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A genome editing vector, its composed genome editing system and application

A genome editing and carrier technology, applied in vectors, nucleic acid carriers, genetic engineering, etc.

Active Publication Date: 2021-05-28
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 Three types of promoters expressed in vivo, especially whether it is possible to find an RNA polymerase type III promoter that can efficiently induce the expression of guide RNA (gRNA) in thermophilic fungi, including whether Cas9 and gRNA can be introduced into thermophilic fungi through in vivo transcription The successful editing of target loci in Mycetophthora cells and whether it can specifically target the genomic DNA of thermophilic fungi have not been reported and elucidated so far

Method used

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  • A genome editing vector, its composed genome editing system and application
  • A genome editing vector, its composed genome editing system and application
  • A genome editing vector, its composed genome editing system 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 14-3-3proteins for morphogenesis, conidiation, stress tolerance and virus of an insect pathogen .EnvironMicrobiol.17:1444–1462) as the backbone to construct expression vectors. 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 tra...

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-Ptef1...

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 present invention relates to a genome editing carrier, its composed genome editing system and its application, in particular to a thermophilic fungal genome editing carrier, its composed genome editing system CRISPR / Cas9 and its editing method and application, the genome editing carrier Including the promoter that starts the transcription of the coding DNA of the sgRNA, the promoter is the RNA polymerase III type U6 type promoter, and the application of the genome editing system can significantly improve the genome editing efficiency of Myceliophthora strains M.thermophile and M.heterothallica, Simultaneous editing of multiple sites in the Myceliophthora genome can be realized, and then multi-gene mutant strains can be obtained. This series of mutant strains can significantly improve the production capacity of cellulase, and can be used for the genetic engineering of thermophilic fungi with high cellulase production; The genome editing system can also promote the research on the function of the Myceliophthora thermophila gene, and at the same time, it is of great significance to the genome-directed editing and metabolic engineering transformation of the thermophilic industrial cellulase production strain.

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, and to a highly efficient induction guide RNA (gRNA) in thermophilic The discovery of the three-type promoter R3P1 expressed in fungi, especially relates to a thermophilic fungal genome DNA editing vector, the genome editing system CRISPR / Cas9 composed of it, 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 a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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