Genome editing method based on zymomonas mobilis endogenous CRISPR-Cas system and application thereof

A Zymomonas, genome editing technology, applied in the field of genetic engineering, can solve the problems of host large cells, toxicity, nuclease inactivity, etc., to avoid cytotoxicity, strong positive selection pressure, and avoid limited available selection markers.

Active Publication Date: 2019-10-22
武汉睿嘉康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, no related applications have been implemented in Zymomonas mobilis. The main reasons may be: 1) Both Cas9 and Cpf1 are large nucleic acid proteins (greater than 1000 amino acids) with multiple domains, which can be expressed in prokaryotic cells. 2) Exogenously expressed nucleases such as Cas9 or Cpf1 may be inactive, or active but have greater cytotoxicity to the host

Method used

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  • Genome editing method based on zymomonas mobilis endogenous CRISPR-Cas system and application thereof
  • Genome editing method based on zymomonas mobilis endogenous CRISPR-Cas system and application thereof
  • Genome editing method based on zymomonas mobilis endogenous CRISPR-Cas system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of Zymomonas mobilis Endogenous CRISPR-Cas Genome Editing System

[0040] (1) CRISPR-Cas phylogenetic analysis of the genome encoding of Zymomonas mobilis

[0041] The premise of the present invention is that the research strain needs to encode the CRISPR-Cas system itself and have DNA splicing activity, which requires the host genome to encode the CRISPR cluster and the complete Cas protein system.

[0042]Taking Zymomonas mobilis Z.mobilis 4 as the model strain, the sequencing data of Z.mobilis 4 were analyzed. The results showed that the genome of the strain encoded four CRISPR structural sequences. According to their sequence in the genome, we sequenced them named CRISPR1-CRISPR4, see figure 1 . CRISPR1 occupies the 113,783-114,170 region of the genome and contains 7 repeat sequences; CRISPR2 occupies the 1,244,355-1,245,866 region and contains 9 repeat sequences; CRISPR3 occupies the 1,598,754-1,599,144 region and contains 7 repeat sequence...

Embodiment 2

[0074] Example 2 Application of Genome Editing Method Based on Zymomonas mobilis Endogenous CRISPR-Cas System in Gene Knockout

[0075] In this embodiment, the selected target gene is the ZMO0038 gene encoded by the Zymomonas mobilis genome, and the specific steps are as follows:

[0076] (1) GuideRNA sequence selection: The 32 bp sequence immediately downstream of any 5'-CCC-3' was intercepted from the target gene sequence ZMO0038 as a guideRNA, and the sequence could be located on any strand of the genome.

[0077] ZMO0038-guideRNA primer sequence:

[0078] 0038-gRNA1-F: GAAAATGCCGCTTTCGATCAGGCCGCCGCCAAGATT, see SEQ ID NO:9;

[0079] 0038-gRNA1-R: GAACAATCTTGGCGGCGGCCTGATCGAAAGCGGCAT, see SEQ ID NO: 10;

[0080] (2) guideRNA primer sequence is constructed on the vector, see Figure 5 : The above-mentioned plasmid containing the artificial CRISPR expression unit was linearized with BsaI (Table 5), annealed with the guideRNA primer (same as above, Table 2) and ligated with ...

Embodiment 3

[0103] Example 3 Application of Genome Editing Method Based on Zymomonas mobilis Endogenous CRISPR-Cas System in Site-directed Insertion of DNA Sequence

[0104] In this embodiment, a His-Tag tag is inserted at a fixed point after the start code ATG of the target gene ZMO0038, see Figure 8 , so that the encoded protein can be purified through a nickel column. This method can be extended to any protein to be purified. Specific steps are as follows:

[0105] (1) ZMO0038(His-Tag)-guideRNA sequence selection: intercept the 32 bp sequence immediately downstream of the 5'-TCC-3' near the start codon ATG of the target gene sequence ZMO0038 as a guideRNA, and this sequence can be located on any strand of the genome.

[0106] ZMO0038(His-Tag)-guideRNA primer sequence:

[0107] 0038His-gRNA-F: GAAAATCTTGACTCCCTCCATGCACTTAAAAAATCT, see SEQ ID NO: 18;

[0108] 0038 His-gRNA-R: GAACAGATTTTTTAAGTGCATGGAGGGAGTCAAGAT, see SEQ ID NO:19.

[0109] (2) GuideRNA is constructed on the vector:...

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Abstract

The invention belongs to the technical field of genetic engineering, particularly relates to a genome editing method based on a zymomonas mobilis endogenous CRISPR-Cas system and application thereof and aims at taking Z.mobilis 4 as a type strain and utilizing the I-F type CRISPR-Cas system coded by its genome to establish a genome editing platform. A powerful tool is provided for basic research and application research in the strain and similar cells, and the development of metabolic engineering, system biology and synthetic biology is promoted. According to the technical scheme, the genome editing method is characterized by comprising the steps of constructing a plasmid containing an artificial CRISPR expression unit; selecting a guide RNA sequence aiming at a target site; constructing aguide RNA primer sequence on the plasmid containing the artificial CRISPR expression unit; constructing a donor DNA sequence on a vector to obtain an editing plasmid; transforming the editing plasmidinto competent cells for editing.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genome editing method based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application. Background technique [0002] In recent years, the use of microorganisms for metabolic engineering, systems biology, and synthetic biology has made good progress. For the rational design and construction of microbial cell factories, living cells or enzymes are used to treat renewable biomass, such as It provides an important theoretical basis for the transformation of cellulose and other substances, the production of bioenergy, and the industrialization of biosmelting. Bioenergy regeneration is one of the effective means to solve the problems of resource and energy shortage and serious environmental pollution that human beings are currently facing. [0003] Zymomonas mobilis (Zymomonas mobilis, Z.mobilis) has the relevant characteristics of an ideal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N1/21C12R1/01
CPCC12N15/113C12N15/902C12N2310/10C12N2310/20C12N1/205C12R2001/01
Inventor 杨世辉彭文舫郑艳丽易犁马立新
Owner 武汉睿嘉康生物科技有限公司
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