阅读说明：本技术 用于毕赤酵母和其他宿主细胞基因组整合的方法 (Methods for pichia and other host cell genome integration ) 是由 江汉笑 于 2018-09-12 设计创作，主要内容包括：本发明提供了通过在宿主细胞(包括毕赤酵母(Pichia))中使用CRISPR高效靶向且无标志物的单、双、三、四和五重整合。(The present invention provides for single, double, triple, quadruple and quintuple integration with efficient targeting and marker-free by using CRISPR in host cells, including Pichia (Pichia).)

1. A method of disrupting a target site in the genome of a host cell, the method comprising:

(a) contacting a host cell comprising 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 other linear nucleic acids with which the host cell is contacted, whereby homologous recombination in the host cell results in the formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker

(ii) A second linear nucleic acid comprising, from 5 'to 3', an 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 directs a DNA nuclease to the target site; and

(b) selecting a transformed host cell expressing the selectable marker, wherein the host cell has reduced NHEJ activity.

2. The method of claim 1, wherein NHEJ activity in the host cell is decreased prior to or concurrently with contacting the cell with the first linear nucleic acid and/or the second linear nucleic acid.

3. The method of claim 1 or 2, wherein the nucleic acid encoding the RNA-guided DNA endonuclease is pre-integrated into the host cell genome.

4. A method according to any one of the preceding claims wherein 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.

5. The method of claim 4, wherein the contacting step comprises contacting the cell with two or more donor DNA molecules capable of homologous recombination with different target sites, such that homologous recombination in the host cell results in integration of the donor DNA molecules at different target sites.

6. The method of claim 4 or 5, wherein the donor DNA molecule comprises a nucleic acid sequence encoding an antibody.

7. The method of any one of the preceding claims, wherein the host cell is a non-conventional yeast cell.

8. The method of any one of the preceding claims, wherein the host cell is Pichia pastoris (Pichia).

9. The method of claim 8, wherein the host cell is pichia pastoris (Pichiapastoris).

10. The method of any one of the preceding claims, wherein the contacting step comprises contacting the cell with two or more second linear nucleic acid molecules, wherein the second linear nucleic acid molecules each comprise a nucleic acid encoding a different guide RNA that directs a DNA nuclease to a different target site.

11. The method of any one of the preceding claims, wherein the nucleic acid encoding the RNA-guided DNA endonuclease is operably linked to the pichia pastoris pgk1 promoter.

12. The method of any one of the preceding claims, wherein the nucleic acid encoding the RNA-guided DNA endonuclease is integrated into the YKU70 gene, thereby reducing NHEJ activity in the host cell.

13. The method of any one of the preceding claims, wherein the RNA-guided DNA endonuclease is Cas 9.

14. The method of claim 13, wherein the nucleic acid sequence encoding the Cas9 is codon optimized for expression in yeast (Saccharomyces).

15. A host made by the method of any one of the preceding claims.

16. The host cell of claim 15, comprising a donor DNA molecule comprising a nucleic acid sequence encoding an antibody.

17. A method of producing an antibody, the method comprising culturing the host cell of claim 16 under conditions suitable for production of the antibody and recovering the antibody produced by the host cell.

18. The method of claim 17, wherein the host cell is pichia pastoris.

Background

Typically, pichia pastoris (p. pastoris) is transformed with vectors or linear constructs with drug or auxotrophic markers. To improve protein production from the integrated construct, clones were passaged in a manner that increased drug concentration and randomly selected construct amplification. Targeted integration is possible and greatly improved when YKU70 is deleted in yeast cells to reduce the NHEJ (non-homologous end joining) repair mechanism. However, available markers are limited, and marker recycling (i.e., reuse of the same marker) is necessary for larger scale engineering attempts. For rapid strain engineering, such as in pichia pastoris, a highly efficient, marker-free and targeted homologous integration transformation method is needed. Recently, Weningger et al (Journal of Biotechnology 235: 139-1492016) reported the CRISPR protocol in Pichia pastoris using a strong constitutive promoter for Cas9 expression, and an RNA polymerase II promoter driving expression of gRNAs, and all components contained on a large plasmid. This study reported that insertions and deletions (indels) were efficiently introduced into a single gene or multiple genes via NHEJ, which often resulted in loss of function, corresponding to knockouts. However, when providing marker-free donor DNA for targeted integration, the observed ratio was only 2.4%.

Therefore, there is a need for improvement in the currently known methods. The present invention addresses these and other needs.

Summary of The Invention

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

In some embodiments, the method comprises (a) contacting a host cell comprising 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 other linear nucleic acids with which the host cell is contacted, whereby homologous recombination in the host cell results in the formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker, and (II) a second linear nucleic acid comprising, from 5 'to 3', an 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. Transformed host cells expressing the selectable marker are then selected. In some embodiments, NHEJ is decreased in the host cell. In some embodiments, the nucleic acid encoding the 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, such that 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 contacting step comprises contacting the cell with two or more donor DNA molecules capable of homologous recombination with different target sites, such that homologous recombination in the host cell results in integration of the donor DNA molecules at the different target sites.

For example, a host cell used in any of the methods provided herein can be an unconventional yeast cell. In some embodiments, the host cell is Pichia (Pichia), in particular Pichia pastoris (Pichiapastoris).

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

In some embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease can be operably linked to the pichia pastoris pgk1 promoter. In addition, a nucleic acid encoding an RNA-guided DNA endonuclease can be integrated into the 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 into another genomic locus (such as the YKU80 gene) to reduce NHEJ. In other embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease can be integrated into a different genomic locus and can separately functionally disrupt one or more genes involved in the NHEJ process. The RNA-guided DNA endonuclease may be Cas 9. In some embodiments, the nucleic acid sequence encoding Cas9 is codon optimized for expression in yeast (Saccharomyces).

