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CRISPR/Cpf1 gene editing system and application of system in mycobacteria

A technology of mycobacteria and cpf1, applied in the field of gene editing system, can solve the problems of cumbersome construction process, toxicity of Cas9 protein, and long time consumption, and achieve high recombination efficiency

Active Publication Date: 2017-08-22
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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Problems solved by technology

[0009] In view of the cumbersome and time-consuming construction process of the traditional homologous recombination system, and the toxicity of the Cas9 protein in mycobacteria, the present invention optimizes the CRISPR / Cpf1 system and uses it in conjunction with the recombination system , applied to the genome editing of mycobacteria, so as to conduct genetic manipulation in mycobacteria more conveniently and quickly, so as to study the function of related genes and the mechanism of drug resistance

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  • CRISPR/Cpf1 gene editing system and application of system in mycobacteria

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Embodiment Construction

[0031] The present invention is described in further detail by the following examples, but the scope of the present invention is not limited in any way.

[0032] First, the FnCpf1 gene sequence was optimized with reference to the characteristics of high GC and codon preference of mycobacteria (the optimized sequence is shown in SEQ ID No: 1 in the sequence listing), and it was cloned into a gene that can be used in Mycobacterium smegmatis On the replicated shuttle vector, FnCpf1 was induced to express under the regulation of TetO promoter. The plasmid was then transformed into Mycobacterium smegmatis mc 2 In 155, although FnCpf1 is a foreign protein, Cpf1 induced by high concentration of ATc did not cause the growth restriction of M. smegmatis (see figure 2 Middle A). Cpf1 was demonstrated to be applicable to M. smegmatis.

[0033] In order to further verify the activity of FnCpf1 in Mycobacterium smegmatis, that is, whether it has a cutting function, we verified it by pla...

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Abstract

The invention discloses a CRISPR / Cpf1 gene editing system and application of the system in mycobacteria. The CRISPR / Cpf1 gene editing system comprises a recombinant vector carrying optimized FnCpf1 genes and pCR plasmids, wherein the sequence of the optimized FnCpf1 genes is shown in SEQ ID No:1, and the recombinant vector is a colibacillus-mycobacterium shuttle vector containing recombinant enzyme gp60 and gp61 genes; the pCR plasmids contain promoter-driven direct repeat sequence-spacer sequence-direct repeat sequence units, wherein the spacer sequence contains target sequence connection sites. When the CRISPR / Cpf1system is used in the mycobacteria for CRISPR / Cpf1 assisted homologous recombination, high recombination efficiency can be obtained, and gene editing of the mycobacteria can be achieved.

Description

technical field [0001] The present invention relates to a gene editing system, in particular to a CRISPR / Cpf1 system for mycobacterium gene editing. Background technique [0002] Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis, which kills 1.5 million people worldwide each year. The tuberculosis threat is growing due to the emergence of multidrug-resistant mutant strains of Mycobacterium tuberculosis. Therefore, genetic manipulation in mycobacteria, especially the construction of gene knockout strains and point mutation strains, is essential for understanding the mechanism of mycobacterial growth and development, differentiation, functions of virulence-related genes and drug resistance genes, and then for the development of attenuated viruses. Both vaccines and therapeutic drugs are of great significance. However, due to the slow growth of Mycobacterium tuberculosis (about 16-24 hours to reproduce one generation), pathogenicity (need to be o...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/74C12N15/90C12R1/32
CPCC12N9/22C12N15/74C12N15/902C12N2800/22
Inventor 闫玫漪郭晓鹏孙义成金奇
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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