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Promoter and expression vector for adjusting and controlling sgRNA transcription, genome editing system and application

A technology for genome editing and expression vectors, applied in the field of genome editing systems based on CRISPR/Cas9, which can solve problems such as lack of promoters and actual effects to be verified, and achieve the effects of improving editing efficiency and organic acid production

Active Publication Date: 2019-10-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Functionally active RNA requires specific eukaryotic type III promoters to initiate its transcription, and such promoters that can be used to initiate transcription of sgRNA-encoding DNA are scarce
However, Chinese patent 201611083079.4 intercepted the 501bp sequence near the two U6 coding regions of Aspergillus niger as the U6 promoter, hoping to use the sgRNA promoter derived from Aspergillus niger to correctly guide the synthesis of sgRNA in vivo, but the actual effect has yet to be verified.

Method used

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  • Promoter and expression vector for adjusting and controlling sgRNA transcription, genome editing system and application
  • Promoter and expression vector for adjusting and controlling sgRNA transcription, genome editing system and application
  • Promoter and expression vector for adjusting and controlling sgRNA transcription, genome editing system and application

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Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1: Cloning of Anp promoter

[0050] In natural evolution, the promoter sequence of U6 small nuclear RNA varies greatly among different species, but the nucleic acid sequence of U6 small nuclear RNA itself has a certain degree of conservation. Therefore, the U6 small nuclear RNA sequence of Myceliophthora thermophila is used as the For reference, nucleic acid sequence analysis and comparison were carried out in the genome of Aspergillus niger ATCC1015, in which Identities were greater than 90%, and E-value was less than 1e-10. After comparison, two RNA polymerase type III small nucleoRNA (snRNA) candidate genes were found. The characteristic of U6 small nuclear RNA promoter is that the initial transcription site is "G", and there is a certain number of nucleotides (>30bp) between this nucleotide and the second upstream "G". Through the analysis and research of the present invention, it is found that the upstream promoter sequence of one RNA polymerase type III...

Embodiment 2

[0055] Example 2: Construction of CRISPR / Cas9-mediated Aspergillus niger genome editing vector

[0056] (1) Cas9 protein expression vector construction

[0057] The Cas9 protein coding gene from Streptococcus pyogenes was codon-optimized and artificially synthesized, and three purification tags and the nuclear localization signal sequence (PPRKRAKTEDE) of Myceliophthora thermophila transcription factor HacI were added to its N-terminus. Two side-by-side SV40 nuclear localization signal sequences (DPKKKRKVDPKKKRKV) and one nuclear localization signal sequence for HacI (PPRKRAKTEDE) as described above were added at the end.

[0058] The coding nucleotide sequence of the Cas9 protein is shown in SEQ ID No.8, and the amino acid sequence of the Cas9 protein is shown in SEQ ID No.9.

[0059] The promoter of the translation elongation factor TEF1 (as shown in SEQ ID NO.5), the promoter of glyceraldehyde phosphate dehydrogenase GpdA (as shown in SEQ ID NO.6), and the glycoside hydrol...

Embodiment 3

[0074] Embodiment 3: CRISPR / Cas9 system is to Aspergillus niger under the condition of not adding homologous donor DNA pyrG Editing of genetic loci

[0075] (1) Aspergillus niger protoplasts

[0076] A. Mycelium preparation

[0077] Mature Aspergillus niger ATCC1015 spores were collected with 0.05% Tween-80 sterilized water, filtered through lens paper to remove mycelium, inoculated in MM liquid medium, and cultured at 30°C and 200rpm for 16h.

[0078] B. Protoplast preparation

[0079] After the hyphae were collected by filtration, they were placed in 30mL lysate (recipe: 0.15g lyase, 30mL solution A, sterilized by filtration; solution A: potassium dihydrogen phosphate 1.0361g, sorbitol 21.864g, dissolved in 90mL deionized water, pH Adjust to 5.6, quantify to 100mL, high-temperature sterilization), lyse at 30°C for 2h, and shake gently every 30min. After filtering with lens tissue, centrifuge at 2000rpm at 4°C for 10min, discard the supernatant, add 4mL solution B (recipe...

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Abstract

The invention relates to a promoter and expression vector for adjusting and controlling sgRNA transcription, a genome editing system and application. The promoter with the function of adjusting and controlling sgRNA encoding DNA transcription comprises a nucleotide sequence as shown in SEQ ID NO.1, the promoter can be used for constructing the expression vector for adjusting and controlling sgRNAencoding DNA transcription and used in the CRISPR / Cas9 genome editing system, and then the genome editing system can be used for significantly improving the editing efficiency of fungal genomes. By utilizing the promoterand expression vector for adjusting and controlling sgRNA transcription, the genome editing system and application, fungi, especially filamentous fungusstrains can be more efficiently transformed, for example, the level of producing malic acid by aspergillusniger is significantly improved, and great application andpromotion value is achieved.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetic engineering. Specifically, the present invention relates to a DNA fragment with the promoter function of regulating the transcription of sgRNA-encoded DNA, and an sgRNA expression vector containing the DNA fragment, a CRISPR / Cas9-based genome editing system and its application. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats) is an acquired self-immune defense system formed during evolution. It consists of highly conserved repeat sequences and completely different spacer sequences alternately, and is widely found in bacteria and archaea. It mainly relies on the foreign fragment integrated in its own genome to resist the re-invasion of the foreign DNA. In 2013, Zhang Feng et al. first applied CRISPR / Cas9 technology to gene editing of mammalian cells (Le Cong et al. Multiplex genome engineering using CRISPR / Cassyetems. Science, 2013,339(612...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/90C12N1/15C12P7/46C12R1/685
CPCC07K14/38C12N9/1007C12N9/88C12N15/113C12N15/63C12N15/902C12N2310/10C12N2310/20C12P7/46C12Y401/01023
Inventor 田朝光李金根顾淑莹赵祯刘倩孙文良
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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