Increased nucleic-acid guided cell editing in yeast

Patent metadata

Field	Value
Publication number	US-11066675-B2
Application number	US-202117140056-A
Country	US
Kind code	B2
Filing date	Jan 2, 2021
Priority date	Jun 25, 2019
Publication date	Jul 20, 2021
Grant date	Jul 20, 2021

Abstract

The present disclosure provides methods to increase the percentage of edited yeast cells in a cell population using nucleic-acid guided editing, and automated multi-module instruments for performing these methods.

First claim

We claim:

1. A method for performing editing in yeast comprising:
providing a population of yeast cells;
transforming the population of yeast cells with a population of editing vectors, wherein each editing vector comprises: a yeast 2μ backbone; a 2μ origin of replication; a first non-minimal or non-core constitutive promoter driving transcription of a gRNA sequence and donor DNA sequence with followed by a terminator element 3′ to the gRNA and donor DNA sequences; a second non-minimal or non-core constitutive promoter driving transcription of a coding sequence for a degron-survival marker fusion gene followed by a terminator element 3′ to the degron-survival marker fusion gene; a third non-minimal or non-core constitutive promoter driving transcription of a nuclease coding sequence with a terminator element 5′ to the nuclease coding sequence; and an origin of replication for propagation of the editing vector in bacteria;
growing the transformed yeast cells in selective medium to select for cells expressing a degron-survival marker fusion protein;
providing conditions to allow the transformed yeast cells to edit nucleic acid sequences in the yeast cells; and
growing the edited yeast cells.

2. The method of claim 1 , wherein the degron is an ubiquitin-dependent degron.

3. The method of claim 1 , wherein the degron is selected from Ura3-d degon, Ubi-R degron, Ubi-M degron, Ubi-Q degron, Ubi-E degron, ZF1 degron, C-terminal phosphodegron; Ts-degron; lt-degron; auxin inducible degron; DD-degron, LID-degron; PSD degron, B-LID degron; and a TIPI degron.

4. The method of claim 3 , wherein the degron is the Ura3-d degron.

5. The method of claim 1 , wherein the survival marker is selected from the group of hygromycin, blasticidin, kanamycin, and nourseothricin.

6. The method of claim 1 , wherein the first, second and third constitutive promoters are the same constitutive promoter.

7. The method of claim 1 , wherein two of the first, second and third constitutive promoters are different constitutive promoters.

8. The method of claim 1 , wherein all of the first, second and third constitutive promoters are different constitutive promoters.

9. A method for performing editing in yeast comprising:
providing a population of yeast cells;
transforming the population of yeast cells with a population of editing vectors, wherein each editing vector comprises: a yeast 2μ backbone; a 2μ origin of replication; a first non-minimal or non-core constitutive promoter driving transcription of a gRNA sequence and donor DNA sequence with followed by a terminator element 3′ to the gRNA and donor DNA sequences; a minimal or weak promoter driving transcription of a coding sequence for a survival marker gene followed by a terminator element 3′ to the survival marker gene; a second non-minimal or non-core constitutive promoter driving transcription of a nuclease coding sequence with a terminator element 5′ to the nuclease coding sequence; and an origin of replication for propagation of the editing vector in bacteria;
growing the transformed yeast cells in selective medium to select for cells expressing a survival marker protein;
providing conditions to allow the transformed yeast cells to edit nucleic acid sequences in the yeast cells; and
growing the edited yeast cells.

10. The method of claim 9 , wherein if the minimal or weak promoter driving transcription of a coding sequence for a survival marker gene is a minimal promoter, the minimal promoter is selected from a URA3-d promoter, a pHIS3 promoter, a pTRP1 promoter, a pLEU2 promoter, a pURA3 promoter, a pTEF1 promoter, and a pHXT7 promoter.

11. The method of claim 10 , wherein the minimal or weak promoter driving transcription of the coding sequence for a survival marker gene is a URA3-d promoter.

12. The method of claim 9 , wherein if the minimal or weak promoter driving transcription of a coding sequence for a survival marker gene is a weak promoter, the weak promoter is selected from a pSSA1 promoter, a pPDA1 promoter, a pCYC1 promoter, a pTPS1 promoter, and a pSSB1 promoter.

13. The method of claim 9 , wherein the survival marker is selected from the group of hygromycin, blasticidin, kanamycin, and nourseothricin.

14. The method of claim 9 , wherein the first and second constitutive promoters are the same constitutive promoter.

15. The method of claim 9 , wherein the first and second constitutive promoters are different constitutive promoters.

Assignees

• Inscripta Inc

Inventors

• Kannan Krishna
• Gander Miles
• Spindler Eileen
• Hardenbol Paul

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C12N15/80 Primary

  for fungi · CPC title