REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file 101928-1100559 SL.txt created on Mar. 5, 2020, 68,581 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

TypicallyP. pastorisis transformed using vectors or linear constructs with drug or auxotrophic markers. To improve protein production from integrated constructs, clones are passaged on increasing concentrations of a drug, selecting for amplification of the construct in a random fashion. Targeted integration is possible, and greatly improved when YKU70 is deleted in yeast cells to reduce NHEJ (non-homologous end joining) repair mechanisms. However, available markers are limited, and marker recycling (i.e., reusing of the same marker) is necessary for more ambitious engineering efforts. For rapid strain engineering, for example, inP. pastoris, a highly efficient, marker-less and targeted homologous integration transformation method is desired. Recently, Weninger et al. (Journal of Biotechnology235:139-149 2016) reported a CRISPR protocol inP. pastorisusing a strong constitutive promoter for Cas9 expression, and an RNA polymerase II promoter driving expression of the gRNA, with all components contained on a large plasmid. The study reported high efficiency of insertion and deletion (indel) introduction by NHEJ into a single gene, or multiple genes, which usually results in loss of function, equivalent to a knockout. However, when marker-less donor DNA was provided for targeted integration, a rate of only 2.4% was observed.

Thus, current known methods are in need of improvement. The present invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of disrupting or inserting a desired donor DNA molecule into one or more target sites in a host cell genome.

In some embodiments, the methods comprise (a) contacting a host cell, which comprises a nucleic acid encoding an RNA-guided DNA endonuclease, with: (i) a first linear nucleic acid capable of homologous recombination with itself or with one or more additional linear nucleic acids contacted with the host cell, whereby homologous recombination in the host cell results in formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker and (ii) a second linear nucleic acid which comprises, from 5′ to 3′, a RNA polymerase II promoter, a first nucleic acid encoding a first ribozyme, a nucleic acid encoding a guide RNA, a second nucleic acid encoding a second ribozyme, and a terminator, wherein the guide RNA guides the DNA nuclease to the target site. A transformed host cell expressing the selectable marker is then selected. In some embodiments, NHEJ is reduced in the host cell. In some embodiments, the nucleic acid encoding an RNA-guided DNA endonuclease is integrated into the host cell genome.

In some embodiments, the method further comprises contacting the host cell with a donor DNA molecule capable of homologous recombination with the target site, whereby homologous recombination in the host cell results in integration of the donor DNA molecule at the target site. In some embodiments, the donor DNA molecule comprises a nucleic acid sequence encoding an antibody. In some cases, the step of contacting includes contacting the cell with two or more donor DNA molecules capable of homologous recombination with different target sites, whereby homologous recombination in the host cell results in integration of the donor DNA molecules at the different target sites.

The host cell used in any of the methods provided herein may be, for example, a non-conventional yeast cell. In some embodiments, the host cell isPichia, in particular,Pichia pastoris.

In some embodiments, the step of contacting includes contacting the cell with two or more second linear nucleic acid molecules, wherein each second linear nucleic acid molecule comprises a nucleic acid encoding a different guide RNA, which guides the DNA nuclease to a different target site. The methods according to the present invention result in high efficiency for single, double, or multiple efficiency for targeted integration of donor nucleic acids into the host cell genome. As used herein, the targeting efficiency refers to a percentage of transformed cells comprising a successful integrated donor nucleic acids among screened cells.

In some embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease can be operably linked to aPichiapPGK1 promoter. In addition, the nucleic acid encoding the RNA-guided DNA endonuclease can be integrated in a YKU70 gene, thereby reducing NHEJ activity in the host cell. In certain embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease can be integrated at another genomic locus to reduce NHEJ, such as in a YKU80 gene. In other embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease can be integrated at a different genomic locus, and one or more genes involved in the NHEJ process can be functionally disrupted separately. The RNA-guided DNA endonuclease can be Cas9. In some embodiments, the nucleic acid sequence encoding the Cas9 is codon optimized for expression inSaccharomyces.

The invention also provides host cells made by the methods of the invention. The host cell may comprise a donor DNA molecule comprising a nucleic acid sequence encoding an antibody. Thus, the invention also provides methods of producing an antibody. The methods comprise culturing the host cell under conditions suitable for production of the antibody and recovering the antibody produced by the host cell. The host cell can bePichia.

