But it does not exhibit cognitive impairment and social behavioral abnormalities nor typical repetitive plating behavior. In addition, the absorption and metabolism modes of the drug of the drosophila and the zebra fish are very different from those of human beings, so that the application of the drosophila and the zebra fish in FXS drug development has very large limitation. The Fmr knockout mouse model is the most used FXS animal model, the Fmr knockout mouse presents phenotypes of some FXS patients, has abnormal development of neuron dendritic spines and large testes, the Fmr knockout mouse also has abnormal behaviors such as overactivity, auditory epileptic seizure (not only sporadic seizure) and the like, but does not reach the typical repeated notch behavior. Furthermore, the autism-like phenotype common in FXS patients was not observed in mice of a few strains, the mouse model also lacked the most typical cognitive impairment in FXS patients, and the manifestation of social behavior (e.g. social impairment) was also lacked. Another non-negligible prob