IP Library › Granted Patent US 12,188,044
Granted Patent B2
US 12,188,044 · App. 18/199,278 · Granted Jan 7, 2025

Methods for genomic integration for

Inventor: Jessica Walter (Albany, CA)
Assignee: Amyris, Inc.
C12N15/905C12N9/22C12N15/11C12N15/815C12N2310/20C12N2800/80
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Quick Facts
Patent No.
US 12,188,044
App. No.
18/199,278
Granted
Jan 7, 2025
Kind
B2
Abstract

The present invention provides high efficiency targeted and marker-less single or simultaneous multiple integrations using nucleases and a stable plasmid in Kluyveromyces host cells.

Claims (21)

1. A method for modifying a target site in a Kluyveromyces host cell genome, the method comprising:

(a) contacting the host cell, which has reduced non-homologous end joining (NHEJ) activity, with:

(i) a nucleic acid molecule comprising a stability element comprising a centromere sequence (CEN) sequence at least 95 % identical to SEQ ID NO: 2 and an autonomously replicating sequence (ARS) consensus sequence at least 90% identical to SEQ ID NO: 3 and nucleic acid sequence encoding a nuclease capable of cleaving the target site; and

(ii) a donor DNA molecule capable of homologous recombination at the cleaved target site;

(b) selecting a transformed host cell in which the donor DNA molecule integrated into the target site.

2. The method of claim 1 , wherein the host cell is K. marxianus.

3. The method of claim 1 , wherein the nuclease is a meganuclease.

4. The method of claim 3 , wherein the meganuclease is F-CphI.

5. The method of claim 1 , wherein the stability element is at least 90% identical to a sequence less than 750bp in length and comprising residues 202 to 876 of SEQ ID NO: 1.

6. The method of claim 1 , wherein the stability element is at least 95% identical to SEQ ID NO: 1.

7. The method of claim 1 , wherein the step of contacting includes contacting the host cell with two or more donor DNA molecules capable of homologous recombination with different target sites in the host cell genome, whereby homologous recombination in the host cell results in integration of the donor DNA molecules at the different target sites.

8. An isolated recombinant host cell made by the method of claim 1 .

9. A recombinant nucleic acid molecule comprising:

(i) a nucleic acid sequence encoding a nuclease or a sequence encoding a crRNA activity and a tracrRNA activity that enables site-specific recognition and cleavage of a target site by an RNA-guided DNA endonuclease; and

(ii) a stability element comprising a centromere sequence (CEN) sequence at least 95% identical to SEQ ID NO: 2 and an autonomously replicating sequence (ARS) consensus sequence at least 90% identical to SEQ ID NO: 3.

10. The recombinant nucleic acid molecule of claim 9 , wherein the stability element is at least 95% identical to a sequence less than 750 bp in length and comprising residues 202 to 876 of SEQ ID NO: 1.

11. The recombinant nucleic acid molecule of claim 9 , wherein the stability element is at least 95% identical to SEQ ID NO: 1.

12. The recombinant nucleic acid molecule of claim 9 , wherein the nuclease is an RNA-guided DNA endonuclease.

13. The recombinant nucleic acid molecule of claim 12 , wherein the RNA-guided DNA endonuclease is a Cas9 endonuclease.

14. The recombinant nucleic acid molecule of claim 9 , wherein the nuclease is a meganuclease.

15. The recombinant nucleic acid molecule of claim 14 , wherein the meganuclease is F-CphI.

Assignments (2)
SECURITY INTEREST Recorded Apr 15, 2025
From: AMYRIS, INC.
To: EUAGORE, LLC
Reel/Frame 070848/0512 →
SECURITY INTEREST Recorded May 24, 2024
From: AMYRIS, INC.
To: EUAGORE, LLC
Reel/Frame 067528/0467 →
Continuity (4)
Division 16646028
Provisional Application 62560029 · Sep 18, 2017
Provisional Application 62667000 · May 4, 2018
Related Publication 20240182927A1 · Jun 6, 2024
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