| CPC C12N 15/85 (2013.01) [C12N 9/22 (2013.01); C12N 15/907 (2013.01); C12N 2310/20 (2017.05); C12N 2800/40 (2013.01); C12N 2800/50 (2013.01); C12N 2810/10 (2013.01)] | 11 Claims |
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1. A two-vector composition comprising:
a first vector comprising:
a first sequence encoding a first component of a DNA sequence modifying complex, wherein the DNA sequence modifying complex induces one or more sequence modifications in an endogenous copy of an essential gene, wherein the first component of the DNA sequence modifying complex is a nuclease, wherein the nuclease is Cas9 nuclease,
a first promoter operably linked to the first sequence encoding the first component of the DNA sequence modifying complex, wherein the first promoter comprises at least one of a germline promoter, a male specific germline promoter, a female specific germline promoter, a cell-type specific promoter, a tissue-specific promoter, a ubiquitous promoter, a promoter activated at a specific stage of mitosis, a promoter activated at a specific stage of meiosis, a viral promoter or prokaryotic promoter;
a rescue transgene sequence;
a rescue transgene promoter operably linked to the rescue transgene sequence, wherein the rescue transgene promoter comprises at least one of a endogenous promoter for the essential gene, germline promoter, a male specific germline promoter, a female specific germline promoter, a cell-type specific promoter, a tissue-specific promoter, a ubiquitous promoter, a promoter activated at a specific stage of mitosis, a promoter activated at a specific stage of meiosis, a viral promoter or prokaryotic promoter; and
further comprising one or more cargo sequences;
a second vector comprising:
a second sequence encoding a second component of the DNA sequence modifying complex, wherein the second component of the DNA sequence modifying complex is Cas9 or a guide RNA, wherein the guide RNA enables the Cas9 nuclease to target specific sequences within the essential gene;
a second promoter operably linked to the second component of the DNA sequence modifying complex, wherein the second promoter comprises at least one of a germline promoter, a male specific germline promoter, a female specific germline promoter, a cell-type specific promoter, a tissue-specific promoter, a ubiquitous promoter, a promoter activated at a specific stage of mitosis, a promoter activated at a specific stage of meiosis, a viral promoter or prokaryotic promoter,
wherein the two-vector composition is configured for reversibly modifying a population of organisms,
wherein the one or more sequence modifications comprise cleavage of the essential gene resulting in the essential gene being rendered partially or wholly non-functional and resulting in a defect in survival, growth control, fertility, or differentiation in absence of the rescue transgene,
wherein a rescue of the defect occurs by the rescue transgene being positioned in any chromosomal or extrachromosomal element that is different from the location of the endogenous copy of the essential gene,
wherein the rescue transgene is either a recoded copy of the essential gene or is a gene of unrelated sequence, wherein the rescue transgene encodes a protein that is functionally equivalent to a protein encoded by the essential gene, and wherein the DNA sequence modifying enzyme does not modify the rescue transgene, and
wherein the one or more additional sequences that allow the vectors of the two-vector composition to be positioned in a chromosome or an extra-chromosomal element that is different from the location of the endogenous copy of the essential gene comprise sequences that are not homologous to sequences flanking the endogenous copy of the essential gene.
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