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An Efficient Yeast Chromosome Fusion Method

A chromosome and yeast technology, applied in the field of efficient yeast chromosome fusion, can solve the problems of difficult DNA separation, limited application, etc.

Active Publication Date: 2021-08-27
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Saccharomyces cerevisiae naturally has 16 linear chromosomes, ranging in size from 230kbp to 1530kbp, which makes it difficult to separate large fragments of DNA over 200kbp cloned in yeast from chromosomes
This defect limits the application of Saccharomyces cerevisiae as a host for heterologous gene expression, large fragment DNA cloning, and genome modification

Method used

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  • An Efficient Yeast Chromosome Fusion Method
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  • An Efficient Yeast Chromosome Fusion Method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Example 1 , two chromosomal fusions

[0136] Using Saccharomyces cerevisiae BY4742 as the starting strain, ChrVI (270kbp) and ChrI (230kbp), ChrIX (440kbp) and ChrII (813kbp) were selected for fusion (Table 2).

[0137] Table 2. Two chromosome fusion information

[0138]

[0139] The Cas9 expression plasmid pleucas9 was transformed into BY4742 yeast cells to obtain strain BY4742 / pleucas9. Plasmid pleucas9 contains the promoter Tef1 to allow constitutive expression of Cas9 in yeast. The plasmid contains the yeast replication region (CEN6ARS4) and the selection marker (LEU2), which is single-copy replication in yeast; also contains the E. coli replication region (derived from pBR322) and the selection marker ampicillin (Ampicillin), which is high in E. coli Copy copy.

[0140] When ChrVI and ChrI are fused, the ChrVI centromere is knocked out, and 148410-148459bp, 148725-148774bp, 148775-149002bp on ChrVI are selected as the left homology arm, right homology arm, ...

Embodiment 2

[0144] Example 2 , three chromosome fusions

[0145] In this example, Saccharomyces cerevisiae BY4742 was used as the starting strain, and ChrVI (270kbp), ChrI (230kbp) and ChrII (813kbp) were selected for fusion, as shown in Table 3.

[0146] Table 3. Three chromosome fusion information

[0147]

[0148] ChrVI (270 kbp), ChrI (230 kbp) and ChrII (813 kbp) were fused to knock out the centromere of ChrVI and ChrII. Select 148410-148459bp, 148725-148774bp, 148775-149002bp on ChrVI as the left homology arm, right homology arm and forward repeat sequence of centromere knockout; 269212-269615bp as the left homology arm of the ChrVI-I fusion component . 2893-3294bp and 3295-3519bp on ChrI serve as the right homology arm and forward repeat of ChrVI-I fusion assembly; 202775-203182bp serve as the left homology arm of ChrI-II fusion assembly. 8680-9089bp and 9090-9359bp on ChrII serve as the right homology arm and forward repeat of the ChrI-II fusion component. Select 237974-2...

Embodiment 3

[0152] Example 3 , two sets of two chromosomal fusions

[0153] In this example, Saccharomyces cerevisiae BY4742 was used as the starting strain, and ChrVI (270kbp) and ChrI (230kbp), ChrIX (440kbp) and ChrII (813kbp) were simultaneously fused (Table 4).

[0154] Table 4. Two sets of chromosome fusion information

[0155]

[0156] When ChrVI (270kbp) and ChrI (230kbp), ChrIX (440kbp) and ChrII (810kbp) are fused at the same time, pgRNA1-6, ChrVI centromere knockout module (URA3), ChrVI-I fusion module (URA3), ChrIX The centromeric knockout module (LYS2) and the ChrIX-II fusion module (LYS2) were transferred into BY4742 / pleucas9 using the protoplast fusion transformation method described in Example 1 (see Example 1 for the selection of homology arms).

[0157] Preliminary verification was carried out according to the colony PCR method described in Example 1 (see Example 1 for verification primers), two experiments were carried out, the number of fusion transformants was 1...

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Abstract

The invention relates to an efficient yeast chromosome fusion method. The invention discloses a method for performing yeast chromosome fusion by using a CRISPR / Cas9 system and a homologous recombination system in yeast. The method of the invention can realize rapid and efficient chromosome fusion.

Description

technical field [0001] The invention relates to the fields of microbial synthetic biology, genome engineering and molecular biology, in particular to an efficient yeast chromosome fusion method. Background technique [0002] Studies have shown that yeast can be used as a heterologous expression host for rare drug precursors artemisinic acid, taxene, hydrocortisone and new energy butanol. Because of its efficient homologous recombination ability, it can be used as a host strain for cloning of large DNA fragments and for the assembly and modification of other microbial genomes, especially for strains that are difficult to cultivate under laboratory conditions and lack effective genetic manipulation methods. [0003] Saccharomyces cerevisiae is one of the model organisms with the most thorough basic research and the most extensive industrial application. However, Saccharomyces cerevisiae naturally has 16 linear chromosomes, ranging in size from 230kbp to 1530kbp, which makes i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/113C12R1/865
CPCC12N15/113C12N15/905C12N2310/10
Inventor 覃重军邵洋洋薛小莉鲁宁
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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