The Construction And Phenotypic Study Of A Novel Humanized ATXN3 Knock-in Mouse Model Using CRISPR/Cas9

Background:Spinocerebellar ataxia type 3/Machado-Joseph disease(SCA3/MJD)is the most common autosomal dominant ataxia caused by abnormal expansion of CAG repeats in the ATXN3 gene.The pathogenesis of the disease is complex,and there is no specific treatment.Mouse models that effectively mimic disease phenotypes are essential for studying disease pathogenesis and treatment.More than ten SCA3 mouse models have been constructed based on transgenic,gene targeting,and other technologies.However,there are still limitations,such as random integration of target genes and low efficiency of homologous recombination.CRISPR/Cas9-based gene-editing technology can accurately and efficiently realize the targeted knock-in of exogenous target genes through homologous recombination,which significantly improves the accuracy and safety of target gene integration and provides insights into the molecular mechanism and treatment of such diseases.Objective:By using CRISPR/Cas9 technology,we will construct a humanized ATXN3 knock-in(knock-in,KI)mouse model with 90-100 copies of CAG repeats.We will conduct phenotypic verification at the molecular level,behavioral phenotype,neuropathology,and neuroimaging to explore whether the model can better simulate the human pathogenic process to lay the foundation for subsequent research on the pathogenesis and treatment of SCA3.Methods:A plasmid carrying the coding region sequence of the human ATXN3 gene was constructed(the number of CAG repeats is 90-100 copies),and it was introduced into HEK293 T cells by transient transfection technology to verify whether the cells could express the ataxin-3 mutant protein.Using CRISPR/Cas9 technology,the donor vector,g RNA and Cas9 were microinjected into fertilized eggs and transplanted into pseudopregnant female mice to construct F0 generation first-born mice.The mice were genotyped by PCR,agarose electrophoresis,Sanger sequencing,and capillary electrophoresis.The expression of mutant ataxin-3 protein and poly Q protein in the brain,cerebellum,and brainstem of mice was detected by Western Blot.The ataxia-related motor function was seen in KI and WT mice from 2 months to 12 months old using behavioral methods such as rotarod tests,balance beam tests,grip force tests,footprint tests,and paw-clasping tests.The pathological changes of cerebellar Purkinje cells and the formation of inclusion bodies were observed by immunofluorescence,immunohistochemistry,and hematoxylin-eosin staining.The coronal and sagittal MRI images were obtained by 7.0 T animal magnetic resonance imaging.The volume changes of each brain region were analyzed by voxel-based morphometry(VBM).Results:A plasmid containing the coding region sequence of the full-length human ATXN3 gene(96 copies of CAG repeats)was successfully constructed.The transiently transfected 293 T cells could express the mutant ataxin-3 protein.Five new humanized ATXN3 knock-in mouse models based on CRISPR/Cas9 were successfully built.Sanger sequencing and capillary electrophoresis showed that the copy number of CAG repeats in KI mice was 96 times.Mutant ataxin-3 protein and mutant poly Q protein were detected in the brain,cerebellum,and brain stem of 1-month-old KI mice.Behavioral experiments showed that compared with WT mice of the same age,the time of 2-month-old KI mice passing through the balance beam was significantly increased(p<0.05);Rotarod residence time was significantly reduced in6-month-old KI mice(p<0.05);12-month-old KI mice had apparent footprint abnormalities and limb clasping responses.The results of hematoxylin-eosin staining and immunofluorescence showed that the number of Purkinje cells in 12-month-old KI mice was reduced,the morphology was abnormal,and the thickness of the cerebellar molecular layer was reduced(p<0.05).The cerebellum and brain of 2-month-old KI mice Early intranuclear inclusion bodies can be seen in the stem neurons,and the number of intranuclear inclusion bodies in the cerebellum and brainstem of 12-month-old KI mice was significantly increased.Imaging results showed that the whole brain of 12-month-old KI mice was atrophied,mainly decreased in cerebellum and pons,and was accompanied by enlargement of the fourth ventricle(p<0.05,FDR corrected).Conclusions:In this study,a novel humanized ATXN3 knock-in mouse model was successfully constructed for the first time based on CRISPR/Cas9 technology.The model can effectively simulate the phenotype of SCA3 patients at the molecular,behavioral,neuropathological,and imaging levels,which provides a more accurate and effective tool for the molecular mechanism exploration and treatment research of SCA3 in different disease stages.