Methods for genomic integration in Pichia and other host cells
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Publication Number
US-12168769-B2
Publication Date
2024-12-17
Expiration Date
Abstract
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.
Core Innovation
The invention relates to producing an antibody using a yeast host cell engineered for CRISPR-based, high-efficiency targeted marker-less genomic integration. A yeast host cell is cultured under conditions suitable for production of the antibody, where the host cell comprises a nucleic acid sequence encoding an antibody integrated in the yeast host cell genome and a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker.
The engineered yeast host cell further includes a nucleic acid encoding an RNA-guided DNA endonuclease, with the nucleic acid integrated in a YKU70 gene to reduce non-homologous end joining (NHEJ) activity compared to a host cell in which YKU70 is intact. This design supports targeted marker-less integration together with homologous recombination using donor DNA, while enabling recovery of the antibody produced by the yeast host cell.
In the disclosed embodiments, the method is applied to Pichia host cells, including Pichia pastoris, where the nucleic acid encoding the RNA-guided DNA endonuclease is operably linked to a Pichia pPGK1 promoter. The document further describes antibody production for HERCEPTIN (trastuzumab), RITUXAN (rituximab), and BIIB antibody, including improved secretion/titers and characterization by Western blot and mass spectrometry/glycosylation analysis.
Claims Coverage
The document includes two independent claims, each focused on antibody production in yeast using genome-encoded antibody sequences plus a circular extrachromosomal nucleic acid for a selectable marker, and an RNA-guided DNA endonuclease integrated in a YKU70 gene to reduce NHEJ; claim 18 additionally requires operable linkage to a pPGK1 promoter.
Antibody production in yeast with genome-encoded antibody and selectable marker on a circular extrachromosomal nucleic acid
A method of producing an antibody comprising culturing a yeast host cell comprising a nucleic acid sequence encoding an antibody in the yeast host cell genome and a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker, under conditions suitable for production of the antibody.
RNA-guided DNA endonuclease nucleic acid integrated in YKU70 to reduce NHEJ
The method 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.
Pichia host cell with genome-encoded antibody and circular extrachromosomal selectable marker nucleic acid
A method of producing an antibody comprising culturing a Pichia host cell comprising a nucleic acid sequence encoding an antibody in the Pichia host cell genome and 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.
RNA-guided DNA endonuclease nucleic acid integrated in YKU70 and operably linked to pPGK1 promoter
The method wherein the nucleic acid is integrated in a YKU70 gene to reduce 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.
Across the independent claims, the inventive thrust is culturing engineered yeast, including Pichia, with a genome-encoded antibody and a circular extrachromosomal nucleic acid carrying a selectable marker, while reducing NHEJ by integrating an RNA-guided DNA endonuclease nucleic acid into a YKU70 gene; claim 18 further specifies operable linkage of the endonuclease nucleic acid to a pPGK1 promoter.
Stated Advantages
Reducing non-homologous end joining (NHEJ) activity in the host cell as compared to a host cell in which YKU70 is intact.
Production of antibody by culturing an engineered yeast host cell followed by recovering the antibody produced by the yeast host cell.
Production in Pichia host cells including operable linkage of the RNA-guided DNA endonuclease nucleic acid to a Pichia pPGK1 promoter.
Documented Applications
Producing antibodies, including HERCEPTIN (trastuzumab), RITUXAN (rituximab), and BIIB antibody, by culturing engineered yeast host cells such as Pichia/Pichia pastoris and recovering the produced antibodies.
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