Method for modulating RNA splicing by inducing base mutation at splice site or base substitution in polypyrimidine region

A method for regulating RNA splicing of a gene in a cell, comprising expressing a targeting cytosine deaminase and an sgRNA in the cell to induce mutation of 3′ splice site AG to AA of an intron of the gene in the cell, or mutation of 5′ splice site GT to AT of an intron of the gene in the cell, or mutation of multiple Cs to Ts in a polypyrimidine region upstream of 3′ splice site of an intron of the gene in the cell;

• wherein the targeting cytosine deaminase is selected from the group consisting of:
• (1) a fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and a Cas enzyme with helicase activity and partial or no nuclease activity;
• wherein partial nuclease activity refers to only having DNA single-strand break ability, and no nuclease activity refers to having no DNA double-strand break ability;
• and
• (2) a fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and a Cpf enzyme with helicase activity and partial or no nuclease activity;
• wherein partial nuclease activity refers to only having DNA single-strand break ability, and no nuclease activity refers to having no DNA double-strand break ability;
• and wherein the sgRNA binds to a sequence having the 5′ splice site of the intron of the gene, and the fusion protein mutates the GT to AT at the 5′ splice site of the intron of the gene, thereby inducing exon skipping, activating alternative splice sites, inducing mutually exclusive exon switching or intron retention;
• or
• the sgRNA binds to a sequence having the 3′ splice site of the intron of the gene, and the fusion protein mutates the AG to AA at the 3′ splice site of the intron of the gene, thereby inducing exon skipping, activating alternative splice sites, inducing mutually exclusive exon switching or intron retention;
• or
• the sgRNA binds to a complementary sequence of the polypyrimidine region of the intron of the gene, and induces the Cs to Ts at the polypyrimidine region of the intron of the gene, thereby enhancing the strength of the 3′ splice site and enhancing exon inclusion;
• and the method includes expressing the targeting cytosine deaminase and an sgRNA in the cell, wherein the sgRNA is specifically recognized by the Cas enzyme or Cpf enzyme and binds to the sequence having the 5′ splice site or the 3′ splice site of the intron of the gene, or binds to the complementary sequence of the polypyrimidine region of the intron of the gene.