The invention also provides host cells prepared by the methods of the invention. The host cell may comprise a donor DNA molecule comprising a nucleic acid sequence encoding an antibody. Accordingly, the invention also provides methods of producing antibodies. The method comprises culturing the host cell under conditions suitable for production of the antibody and recovering the antibody produced by the host cell. The host cell may be pichia pastoris.

Drawings

FIGS. 1A and 1B show engineered Pichia pastoris strains secreting full-length Herceptin (Herceptin) and Rituxan (Rituxan). Protein a purified samples were tested by Western blotting under non-reducing (a) and reducing (B) conditions. Lanes 1-4, protein a purified sample; lanes 5-8, protein A purification and Endo H_fTreated samples, lanes 1 and 5, herceptin with pre- α secretory leader sequence (pre-alpha secretory leader sequence), lanes 2 and 6, herceptin with pre- α secretory leader sequence and mutation of KR to TR in L C, lanes 3 and 7, meruhua with Kar2 leader sequence, lanes 4 and 8, meruhua with Kar2 sequence and mutation of KR to TR in L C, MW, molecular weight scale, and number left of each gel ++, BIIB antibody standard.

FIG. 2 shows the results of mass spectrometry of a sample from Pichia pastoris for production of herceptin. The samples were analyzed to determine the N-glycan profile of herceptin produced by pichia pastoris. Mass spectrometry results As expected, the Pichia pastoris produced herceptin with a glycosylation pattern with high mannose content.

Definition of

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

The term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-or double-stranded form, and polymers thereof. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260:2605-2608 (1985); and Rossolini et al, mol.cell.Probes 8:91-98 (1994)).

The term "gene" may refer to a segment of DNA involved in the production or encoding of a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" may refer to a gene involved in producing or encoding an untranslated RNA, such as an rRNA, tRNA, guide RNA, or microRNA.

A "promoter" is defined as one or more nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the start site of transcription. Promoters also optionally include distal enhancer or repressor elements, which can be located up to several thousand base pairs from the transcription start site.

As used herein, the term "marker-less" refers to the integration of donor DNA into a target site within the host cell genome without the simultaneous integration of a selectable marker. The term also refers to the situation where no selectable marker gene is integrated into the host cell genome for recovery of the host cell in which the donor DNA is integrated into the host cell genome. In some embodiments, the term also refers to recovery of such host cells without the use of a selection scheme that relies on integration of a selectable marker into the host cell genome. For example, in certain embodiments, episomal or extrachromosomal selectable markers can be used to select cells that comprise a plasmid that comprises a gRNA. Such use is considered marker-free as long as the selectable marker is not integrated into the host cell genome.

As used herein, the term "operably linked" refers to a functional linkage between nucleic acid sequences such that the sequences encode a desired function. For example, when the promoter and/or regulatory region to which it is linked functionally controls the expression of the coding sequence, the coding sequence of the gene of interest (e.g., selectable marker) is operably linked to its promoter and/or regulatory sequence. It also refers to the linkage between coding sequences such that they may be controlled by the same linked promoter and/or regulatory regions; such joining between coding sequences may also be referred to as in-frame or in-frame joining in the same coding sequence. "operably linked" also refers to a linkage of functional but non-coding sequences, such as an autonomously propagating sequence or an origin of replication. Such sequences are operably linked when they are capable of performing their normal function, e.g., enabling replication, propagation and/or isolation of a vector carrying the sequence in a host cell.

As used herein, the term "transformation" refers to a genetic alteration of a host cell resulting from the introduction of exogenous genetic material into the host cell.

As used herein, the term "selecting a host cell that expresses a selectable marker" also includes enriching a population of transformed cells for host cells that express the selectable marker.

As used herein, the term "selectable marker" refers to a gene that is used as a guide for selecting a host cell comprising a marker as described herein (e.g., a marker expressed by a circular extrachromosomal nucleic acid in a host cell). Selectable markers include, but are not limited to: fluorescent markers, luminescent markers, drug-selective markers, and the like. Fluorescent markers may include, but are not limited to, genes encoding fluorescent proteins such as Green Fluorescent Protein (GFP), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), red fluorescent protein (dsRFP), and the like. Luminescent markers may include, but are not limited to, genes encoding luminescent proteins such as luciferase. Drug-selective markers suitable for use in the methods and compositions provided herein include, but are not limited to, genes that are resistant to antibiotics such as ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, tetracycline, chloramphenicol, and neomycin, for example. In some embodiments, the selection may be a positive selection; that is, cells expressing the marker are isolated from the population, e.g., to produce an enriched population of cells comprising the selectable marker. In other cases, the selection may be a negative selection; that is, the population is separated from the cells, e.g., to produce an enriched population of cells that does not comprise a selectable marker. The separation can be carried out by any conventional separation technique suitable for the selective marker used. For example, if a fluorescent marker is used, cells can be isolated by fluorescence-activated cell differentiation, whereas if a cell surface marker has been inserted, cells can be isolated from a heterogeneous population by affinity separation techniques, e.g., magnetic separation, affinity chromatography, using an affinity reagent "panning" linked to a solid substrate, or other conventional techniques.

"polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

As used herein, "complementary" or "complementarity" refers to specific base pairing between nucleotides or between nucleic acids. Some embodiments, by way of example and not limitation, describe base pairing between a target site or region and a guide RNA in a host cell genome. Complementary nucleotides are typically A and T (or A and U) and G and C. The guide RNAs described herein may comprise sequences, e.g., DNA targeting sequences that are perfectly or substantially complementary (e.g., having 1-4 mismatches) to genomic sequences in the host cell.

