Constructs and methods for genome editing and genetic engineering of fungi and protists
AssigneeWisconsin Alumni Research Foundation
InventorsChristopher Todd Hittinger, William Gerald Alexander
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Abstract
Provided herein are constructs for genome editing or genetic engineering its fungi or protists, methods of using the constructs and media for use in selecting cells. The construct include a polynucleotide encoding a thymidine kinase operably connected to a promoter, suitably a constitutive promoter; a polynucleotide encoding an endonuclease operably connected to an inducible promoter; and a recognition site for the endonuclease. The constructs may also include selectable markers for use in selecting recombinations.
Claims11
A single construct for simultaneously editing a target allele on both chromosomes in diploid fungi or protists comprising:
- a first polynucleotide encoding a thymidine kinase operably connected to a first promoter;
- a second polynucleotide encoding a 5′ portion of a first selectable marker operably connected to a second promoter;
- a third polynucleotide encoding a second selectable marker operably connected to a third promoter;
- and a fourth polynucleotide encoding a 3′ portion of the first selectable marker, wherein the second polynucleotide and the fourth polynucleotide encoding the 5′ portion and the 3′ portion of the first selectable marker contain a repeated polynucleotide sequence to allow for recombination, and wherein the third polynucleotide is positioned between the second polynucleotide and the fourth polynucleotide in the construct.
Dependencies are inferred from the claim text.