CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat sequences)-Cas (CRISPR-associated proteins) system in Streptomyces virginiae IBL14 and method for carrying out gene editing by using CRISPR-Cas system

A technology of IBL14, gene editing, applied in the field of gene editing, to achieve the effect of improving production level

Inactive Publication Date: 2016-05-04
ANHUI UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular: The CRISPR-Cas system and gene editing methods in Streptomyces virginia IBL14 have not been reported

Method used

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  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat sequences)-Cas (CRISPR-associated proteins) system in Streptomyces virginiae IBL14 and method for carrying out gene editing by using CRISPR-Cas system
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat sequences)-Cas (CRISPR-associated proteins) system in Streptomyces virginiae IBL14 and method for carrying out gene editing by using CRISPR-Cas system
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat sequences)-Cas (CRISPR-associated proteins) system in Streptomyces virginiae IBL14 and method for carrying out gene editing by using CRISPR-Cas system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 (svu016 gene knockout)

[0048] (1) Gene svu016 primer design and DNA amplification

[0049] According to the whole genome sequencing information, gene svu016 specific primers 016-F and 016-R were designed (Table 3). Genomic DNA of Streptomyces virginia IBL-14 was extracted, and PCR amplification of svu016 gene was performed using PfuDNAPolymerase produced by Shanghai Sangon Bioengineering Co., Ltd., reaction conditions: 95°C for 5min, 94°C for 30s, 58°C for 30s, 72°C for 2min , 2.5U PfuDNAPolymerase (50μl reaction system) produced by Sangon, 30 cycles, 72°C for 10min. The PCR product was detected by 1% agarose electrophoresis, the kit was recovered, and the purified svu016 full-length gene fragment was obtained for future use.

[0050] (2) Preparation of upstream and downstream homology arms

[0051] According to the full sequence of the svu016 gene (Table 2), the upstream homology arm primers 016-UF and 016-UR of the svu016 gene, and the downstream hom...

Embodiment 2

[0065] Embodiment 2 (insertion of chloramphenicol resistance gene)

[0066] (1) Gene svu016 primer design and DNA amplification

[0067] With embodiment 1 step (3).

[0068] (2) Preparation of upper and lower homology arms and chloramphenicol gene

[0069] The amplification of the upstream and downstream homology arms is the same as step (2) of Example 1. Chloramphenicol gene primers cm-F and cm-R (Table 3). Use the purified chloramphenicol gene DNA as a template to amplify the chloramphenicol gene. The reaction conditions are: 95°C for 5min, 94°C for 30s, 60°C for 30s, 72°C for 1min, 2.5U PfuDNA Polymerase (50μl reaction system ), 30 cycles, 10min at 72°C. The PCR product was detected by 1% agarose electrophoresis, recovered by the kit, and the purified chloramphenicol DNA fragment was obtained for future use.

[0070] (3) Preparation of editing template fragments

[0071] Mix 0.4 μl of the purified product of the upper homology arm and the purified product of the chlor...

Embodiment 3

[0082] Embodiment 3 (svu016 gene traceless point mutation)

[0083] (1) Gene svu016 primer design and DNA amplification

[0084] With embodiment 1 step (3).

[0085] (2) Preparation of point mutation editing template fragments

[0086] The point mutation editing template fragment SVU016C352T was directly synthesized by Chuzhou General Biology Co., Ltd., with BamHI and EcoRI restriction sites added at the beginning and end respectively. On this editing template fragment, the cysteine ​​codon sequence at position 352 of the protein active site was mutated into Su amino acid codon sequence, and in order to ensure correct targeting, the amino acid codon sequence near the active site was modified to its degenerate sequence (Table 3).

[0087] (3) Preparation of targeted gene fragments

[0088] The target gene guideDNA-SVU016C352T was prepared in the same way as in step (4) of Example 1. (table 3)

[0089] (4) Construction of gene editing plasmid pKCSV14-SVU016C352T

[0090] W...

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Abstract

The invention discloses a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat sequences)-Cas (CRISPR-associated proteins) system in Streptomyces virginiae IBL14 and a gene editing operation method using the CRISPR-Cas system. The method comprises the processes of carrying out design and plasmid construction on features, targeted genes and gene editing templates of a CRISPR-Cas I-B type gene editing system and carrying out gene editing operation in the Streptomyces by a targeted gene and gene editing template recombinant plasmid. Through carrying out knockout, insertion, traceless point mutation and RNA (Ribonucleic Acid) level regulation and control on genetic genes by using the CRISPR-Cas I-B type gene editing system in the Streptomyces virginiae IBL14, the hereditary features of organisms can be conveniently changed; according to the CRISPR-Cas system and the method for carrying out gene editing by using the CRISPR-Cas system, novel technologies and methods are provided for improving the production level of products of medicine, food and other industries and improving the quality of the products.

Description

technical field [0001] The invention relates to a gene editing technology in the field of biotechnology, specifically a CRISPR-Cas system in Streptomyces virginia IBL14 and a gene editing method using it. Background technique [0002] CRISPR (clusteredregμlarlyinterspacedshortpalindromicrepeatsequences) is called clustered regularly spaced short palindromic repeat sequences, which were first discovered in Escherichia coli in 1987 (Nakata, A., Amemura, M. and Makino, K. (1989) Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12chromosome171Bacteriol. 3553-3556), and subsequently found to exist in the genomes of many eubacteria and archaea. In 2002, R. Jansen et al. analyzed and compared this repetitive sequence in the prokaryotic genome in detail, and more importantly found that the side of this repetitive sequence is often accompanied by conserved gene sequences (Jansen, R., Embden, J.D., Gaastra, W. and Schouls, L.M. (2002) Identification of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/76
CPCC12N15/76C12N2800/101C12N2800/80
Inventor 童望宇雍德祥李雪
Owner ANHUI UNIVERSITY
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