The "CRISPR/Cas" system refers to a broad class of bacterial systems used to defend against foreign nucleic acids. CRISPR/Cas systems are present in a wide range of eubacteria and archaea. CRISPR/Cas systems include the following types: I. subtypes II and III. The wild type II CRISPR/Cas system utilizes an RNA-guided endonuclease, Cas9, along with a guide RNA, to recognize and cleave foreign nucleic acids.

As used herein, with respect to RNA-guided endonucleases (e.g., Cas9), the terms "cleave," "cleaved," and/or "cleavage" refer to the act of creating a break in a particular nucleic acid. As understood by those skilled in the art, the cleavage can leave blunt ends or sticky ends (i.e., 5 'or 3' overhangs). The term also includes single-stranded DNA breaks ("nicks") and double-stranded DNA breaks.

As used herein, the term "Cas 9" refers to an RNA-guided nuclease (e.g., from, or derived from, a bacterial or archaeal source). RNA-guided nucleases include the aforementioned Cas9 protein and homologs thereof, and include, but are not limited to, Cpf1 (see, e.g., Zetsche et al, Cell, volume 163, phase 3, page 759-.

Cas9 homologs are present in a variety of eubacteria, including, but not limited to, bacteria of the following taxonomic groups: actinomycetes (actinobacilla), aquatics (Aquificae), bacteroides-chloromycetes (bacteroides-Chlorobi), chlamydia-Verrucomicrobia (chlamydia-Verrucomicrobia), chloroflexus (chloflexi), Cyanobacteria (Cyanobacteria), Firmicutes (Firmicutes), Proteobacteria (Proteobacteria), spirochetes (Spirochaetes), and thermomyces (thermoanaerobae). An exemplary Cas9 protein is the Streptococcus pyogenes (Streptococcus pyogenes) Cas9 protein. Other Cas9 proteins and their homologs are described, for example, in Chylinksi, et al, RNA biol.2013, 5 months and 1 days; 10(5) 726-; nat. rev. microbiol.2011 for 6 months; 9(6) 467-477; hou, et al, Proc Natl Acad Sci U S A.2013, 24.9 months; 110(39) 15644-9; sampson et al, Nature.2013, 5, month, 9; 497(7448) 254-7; and Jinek, et al, science.2012 8 month 17; 337(6096):816-21. Any of the variants of Cas9 nuclease provided herein can be optimized to provide high efficiency activity or enhance stability in a host cell. Thus, engineered Cas9 nuclease for expression in pichia or yeast, e.g., codon optimized Cas9 nuclease, is also contemplated.

As used herein, in the context of disrupting a target site in the genome of a host cell, the phrase "disrupting" refers to inducing nucleic acid fragmentation in the target site. Disruption can be used to edit the genome. As used herein, the term "editing" refers to structural changes in the genomic sequence at a target site. For example, the host cell genome can be edited by deleting or inserting a nucleotide sequence in the genome of the cell. The nucleotide sequence may encode a polypeptide or a fragment thereof. Such editing may be performed, for example, by inducing a double-stranded break within a target site in the host cell genome, or a pair of single-stranded nicks on opposing strands, and flanking the target site in the host cell genome. Methods for inducing single-or double-stranded breaks at or within a target site include the use of an RNA-guided DNA endonuclease or derivative thereof and a guide RNA directed to the target site in the genome of the host cell.

The phrase "heterologous" as used herein refers to a substance not normally found in nature. The term "heterologous nucleotide sequence" refers to a nucleotide sequence that does not normally occur naturally in a given cell. Thus, the heterologous nucleotide sequence may be: (a) is foreign to its host cell (i.e., is exogenous to the cell); (b) naturally occurring in a host cell (i.e., endogenous), but not naturally occurring in the cell (i.e., greater or lesser than the amount naturally occurring in the host cell); or (c) occurs naturally in the host cell but is outside its native locus.

As used herein, the term "homologous recombination" refers to a molecular process in which nucleotide sequences are exchanged between two DNA molecules that are similar or identical.

The term "non-homologous end joining" or NHEJ as used herein refers to a cellular process in which the cut or nicked ends of DNA strands are joined directly without the need for a homologous nucleic acid template. NHEJ may result in the addition, deletion, substitution, or a combination thereof of one or more nucleotides at the repair site.

The term Homology Directed Repair (HDR) as used herein refers to a cellular process in which the cut or nicked ends of DNA strands are repaired by polymerization from a homologous template nucleic acid, e.g., a donor DNA molecule. Thus, the original sequence is replaced by the sequence of the template. Homologous template nucleic acids may be provided by homologous sequences elsewhere in the genome (sister chromatids, homologous chromosomes or repeated regions on the same or different chromosomes). Alternatively, an exogenous template nucleic acid, e.g., a donor DNA molecule, can be introduced to obtain a specific HDR-induced sequence change at a target site. In this way, specific sequences can be introduced into the cleavage site.

As used herein, the phrase "introducing" or "contacting," in the context of introducing a nucleic acid or protein into a host cell, refers to any process that results in the presence of a heterologous nucleic acid or polypeptide within the host cell. For example, the term includes the introduction of a nucleic acid molecule (e.g., a plasmid or linear nucleic acid) that encodes a nucleic acid of interest (e.g., an RNA molecule) or a polypeptide of interest and results in transcription of the RNA molecule and translation of the polypeptide. The term also includes the integration of a nucleic acid encoding an RNA molecule or polypeptide into the genome of the progenitor cell. The nucleic acid is then passed to the host cell through subsequent generations, for example, to "pre-integrate" the nucleic acid encoding the RNA-guided endonuclease into the host cell genome. In some cases, introduction refers to the translocation of a nucleic acid or polypeptide from outside the host cell to inside the host cell. A variety of methods for introducing nucleic acids, polypeptides, and other biomolecules into host cells are contemplated, including, but not limited to, electroporation, contact with nanowires or nanotubes, spheroplasty, PEG 1000-mediated transformation, gene gun, lithium acetate transformation, lithium chloride transformation, and the like.

