Method for multicopy integration of target gene to saccharomyces cerevisiae genome

A Saccharomyces cerevisiae, multi-copy technology, applied in the field of microorganisms, can solve the problems of difficult to achieve gene integration, affect the target sequence, and cumbersome steps, and achieve the effects of reducing the number of PCR times, short time consumption, and simple operation

Inactive Publication Date: 2020-01-17
SHANGHAI ACAD OF AGRI SCI
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Problems solved by technology

[0007] In the current method of high-copy integration of the target gene using CRISPR/Cas9 technology, the homology arms of the donor DNA are mostly 500bp or even more than 1000bp, and it is necessary to PCR amplify the upper and lower homology arms and the gene expre

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  • Method for multicopy integration of target gene to saccharomyces cerevisiae genome
  • Method for multicopy integration of target gene to saccharomyces cerevisiae genome
  • Method for multicopy integration of target gene to saccharomyces cerevisiae genome

Examples

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Embodiment

[0034] Example Green Fluorescent Protein Gene (gfp) Multi-copy Integration at Saccharomyces cerevisiae S288c Strain rDNA Site

[0035] 1. Expression of Cas9 nuclease in Saccharomyces cerevisiae strain

[0036]Transform the Cas9 nuclease expression vector pHCas9 into the Saccharomyces cerevisiae strain by the lithium acetate method (other methods such as electroporation are also available), screen the transformants on a YPD solid plate containing noursstatin (100 μg / mL), and extract the transformants The genome was verified by PCR, and the PCR products were purified and sent for sequencing, and the strains with correct sequencing results were cultured in YPD medium containing noerscencin (100 μg / mL) for later use.

[0037] 2. Select the target sequence in the non-transcribed region of rDNA repeat unit NTS2 and extract the vector plasmid for corresponding transcribed gRNA

[0038] The selected target sequence of the present invention is located in the NTS2 non-transcribed regio...

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Abstract

A method for multicopy integration of a target gene to a saccharomyces cerevisiae genome includes: selecting target sequences, designing homologous arm primers in length of 15-90bp on two sides of each cleavage site in the corresponding target sequences to primer front ends of amplification target gene cassettes to synthesize homologous arm loaded primers, and amplifying the target gene cassettesto obtain donor DNA fragments; subjecting transcribed gRNA vectors in corresponding target sequences and the donor DNA fragments to co-transformation into Cas9 nuclease expressing saccharomyces cerevisiae to realize gene integration; removing Cas9 nuclease expression vectors and the transcribed gRNA vectors by a non-resistant flat plate. The method has advantages that the homologous arm primers ontwo sides of each cleavage site is in sequence length of 15-90bp, and by respective adding of the homologous arm primers to upstream and downstream primers of the target gene cassettes, an integratorgene template can be obtained directly through once PCR, so that multi-copy integration of target genes is realized. Compared with an existing method, the method has advantages of low time consumption and simplicity in operation.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for integrating multiple copies of a target gene into the genome of Saccharomyces cerevisiae. Background technique [0002] The Saccharomyces cerevisiae system is one of the most important exogenous gene expression systems and has been widely used in the synthesis of biological agents. Saccharomyces cerevisiae has an efficient homologous recombination mechanism, as long as there is a 30-50bp homologous segment between two DNA molecules, homologous recombination can be carried out accurately and effectively. According to the characteristics of Saccharomyces cerevisiae that is prone to homologous recombination, using repetitive sequences as homologous recombination sites of integration vectors is an effective way to increase the copy number of exogenous genes in the yeast genome. [0003] The rDNA in the yeast genome has 100-200 repeating units, and the copy number has a sig...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12R1/865
CPCC12N9/22C12N15/905
Inventor 蒋玮唐雪明
Owner SHANGHAI ACAD OF AGRI SCI
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