CRISPR-CAS SYSTEM FOR AN ALGAL HOST CELL

• US 20200131511A1
• Filed: 07/13/2017
• Published: 04/30/2020
• Est. Priority Date: 07/13/2016
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: LIPPMEIER, James Casey, BETZ, Yelena, ROUBOS, Johannes Andries, VERWAAL, Rene
• Granted Patent: US 11,466,269 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Q 2521/301 Endonuclease

Abstract

The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention trelates to a CRISPR-Cas system for a Labyrinthulomycetes host cell.

Claims

1. A non-naturally occurring or engineered composition comprising a source of a CRISPR-Cas system comprising a guide-polynucleotide and a Cas protein, wherein the guide-polynucleotide comprises a guide-sequence that essentially is the reverse complement of a target-polynucleotide in a host cell and the guide-polynucleotide can direct binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex, wherein the guide-sequence is essentially the reverse complement of the (N)y part of a 5′

-(N)yPAM-3′

polynucleotide sequence target in the genome of the host cell, wherein y is an integer of 8-30, wherein PAM is a protospacer adjacent motif, wherein the host cell is a Labyrinthulomycete, preferably a member of the order Thraustochytriales, preferably a member of the family Thraustochytriaceae, more preferably a member of a genus selected from the group consisting of Aurantiochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium, and Ulkenia, even more preferably Schizochytrium sp. ATCC #20888, and wherein PAM is preferably a sequence selected from the group consisting of 5′

-XGG-3′

, 5′

-XGGXG-3′

, 5′

-XXAGAAW-3′

, 5′

-XXXXGATT-3′

, 5′

-XXAGAA-3′

, 5′

-XAAAAC-3′

, wherein X can be any nucleotide or analog thereof, preferably X can be any nucleotide; and

W is A or T.

2. The composition according to claim 1, wherein the Cas protein is encoded by a polynucleotide and/or the guide-polynucleotide is encoded by or present on a polynucleotide.

3. The composition according to claim 2, wherein the Cas protein is encoded by a polynucleotide and/or the guide-polynucleotide is encoded by or present on another polynucleotide and the polynucleotide or polynucleotides are comprised in a vector.

4. The composition according to claim 3, wherein the guide polynucleotide is encoded by a polynucleotide that is transcribed to provide for the actual guide-polynucleotide.

5. The composition according to claim 3, wherein the Cas protein is encoded by a polynucleotide and the guide-polynucleotide is encoded by or present on another polynucleotide and the polynucleotides are comprised in one vector.

6. The composition according to claim 3, wherein the Cas protein is encoded by a polynucleotide comprised in a vector and the guide-polynucleotide is encoded by or present on another polynucleotide comprised in another vector, wherein preferably the vector encoding the Cas protein is driven by a low strength promoter and the vector encoding the guide-polynucleotide is driven by a high strength promoter.

7. The composition according to claim 6, wherein one or more or all vectors comprise a selectable marker, preferably each vector comprising a distinct selectable marker.

8. The composition according to claim 1, wherein one or more exogenous polynucleotides are operably linked to the guide-polynucleotide.

9. The composition according to claim 1, wherein the Cas protein comprises at least one nuclear localization sequence, preferably a heterologous nuclear localization sequence.

10. The composition according to claim 1, wherein the Cas protein has activity for directing cleavage of both polynucleotide strands at the location of the target-sequence.

11. The composition according to claim 1, wherein the Cas protein encoding polynucleotide is codon optimized for the host cell, preferably codon pair optimized.

12. The composition according to claim 8, wherein the guide-polynucleotide is encoded by a polynucleotide that is operably linked to a RNA polymerase II or III promoter, preferably to a Labyrinthulomycete EF-1 promoter.

13. The composition according to any one of claims 1-12, wherein a polynucleotide that is operably linked to an RNA polymerase II promoter encodes a pre-guide-polynucleotide comprising the guide-polynucleotide and self-processing ribozymes, wherein, when transcribed, the guide-polynucleotide is released by the self-processing ribozymes from the pre-guide-polynucleotide transcript.

14. A method of modulating expression of a polynucleotide in a cell, comprising contacting a host cell with the composition according to any one of claims 1-12, wherein the guide-polynucleotide directs binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex.

15. The method according to claim 14, wherein the host cell comprises a polynucleotide encoding a compound of interest.

16. The method according to claim 14, wherein the host cell is a recombinant host cell.

17. A host cell comprising the composition according to any one of claims 1-12.

18. A method of producing a host cell, comprising contacting a host cell with the composition according to any one of claims 1-12, wherein the guide-polynucleotide directs binding of the Cas protein at the target-polynucleotide in the host cell to form a CRISPR-Cas complex.

19. The method according to claim 18, wherein the host cell is first contacted with a source of a Cas protein and subsequently is contacted with a source of a guide-polynucleotide and optionally an exogenous polynucleotide.

20. The method according to claim 19 or the host cell according to claim 17, wherein the host cell comprises a polynucleotide encoding a compound of interest.

21. The method according to any one of claims 18-20 or a host cell according to claim 17, wherein the host cell is a recombinant host cell.

22. A method for the production of a compound of interest, comprising culturing under conditions conducive to the production of the compound of interest a host cell obtainable by the method of any one of claims 18-20 or a host cell according to claim 17 or a host cell produced according to the method of any one of claims 18-20 and optionally purifying or isolating the compound of interest.

23. The method according to any one of claims 14-16 and 18-22, wherein the Cas protein is a Cas9 protein.

24. The method according to any one of claims 18-20, wherein expression of the Cas protein is driven by a medium strength promoter.

25. The method according to claim 24, wherein the medium strength promoter is an alpha tubulin promoter.