As used herein, the term "full-length antibody" refers to an antibody having a structure that is substantially similar to a native antibody structure. Full-length antibodies comprise four polypeptides-two light chains and two heavy chains linked by disulfide bonds to form a "Y" shaped molecule. Each heavy chain includes a constant region and a variable region connected by a hinge region. The two constant regions of the two heavy chains form an Fc domain. The full-length antibody can be of any isotype defined by the antibody heavy chain (e.g., IgA, IgD, IgE, IgG, and IgM).

Detailed Description

Method for disrupting a target site in the genome of a host cell

Provided herein are methods of disrupting one or more target sites in the genome of a host cell. These methods allow for efficient, simultaneous integration of one or more donor DNA molecules into the host cell genome. In certain methods, one or more donor DNA molecules are integrated into the host cell genome without concomitant integration of the selectable marker into the host cell genome.

In some embodiments, disrupting the one or more target sites comprises (a) contacting a host cell expressing an RNA-guided DNA endonuclease with: (i) a first linear nucleic acid capable of homologous recombination with itself or with one or more other linear nucleic acids with which the host cell is contacted, such that homologous recombination in the host cell results in the formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker, and (II) a second linear nucleic acid comprising, from 5 'to 3', an 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. Transformed host cells expressing the selectable marker are then selected.

In some embodiments, disrupting the one or more target sites comprises (a) contacting a host cell expressing an RNA-guided DNA endonuclease with: (i) a first linear nucleic acid capable of homologous recombination with itself or with one or more other linear nucleic acids with which the host cell is contacted, such that homologous recombination in the host cell results in the formation of a circular extrachromosomal nucleic acid comprising a coding sequence for a selectable marker, and (II) a second linear nucleic acid comprising, from 5 'to 3', an 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 a target site, wherein the NHEJ activity of the host cell is reduced. Transformed host cells expressing the selectable marker are then selected.

In some embodiments, the method further comprises contacting the host cell with a donor DNA molecule capable of homologous recombination with the target site, such that 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 is a heterologous donor DNA molecule. In some embodiments, the donor DNA molecule is flanked by nucleotide sequences that are homologous to genomic sequences flanking the target site. In some embodiments, the donor DNA molecule comprises a homologous sequence at the 5 'end that is about 70%, 75%, 80%, 85%, 90%, 95%, or 100% homologous to the 5' region of the selected genomic target site. In some embodiments, the donor DNA molecule comprises a homologous sequence at the 3 'end that is about 70%, 75%, 80%, 85%, 90%, 95%, or 100% homologous to the 3' region of the selected genomic target site. In some cases, the homologous sequences flanking the donor DNA molecule each comprise about 50 to about 5,000 nucleotides, about 100 to 2500 nucleotides, about 200 to 1500 nucleotides, about 500 to about 1000 nucleotides, or any number of nucleotides within these ranges. See, for example, U.S. patent No. 9,476,065.

In some embodiments, the NHEJ in the host cell is decreased prior to contacting the host cell with the first linear nucleic acid, the second linear nucleic acid, and/or the donor DNA molecule. In some embodiments, the NHEJ in the host cell is decreased while the host cell is contacted with the first linear nucleic acid, the second linear nucleic acid, and/or the donor DNA molecule. In some embodiments, the NHEJ in the host cell is decreased after contacting the host cell with the first linear nucleic acid, the second linear nucleic acid, and/or the donor DNA molecule.

In some embodiments, the donor DNA molecule comprises a nucleic acid of interest. For example, the donor DNA molecule comprises a gene of interest that can be knocked into the host genome. In other embodiments, the donor DNA molecule acts as a knock-out construct that is capable of specifically disrupting the target gene upon integration of the construct into a target site in the host cell genome, thereby rendering the disrupted gene non-functional. Examples of nucleic acids of interest include, but are not limited to, protein coding sequences, promoters, enhancers, terminators, transcriptional activators, transcriptional repressors, transcriptional activator binding sites, transcriptional repressor binding sites, introns, exons, poly-a tails, multiple cloning sites, nuclear localization signals, mRNA stabilization signals, integration loci, epitope tag coding sequences, degradation signals, or any other naturally occurring or synthetic DNA molecule. In particular embodiments, the nucleic acid of interest does not comprise a nucleic acid encoding a selectable marker.

In some embodiments, the nucleic acid of interest encodes an antibody, such as, but not limited to, a monoclonal antibody, a Fab fragment, a single chain variable fragment (scFv), a dimeric single chain variable fragment (di-scFv), or a single domain antibody (sdAb). In some embodiments, the nucleic acid of interest encodes the full-length antibody herceptin (trastuzumab). In some embodiments, the nucleic acid of interest encodes the full-length antibody rituximab (rituximab). In some embodiments, the nucleic acid of interest does not include a nucleic acid encoding the full length antibody herceptin (trastuzumab), the full length antibody rituximab (rituximab), or the full length antibody BIIB. In other embodiments, the nucleic acid of interest encodes an enzyme, hormone, growth factor, anticoagulant, blood factor, engineered protein, interferon, interleukin, thrombolytic agent, viral protein, or bacterial protein.

