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Method for carrying out gene mutation on rhodobacter sphaeroides

A technology of Rhodobacter sphaeroides and encoding genes, which is applied in the field of gene manipulation technology and can solve problems such as application limitation, limited number of DNA sequences, and complicated operation process.

Active Publication Date: 2020-02-21
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this technology patent is monopolized by the company, and the number of zinc finger proteins is limited, and the number of DNA sequences that can be identified is also limited, so its application is greatly restricted
[0004] The second generation is the transcription activator-like effector nuclease (TALEN), which uses TAL effector: a natural protein secreted by plant bacteria to recognize specific DNA base pairs, which can theoretically be Designed to recognize and bind all target DNA sequences, but the operation process is cumbersome, which limits its application to a certain extent
[0015] Therefore, from the perspective of genetic manipulation, up to now there is no universal high-efficiency, high-throughput bacterial gene editing technology; from the perspective of industrial production, the output, properties and market share of secondary metabolites in China are not as good as Japan, Europe and the United States and other countries urgently need to develop new genome modification patents for microorganisms with great industrial value

Method used

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  • Method for carrying out gene mutation on rhodobacter sphaeroides
  • Method for carrying out gene mutation on rhodobacter sphaeroides
  • Method for carrying out gene mutation on rhodobacter sphaeroides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1. Using the CRISPR-Cas9 system to mutate a single gene of Rhodobacter sphaeroides

[0072] In this example, the effectiveness of the mutation method described in the present invention was verified by constructing appA, ppsR, and crtB gene mutant strains. Edit the plasmid map as figure 1 As shown, the experimental operation flow chart is as follows figure 2 shown.

[0073] 1. Construction of editing plasmid

[0074] 1. With dCas9 or nCas9 of Streptococcus pyogenes (nCas9 protein is divided into two kinds, i.e. nCas9 (D10A) protein and nCas9 (H840A) protein), the cytosine deaminase pmCDA1 of seven gill mantle (or the adenosine monophosphate of Escherichia coli Deaminase TadA), uracil DNA glycosidase inhibitor UGI of Bacillus subtilis bacteriophage based on the original sequence, codon optimization was carried out on the IDT website (http: / / sg.idtdna.com / codonopt) according to the codon table of Escherichia coli , form new dCas9 or nCas9, pmCDA1 (or TadA), UG...

Embodiment 2

[0113] Example 2. Using the CRISPR-Cas9 system to simultaneously mutate multiple genes of Rhodobacter sphaeroides

[0114] The experimental steps involved in Example 2 are the same as in Example 1, except that the sgRNA in Example 2 is a series of multiple sgRNA expression cassettes, as follows:

[0115] When constructing two or more sgRNAs in tandem, first mix the first two sgRNA1 and sgRNA2 as templates in order, use primers at both ends (sgRNA1-2F / sgRNA2-R) to amplify sgRNA1-sgRNA2, and then use the tandem sgRNA1-sgRNA2 and the following sgRNA3 are mixed as templates, amplified with new double-ended primers (sgRNA1-2F / sgRNA3-R) to obtain sgRNA1-sgRNA2-sgRNA3, and then the tandem sgRNA1-sgRNA2-sgRNA3 and subsequent sgRNA4 are mixed as templates , amplified with new two primers (sgRNA1-2F / sgRNA4-R) to obtain sgRNA1-sgRNA2-sgRNA3-sgRNA4, and so on to obtain several sgRNA series.

[0116] In this embodiment, the specific operation is as follows: use the primer appAsgRNA1-N in ...

Embodiment 3

[0128] Example 3. Using the CRISPR-Cpf1 system to mutate a single gene of Rhodobacter sphaeroides

[0129] This example is the same as Example 1, except that Cas9 is replaced by dCpf1, and sgRNA is replaced by crRNA.

[0130] Wherein the dCpf1 protein is from Francisella noobacillus, the amino acid sequence of the protein is SEQ ID No.2, and the corresponding nucleotide sequence (Escherichia coli codon optimization) is SEQ ID No.8.

[0131] The sequence of the crRNA expression cassette is specifically as follows:

[0132] TTGACAGCTAGCTCAGTCCTAGGTATAATGGATCCGAATTTCTACTGTTGTAGATNNNNNNNNNNNNNNNNNNNNNNNTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT (SEQ ID No. 14). Wherein, N represents A or T or G or C.

[0133] Wherein, the 1st-35th of SEQ ID No.14 is a promoter; the 36th-55th of SEQ ID No.14 is a repeat sequence; the 56th-79th of SEQ ID No.14 is for expressing spacer DNA sequence; positions 80-119 of SEQ ID No.14 are terminator regions.

[0134] When fused with cytosine deaminase...

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Abstract

The invention discloses a method for carrying out gene mutation on rhodobacter sphaeroides. The invention provides a method for carrying out gene point mutation on rhodobacter sphaeroides to obtain amutant strain. The method comprises the following steps: on basis of a gene editing technology, editing a genome of rhodobacter sphaeroides by using a fusion protein, and obtaining the mutant strain through screening, wherein the fusion protein contains a protein with a DNA targeting function and an enzyme with a mononucleotide directional mutation function. The method disclosed by the invention has the advantages that the whole flow is simple to operate and steps are few; regulation aiming at a specific metabolic pathway is more accurate and strict and the miss rate is low; and the method hasuniversal applicability and is a novel microbial gene transformation technology with an important application value in the aspects of industrial production, environmental protection and the like.

Description

technical field [0001] The invention relates to the technical field of gene manipulation, in particular to a method for gene mutation of Rhodobacter sphaeroides, in particular to a method for high-throughput gene point mutation in Rhodobacter sphaeroides, which is a gene editing technology based on , a method for fusing proteins with DNA targeting functions and enzymes with single-nucleotide directed mutation functions to construct editing plasmids and transform them into Rhodobacter sphaeroides for screening and obtain mutant strains. Background technique [0002] Gene editing refers to the purposeful "modification" of the target gene sequence, including the insertion, deletion, single or multiple base mutations of DNA fragments, etc. The principle is that under the action of specific nucleases, the double-strand break (Double-stranded break, DSB) of the target gene is caused, and the natural repair mechanism of the cell is activated, through non-homologous end joining (NHE...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N9/22C12N15/63
CPCC12N15/102C12N9/22C12N15/63
Inventor 席建忠骆宇峰
Owner PEKING UNIV
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