Evolution of whole cells and organisms by recursive sequence recombination

A method of enhancing tissue-specific expression of a protein in a transgenic animal, comprising

• (i.) recombining at least first and second forms of a gene encoding a protein, the forms differing from each other in at least two nucleotides, to produce a library of chimeric genes,
• (ii) screening the library to identify at least one chimeric gene, which as a component of a transgene, confers enhanced expression of the protein in cells from the tissue relative to a transgene containing the wildtype form of the gene,
• (iii.) recombining the at least one chimeric gene with a further form of the gene, the same or different from the first and second forms, to produce a further library of chimeric genes,
• (iv.) screening the further library for at least one further chimeric gene that as a component of a transgene confers enhanced expression of the protein in cells from the tissue relative to a transgene comprising the chimeric gene in the previous screening step,
• (v) repeating (iii) and (iv), as necessary until the further chimeric gene confers a desired level of expression in cells from the tissue

A method of performing in vivo recombination, the method comprising:

• providing a cell incapable of expressing a cell septation gene,
• introducing at least first and second segments from at least one gene into a cell, the segments differing from each other in at least two nucleotides, whereby the segments recombine to produce a library of chimeric genes;
• and,
• selecting a chimeric gene from the library having an acquired function.

A method of predicting efficacy of a drug in treating a viral infection, the method comprising

• (i) recombining a nucleic acid segment from a virus, whose infection is inhibited by a drug, with at least a second nucleic acid segment from the virus, the second nucleic acid segment differing from the nucleic acid segment in at least two nucleotides, to produce a library of recombinant nucleic acid segments.
• (ii) contacting host cells with a collection of viruses having genomes including the recombinant nucleic acid segments in a media containing the drug, and collecting progeny viruses resulting from infection of the host cells,
• (iii.) recombining a recombinant DNA segment from a first progeny virus with at least a recombinant DNA segment from a second progeny virus to produce a further library of recombinant nucleic acid segments,
• (iv.) contacting host cells with a collection of viruses having genomes including the further library or recombinant nucleic acid segments, in media containing the drug, and collecting further progeny viruses produced by the host cells, and,
• (v) repeating (iii) and (iv), as necessary, until a further progeny virus has acquired a desired degree of resistance to the drug, whereby the degree of resistance acquired and the number of repetitions of (iii.) and (iv) needed to acquire it provide a measure of the efficacy of the drug in treating the virus

A method of predicting efficacy of a drug in treating an infection by a pathogenic microorganism, the method comprising.

• (i.) transforming a plurality of cells of the microorganism with a library of DNA fragments at least some of which undergo recombination with segments in the genome of the cells to produce modified microorganism cells,
• (ii) propagating modified microorganisms in a media containing the drug, and recovering surviving microorganisms,
• (iii) recombining DNA from surviving microorganisms with a further library of DNA fragments at least some of which undergo recombination with cognate segments in the DNA from the surviving microorganisms to produce further modified microorganisms cells,
• (iv) propagating further modified microorganisms, in media containing the drug and collecting further surviving microorganisms,
• (v) repeating (iii) and (iv), as necessary, until a further surviving microorganism has acquired a desired degree of resistance to the drug, whereby the degree of resistance acquired and the number of repetitions of (iii) arid (iv) needed to acquire it provide a measure of the efficacy of the drug in killing the pathogenic microorganism

A method of evolving a cell to acquire a desired function, the method comprising:

• (a) providing a population of different cells,
• (b) culturing the cells under conditions whereby DNA is exchanged between cells, forming cells with hybrid genomes,
• (c) screening or selecting the cells for cells that have evolved toward acquisition of a desired property, and,
• (d) repeating steps (b) and (c) with the selected or screened cells forming the population of different cells until a cell has acquired the desired property.

A method of evolving a cell to acquire a desired property, comprising:

• (i.) forming protoplasts of a population of different cells,
• (ii.) fusing the protoplasts to form hybrid protoplasts, in which genomes from the protoplasts recombine to form hybrid genomes;
• (iii.) incubating the hybrid protoplasts under conditions promoting regeneration of cells, thereby producing regenerated cells.
• (iv) optionally repeatedly forming protoplasts from the regenerated cells, fusing the protoplasts to form hybrid protoplasts, in which genomes from the protoplasts recombine to form additional hybrid genomes, incubating the additional hybrid protoplasts under conditions promoting regeneration of cells, thereby producing additional regenerated cells,
• (v) selecting or screening to isolate regenerated cells or additionally regenerated cells that have evolved toward acquisition of the desired property, and,
• (vi) repeating steps (i)-(v) with regenerated cells in step (iii) or additional regenerated cells in step (iv) being used to form the protoplasts in step (i) until the regenerated cells have acquired the desired property

A method of evolving a cell to acquire a desired property, comprising

• (i.) forming protoplasts of a population of different cells
• (ii.) fusing the protoplasts to form hybrid protoplasts, in which genomes from the protoplasts recombine to form hybrid genomes,
• (iii) incubating the hybrid protoplasts tinder conditions promoting regeneration of cells, thereby producing regenerated cells,
• (iv) repeatedly forming protoplasts from the regenerated cells, fusing the protoplasts to form hybrid protoplasts. in which genomes from the protoplasts recombine to form additional hybrid genomes, incubating the additional hybrid protoplasts under conditions promoting regeneration of cells, thereby producing additional regenerated cells, and,
• (v) selecting or screening to isolate regenerated cells or additionally regenerated cells that have evolved toward acquisition of the desired property

