A method for targeted knockout of extended mutant polyq sequences in the ATXN3 gene

Abstract

The invention discloses a method for targeting knockout of an extended mutant type polyQ sequence in an ATXN3 gene; a plasmid containing a first guide RNA and a Cas9 nuclease expression gene and a plasmid containing a second guide RNA and a Cas9 nuclease expression gene are transfected into a cell containing a polyQ expression sequence, wherein the first guide RNA can be targetedly combined to a following structure contained in a 9th intron of the ATXN3 gene by base complementary pairing: 5'-GNx-NGG-3'; the second guide RNA can be targetedly binded to a following structure contained in a 10thexon of the ATXN3 gene by base complementary pairing: 5'-GNx-NGG-3'; N represents any one of A, T, C or G; x is 19 or 20. The method completes the knockout of the expanded mutant type polyQ sequence at one time, has stable effect, and does not destroy functional fragments of the ATXN3 gene.

Description

technical field [0001] The invention relates to the technical field of gene modification, in particular to a method for targeted knockout of extended mutant polyQ sequence in ATXN3 gene. Background technique [0002] Polyglutamine (polyQ) diseases are a group of common neurodegenerative diseases, which are caused by abnormal expansion of CAG repeats in the coding regions of their respective pathogenic genes and encoding polyglutamine peptide chains. At present, it has been found that it includes Huntington's disease, spinobulbar muscular atrophy, dentate nucleus rubrum pallidus subthalamus atrophy and spinocerebellar ataxia type 1 (spinocerebellar ataxia, SCA1), SCA2, SCA3, SCA6, SCA7 and SCA17 9 polyglutamine disorders. Most patients with PolyQ diseases have adult onset, mainly manifested as progressive nervous system dysfunction. Except for spinal bulbar muscular atrophy, the age of onset and severity of the other 8 PolyQ diseases are related to the length of CAG repeat e...

AI Technical Summary

Problems solved by technology

For example, techniques such as RNA interference and exon skipping are used to reduce the expression of the diseased ATXN3 gene, thereby alleviating the disease phenotype, but these methods have limitations such as low specificity and unstable expression, etc.

There are also traditional techniques that use antisense oligonucleotide chains to silence excessively grown polyQ sequences or exons skip exon 10 containing polyQ sequences, thereby achieving the purpose of alleviating disease phenotypes in cell or animal models, but these The method has the following problems: the target sequence is not high enough, and it is easy to cause non-specific interference to the target sequence in the non-ATXN3 gene; the interference effect is not long-lasting, and a certain interference effect needs to be achieved with an appropriate concentration and frequency

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