Increased nucleic-acid guided cell editing in yeast

Patent metadata

Field	Value
Publication number	US-10927385-B2
Application number	US-202016904405-A
Country	US
Kind code	B2
Filing date	Jun 17, 2020
Priority date	Jun 25, 2019
Publication date	Feb 23, 2021
Grant date	Feb 23, 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. An editing vector for performing nucleic add-guided nuclease editing in yeast comprising: a yeast 2μ backbone; a 2μ origin of replication; a first constitutive non-minimal or non-core promoter driving transcription of a gRNA sequence and donor DNA sequence followed by a terminator element 3′ to the gRNA and donor DNA sequences; a second constitutive non-minimal or non-core 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 constitutive non-minimal or non-core 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.

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

3. The editing vector of claim 2 , wherein the degron is the Ura3-d degron.

4. The editing vector 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.

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

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

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

8. An editing vector for performing nucleic acid-guided nuclease editing in yeast comprising: a yeast 2μ backbone; a 2μ origin of replication; a first constitutive non-minimal or non-core promoter driving transcription of a gRNA sequence and donor DNA sequence followed by a terminator element 3′ to the gRNA and donor DNA sequences; a minimal 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 constitutive non-minimal or non-core 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.

9. The editing vector of claim 8 , wherein the minimal promoter driving transcription of a coding sequence for a survival marker gene is the URA3-d promoter.

10. The editing vector of claim 8 , wherein the survival marker is selected from the group of hygromycin, blasticidin, kanamycin, and nourseothricin.

11. The editing vector of claim 8 , wherein the first and second constitutive promoters are the same constitutive promoter.

12. The editing vector of claim 8 , wherein the first and second constitutive promoters are different constitutive promoters.

Assignees

• Inscripta Inc

Inventors

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

Classifications

• C12N15/80 Primary

  for fungi · CPC title

• C12N2310/20

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

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/81 Primary

  for yeasts · 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