A method of evolving a cell toward acquisition of a desired property comprising

• (a) providing a population of different cells,
• (b) isolating DNA from a first subpopulation of the different cells and encapsulating the DNA in liposomes,
• (c) forming protoplasts from a second subpopulation of the different cells,
• (d) fusing the liposomes with the protoplasts whereby DNA from the liposomes is taken Lip by the protoplasts and recombines with the genomes of the protoplasts,
• (e) incubating the protoplasts under regenerating conditions,
• (f) selecting or screening for regenerating or regenerated cells that have evolved toward the desired property, and,
• (g) repeating steps (a)-(t) with the cells that have evolved toward the desired property forming the population of different cells in step (a)

A method of evolving a DNA segment for acquisition of a desired properly the method comprising

• (a) providing a library of variants of the segment, each variant cloned into separate copies of an artificial chromosome,
• (b) introducing the copies of the artificial chromosome into a population of cells,
• (c) culturing the cells under conditions whereby sexual recombination occurs between cells and homologous recombination occurs between copies of the artificial chromosome bearing the variants, and,
• (d) screening or selecting for variants that have evolved toward acquisition of the desired property

A method of poolwise recombination and selection of cells, the method comprising:

• (i) introducing a library of DNA fragments into a plurality of recipient cells, whereby a plurality of fragments of the library undergo recombination with a genome or an episome of a plurality of recipient cells to produce a plurality of modified cells, whereby the plurality of modified cells comprise a plurality of different modified fragments;
• (ii) fusing at least two of the modified cells, whereby genomes of the at least two modified cells recombine,
• (iii) segregating the resulting fused cell to produce a plurality of regenerated cells, each with a single genome, and,
• (iv) selecting the regenerated cells for a desired properly

A method of increasing resistance to virus infection in cells comprising

• introducing a shuffled library comprising at least one shuffled interferon gene into animal cells, thereby creating an initial library of animal cells or animals,
• challenging the initial library with the virus,
• selecting animal cells or animals from the initial library which are resistant to the virus,
• recovering a plurality of transgenes from a plurality of animal cells or animals which are resistant to the virus;
• shuffling the plurality of transgenes to produce an evolved library of animal cells or animals
• challenging the evolved library of animal cells or animals with the virus;
• and,
• selecting cells or animals from the evolved library the which are resistant to the virus

A method of whole genome shuffling in plants, comprising:

• isolating genomic DNA from a source plant and fragmenting the genomic DNA;
• cloning the resulting fragmented genomic DNA into a cloning vector to produce a primary genomic library;
• transducing the primary genomic library into target plant cells,
• selecting the resulting transduced target plant cells, or a plant comprising transduced target plant cells for acquisition of a desired trait or property,
• isolating genomic DNA from a plurality of target cells which display the desired property,
• cloning the isolated genomic DNA from the plurality of target cells into a cloning vector to produce a secondary genomic library,
• transducing the secondary genomic library into the target plant cells;
• and,
• selecting the resulting transduced target plant cells, or a plant comprising transduced target plant cells for acquisition of a desired trait or property, thereby providing an improved plant with a new phenotype

A method of selecting for increased copy number of a nucleic acid sequence contributing to a desired phenotype of a target cell comprising:

• providing a genomic library in a suicide vector comprising a dose-sensitive selectable market,
• transducing the genomic library into a population of target cells;
• selecting the population of target cells for increasing doses of the selectable marker under conditions in which the suicide vector does not replicate episomally,
• selecting a plurality of target cells for the desired phenotype;
• recombining the selected plurality of target cells, and,
• selecting the selected plurality of target cells for the desired phenotype

A method of producing a library of diverse multicellular organsims, the method comprising:

• providing a pool of male gametes and a pool of female gametes, wherein at least one of the male pool or the female pool comprises a plurality of different gametes derived from different strains of a species or different species, wherein the male gametes fertilize the female gametes,
• permitting at least a portion of the resulting fertilized gametes to grow into reproductively viable organisms,
• repeatedly crossing the reproductively viable organisms to produce a library of diverse organisms, and,
• selecting the library for a desired trait or property.

A method for whole genome shuffling through organized heteroduplex shuffling, the method comprising

• (a). providing chromosomal DNA of an organism which is targeted for shuffling, digesting the chromosomal DNA with one or more restriction enzymes, ligating the chromosomal DNA into a cosmid, the cosmid comprising at least two rare restriction enzyme recognition sites, aliquoting, purifying, and storing sufficient cosmids to represent a complete chromosome,
• (b) mutagenizing aliquots of the library in vitro using a mutagen,
• (c). transfecting a sample from a plurality of the mutagenized aliquots into a population of target cells,
• (d) assaying resulting transfectants for phenotypic improvements;
• (e) growing transfected cells harboring a mutant library of the identified cosmid(s) on media and screening the resulting cell colonies for independent mutants conferring an desired phenotype;
• (f) isolating and pooling DNA from cells identified in the screening,
• (g). dividing the selected pools and digesting at least one sample with a rare-cutting restriction enzyme, pooling the cleaved samples, denaturing the samples, reannealing the samples and religating the samples, and,
• (h). transfecting target cells with the resulting heteroduplexes and propagating the cells to allow recombination to occur between the strands of the heteroduplexes in vivo