Trans-splicing methods and compositions for generation of single sex offspring

A method of producing a single sex population of non-human vertebrate animals, the method comprising:

• obtaining (i) a first non-human vertebrate animal comprising one or more first sequence variants of an autosomal gene, and a modified allosome comprising one or more expression cassettes, wherein the one or more expression cassettes comprise the following elements:
• a nucleic acid encoding a Cas polypeptide (dCas) linked to an RNA Binding Protein (RBP);
• repRNA comprising an open reading frame encoding one or more transgenic proteins, a splice site, an intron with RBP-binding hairpins, and a polyadenylation signal;
• and
• guide RNA capable of directing sequence specific binding of one or more CRISPR RNA-guided complexes encoded by the one or more expression cassettes to one or more second sequence variants of the gene;
• obtaining (ii) a second non-human vertebrate animal comprising the one or more second variants of an autosomal gene;
• and
• crossing the first non-human vertebrate animal and the second non-human vertebrate animals, wherein a resulting progeny comprising the one or more second variants of a gene and the modified allosome expressing the one or more transgenic proteins is not viable;
• thereby creating a single sex population.

A non-human vertebrate animal having a modified genotype comprising:

• one or more nucleotide modifications in a sequence of an intron of a gene;
• and
• one or more expression cassettes comprising a nucleic acid encoding a Cas polypeptide linked to an RNA Binding Protein (RBP), repRNA comprising an open reading frame encoding a transgenic protein, a splice site, an intron with RBP-binding hairpins, and a polyadenylation signal, and guide RNA capable of directing sequence specific binding of one or more CRISPR RNA-guided complexes encoded by the one or more expression cassettes to one or more second sequence variants of the gene,
• wherein the one or more nucleotide modifications in the sequence of the intron cannot splice to the splice site, and
• wherein the intron of the gene and the one or more expression cassettes are located on a single allosome.

A method of producing a single sex population of non-human vertebrate animals, the method comprising:

• crossing (i) a first non-human vertebrate animal having a first genotype comprising one or more nucleotide modifications in a sequence of an intron of a gene;
• and one or more expression cassettes comprising a nucleic acid encoding a Cas polypeptide linked to an RNA Binding Protein (RBP), repRNA comprising an open reading frame encoding a transgenic protein, a splice site, an intron with RBP-binding hairpins, and a polyadenylation signal, and guide RNA capable of directing sequence specific binding of one or more CRISPR RNA-guided complexes encoded by the one or more expression cassettes to one or more second sequence variants of the gene wherein the one or more nucleotide modifications in the sequence of the intron cannot splice to the splice site, and wherein the intron of the gene and the one or more expression cassettes are located on a single allosome;
• with (ii) a second transgenic non-human vertebrate animal having a second genotype comprising a second sequence variant of the intron of the gene, wherein the second sequence variant of the intron of the gene is capable of splicing to the splice site and homozygous allosomes;
• wherein a resulting progeny having a genotype comprising the second sequence variant of the intron of the gene and the one or more expression cassettes is visually identifiable.