# US 2014/0154807 A1 · Method for homologous recombination

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- Number: US 2014/0154807 A1
- Document ID: US20140154807A1
- Status: Published application
- Kind: A1
- Application number: 14/146,858
- Filed: 2014-01-03
- Published: 2014-06-05
- Earliest claimed: 2007-03-21
- Inventors: Hesselien TOUW-RIEL; Marco Alexander BERG VAN DEN
- Web page: https://patentgrep.com/patent/US20140154807A1

## CPC

- C12N 15/80: for fungi
- C07K 14/385: from Penicillium
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12N 15/902: {using homologous recombination}
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention discloses a method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a replacement sequence of interest comprising: modifying a parent eukaryotic cell with a preference for NHR to provide a eukaryotic cell having an increased HR/NHR ratio as compared to the parent cell, providing two sets of DNA molecules of which the first set comprises DNA molecules each comprising a first non-functional fragment of the replacement sequence of interest flanked at its 5′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence and the second set comprises DNA molecules each comprising a second non-functional fragment of the DNA replacement sequence of interest overlapping with the first non-functional fragment and flanked at its 3′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence, wherein the first and second non-functional fragments become functional upon recombination; transforming the modified eukaryotic cells with both sets of DNA molecules; and growing the cells to obtain transformed progeny cells having a DNA molecule from the first set recombined with a DNA molecule from the second set into a DNA molecule comprising a functional replacement sequence and having the target sequence replaced by the functional replacement sequence.

## Claims

### Claim 1

A method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a replacement sequence of interest comprising:
modifying a parent eukaryotic cell with a preference for non-homologous recombination to provide a eukaryotic cell having an increased homologous to non-homologous recombination ratio as compared to the parent cell,
providing two sets of DNA molecules of which the first set comprises DNA molecules each comprising a first non-functional fragment of the replacement sequence of interest flanked at its 5′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence and the second set comprises DNA molecules each comprising a second non-functional fragment of the DNA replacement sequence of interest overlapping with the first non-functional fragment and flanked at its 3′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence, wherein the first and second non-functional fragments become functional upon recombination;
transforming the modified eukaryotic cells with both sets of DNA molecules; and
growing the cells to obtain transformed progeny cells having a DNA molecule from the first set recombined with a DNA molecule from the second set into a DNA molecule comprising a functional replacement sequence and having the target sequence replaced by the functional replacement sequence.

### Claim 2 (depends on 1)

The method of claim 1, wherein modifying the parent eukaryotic cell is achieved by increasing the efficiency of the homologous recombination pathway and/or decreasing the efficiency of the non-homologous recombination pathway.

### Claim 3 (depends on 1)

The method of claim 1, wherein modifying the parent eukaryotic cell is achieved by down regulating, preferably inactivating, equivalents of the yeast genes KU70, KU80, RAD50, MRE11, XRS2, LIG4, LIFL, NEIL and/or SIR4, involved in the non-homologous recombination pathway.

### Claim 4 (depends on 1)

The method of claim 1, wherein the two sets of DNA molecules are labeled with different fluorescent dyes for each set and the step of growing the cells to obtain transformed progeny cells comprises subjecting the cells to selection using fluorescence activated cell sorting.

### Claim 5 (depends on 1)

The method of claim 1, wherein the replacement sequence of interest comprises a selection marker, a modified version of the target sequence and/or additional copies of a sequence of interest being present in the genome of the eukaryotic cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the replacement sequence of interest comprises a selection marker and the step of growing the cells to obtain transformed progeny cells comprises selecting the cells by expression of the functional selection marker.

### Claim 7 (depends on 6)

The method of claim 6, wherein the DNA molecules of the first set and/or the DNA molecules of the second set comprise an additional replacement sequence of interest.

### Claim 8 (depends on 6)

The method of claim 6, wherein the selection marker is amdS.

### Claim 9 (depends on 1)

The method of claim 1, wherein the DNA sequences flanking the functional replacement sequence have a degree of identity to a chromosomal DNA sequence flanking the target sequence of at least 80%.