Hansenula polymorpha gene editing system and application thereof, and gene editing method

A Hansenula polymorpha gene editing technology, applied in the field of Hansenula polymorpha gene editing system, can solve the problems of low efficiency and homologous recombination efficiency, achieve high genome editing efficiency, expand application potential, The effect of improving vitality

Active Publication Date: 2021-08-17
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the CRISPR-Cas9 system can be used to realize the genome editing process, the efficiency is relatively low, especially the efficiency of homologous recombination is far from meeting the requirements of host bacteria for cell factories

Method used

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  • Hansenula polymorpha gene editing system and application thereof, and gene editing method
  • Hansenula polymorpha gene editing system and application thereof, and gene editing method
  • Hansenula polymorpha gene editing system and application thereof, and gene editing method

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Experimental program
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Effect test

Embodiment 1

[0060] CRISPR / Cas9 system construction

[0061] (1) Integrated expression of CAS9 gene

[0062] The starting strain of the present invention, Hansenula polymorpha, was purchased from China General Microorganism Culture Collection Center (CGMCC), and the strain preservation number is CGMCC No. 2.2412.

[0063] Routine molecular biology operations such as PCR amplification, enzyme digestion and connection in the present invention follow standard procedures. High-fidelity PCR enzyme PrimeSTAR, DNA maker (DL2,000, DL10,000 and DL15,000), Q.cut restriction endonuclease, etc. were purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0064] High-fidelity PCR enzyme (Phanta Super-Fidelity DNA Polymerase), rapid verification taq enzyme (2×Taq Master Mix (Dye Plus)) and ClonExpress II One Step Cloning Kit were purchased from Vazyme Biotech Co., Ltd. ., Ltd).

[0065] Master Mix was purchased from NEB.

[0066] Plasmid extraction kits, PCR purification kits and gel recove...

Embodiment 2

[0078]Promotes homologous recombination-mediated DNA repair mechanisms in Hansenula

[0079] In Saccharomyces cerevisiae, when a double-stranded DNA molecule breaks, proteins such as Mre11 and Sae2 form a complex to cut the DNA end at the break to form a single-stranded DNA. Subsequently, the homologous recombination-related protein Rad52 binds to single-stranded DNA molecules, recruits various HR-related proteins including Rad51 to form a complex, and prepares for chain expansion and D-loop formation. Finally, through the search of the homologous template sequence, on the effect of DNA polymerase (Pol) and ligase, realize the homologous integration of template DNA molecule (the reaction process is as follows Figure 4 shown).

[0080] Therefore, the inventors expressed HR-related proteins (Rad51, Rad51 and Sae2) from Saccharomyces cerevisiae and HR-related proteins (HpRad51 and HpRad52) from Hansenula cerevisiae individually and in combination. Such as Figure 5 As shown, ...

Embodiment 3

[0082] Impaired Hansenula non-homologous end-joining levels

[0083] In order to down-regulate the expression intensity of the KU80 gene, the inventors integrated the 504bp MET3 gene promoter (pHpMET3) from Hansenula itself into the Hansenula genome by homologous recombination to replace the promoter of the KU80 gene in situ. The obtained transformants were verified by PCR and sequenced correctly, and were used for subsequent experimental analysis. The pHpMET3 promoter is repressed by methionine, therefore, a series of concentrations of methionine were added during the YPD incubation as well as in the selection plate during transformation.

[0084] The specific operation steps include:

[0085] (1) Using the conventional electric shock transformation method, after the electric shock, the cells were incubated with 1 mL of YPD liquid medium containing 1.7mM, 2.5mM, 5.0mM and 10mM methionine for 1h;

[0086] (2) Add methionine at the concentration of the above-mentioned step (1...

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Abstract

The invention discloses a hansenula polymorpha CRISPR / Cas9 gene editing system. The system comprises the following components: (1) Cas9 protein, which is integrated in a hansenula polymorpha genome; and (2) an sgRNA expression vector, which comprises an sgRNA expression cassette, wherein the sgRNA expression cassette is composed of an RNA pol II type promoter, an RNA pol II type terminator, a corresponding ribozyme recognition sequence HH and a corresponding ribozyme recognition sequence HDV. The gene editing system disclosed by the invention has extremely high genome editing efficiency and relatively high homologous integration capability, so the application potential of hansenula polymorpha as a cell factory is further expanded, and the hansenula polymorpha is simpler, quicker, more convenient and more accurate in a genetic modification process.

Description

technical field [0001] The invention belongs to the application field of microbial genetic engineering, and in particular relates to a Hansenula polymorpha gene editing system and its application. Background technique [0002] In nature, there is a class of "methylotrophic" microorganisms that can use C1 compounds (methane, methanol, etc.) as carbon sources for growth and metabolism, and they mainly include some prokaryotic bacteria and methanolic yeasts. Among them, Hansenula polymorpha is an important methylotrophic yeast, which has a wide spectrum of substrates, can naturally utilize carbon sources such as xylose and methanol, and can tolerate high temperatures above 50°C. These excellent characteristics make it Become a potential excellent microbial cell factory. For example, Hansenula's natural xylose utilization and high temperature tolerance have long served as excellent hosts for protein expression and cellulosic ethanol fermentation. As a non-traditional yeast, al...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/113C12N15/81C12N15/90C12N1/19C12R1/78
CPCC12N9/22C12N15/113C12N15/815C12N15/905C12N2310/20
Inventor 周雍进高教琪高宁
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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