CRISPR/Cas9 gene editing system and application thereof

A gene editing and gene technology, applied in the field of genetic engineering and bioengineering, can solve the problems of no advantages, low occurrence rate, low efficiency, etc., and achieve the effect of high knockout efficiency

Active Publication Date: 2019-07-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to the strong exclusiveness of the protective mechanism of M. aureus itself, it is difficult for foreign genes to stably exist and express in M. aureus, which makes it applicable to the gene editing method of M. aureus Fewer, for example, due to the significantly lower incidence of homologous recombination in mycobacteria than in other bacteria, only 10 -6 ~10 -5 , and the use of homologous recombination to edit the genes of Mycobacterium aureus needs to bring resistance tags into the genome. Therefore, the use of homologous recombination to edit the genes of Mycobacterium aureus has low efficiency, long cycle and screening However, it has disadvantages such as large quantity and complex operation; although other gene editing methods such as suicide vector system (pNIL / pGOAL series plasmids) can edit the genes of M. Advantage

Method used

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  • CRISPR/Cas9 gene editing system and application thereof
  • CRISPR/Cas9 gene editing system and application thereof
  • CRISPR/Cas9 gene editing system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Construction and use of CRISPR / Cas9 gene editing system

[0064] Specific steps are as follows:

[0065] 1. Construction of CRISPR / Cas9 gene editing system

[0066] (1) Replace the Hsp60 promoter on the pMV261 expression vector with the Tac promoter by restriction enzymes Xba I and BamH I and T4 ligase, and enzyme the pMV261 expression vector by restriction enzymes Xba I and Hind III Cut linearization to obtain the basic skeleton;

[0067] (2) obtain the Cas9 gene (nucleotide sequence as shown in SEQ ID NO: 1) by chemical synthesis, namely Frag1;

[0068] (3) obtain the gene (nucleotide sequence as shown in SEQ ID NO:3) and the gene (nucleotide sequence as shown in SEQ ID NO:4) of coding DNA ligase LigD by chemical synthesis ; The gene encoding DNA end assembly protein mku obtained by chemical synthesis and the gene encoding DNA ligase LigD were used as templates, respectively using P1 primers, P2 primers, P3 primers, and P4 primers in Table 1 to clone to ...

Embodiment 2

[0083] Embodiment 2: Application of CRISPR / Cas9 gene editing system

[0084] Specific steps are as follows:

[0085] (1) Construction of CRISPR / Cas9 gene editing system

[0086] The ksdd gene of Mycobacterium aureus JC-12 is used as the gene to be knocked out, and the nucleotide sequence of the gene to be knocked out is used as the target sequence, and the sgRNA (nucleotide sequence) for specifically targeting the target sequence is designed according to the target sequence As shown in SEQ ID NO: 24); according to Example 1, the CRISPR / Cas9 gene editing system was constructed according to the designed sgRNA;

[0087] (2) Gene editing of neomycobacterium aureus JC-12

[0088] Using the new Mycobacterium aureus JC-12 that was not edited by the CRISPR / Cas9 gene editing system as a blank control, the pML-Cas9 plasmid in the CRISPR / Cas9 gene editing system obtained in (1) was transformed into the new Mycobacterium aureus JC- 12 Competent cells, spread on a slant / solid medium (co...

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Abstract

The invention discloses a CRISPR / Cas9 gene editing system and application thereof, and belongs to the technical field of gene engineering and the technical field of bio-engineering. By means of the system, the purpose of gene editing of mycobacterium neoaurum can be achieved. pML-Cas9 plasmid and pJM-sgRNA plasmid used in the system can stably exist and can be stably expressed in the mycobacteriumneoaurum, and cannot be lost along with passage of the mycobacterium neoaurum or repelled by a protection mechanism of the mycobacterium neoaurum. The CRISPR / Cas9 gene editing system is used for carrying out gene editing on the mycobacterium neoaurum, and the knockout efficiency is high, and can reach 50%.

Description

technical field [0001] The invention relates to a CRISPR / Cas9 gene editing system and an application thereof, belonging to the technical fields of genetic engineering and bioengineering. Background technique [0002] Steroid drugs belong to hormone drugs, which are endogenous substances discovered in the study of mammalian endocrine system. aspects play an important role. These effects make steroid drugs become the second largest class of drugs after antibiotics in the chemical industry, and also make steroid drugs become one of the key points in the development of new drug resources in my country, and make steroid drugs and steroid drugs intermediate The body has become an important variety of my country's drug export. [0003] However, due to the fact that there is still a certain gap between our country and the advanced countries in the world in terms of the production level of steroid drugs, the types of steroid drugs available in my country are only one-third of the st...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N1/21C12R1/32
CPCC12N15/902C07K14/35C12N2800/80C12N2810/10Y02A50/30
Inventor 饶志明林春邵明龙张显杨套伟徐美娟
Owner JIANGNAN UNIV
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