The endogenous U6 promoter of Fusarium venezia and its CRISPR/Cas9-based gene editing method

A Fusarium and promoter technology, applied in the direction of microorganism-based methods, genetic engineering, plant genetic improvement, etc., to achieve the effect of efficient gene editing and high application value

Active Publication Date: 2022-06-14
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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  • The endogenous U6 promoter of Fusarium venezia and its CRISPR/Cas9-based gene editing method
  • The endogenous U6 promoter of Fusarium venezia and its CRISPR/Cas9-based gene editing method
  • The endogenous U6 promoter of Fusarium venezia and its CRISPR/Cas9-based gene editing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0024] The present invention excavates and clones into the endogenous U6 promoter U6 in Fusarium veniceum TB01 through sequence comparison and retrieval with other species 94 and U6 374 (Sequences such as SEQ ID NO: 1 and SEQ ID NO: 2, respectively), which belong to class II promoters, are therefore particularly 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

ATGCGGGCGGATGTTTTGCGAGTCT U6 374 -2

TGATATAGACGTTGTGGCTGCGAAGGGTTGGTCTTATTTTAAC sgRNA-1 GCAGCCACAACGTCT...

Embodiment 2

[0032] Embodiment 2: Cas9 protein expression vector construction

[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 Transformant Verification 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 (SEQ ID NO: 5) of endogenous strong promoter PgpdA is amplified from the DNA genome of Fusarium veniceum TB01 with primer pair PgpdA1 / 2, with primer pai...

Embodiment 3

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

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

[0042] 1. Medium

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

[0044] Buffer for lysing 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, dilute to 1L.

[0049] 2. Experimental method

[0050] 1) Tween 80 to prepare the spore suspension of Fusarium venezia, apply it...

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Abstract

The invention discloses a U6 promoter derived from Fusarium venetica and a gene editing method applicable to Fusarium venetica. The present invention has successfully established a CRISPR / Cas9 gene editing system based on the endogenous U6 promoter in Fusarium venezia, and realized the fixed-point editing of the target gene, thus realizing convenient and rapid , Efficient gene editing, with high application value.

Description

technical field [0001] The present invention relates to the field of genetic engineering of Fusarium veniceum, in particular to the endogenous U6 promoter of Fusarium venezia and its gene editing method based on CRISPR / Cas9. Background technique [0002] Fusarium venetica is an industrial strain that can be used to ferment and produce mycelium protein from more than 3,000 strains of fungi. Compared with single-cell protein, the mycelium protein produced by fermentation of this strain is more delicious, has a tissue structure similar to meat, 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 venetica mycelium protein has good safety and has been approved as a food raw material in 18 countries around the world. Therefore, it has the potential to partially or completely replace animal and vegetable protein...

Claims

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Application Information

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Patent Type & Authority Patents(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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