In the methods and compositions provided herein, the host cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is selected from the group consisting of: fungal cells, bacterial cells, plant cells, insect cells, avian cells, fish cells and mammalian cells. In some embodiments, the mammalian cell is selected from the group consisting of: rodent cells, primate cells, and human cells. In some embodiments, the fungal cell is a yeast cell. In some embodiments, the yeast cell is a non-conventional yeast cell. Non-conventional yeast cells refer to yeast species that utilize non-homologous end joining as a primary mechanism of a DNA repair system, in contrast to conventional yeast cells (e.g., yeast (Saccharomyces) or schizosaccharomyces (schizosaccharomyces)) that utilize homologous recombination as a primary mechanism of a DNA repair system. Examples of the non-conventional yeast cell include Pichia (Pichia) (e.g., Pichia pastoris (p. pastoris)), Kluyveromyces (Kluyveromyces marxianus (k. marxianus) or Kluyveromyces lactis (k. lactis)), Hansenula (e.g., Hansenula polymorpha (h. polymorpha)) or Arxula (Arxula) akanei (a. adenitivorans)). In some embodiments, the yeast cell is a pichia cell. In a specific embodiment, the yeast cell is a pichia pastoris cell. Examples of host cells that can be used in the methods described herein are described in international application publication No. WO 2015/095804. In some embodiments, the host cell does not comprise a nucleic acid encoding the full length antibody herceptin (trastuzumab), the full length antibody rituximab (rituximab), or the full length antibody BIIB. In some embodiments, the host cell does not express the full length antibody herceptin (trastuzumab), the full length antibody rituximab (rituximab), or the full length antibody BIIB.

In some embodiments, a host cell with decreased NHEJ activity is a cell with a disruption in a locus involved in cellular NHEJ activity (i.e., a disruption in one or more genes encoding proteins driving the NHEJ pathway or causing NHEJ, examples of NHEJ pathway genes for pichia pastoris include, but are not limited to, YKU70, YKU, DN L4, Rad50, Rad 27, MRE11, and PO L. for different host cells, the names of genes may differ. suitable NHEJ pathway genes for disruption may be present, for example, in KEGG Non-homologous end joining pathways (KEGG Non-homologous end-joinpathway), see http:// www.genome.jp/KEGG-bin/showthway?03450 & show _ depiction ═ show ═ shiw, in some embodiments, the percentage of NHEJ pathway genes disrupted by NHEJ/chromosome 20%, the percentage of NHEJ genes in a host cell disruption in a locus is reduced by at least 20%, the percentage of NHEJ genes inserted into a pichia host cell, such as the host cell has a reduced NHEJ activity in a host cell, or a host cell with a reduced percentage of NHEJ gene deletion in a host cell, such as compared to No. 20%, the percentage of NHEJ gene targeting NHEJ 20%, e.g 21.

In some embodiments, the RNA-guided DNA endonuclease is provided by introducing a nucleic acid encoding the endonuclease into a host cell. For example, a plasmid or vector comprising a nucleic acid encoding an RNA-guided DNA endonuclease can be introduced into a cell. In some embodiments, the plasmid may further comprise a nucleic acid sequence encoding a selectable marker for maintenance of the plasmid in a host cell. In some embodiments, the nucleic acid encoding the endonuclease further comprises a promoter sequence. In some embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease is integrated into the genome of the host cell. In certain embodiments, an RNA-guided DNA endonuclease, e.g., Cas9, is integrated into the YKU70 gene of a yeast cell, thereby reducing NHEJ activity in the yeast cell. In some embodiments, the nucleic acid encoding the RNA-guided DNA endonuclease is under the control of a constitutive promoter. In a specific embodiment, the nucleic acid encoding the RNA-guided DNA endonuclease is under the control of the medium strength pichia pastoris pgk1 promoter. Examples of suitable promoters include, but are not limited to: pYPT1, pTEF1, pSSA3, pGPM1, pENO 1. In some embodiments, the RNA-guided DNA endonuclease can be introduced into the host cell prior to, simultaneously with, or subsequent to the introduction of the first and second linear nucleic acids. In other embodiments, the RNA-guided DNA endonuclease can be introduced into the host cell prior to, simultaneously with, or subsequent to the introduction of the first linear nucleic acid, the second linear nucleic acid, and the donor DNA molecule. In some embodiments, RNA encoding an RNA-guided DNA endonuclease can be introduced into a host cell. In other embodiments, an RNA-guided DNA endonuclease protein or functional fragment thereof can be introduced into a host cell.

In some embodiments, the first linear nucleic acid comprises two internal homologous sequences capable of homologous recombination with each other, such that homologous recombination of the internal homologous sequences results in formation of a circular extrachromosomal nucleic acid that expresses the selectable marker. In some embodiments, the first linear nucleic acid is capable of recombining with the second linear nucleic acid. In some embodiments, the first linear nucleic acid comprises a selectable marker, such that upon introduction of the first and second linear nucleic acids, the first and second linear nucleic acids undergo homologous recombination to form a circular, episomal or extrachromosomal nucleic acid comprising the selectable marker and the coding sequence for the guide RNA, e.g., via nick repair. Once circularized, the extrachromosomal nucleic acid comprises the coding sequence for the selectable marker, as well as appropriate regulatory sequences, such as promoters and/or terminators, that enable expression of the marker in the host cell. Providing a selectable marker on the circular extrachromosomal nucleic acid allows marker-free integration of one or more donor DNA molecules in the host cell genome, while avoiding integration of unrelated sequences (i.e., selectable markers) in the genome and any deleterious effects associated with prolonged marker expression. See, e.g., U.S. patent No. 9,476,065 for a gap repair mechanism that can be used in the methods described herein.

