Site-directed mutation method for genomes of saccharomyces cerevisiae

A genome-specific, Saccharomyces cerevisiae technology, applied in the field of Saccharomyces cerevisiae genome site-directed mutation, can solve the problems of long time, limited success rate, high cost, etc.

Inactive Publication Date: 2016-06-01
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the traditional gene knockout method requires a series of steps such as complex targeting vect...

Method used

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  • Site-directed mutation method for genomes of saccharomyces cerevisiae
  • Site-directed mutation method for genomes of saccharomyces cerevisiae
  • Site-directed mutation method for genomes of saccharomyces cerevisiae

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Using CRISPR / Cas9 technology, a single point mutation was introduced into sites 14 and 16 on chromosome V of S. figure 1 ), including the following steps:

[0042] 1. The sequence at position 14 is "ctggagatgAaccaatgattTGG" (as shown in SEQIDNo.1), wherein TGG is a PAM sequence, and A is the site to be mutated; the sequence after introducing a single point mutation is "ctggagatgCaccaatgattTGG" (as shown in SEQIDNo.2 Shown), wherein TGG is the PAM sequence, and C is the site after mutation. The sequence at position 16 is "ttagctagcaaAcccttagaac" (as shown in SEQIDNo.3), where A is the site with a mutation, and the sequence after introducing a single point mutation is "ttagctagcaaCcccttagaac" (as shown in SEQIDNo.4), where C is the mutation post site. Site 14 was selected as the Cas9 cleavage target site, and site 16 was selected as the co-transformation mutation site.

[0043] 2. Construct the guide-RNA plasmid of site 14 to be repaired, and its construction steps are...

Embodiment 2

[0068] Using CRISPR / Cas9 technology, the 9 sites on chromosome V of Saccharomyces cerevisiae synthetic type ( image 3 ) introducing a single point mutation, comprising the following steps:

[0069] 1. The sequence to be introduced into the single point mutation is "aaatacgaagaacCattttgCGG" (as shown in SEQIDNo.15), wherein CGG is a PAM sequence, and C is the site to be repaired; the sequence after introducing the single point mutation is "aaatacgaagaacGattttgCGG" (as shown in SEQIDNo. .16), where CGG is the PAM sequence, and G is the repaired site. Site 9 was selected as the Cas9 cleavage target site, and site 15-2 was selected as the co-transformation mutation site.

[0070] 2. Construct the guide-RNA plasmid of site 9 to be repaired, the construction steps are as follows:

[0071] a) Select the protospacer as aaatacgaagaacCattttg;

[0072] b) synthesize primers "GCAGTGAAAGATAAATGATCaaatacgaagaacCattttgGTTTTAGGCTAGAAATAGC" (as shown in SEQ ID No. 17) and "GCTATTTCTAGCTCTA...

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Abstract

The invention relates to the field of microorganisms, in particular to a site-directed mutation method for genomes of saccharomyces cerevisiae. To-be-repaired single-base mutation sites on the genomes of the saccharomyces cerevisiae can be repaired by the aid of CRISPR/Cas9 technologies. Effects can be realized near the to-be-repaired sites by Cas9 proteins under the guidance actions of guide RNA (ribonucleic acid) if the mutation sites are positioned in PAM sequences (NGG) or front 11bp of the PAM sequences, and the genomes of the saccharomyces cerevisiae can be subjected to double-strand break at cut positions, so that donor DNA (deoxyribonucleic acid) can be efficiently recombined. Compared with existing results, the site-directed mutation method has the advantages that the sites of the genomes of the saccharomyces cerevisiae can be efficiently and quickly mutated, and screening markers do not need to be integrated for the genomes of the saccharomyces cerevisiae.

Description

technical field [0001] The invention relates to the field of microbes, in particular to a method for site-directed mutation of the brewer's yeast genome. Background technique [0002] As a model strain of eukaryotes, Saccharomyces cerevisiae is widely used in many fields such as medicine and food, and the site-directed mutation of Saccharomyces cerevisiae genes or chromosomal DNA sequences provides a great reference for the study of eukaryotic gene functions. The introduction of site-directed mutations in the Saccharomyces cerevisiae genome is generally accomplished by a two-step method: 1. Use the homologous recombination of Saccharomyces cerevisiae to insert a selection marker at the position to be introduced; Genomic single point mutation. However, this method needs to use PCR to construct an expression cassette carrying a screening marker, and the experiment cycle and operation are cumbersome, and generally only one target site can be subjected to site-directed mutation...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12R1/865
CPCC12N1/185C12R2001/865
Inventor 元英进谢泽雄李炳志
Owner TIANJIN UNIV
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