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 in 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.
Claims20
A method of genome editing, allele replacement or genetic engineering of a fungus or protist, the method comprising:
- generating or obtaining a construct, the construct comprising a first polynucleotide encoding a thymidine kinase operably connected to a first promoter;
- a second polynucleotide encoding an endonuclease operably connected to a second promoter;
- and a third polynucleotide comprising a recognition site for the endonuclease, wherein the second promoter is an inducible promoter;
- introducing the construct into the fungus or protist cells;
- and
- selecting for thymidine kinase by growing the fungus or protists on antifolate containing medium.
Dependencies are inferred from the claim text.