After forming the extrachromosomal nucleic acid comprising the selectable marker and the guide RNA coding sequence, the guide RNA is transcribed from the extrachromosomal nucleic acid and the RNA-guided DNA endonuclease expressed in the host cell is guided to a target site in the genome of the host cell, wherein the endonuclease generates a break at the target site. In some embodiments, once the endonuclease generates a break at the target site, the donor DNA molecule is integrated into the host cell genome via homologous recombination.

In some embodiments, the method comprises integrating multiple (i.e., two or more) donor DNA molecules into multiple target sites of the host cell genome. In some embodiments, the host cell is contacted with the first linear nucleic acid and two or more second linear nucleic acid molecules, wherein the second linear nucleic acid molecules each comprise a nucleic acid encoding a different guide RNA that targets a different site in the genome of the host cell. The second, different linear nucleic acids can each recombine with the first linear nucleic acid to form two or more different circular extrachromosomal nucleic acids in the host cell. It is understood that the terms "first linear nucleic acid" and "second linear nucleic acid" include multiple copies of the same nucleic acid molecule. For example, the host cell may be contacted with two or more second linear nucleic acid molecules, wherein the second linear nucleic acid molecules each comprise nucleic acids encoding different guide RNAs to target 2, 3, 4, 5, 6, 7 or more different sites in the host cell genome. In some embodiments, once the guide RNA directs the RNA-guided endonuclease to two or more target sites, the endonuclease generates breaks at the two or more target sites and the two or more donor DNA molecules integrate into the host cell genome via homologous recombination.

In some embodiments, the circular extrachromosomal nucleic acid comprises a coding sequence for a selectable marker and a guide RNA cassette comprising, from 5 'to 3', an 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.

Examples of promoters that can be used in any of the methods provided herein to control expression of guide RNAs include, but are not limited to, the pichia pastoris Pol II promoter (pHTA1), the yeast promoter pPGK1, the yeast promoter pTDH3, and the yeast promoter pACT 1. In some embodiments, the promoter, e.g., RNA polymerase II promoter, is from the same species as the host cell. In some embodiments, the promoter, e.g., RNA polymerase II promoter, is from a different species than the host cell.

By using first and second ribozymes flanking the guide RNA, after transcription of the gRNA cassette, under the control of the RNA polymerase II promoter, the ribozymes self-cleave the transcript to produce the desired guide RNA sequence. See, for example, Gao and ZHao (J.Integr. plant biol.56(4):343-349 (2014)). In some embodiments, the guide RNA flanks a hammer-headed (HH) and Hepatitis Delta Virus (HDV) ribozyme sequence. In particular embodiments, one or both ribozymes flank a linker sequence to facilitate release of the guide RNA following cleavage. In some embodiments, the linker sequence is at least 5, 6, 7, or 8 nucleotides in length. Exemplary linker sequences are provided in the examples.

In some embodiments, the first linear nucleic acid comprising the selectable marker is a gapped vector (gapped vector) comprising a pair of homologous flanking sequences that recombine with a pair of homologous sequences flanking a gRNA cassette in the second linear nucleic acid to form a larger circular vector, wherein the gap has been repaired by insertion of the second linear nucleic acid into the gapped vector. In some embodiments, the homologous flanking sequences of the pair of homologous flanking sequences in the first nucleic acid each comprise a recombination region comprising a nucleotide sequence of sufficient length and sequence identity to allow homologous recombination with the pair of homologous flanking sequences in the second linear nucleic acid, but not with other regions of the first or second linear nucleic acid and any genomic regions of the host cell involved in vivo assembly. For in vivo assembly of marker/gRNA vectors via gap repair and for selection of cells capable of homologous recombination and gap repair, see, e.g., Horwitz et al (CellSystems 1:88-96(2015)) and international application publication No. WO2015/095804, each of which is incorporated herein by reference in its entirety.

In some embodiments, "sufficient sequence identity" refers to sequences that have at least 70%, at least 75% >, at least 80% >, at least 85% >, at least 90% >, at least 95% >, at least 99% >, or 100% identity between recombined regions over a length of, for example, at least 15 base pairs, at least 20 base pairs, at least 50 base pairs, at least 100 base pairs, at least 250 base pairs, at least 500 base pairs, or more than 500 base pairs. One skilled in the art readily understands how to determine the identity of two nucleic acids. For example, identity can be calculated after aligning the two sequences so that identity is at its highest level. Another method of calculating identity may be performed by published algorithms. For example, the algorithm of Needleman and Wunsch, J.Mol.biol.48:443(1970) can be used to perform optimal alignments of sequences for comparison. For a discussion of homology in effective length between recombination regions, see Hasty et al (Mol Cell Biol 11:5586-91 (1991)).

Using the methods provided herein, one or more target sites in a host cell genome can be modified with surprisingly high efficiency compared to conventional CRISPR/Cas systems. The efficiency of the alteration in the cell population can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, or any percentage between these percentages.

In some embodiments, the methods of the invention provide marker-free recovery (marker recovery) of transformed host cells comprising successfully integrated donor nucleic acids. Such cells occur at a frequency of about one per 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 screened contacted host cells or clonal populations thereof. In particular embodiments, marker-free recovery of transformed host cells comprising successfully integrated donor nucleic acid occurs at a frequency of about every 90, 80, 70, 60, 50, 40, 30, 20, or 10 contacted host cells or clonal populations thereof (screened). In more specific embodiments, marker-free recovery of transformed cells comprising successfully integrated donor nucleic acid occurs at a frequency of about every 9, 8, 7, 6, 5, 4, 3, or 2 contacted host cells or clonal populations thereof (screened).

