# US 10,870,858 B2 · Constructs and methods for genome editing and genetic engineering of fungi and protists

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- Number: US 10,870,858 B2
- Document ID: US10870858B2
- Status: Granted
- Kind: B2
- Application number: 15/878,698
- Filed: 2018-01-24
- Published: 2018-06-07
- Granted: 2020-12-22
- Earliest claimed: 2014-08-15
- Assignees: Wisconsin Alumni Research Foundation
- Applicants: Wisconsin Alumni Research Foundation
- Inventors: Christopher Todd Hittinger; William Gerald Alexander
- Web page: https://patentgrep.com/patent/US10870858B2

## CPC

- C12N 15/80: for fungi
- C12Q 1/045: {Culture media therefor}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 1

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.

### Claim 2 (depends on 1)

The construct of claim 1, wherein the repeated polynucleotide sequence is at least 20 nucleotides in length.

### Claim 3 (depends on 1)

The construct of claim 1, further comprising 20 to 2000 nucleotides homologous to the fungi or protists, wherein the homologous nucleotides are homologous to the target allele.

### Claim 4 (depends on 1)

The construct of claim 1, wherein the thymidine kinase is a Herpes Simplex Virus thymidine kinase.

### Claim 5 (depends on 1)

The construct of claim 1, wherein the first promoter is a constitutive promoter functional in fungi or protists.

### Claim 6 (depends on 1)

A method of editing at least one allele on a chromosome in a diploid fungus or protist using a single construct, the method comprising: generating or obtaining the construct of claim 1; introducing the construct into the fungus or protist cells; selecting for thymidine kinase by growing the fungus or protists on antifolate containing medium and selecting for the second selectable marker by growing the fungus or protists on media containing an agent to which the second selectable marker confers resistance, wherein the selecting steps result in recombination of at least a portion of the construct into at least one allele on one chromosome of the genome of the fungus or protist resulting in genome editing, allele replacement or genetic engineering of the fungus or protist.

### Claim 7 (depends on 6)

The method of claim 6, further comprising simultaneously selecting the engineered fungus or protist for the first selectable marker and the second selectable marker, wherein the selecting step for both markers results in recombination of at least a portion of the construct into the homozygous position on the second chromosome of the selected engineered fungus or protist, and wherein the selected engineered fungus or protist—contains at least a portion of the construct in homozygous positions on both chromosomes of the fungus or protist.

### Claim 8 (depends on 6)

The method of claim 6, wherein the antifolate containing medium comprises thymidine and at least one agent selected from the group consisting of methotrexate and sulfanilamide.

### Claim 9 (depends on 6)

The method of claim 6, wherein the carbon source in the antifolate containing medium is a non-fermentable carbon source.

### Claim 10 (depends on 6)

The method of claim 6, wherein the construct further comprises a target polynucleotide for insertion into the genome of the fungi or protist.

### Claim 11 (depends on 10)

The method of claim 10, wherein more than one construct each comprising a different target polynucleotide are incorporated into more than one fungus or protist cells.