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Method for constructing thermophilic mycelia strain with high homologous recombination efficiency and application of thermophilic mycelia strain

A technology for Myceliophthora thermophila and Myceliophthora thermophila is applied in the field of genetic engineering and can solve the problems of not improving the efficiency of mammalian cells, increasing the efficiency of site-specific integration of DNA sequences, and unclear knockout.

Inactive Publication Date: 2022-06-07
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In recent years, although some studies have reported that the deletion of key genes in the non-homologous end-joining pathway can improve the efficiency of homologous recombination, the deletion of the same functional gene has completely different effects in different eukaryotic host cells, such as the deletion of Ku70 in Saccharomyces cerevisiae strains It has the effect of increasing the efficiency of site-directed integration of DNA sequences into its genome, while the efficiency of site-specific integration of target genes in mammalian cells lacking Ku70 is not improved
Furthermore, considering that the genetic transformation efficiency of filamentous fungi is low, it is not yet clear whether knocking out key genes in the non-homologous end-joining pathway of filamentous fungi will indirectly affect their genetic transformation

Method used

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  • Method for constructing thermophilic mycelia strain with high homologous recombination efficiency and application of thermophilic mycelia strain
  • Method for constructing thermophilic mycelia strain with high homologous recombination efficiency and application of thermophilic mycelia strain
  • Method for constructing thermophilic mycelia strain with high homologous recombination efficiency and application of thermophilic mycelia strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Construction of Myceliophthora thermophila Genetically Engineered Strain ΔKu80 Knockout of Ku80 Gene

[0029] 1. Construction of Ku80 gene knockout cassette

[0030] To construct the Ku80 gene knockout cassette, the primers are as follows:

[0031] Ku80-F1 (SEQ ID NO: 4), Ku80-R1 (SEQ ID NO: 5);

[0032] Ku80-F2 (SEQ ID NO: 6), Ku80-R2 (SEQ ID NO: 7);

[0033] G418-F1 (SEQ ID NO: 8), G418-R1 (SEQ ID NO: 9).

[0034] Using the genome DNA of Myceliophthora thermophila as the template, the Ku80 gene fragment was amplified with the primers Ku80-F1 / Ku80-R1, and the amplified product was recovered and connected to the pEASY-Blunt vector, transformed into the Trans I clone host, and sequenced to verify the intermediate vector. pEASY-Blunt-Ku80; secondly, using pEASY-Blunt-Ku80 as a template, primers Ku80-F2 / Ku80-R2 were used to amplify the upstream and downstream of the Ku80 gene fragment, and the amplified product was recovered and passed through the geneticin G4...

Embodiment 2

[0039] Example 2 Construction of Myceliophthora thermophila Genetically Engineered Strain ΔKu70 Knockout of Ku70 Gene

[0040] 1. Construction of Ku70 knockout cassette

[0041] The primers used to construct the Ku70 knockout cassette are as follows:

[0042] Ku70-F1 (SEQ ID NO: 10), Ku70-R1 (SEQ ID NO: 11);

[0043] Ku70-F2 (SEQ ID NO: 12), Ku70-R2 (SEQ ID NO: 13);

[0044] G418-F1 (SEQ ID NO: 14), G418-R1 (SEQ ID NO: 15).

[0045]Using the genomic DNA of Myceliophthora thermophila as a template, the Ku70 gene fragment was amplified with the primers Ku70-F1 / Ku70-R1, and the amplified product was recovered and connected to the pEASY-Blunt vector, transformed into a Trans I clone host, and sequenced to verify the intermediate vector. pEASY-Blunt-Ku70; secondly, using pEASY-Blunt-Ku70 as a template, primers Ku70-F2 / Ku70-R2 were used to amplify the upstream and downstream of the Ku70 gene fragment, and the amplified product was recovered and passed through the geneticin G418 r...

Embodiment 3

[0050] Example 3 Homologous recombination efficiency verification in Myceliophthora thermophila genetically engineered strains ΔKu80 and ΔKu70

[0051] 1. Construction of alp1 knockout cassette

[0052] The primers for the alp1 knockout cassette were constructed as follows:

[0053] alp1-F1 (SEQ ID NO: 16), alp1-R1 (SEQ ID NO: 17);

[0054] alp1-F2 (SEQ ID NO: 18), alp1-R2 (SEQ ID NO: 19);

[0055] HygR-F1 (SEQ ID NO: 20), HygR-R1 (SEQ ID NO: 21).

[0056] Using the genome DNA of Myceliophthora thermophila as a template, the alp1 gene fragment was amplified by primers alp1-F1 / alp1-R1, and the amplified product was recovered and connected to the pEASY-Blunt vector, transformed into a Trans I clone host, sequenced and verified to obtain an intermediate vector pEASY-Blunt-alp1; secondly, using pEASY-Blunt-alp1 as a template, primers alp1-F2 / alp1-R2 were used to amplify the upstream and downstream of the alp1 gene fragment, and the amplified product was recovered and passed thr...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a method for constructing a thermophilic mycelia strain with high homologous recombination efficiency and application of the thermophilic mycelia strain. According to the mutant strain delta Ku80 obtained by the invention, the homologous recombination efficiency of a trichoderma thermophile CRISPR-Cas9 gene editing system can be improved to 86% from 57% of a wild type, the workload of genetic transformation of the trichoderma thermophile is reduced, and the homologous recombination efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing a Myceliophthora thermophila strain with high homologous recombination efficiency and its application. Background technique [0002] Myceliophthora thermophila is a thermophilic filamentous ascomycete fungus that can rapidly degrade cellulose and has a high ability to express and secrete cellulase. Compared with the filamentous fungi Trichoderma reesei and Penicillium reesei produced by industrial cellulase, Myceliophthora thermophila has high enzyme activity and strong thermal stability, and has broad application prospects in industrial production. At the same time, the glycan structure produced by Myceliophthora thermophila is more similar to the human glycan form than most other non-mammalian production hosts, showing great application potential in the field of medicine. Therefore, the development of Myceliophthora thermophila as a novel ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/113C12N9/22C12R1/645
CPCC12N15/113C12N9/22C07K14/37C12N2310/20Y02E50/10
Inventor 秦星罗会颖杨坤李金阳王晓璐涂涛苏小运张杰黄火清柏映国王苑王亚茹姚斌
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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