Methods for genomic integration in
The present invention provides high efficiency targeted and marker-less single, double, triple, quadruple, and quintuple integrations by using CRISPR in host cells, including Pichia.
1. A method of producing an antibody, the method comprising,
culturing a yeast host cell comprising (i) a nucleic acid sequence encoding an antibody in the yeast host cell genome and (ii) a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker, under conditions suitable for production of the antibody, wherein the host cell further comprises a nucleic acid that encodes an RNA-guided DNA endonuclease, wherein the nucleic acid is integrated in a YKU70 gene, thereby reducing non-homologous end joining (NHEJ) activity in the host cell as compared to a host cell in which YKU70 is intact; and
recovering the antibody produced by the yeast host cell.
2. The method of claim 1 , wherein the host cell is Pichia.
3. The method of claim 2 , wherein the host cell is Pichia pastoris.
4. The method of claim 2 , wherein the nucleic acid encoding the RNA-guided DNA endonuclease is operably linked to a Pichia pPGK1 promoter.
5. The method of claim 1 , wherein the RNA-guided DNA endonuclease is Cas9.
6. The method of claim 5 , wherein the yeast host cell is a Saccharomyces yeast host cell and the nucleic acid sequence encoding the Cas9 is codon optimized for expression in Saccharomyces.
7. The method of claim 5 , wherein the yeast host cell is a Pichia yeast host cell and the nucleic acid sequence encoding the Cas9 is codon optimized for expression in Pichia.
8. The method of claim 7 , wherein the Pichia yeast host cell is a Pichia pastoris yeast host cell.
9. The method of claim 3 , wherein BMT1, BMT2, BMT3, BMT4, PNO1, MNN4-1, MNN4-2, MNN4-3, PRB1, PEP4, AOX1, or DNL4 are deleted.
10. The method of claim 3 , wherein PEP4 is deleted.
11. The method of claim 3 , wherein VTH1, CNE1, ECM13, and ERO1 are overexpressed.
12. The method of claim 1 , wherein the antibody is trastuzumab.
13. The method of claim 12 , wherein the nucleic acid sequence encoding the antibody coding sequence is codon-optimized for the yeast host cell.
14. The method of claim 1 , wherein the antibody is rituximab.
15. The method of claim 14 , wherein the nucleic acid sequence encoding the antibody coding sequence is codon-optimized for the yeast host cell.
16. The method of claim 1 , wherein the antibody is BIIB.
17. The method of claim 16 , wherein the nucleic acid sequence encoding the antibody coding sequence is codon-optimized for the yeast host cell.
18. A method of producing an antibody, the method comprising,
culturing a Pichia host cell comprising (i) a nucleic acid sequence encoding an antibody in the Pichia host cell genome and (ii) a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker, under conditions suitable for production of the antibody, wherein the Pichia host cell further comprises a nucleic acid that encodes an RNA-guided DNA endonuclease, wherein the nucleic acid is integrated in a YKU70 gene, thereby reducing non-homologous end joining (NHEJ) activity in the Pichia host cell as compared to a Pichia host cell in which YKU70 is intact and wherein the nucleic acid encoding the RNA-guided DNA endonuclease is operably linked to a Pichia pPGK1 promoter; and
recovering the antibody produced by the Pichia host cell.
19. The method of claim 18 , wherein the Pichia host cell is a Pichia pastoris host cell.