Genome editing vector, genome editing system constituted by genome editing vector and application

A genome editing and vector technology, applied in the fields of genetic engineering and biology, to achieve the effect of improving editing efficiency

Active Publication Date: 2018-06-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY 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 has been successfully developed in some mesophilic fungi, whether the CRISPR / Cas9 system can work in thermostable filamentous fungal cells, especially whether it is possible to find highly efficient inducible guide RNA (gRNA) in thermophilic fungi Whether the RNA polymerase type III promoter expressed in vivo, including Cas9 and gRNA, can be introduced into Myceliophthora thermophila cells through in vivo transcription to successfully edit the target site, and whether it can specifically target the genomic DNA of the thermophilic fungus , so far these studies have not been reported and explained

Method used

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  • Genome editing vector, genome editing system constituted by genome editing vector and application
  • Genome editing vector, genome editing system constituted by genome editing vector and application
  • Genome editing vector, genome editing system constituted by genome editing vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: Construction of CRISPR-Cas9-mediated Myceliophthora genome editing vector

[0091] (1) Construction of Cas9 expression cassette vector

[0092] Taking p0380-bar (Liu Q, Li J, Ying S, Wang J, Sun W, Tian C, FengM. 2015. Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virus of an insect Pathogen.EnvironMicrobiol.17:1444–1462) as the backbone to construct the expression vector. Referring to the genome of Myceliophthora thermophila, the codon preference of the Cas9 protein from Streptococcus pyogenes was optimized, and the transcription factor hacI (MYCTH_2310995) was added to the N-terminal and C-terminal of the Cas9 protein. The nuclear localization sequence (PPRKRAKTEDE), its amino acid sequence and nucleotide sequence are respectively shown in SEQ ID No.8 and SEQ ID No.9.

[0093] The codon-optimized Cas9 was placed under the promoter Ptef1 of the translation elongation factor TEF1A (MYCTH_2298136) for ...

Embodiment 2

[0106] Example 2: Stable expression of Cas9 in Myceliophthora thermophila

[0107] The plasmid p0380-bar-Ptef1-Cas9-TtprC with Cas9 expression cassette and the plasmid p0380-bar-Ptef1-Cas9-eGFP-TtprC with green fluorescent protein fusion protein were introduced into In Myceliophthora thermophilis.

[0108] (1) Cultivation of Myceliophthora thermophila strain

[0109] Myceliophthora thermophila ATCC 42464 was cultured on MM slant medium] at 45°C for 10 days before use.

[0110] MM slant medium: 50×Vogel’s salt 20mL, sucrose 20g, agar 15g, constant volume to 1L, autoclaved. 50 x Vogel's Salt (1L): Trisodium Citrate (1 / 2H 2 O) 150g, anhydrous KH 2 PO 4 250g, anhydrous NH 4 NO 3 100g, MgSO 4 ·7H 2 O 10g, CaCl 2 2H 2 O 5g, trace element salt solution 5mL, biotin (0.1mg / mL) 2.5mL, constant volume to 1L.

[0111] (2) Transformation of Myceliophthora thermophila mediated by Agrobacterium tumefaciens

[0112] The vector Agrobacterium transformation plasmids p0380-bar-Pte...

Embodiment 3

[0150] Embodiment 3: The acquisition of the mutant strain of Myceliophthora genome edited by CRISPR-Cas9 system

[0151] (1) Myceliophthora protoplast transformation

[0152] 1) Mycelium preparation

[0153] Mature Myceliophthora spores were collected with 0.05% Tween-80 sterilized water, filtered through lens paper to remove mycelia, spread on MM plates covered with cellophane, and incubated at 45°C for 16h.

[0154] 2) Protoplast preparation

[0155] Place the cellophane with hyphae in 30mL lysate (recipe: 0.15g lyase, aseptically add 30mL solution A, filter and sterilize; solution A: 1.0361g potassium dihydrogen phosphate, 21.864g sorbitol, dissolve in 90mL Deionized water, potassium hydroxide to adjust the pH to 5.6, quantify to 100mL, high-temperature sterilization), lyse at 30°C for 2h, and shake gently every 20min. After filtering with cellophane, centrifuge at 2000rpm at 4°C for 10min, discard the supernatant, add 4mL solution B (0.735g calcium chloride, 18.22g sorb...

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Abstract

The invention relates to a genome editing vector, a genome editing system constituted by the genome editing vector and an application, and in particular relates to a thermophilic fungi genome DNA editing vector, a genome editing system CRISPR / Cas9 constituted by the genome editing vector as well as an editing method an application. The genome editing vector comprises a promoter for promoting coding DNA transcription of sgRNA, wherein the promoter is an RNA polymerase III type U6 promoter. With the application of the genome editing system, genome editing efficiency of myceliophthora strains M.thermophile and M.heterothallica can be obviously improved, and simultaneous editing of multiple sites of myceliophthora genome can be implemented, so that polygenic mutant strains are obtained; the series of mutant strains can obviously improve production capacity of cellulase and can be applied to transformation of thermophilic fungi genetic engineering for high yield of the cellulase; the genomeediting system can also promote researches on gene functions of myceliophthora thermophila; and meanwhile, the genome editing system is of significance to genome directional editing and metabolic engineering transformation of thermophilic industrial cellulase production strains.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, and relates to the development of a CRISPR / Cas9 system, in particular to a genome editing carrier, its composed genome editing system and its application, especially to a thermophilic fungus genome DNA editing carrier, its The composed genome editing system CRISPR / Cas9 and editing methods and applications. Background technique [0002] Myceliophthora is a thermophilic filamentous fungus that can rapidly degrade cellulose. It has a fast metabolic rate and can secrete a large number of lignocellulolytic enzymes. The types and quantities of its enzyme systems are quite rich. Compared with the industrial cellulase producing bacteria Trichoderma reesei and Penicillium decumbens, the high-temperature fermentation of Myceliophthora thermophila and the enzyme system produced have the advantages of high activity and high stability at high temperature, and become natural high-temperatur...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/113C12R1/645
CPCC07K14/37C12N9/2402C12N9/58C12N15/113C12N15/80C12N2310/10C12N2800/60C12N2800/80C12N2810/10
Inventor 田朝光刘倩高染染李金根孙文良林良才高婧芳
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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