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CRISPR/Cas gene editing system applied to trichoderma reesei

A gene editing, Trichoderma reesei technology, applied in microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve the problems of complexity, instability, etc., achieve high enzyme production efficiency, good resistance, improved The effect of enzyme production capacity

Pending Publication Date: 2021-11-05
INST OF BOTANY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to overcome the defects of the prior art, the present invention constructs a novel CRISPR-Cas9 gene editing system, which realizes the simultaneous in vivo transcription of Cas9 and gRNA in Trichoderma reesei for the first time, avoiding the complexity and complexity of retransformation of gRNA after in vitro transcription. Disadvantages such as instability

Method used

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  • CRISPR/Cas gene editing system applied to trichoderma reesei
  • CRISPR/Cas gene editing system applied to trichoderma reesei
  • CRISPR/Cas gene editing system applied to trichoderma reesei

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Construction of gRNA expression cassettes for in vivo transcription of Trichoderma reesei

[0053] Ace1 is a DNA-binding protein containing three Cys(2)-His(2) zinc-finger structures, which is involved in regulating the expression of cellulase and hemicellulase in Trichoderma reesei. After the ace1 gene was knocked out in Trichoderma reesei, the expression levels of cellulase gene and hemicellulase gene of Trichoderma reesei were increased under the same culture conditions.

[0054] The present invention takes knocking out the ace1 gene as an example to construct a Trichoderma reesei CRISPR-Cas9 gene editing system, using a site of the ace1 gene as the target sequence, using the online tool http: / / grna.ctegd.uga.edu / to edit the ace1 gene The gRNA sequence gRNA ace1 (SEQ ID NO: 5) was designed at the front end of the first CDS region. After the gRNA ace1 sequence, the gRNAbackbone sequence was synthesized, and then the viral sequence and the hepatitis virus (...

Embodiment 2

[0063] Embodiment 2 is used for the construction of the CRISPR / Cas9 gene editing system of Trichoderma reesei

[0064] The gRNA expression cassette prepared in Example 1 was inserted into the expression plasmid of the Cas9 gene to obtain the knockout plasmid pCRISPR-ace1 of the ace1 gene, and the CRISPR / Cas9 gene editing system for gene modification of Trichoderma reesei was completed. Wherein, the sequence arrangement of some key elements in the CRISPR / Cas9 gene editing system is as follows: figure 1 shown. The Cas9 gene of the present invention is an unoptimized conventional sequence, and the plasmid construction method is also an operation process well known to those skilled in the art.

[0065] The present invention uses the 4 gRNA expression cassettes prepared in Example 1, 5'TRSV-gRNA ace1-gRNA backbone-3'HDV, 5'ARMV-gRNA ace1-gRNA backbone-3'HDV, 5'CYMVT-gRNA ace1-gRNAbackbone-3'HDV, 5'HH-gRNAace1-gRNA backbone-3'HDV were respectively inserted into the expressi...

Embodiment 3

[0066] Example 3 The method for knocking out the Trichoderma reesei ace1 gene using the CRISPR / Cas9 system

[0067] (1) Plasmid transformation

[0068] The present invention uses Trichoderma reesei RUT-C30 as the starting strain, and uses the pCRISPR-ace1 (a) plasmid constructed in Example 2 to transform the protoplasts of Trichoderma reesei RUT-C30 (the operation method is well known to those skilled in the art, here Steps are omitted here), and the transformants were applied to the screening medium, and after growing for 5-7 days, 50 transformants were picked, transferred to Trichoderma agar medium for propagation, and then the spores were scraped to obtain a spore suspension. The spore suspension was inoculated into the Trichoderma seed medium and cultured overnight to obtain hyphae.

[0069] The screening medium composition described in this embodiment is: 1M sorbitol, 20g / L glucose, 15g / L KH 2 PO 4 , 5g / L (NH 4 ) 2 SO 4 , 0.6g / L MgSO 4 ·7H 2 O, 0.6g / L CaCl 2 , 0....

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Abstract

The invention provides a gRNA expression cassette applied to a CRISPR / Cas gene editing system. The gRNA expression cassette has the following structure from 5'-3' that A-B-C-D, A is a virus sequence with a gRNA transcription starting effect, B is gRNA, C is gRNA backbone, D is a hepatitis virus ribozyme (HDV) sequence, and the gRNA expression cassette can be recognized and transcribed by an RNA polymerase II promoter in trichoderma reesei. According to the gRNA expression cassette applied to the CRISPR / Cas gene editing system, simultaneous in-vivo transcription of Cas9 and gRNA is realized in Trichoderma reesei for the first time, and the defects of complexity, instability and the like of transformation after in-vitro transcription of gRNA are overcome.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a gRNA expression cassette for a Trichoderma reesei CRISPR / Cas gene editing system, and a CRISPR / Cas gene editing system comprising the gRNA expression cassette. Background technique [0002] The development of the global economic system has always depended on the development of fossil energy. However, fossil energy is limited and is a non-renewable disposable resource in the short term. The dependence on fossil energy not only brings potential energy crisis to human society, but also triggers a series of ecological and environmental problems. Therefore, more and more researches have begun to focus on finding raw materials that can replace fossil energy and their usage methods. Lignocellulosic biomass is the largest renewable resource on earth, and it can be used as a substitute for fossil energy, and it is more and more widely used in the development of emerging biorefi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N1/15C12R1/885
CPCC12N15/113C12N9/22C12N2310/20
Inventor 相金悦康丽芳陶程程
Owner INST OF BOTANY CHINESE ACAD OF SCI
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