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Fusarium venanii endogenous U6 promoter and CRISPR/Cas9-based gene editing method thereof

A fusarium and promoter technology, applied in the field of genetic engineering of Fusarium venezia, to achieve high-efficiency gene editing and high application value

Active Publication Date: 2022-04-29
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

There are currently no reports of an endogenous high-efficiency U6 promoter in Fusarium venezia

Method used

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  • Fusarium venanii endogenous U6 promoter and CRISPR/Cas9-based gene editing method thereof
  • Fusarium venanii endogenous U6 promoter and CRISPR/Cas9-based gene editing method thereof
  • Fusarium venanii endogenous U6 promoter and CRISPR/Cas9-based gene editing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Regulation of sgRNA by different endogenous U6 promoters of Fusarium venetianus TB01 GFP Expressed fusion fragment construction

[0024] The present invention excavates and clones the endogenous U6 promoter U6 in Fusarium venezia TB01 through sequence comparison and retrieval with other species 94 and U6 374 (the sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively), which belong to class II promoters, so they are especially suitable for inducing sgRNA expression in gene editing.

[0025] 1. Primers

[0026] U6 98 -1

CTCACGTGCCAGTGCCAAAT U6 98 -2

TGATATAGACGTTGTGGCTGCATATTCGGTCAGTATCGTGATATCAGG U6 94 -1

CCAGGCGGAATTAATCAGTCAG U6 94 -2

TGATATAGACGTTGTGGCTGCGTAGTGATGGTCTTGTGTTAAC U6 86 -1

GCTGGAGTGTGATAGAATAG U6 86 -2

TGATATAGACGTTGTGGCTGCCCATAGACCGCTGGACCAAAC U6 374 -1

ATGCGGCGGATGTTTTGCGAGTCT U6 374 -2

TGATATAGACGTTTGTGGCTGCGAAGGGTTGGTCTTATTTTAAC sgRNA-1 GCAGCCACAACGTCTATATCAGT...

Embodiment 2

[0032] Example 2: Construction of Cas9 protein expression vector

[0033] 1. Primers

[0034] PgpdA1 TATGACCATGATTACgaattcGGTGTTGATCGTCAACCAAGTCC PgpdA2 tatacttcttgtccatAAGCTTTTTGTTAAGGAGGTTCTGTTTGAGG PCas9-T1 atggacaagaagtatagcatcgg PCas9-T2 catcttctgtcgacactagtgCCTCTAAACAAGTGTACCTG Pgc1 TGTGTGTTCGTGGGATGATG Pgc2 gtcagaccaagtgacaacgc

[0035] 2. Fragment Amplification Procedure

[0036] Pgpda fragment 98℃ 10s, 55℃ 15s, 72℃ 50s; 35 cycles primestar, TaKaRa Cas9-T fragment 98℃ 10s, 55℃ 15s, 72℃ 3min; 35 cycles primestar, TaKaRa Transformer Validation 98℃ 10s, 55℃ 15s, 72℃ 50s; 35 cycles primestar, TaKaRa homologous recombination 50℃ 30min Minerva Super Fusion Cloning Kit, US Everbright

[0037] 3. Experimental method

[0038] The sequence of the endogenous strong promoter PgpdA (SEQ ID NO: 5) was amplified from the DNA genome of Fusarium venetianus TB01 with primer pair PgpdA1 / 2, and the seq...

Embodiment 3

[0040] Example 3: Different endogenous U6 promoters regulate sgRNA GFP Co-transformation of protoplasts of the fusion fragment with a Cas9 expression vector of a GFP fluorescent strain of Fusarium venetianus

[0041] This example describes the specific experimental process of CRISPR / Cas9 gene editing in Fusarium venezia. image 3 The schematic diagram of the Fusarium venezia CRISPR / Cas9 gene editing system of the present invention is shown.

[0042] 1. Culture medium

[0043] YEPD: Yeast powder 3g, peptone 10g, glucose 20g, make up to 1L

[0044] Buffer to dissolve enzyme: 0.7 M NaCl

[0045] STC: 0.8 M sorbitol; 50 mM CaCl 2 , 50 mM Tris-HCl (Ph 8.0)

[0046] SPTC: STC with 40% PEG6000

[0047] Regeneration medium: yeast extract 1g, tryptone 1g, sucrose 274g, agarose 10g, constant volume 1L

[0048] Screening medium: glucose 30g, yeast powder 6g, agar powder 15g, make up to 1L.

[0049] 2. Experimental method

[0050] 1) The spore suspension of Fusarium venezia was p...

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Abstract

The invention discloses a U6 promoter derived from fusarium venanii and a gene editing method suitable for the fusarium venanii. The CRISPR / Cas9 gene editing system based on the endogenous U6 promoter is successfully established in the fusarium venanii, and site-directed editing of a target gene is realized, so that convenient, rapid and efficient gene editing is realized in the fusarium venanii with a low homologous recombination rate, and the application value is relatively high.

Description

technical field [0001] The invention relates to the field of genetic engineering of Fusarium venetianus, in particular to an endogenous U6 promoter of Fusarium venetianus and a gene editing method based on CRISPR / Cas9. Background technique [0002] Fusarium venezia is an industrial strain screened from more than 3,000 fungi that can be used for fermentative production of mycelin. Compared with the single-cell protein, the mycelial protein produced by the fermentation of this strain is more delicious, has a meat-like tissue structure, and can provide a good nutritional balance, including low fat content, complete amino acid types, rich in trace elements, vitamins and benefits. Edible crude fiber for human gastrointestinal motility. In addition, Fusarium venezia mycelial protein also has very good safety, and has obtained the marketing license of food raw materials in 18 countries around the world. Therefore, it has the potential to partially or completely replace animal-bas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/80C12N15/55C12N15/65C12N1/15C12R1/77
CPCC07K14/37C12N15/80C12N15/113C12N9/22C12N15/65C12N2310/20
Inventor 马延和童胜李德茂王钦宏陈吴西孙媛霞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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