Extrachromosomal switching auxotrophies progressively by integration (eSwAP-In) for assembly of DNA sequences in yeast

Patent metadata

Field	Value
Publication number	US-11814644-B2
Application number	US-201716315844-A
Country	US
Kind code	B2
Filing date	Jul 7, 2017
Priority date	Jul 7, 2016
Publication date	Nov 14, 2023
Grant date	Nov 14, 2023

Abstract

Compositions, methods and kits are provided. The compositions, methods and kits are for assembly of series of DNA segments in yeast using homologous recombination. The assembled DNA segments are maintained episomally. Yeast made using the methods are included, as are methods of using the yeast to express proteins, and for screening test agents that can affect yeast that are modified to include the assembled DNA segments.

First claim

What is claimed is:

1. A method for producing an extrachromosomal element that is capable of assembly in yeast comprising:
a) providing a vector comprising a bacterial selectable marker, optionally a bacterial artificial chromosome sequence (BAC), and a first yeast selectable marker that is flanked by two restriction endonuclease digestion sites, and a centromere and an autonomously replicating sequence (CEN/ARS) that is functional in yeast, the vector comprising first and second recombination sequences that are non-homologous to the yeast's genome and wherein the first and second recombination sequences flank a second yeast selectable marker that is different from the first yeast selectable marker and the two restriction endonuclease sites when the vector is circular, the vector optionally further comprising a selectable marker that is functional in mammalian cells, and optionally further comprising a self-replicating element for maintaining episomal persistence in human cells;
b) introducing into yeast a linearized vector of a) wherein the vector is linearized with a restriction endonuclease that excises the first yeast selectable marker:
a first set of linear double stranded DNA segments comprising a first DNA segment, a set of interior DNA segments, and a first terminal DNA segment, the first DNA segment comprising at its left end a sequence homologous to the first recombination sequence in the vector and at its right end a sequence that is homologous to a left end of a first interior DNA segment, the set of interior DNA segments having successive left end and right end homology to one another, and the terminal DNA segment having at its right end a sequence that comprises the second yeast selectable marker and a sequence that is homologous to the second recombination sequence in the vector, and

c) allowing homologous recombination of the linearized vector with the first DNA segment, the interior DNA segments and the terminal DNA segment such that the vector is circularized in the yeast by the homologous recombination to produce a first recombined vector comprising a replacement of the first selectable marker with the second selectable marker that is selectable in the yeast using the second yeast selectable marker incorporated into the vector via the terminal DNA segment and further screening for the loss of the first selectable marker;
d) optionally introducing into the yeast comprising the circularized vector of c) a second set of linear double stranded DNA segments comprising interior DNA segments having successive left end and right end homology to one another, and a second terminal DNA segment having at its right end a sequence that comprises the first or a third yeast selectable marker such that the second set of linear double stranded DNA segments and the second terminal DNA segment are homologously recombined into the vector of c) to produce a second recombined vector that is selectable in the yeast by the first or third yeast selectable marker, and subsequently screening for the loss of the first or third selectable marker, and
e) optionally repeating step d) with distinct linear double stranded DNA segments that are selectable by a yeast selectable marker not present in the vector of d).

2. The method of claim 1 , further comprising performing step d).

3. The method of claim 2 , further comprising performing step e).

4. The method of claim 3 , wherein at least the first or the second recombination sequence that is non-homologous to the yeast genome comprises at least 40 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

5. The method of claim 2 , wherein at least the first or the second recombination sequence that is non-homologous to the yeast genome comprises at least 40 contiguous nucleotides of a sequence that is at least 80% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

6. The method of claim 1 , wherein the first and second recombination sequences that are non-homologous to the yeast genome comprise at least 40 nucleotides, and wherein the first and second recombination sequences that are non-homologous to the yeast genome are characterized by:
(i) not being present in the yeast genome wherein the homologous recombination occurs;
(ii) have approximately 45-55% GC content in the at least 40 nucleotides;
(iii) contain no homopolymer segments greater than 5 nucleotides in length;
(iv) do not comprise BsaI, AarI, BceAI, SalI, XhoI, BsmBI, NotI, or I-SceI restriction enzyme recognition sites;
(v) each having distinct sequences from one another.

7. The method of claim 1 , wherein at least the first or the second recombination sequence that is non-homologous to the yeast genome comprises at least 40 contiguous nucleotides of a sequence that is at least 80% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

Assignees

• Univ New York

Inventors

• Boeke Jef D
• Mitchell Leslie A
• Agmon Neta

Classifications

• C12N15/905 Primary

  in yeast · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12N15/64

  General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host · CPC title

• C12N15/65

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title