Methods for genomic integration for Kluyveromyces host cells
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Publication Number
US-12188044-B2
Publication Date
2025-01-07
Expiration Date
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.
Core Innovation
The invention relates to modifying a target site in a Kluyveromyces host cell genome having reduced non-homologous end joining (NHEJ) activity. A nucleic acid molecule is contacted with the host cell that includes a stability element comprising a centromere sequence (CEN) 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, together with nucleic acid sequence encoding a nuclease capable of cleaving the target site. A donor DNA molecule capable of homologous recombination at the cleaved target site is also used.
The method further includes selecting a transformed host cell in which the donor DNA molecule integrated into the target site. The disclosure emphasizes high-efficiency targeted and marker-less single or multiplex integrations, including homologous recombination at nuclease-cleaved target sites in Kluyveromyces. The approach is described as reducing NHEJ by using host cells with reduced NHEJ activity together with stability elements that use CEN/ARS.
In addition, the disclosure provides recombinant nucleic acid molecules that encode a nuclease or encode crRNA activity and tracrRNA activity enabling an RNA-guided DNA endonuclease, and include the same CEN and ARS stability elements meeting the stated identity thresholds. The recombinant nucleic acid molecule is configured for site-specific recognition and cleavage of a target site, supporting the described genome modification. The disclosure also describes a meganuclease landing-pad strategy including F-CphI and multiplexing concepts using nuclease-mediated double-strand breaks.
Claims Coverage
The partial content includes two independent claims. The inventive features center on genome modification in a Kluyveromyces host cell with reduced NHEJ activity using a nuclease-encoding CEN/ARS stability element and homologous recombination donor DNA with selection, and on recombinant nucleic acid molecules that combine nuclease or RNA-guided DNA endonuclease functionality with specified CEN/ARS identity thresholds for site-specific cleavage.
Kluyveromyces genome modification with reduced NHEJ using a CEN/ARS nuclease cassette and donor DNA
A method for modifying a target site in a Kluyveromyces host cell genome with reduced non-homologous end joining (NHEJ) activity by contacting the host cell with a nucleic acid molecule comprising a stability element with a CEN sequence at least 95% identical to SEQ ID NO: 2 and an 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, together with a donor DNA molecule capable of homologous recombination at the cleaved target site, and selecting a transformed host cell in which the donor DNA molecule integrated into the target site.
Recombinant CEN/ARS nucleic acid molecule encoding a nuclease or RNA-guided DNA endonuclease
A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a nuclease or a sequence encoding crRNA activity and tracrRNA activity that enables site-specific recognition and cleavage of a target site by an RNA-guided DNA endonuclease, together with a stability element comprising a CEN sequence at least 95% identical to SEQ ID NO: 2 and an ARS consensus sequence at least 90% identical to SEQ ID NO: 3.
Across the independent claims, the coverage is directed to combining reduced-NHEJ Kluyveromyces genome editing with a nuclease on a CEN/ARS stability element and a homologous recombination donor DNA with selection, and to the corresponding recombinant CEN/ARS nucleic acid molecules that encode the nuclease or RNA-guided DNA endonuclease activity for site-specific cleavage.
Stated Advantages
High-efficiency targeted and marker-less single or multiplex integrations using reduced NHEJ activity.
Improved performance of genome engineering described as high single-locus integration efficiencies and stated multiplex integration efficiencies in the disclosure.
Documented Applications
Genome engineering and targeted genome modification in Kluyveromyces host cell genome at nuclease-cleaved target sites using a stability-element-containing nuclease cassette and donor DNA for homologous recombination.
Marker-less integration and single or multiplex integration at specified loci such as GAS2, NDT80, GAL80, LEU2, and loci associated with YKU80/YYKU70.
Recombinant nucleic acid molecule use for site-specific recognition and cleavage of a target site by an RNA-guided DNA endonuclease or a meganuclease including F-CphI, as part of the described genome modification approach.
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