Method for verifying feasibility of inserting CRISPR-Cas9 system mediated target gene into candida utilis

A technology for producing Candida utilis and target genes, which can be applied to other methods of inserting foreign genetic materials, microorganism-based methods, genetic engineering, etc., and can solve problems such as unidentified

Active Publication Date: 2019-10-08
GUANGDONG QIZHI BIOTECHNOLOGY CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] If the Cas9 system can be applied to the gene insertion operation of Candida utilis, it will help the realization of the new expression system of Candida utilis, combining the According to the characteristics of Candida utilis itself, providing a new method of gene insertion in Candida utilis can provide a new way for exogenous gene expression in Candida utilis and provide a basis for the wide application of Candida utilis. Whether the Cas9 system in the art can be applied to the gene insertion operation of Candida utilis has not yet been confirmed

Method used

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  • Method for verifying feasibility of inserting CRISPR-Cas9 system mediated target gene into candida utilis
  • Method for verifying feasibility of inserting CRISPR-Cas9 system mediated target gene into candida utilis
  • Method for verifying feasibility of inserting CRISPR-Cas9 system mediated target gene into candida utilis

Examples

Experimental program
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Effect test

Embodiment 1

[0097] CRISPR-Cas9-mediated targeted integration and expression of reporter gene GFP

[0098] Candida utilis ATCC22023 used in this example was purchased from Guangdong Microbial Culture Collection Center; yeast Cas9 expression vector p415-GalL-hCas9 and sgRNA expression vector p426-crRNA were provided by Addgene; Candida utilis free expression Vector: pCuARS-GAPGαA, pCuARS-GAPZαAm, the structure and special sequence of pCuARS-GAPGαA are as follows Figure 24 As shown, the structure and special sequence of pCuARS-GAPZαAm are as Figure 25 Shown; Escherichia coli cloning vector pMD19-T was purchased from Dalian Bao Biological Engineering Co., Ltd.; primer synthesis and DNA sequencing were completed by Invitrogen; primer sequence-F represents the upstream primer, and primer sequence-R represents the downstream primer.

[0099] 1. Construction and verification of Candida utilis Cas9 expression vector

[0100] 1. Extract the plasmids p415-GalL-hCas9 and pCuARS-PGAPGαA, and then ...

Embodiment 2

[0236] CRISPR-Cas9-mediated targeted insertion of pectin methylesterase gene into the genome of Candida utilis

[0237] The expression vector used in this example is from the same source as the expression vector in Example 1 above. Primer synthesis and DNA sequencing were completed by Invitrogen Company. Candida utilis episomal expression vector: pCuARS-PGKG-GFP, its specific structure and special sequence such as Figure 26 Shown; Plasmid pCGGαAL-pmeA (containing the GAP promoter of Candida utilis, G418 resistance gene) with pmeA gene, its specific structure and special sequence are as follows Figure 27 shown. Both the pGlu-sgRNA-PGK plasmid and the Candida utilis containing Cas9 were constructed in Example 1; the primer sequence-F represents the upstream primer, and the primer sequence-R represents the downstream primer.

[0238] Based on the existing pGlu-sgRNA-PGK plasmid and Cas9-containing Candida utilis, a recombinant donor fragment containing left and right homology...

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Abstract

The invention discloses a method for verifying the feasibility of inserting a CRISPR-Cas9 system mediated target gene into candida utilis. The method includes the following steps that S1, an expression vector of the candida utilis containing Cas9 segments is constructed; S2, tRNAGlu genes and downstream expression units of sgRNA are amplified, PCR link is overlapped and a restriction enzyme cutting site is introduced, and after connection, the amplified tRNAGlu genes and downstream expression units of the sgRNA are combined with the vector of the candida utilis to obtain sgRNA initial recombinant plasmid; S3, pre-insertion and target sequences are selected, a sticky tail end is added to the 5' end of a target and complementary sequence, and double chain segments are formed by annealing andthen connected with the initial recombinant plasmid after enzyme digestion to obtain sgRNA mature recombinant plasmid; S4, a recombinant donor segment containing a left homologous arm and a right homologous arm of a pre-insertion sequence is constructed; and S5, the sgRNA recombinant plasmid and the recombinant donor segment are used for transforming the candida utilis carrying Cas9. The feasibility of inserting the CRISPR-Cas9 system mediated target gene into the candida utilis is verified by identifying the expression of the donor segment.

Description

technical field [0001] The invention relates to the field of gene insertion, and more specifically, relates to a method for verifying the feasibility of CRISPR-Cas9 system-mediated insertion of a target gene into Candida utilis. Background technique [0002] Yeast is a simple single-celled eukaryote, which has a strict eukaryotic gene expression regulation system, and also has the characteristics of rich species, easy cultivation, and most of them are non-pathogenic. Saccharomyces cerevisiae and Pichia pastoris have been used as cell factories to produce various exogenous proteins, but they still have many shortcomings as hosts, such as the phenomenon of plasmid deletion in a large number of host yeasts; Saccharomyces cerevisiae usually loses protein during fermentation Ethanol is produced, which is not conducive to high-density fermentation; when recombinant proteins are expressed in yeast, hyperglycosylation usually occurs; when Pichia pastoris expresses foreign proteins, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/90C12R1/72
CPCC12N15/815C12N15/905C12N2810/10
Inventor 刘泽寰林蒋海戴钰
Owner GUANGDONG QIZHI BIOTECHNOLOGY CO LTD
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