In certain embodiments, marker-free recovery of transformed cells comprising successfully integrated donor nucleic acid at a single locus occurs at a frequency of at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% of contacted host cells or clonal populations thereof (screened). In certain embodiments, marker-free recovery of transformed cells comprising successfully integrated donor nucleic acid at 2, 3, 4, or 5 loci occurs in at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% of the contacted host cells or clonal populations thereof (screened). In certain embodiments, any suitable number of donor nucleic acids (e.g., n ═ 1 to 20) can be successfully integrated at n loci in the host cell genome.

There are a variety of methods that can be used to identify cells that have genomic alterations at or near the target site without the use of a selectable marker. In some embodiments, such methods attempt to detect any change in the target site and include, but are not limited to, PCR methods, sequencing methods, nuclease digestion, e.g., restriction mapping, Southern blotting, and any combination thereof. Phenotypic readings, e.g., predicted gain or loss of function, may also be used as a surrogate to achieve the desired genomic modification.

Cell culture

In some embodiments of the methods described herein, the host cell is cultured for a period of time sufficient to express the selectable marker from the circularized extrachromosomal vector. In some embodiments where the selectable marker is a drug resistance marker, culturing is conducted for a period of time sufficient to produce an amount of the marker protein that can support survival of cells expressing the marker in a selective nutrient medium. In certain embodiments, these conditions are also selected for the survival of cells that do not express the selectable marker. The selective pressure can be applied to the cells using a variety of compounds or treatments known to those skilled in the art. For example, cells that are tolerant or resistant to these conditions may be selected as compared to cells that are not tolerant or not resistant to these conditions by applying selective pressure to the host cells by exposing them to conditions that are suboptimal or detrimental to growth, progression of the cell cycle, or viability. Conditions that may be used to apply or exert selective pressure include, but are not limited to, antibiotics, drugs, mutagens, compounds that slow or stop cell growth or synthesis of biological modules (building blocks), compounds that disrupt RNA, DNA, or protein synthesis, deprive or limit cell growth or media of nutrients, amino acids, carbohydrates, or compounds required for cell growth and viability, treatments, such as cell growth or maintenance under conditions that are suboptimal for cell growth, such as under suboptimal temperature, atmospheric conditions (e.g., carbon dioxide, oxygen, or nitrogen% or humidity), or under starved media conditions. The level of selection pressure used can be determined by one skilled in the art. This can be accomplished, for example, by performing a lethal curve (kill curve) experiment in which control cells and cells containing a resistance marker or gene are tested with increasing levels, doses, concentrations, or treatment of selection pressure, while the range of selection for negative cells is only or preferentially within the desired time range (e.g., 1 to 24 hours, 1 to 3 days, 3 to 5 days, 4 to 7 days, 5 to 14 days, 1 to 3 weeks, 2 to 6 weeks). The exact level, concentration, dose or treatment of the selection pressure that may be used depends on the cell used, the desired property itself, the marker, factor or gene conferring resistance or tolerance to the selection pressure, and the level of the desired property desired in the selected cell, and one skilled in the art will readily understand how to determine the appropriate range based on these considerations.

The culture may be performed in a suitable medium in a suitable vessel, including but not limited to a cell culture plate, flask, or fermentor. In some embodiments, the medium is an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources. Such media may also include appropriate salts, minerals, metals and other nutrients. In some embodiments, in addition to the selection agent, the suitable medium is supplemented with one or more additional agents, e.g., an inducing agent (e.g., when the one or more nucleotide sequences encoding the gene product are under the control of an inducible promoter), a repressing agent (e.g., when the one or more nucleotide sequences encoding the gene product are under the control of a repressible promoter). Materials and methods for cell culture maintenance or growth are well known to those skilled in the art of microbiology or zymology (see, e.g., Bailey et al, Biochemical Engineering Fundamentals, second edition, McGraw Hill Press, New York, 1986). Depending on the specific requirements of the host cell, fermentation and process, the appropriate medium, pH, temperature and the need for aerobic, micro-aerobic or anaerobic conditions must be considered. In some embodiments, the culturing is performed for a period of time sufficient to subject the transformed population to multiple doublings until the desired cell density is reached. In some embodiments, the culturing is performed for a period of time sufficient for the population of host cells to reach a cell density (OD) of between 0.01 and 400 in the fermentor or vessel in which the culturing is performed₆₀₀). In other embodiments, the culturing is performed for a period of at least 12, 24, 36, 48, 60, 72, 84, 96, or greater than 96 hours. In some embodiments, the culturing is performed for a period of time between 3 and 20 days. In some embodiments, the culturing is performed for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 days.

In some embodiments of the methods described herein, for example, once a selected host cell has been identified as comprising a desired genomic integration, the method further comprises the step of eliminating the circularized extrachromosomal vector from the host cell. Plasmid-based systems typically require the application of selective pressure to the plasmid to maintain the foreign DNA in the cell. In some embodiments, elimination of the plasmid encoding the selectable marker from the selected cells can be achieved by subjecting the selected cells to sufficient mitosis to effectively dilute the plasmid from the population. Alternatively, plasmid-free cells can be selected by selecting for the absence of plasmid, e.g., by selecting for a counter-selectable marker (e.g., URA3) or by plating the same colonies on selective and non-selective media, and then selecting for colonies that do not grow on selective media but do grow on non-selective media.

In any of the methods described herein, disruption of the target site in the host cell genome occurs when the RNA-guided DNA endonuclease cleaves the target site in the host cell genome. Once the RNA-guided DNA endonuclease cuts the target site, the time required for integration of the donor DNA molecule will vary. For example, the encompassed time period may be at least 6, 12, 24, 36, 48, 60, 72, 96 or more than 96 hours of cell culture beginning at the point in time when the host cell is contacted with the first linear nucleic acid, the second linear nucleic acid, and the donor DNA molecule, regardless of whether the nucleic acid encoding the RNA-guided DNA endonuclease is integrated into the host cell genome or introduced into the host cell simultaneously with the first linear nucleic acid, the second linear nucleic acid, and the donor DNA molecule.

Guide RNA

As used throughout, a guide RNA (gRNA) sequence is a sequence that interacts with an RNA-guided DNA endonuclease and specifically binds or hybridizes to a target nucleic acid within a cell genome, thereby co-localizing the gRNA and targeted nuclease to the target nucleic acid in the cell genome. grnas each include a DNA targeting sequence of about 10 to 50 nucleotides in length that specifically binds or hybridizes to a target DNA sequence in the genome. For example, the DNA targeting sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. The grnas each comprise a gRNA scaffold sequence that binds to an RNA-guided DNA endonuclease that does not comprise a DNA targeting sequence. In some embodiments, the gRNA comprises a crRNA sequence and a trans-activating crRNA (tracrrna) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence.

Typically, the DNA targeting sequence is designed to be complementary (e.g., perfectly complementary) or substantially complementary to the target DNA sequence. In some cases, the DNA targeting sequence may incorporate wobble or degenerate bases to bind multiple genetic elements. In some cases, the 19 nucleotides at the 3 'or 5' end of the binding region are perfectly complementary to one or more target genetic elements. In some cases, the binding region may be altered to increase stability. For example, non-natural nucleotides may be included to increase the resistance of the RNA to degradation. In some cases, the binding region may be altered or designed to avoid or reduce secondary structure formation in the binding region. In some cases, the binding region can be designed to optimize the G-C content. In some cases, G-C content is preferably from about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, 60%).

RNA-guided DNA endonucleases

The methods provided herein can use any RNA-guided DNA endonuclease. In some embodiments, the RNA-guided DNA endonuclease is an activated Cas9 endonuclease, thereby introducing a double strand break into the target nucleic acid upon binding to the target nucleic acid as part of a complex with the gRNA. In some embodiments, the double strand break is repaired by HDR to insert the donor DNA molecule into the genome of the host cell. The methods described herein can use a variety of different Cas9 endonucleases. For example, Cas9 nuclease can be utilized that requires a NGG Protospacer Adjacent Motif (PAM) immediately 3' to the region targeted by the guide RNA for Cas9 nuclease. As another example, Cas9 proteins with orthogonal PAM motif requirements can be used to target sequences that do not have adjacent NGG PAM sequences. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Evelt et al (Nature Methods 10: 1116-1121 (2013)).

In some cases, the Cas9 protein is a nickase, thus introducing single strand breaks or nicks into the target nucleic acid upon binding to the target nucleic acid as part of a complex with the guide RNA. A pair of Cas9 nickases, each binding a different guide RNA, can target 2 adjacent (proximal) sites of the target genomic region and thus introduce a pair of adjacent single-strand breaks into the target genomic region. Nickase pairs can provide enhanced specificity because off-target action can result in a single nick, which is usually repaired atraumatically by base excision repair mechanisms. Exemplary Cas9 nickases include Cas9 nuclease with D10A or H840A mutations (see, e.g., Ran et al, "Double nicks formed by RNA-guided CRISPR Cas9 for enhanced genome editing specificity," Cell 154(6):1380-1389 (2013)).

Host cell

In another embodiment, provided herein is a modified host cell produced by any method of disrupting a target site in the genome of a host cell or integrating one or more exogenous nucleic acids described herein genomically. Also provided are populations of modified host cells produced by any of the methods provided herein. In particular embodiments, also provided are host cell populations in which at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, or any percentage therebetween, is altered using any of the methods provided herein.

Suitable host cells include any cell that requires integration of a donor DNA molecule of interest into a target site in the host cell genome. In some embodiments, the host cell is a cell capable of undergoing homologous recombination. In other embodiments, the host cell is a cell capable of gap repair. In some embodiments, the host cell is a yeast cell. In some embodiments, the yeast cell is a pichia cell. In a specific embodiment, the pichia cell is a pichia pastoris cell. In some embodiments, the yeast host cell is a cell suitable for industrial fermentation, such as bioethanol fermentation. In particular embodiments, the cells are adapted to survive high solvent concentrations, high temperatures, increased substrate utilization, nutrient limitation, osmotic pressure, acidity, sulfite and bacterial contamination, or combinations thereof, which are considered stress conditions for industrial fermentation environments. For a list of cell types suitable for integration of one or more donor DNA molecules using the methods described herein, see international application publication No. WO 2015095804.

Method for producing a protein of interest

In another embodiment, provided herein is a method of producing a protein of interest. The method comprises culturing a host cell comprising one or more integrated donor DNA molecules of interest encoding one or more proteins of interest under conditions suitable for the production of the proteins, and recovering the proteins produced by the host cell. In some embodiments, the protein of interest is a protein selected from the group consisting of: antibodies, enzymes, hormones, growth factors, anticoagulants, blood factors, engineered proteins, interferons, interleukins, thrombolytic agents, viral proteins or bacterial proteins.

The materials, compositions, and components disclosed herein can be used in, can be used in conjunction with, can be used in preparation for, or are products of the methods and compositions disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that even if not explicitly recited as each individual or individual combination or collective combination or permutation of these compounds, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of variations which can be made to one or more molecules in the method are discussed, then each and every possible combination and permutation of the variations and the method are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this application including, but not limited to, the steps of methods employing the compositions described herein. As such, if there are a variety of other steps that can be performed it is contemplated that each of these other steps can be performed in conjunction with any specific method step or combination of method steps of the methods described herein, and that each such combination or subset of combinations is specifically contemplated and considered disclosed herein.

The publications cited herein and the materials to which they are cited are specifically incorporated by reference in their entirety.