阅读说明：本技术 一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统及应用 (Dual-function system for simultaneous gene knockout and gene expression inhibition of corynebacterium glutamicum and application ) 是由 徐志南 连佳长 刘伟 杨修亮 于 2019-08-21 设计创作，主要内容包括：本发明公开了一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统及应用。本发明构建的CRISPR/Cas12a系统的载体,包括Cas12a蛋白基因和crRNA的编码DNA。本发明构建的CRISPR/Cas12a基因载体能够对谷氨酸棒杆菌基因组同时进行基因敲除和基因表达抑制,具有实验周期短、节省时间和效率高等优点。(The invention discloses a bifunctional system for simultaneous gene knockout and gene expression inhibition of corynebacterium glutamicum and application thereof. The vector of the CRISPR/Cas12a system constructed by the invention comprises a Cas12a protein gene and a coding DNA of crRNA. The CRISPR/Cas12a gene vector constructed by the invention can simultaneously carry out gene knockout and gene expression inhibition on a corynebacterium glutamicum genome, and has the advantages of short experimental period, time saving, high efficiency and the like.)

1. A dual-function system for simultaneously knocking out genes and inhibiting gene expression of corynebacterium glutamicum is characterized in that:

the bifunctional system comprises a cas12a expression vector and a crRNA expression vector;

the cas12a expression vector comprises a cas12a sequence and a promoter sequence, wherein the cas12a sequence is shown as SEQ ID No.1, the promoter sequence is shown as SEQ ID No.2, and the promoter sequence is connected in front of an initiation codon ATG of a cas12a sequence;

the crRNA expression vector comprises a homologous repair sequence, a crRNA1 sequence and a crRNA2 sequence, wherein the crRNA1 sequence is shown as SEQ ID No.3, the crRNA2 sequence is shown as SEQ ID No.4, the crRNA1 sequence is a 18-24 bp guide sequence which is identical to the N18-N24 sequences of the target gene, and the crRNA2 sequence is a 13-16 bp guide sequence which is identical to the N13-N16 sequences of the target gene.

2. The bifunctional system for simultaneous gene knockout and gene expression suppression of corynebacterium glutamicum of claim 1, wherein: the nucleotide sequence of the cas12a expression vector is shown as SEQ ID No. 5.

3. The bifunctional system for simultaneous gene knockout and gene expression suppression of corynebacterium glutamicum of claim 1, wherein: the cas12a expression vector is prepared by the following method:

a. synthesizing a cas12a gene sequence; PCR amplification promoter P with Corynebacterium glutamicum genome as template_tufA sequence; amplification of kanamycin resistance Gene Kan Using pEC-XK99E plasmid as template^RC.glutamicum replicon and e.coli replicon elements and terminator rrnB;

b. ligation of P by homologous recombination using homologous recombination technique_tufPromoter, cas12a Gene, rrnB terminator element, Kan^RResistance gene, Corynebacterium glutamicum and Escherichia coli replicon elements, to construct the cas12a expression vector.

4. The bifunctional system for simultaneous gene knockout and gene expression suppression of corynebacterium glutamicum of claim 1, wherein: the homologous repair sequence contained in the crRNA expression vector is formed by connecting an upstream sequence of a knocked-out target gene and a downstream sequence of the target gene, and the lengths of the upstream sequence and the downstream sequence of the target gene are 300 bp-1500 bp.

5. The bifunctional system for simultaneous gene knockout and gene expression suppression of corynebacterium glutamicum of claim 1, wherein: the nucleotide sequence of the crRNA expression vector is shown as SEQ ID No. 6.

6. The bifunctional system for simultaneous gene knockout and gene expression suppression of corynebacterium glutamicum of claim 1, wherein: the crRNA expression vector is prepared by the following method:

a. synthesizing crRNA1 sequence, using pXMJ19 plasmid as template, amplifying Cm^RResistance genes, Corynebacterium glutamicum and E.coli replicon elements; constructing a homologous repair sequence, and introducing XhoI, PstI, BamHI, XbaI, EcoRI and HindIII enzyme cutting sites at the 5' end of the homologous repair sequence;

b. homologous recombination using homologous recombination techniques to join the crRNA1 sequence, Cm^RThe resistance gene, the replicon element of the corynebacterium glutamicum and the escherichia coli and the homologous repair sequence are obtained to obtain psg-A1-HR vector;

c. synthesizing a crRNA2 sequence, and introducing two enzyme digestion recognition sequences of XhoI, PstI, BamHI, XbaI, EcoRI and HindIII at two ends of crRNA 2;

d. and (3) digesting psg-A1-HR plasmid and crRNA2 sequence by using two selected restriction enzymes, purifying and then connecting to obtain the crRNA expression vector for gene knockout and gene expression inhibition simultaneously.

7. Use of a bifunctional system according to claims 1 to 6 in Corynebacterium glutamicum, characterized in that: the specific operation of the application is as follows:

a, transferring a cas12a expression vector into corynebacterium glutamicum, and culturing and screening to obtain a transformant;

b, transferring the crRNA expression plasmid into corynebacterium glutamicum containing cas12a expression vector, culturing, screening and obtaining transformant.

Technical Field

The invention relates to the field of gene editing, in particular to a CRISPR/Cas12a system for simultaneously knocking out genes and inhibiting gene expression of corynebacterium glutamicum and application thereof.

Background

CRISPR/Cas (Clustered regularly interspersed short palindromic repeats) system is used as an acquired immune system for bacteria and archaea, and is used for specifically cutting exogenous genetic materials through RNA mediation so as to resist invading viruses. In recent years, the system is successfully applied to species such as escherichia coli, arabidopsis thaliana, saccharomyces cerevisiae and mice, and quick and efficient gene knockout, gene insertion and gene silencing are realized.

CRISPR/Cas12a belongs to the V-a type CRISPR system. The system plays a role of gene editing, is established in a protein nucleic acid complex formed by CRISPRRNA(crRNA) and Cas12a protein, and recognizes a downstream sequence at the 3' end of a T-rich PAM site (TTTN) under the guidance of crRNA; the target fragment is cut by complementing a guide sequence with the length of 20bp on the crRNA with a specific sequence on the target gene and recruiting the Cas12a protein to cut the target gene. Whereas when the length of crRNA is less than 17bp, Cas12a protein is still able to bind to DNA, but lacks the ability to cleave DNA.

Corynebacterium glutamicum (Corynebacterium glutamicum) is a gram-positive bacterium which is generally regarded as safe and non-pathogenic, and is widely used for industrial production of amino acids such as glutamic acid and lysine, and other chemicals applied to food, feed and medicines.

In the early days, strains of corynebacterium glutamicum were obtained as high-yielding industrial bacteria mainly by means of mutagenic breeding. With the development of whole genome sequencing, the oriented modification of the genome of corynebacterium glutamicum by using a genetic engineering method is more and more emphasized. At present, in addition to pK18mobsacB/pK19 mobsacB-mediated homologous recombination, the CRISPR/Cas method is also used for Corynebacterium glutamicum. Because the corynebacterium glutamicum has strong rejection and digestion capacity to exogenous DNA and the activity of homologous recombinase is low, the transformation efficiency and homologous recombination repair efficiency of the corynebacterium glutamicum are low. Although single-function CRISPR systems (e.g. gene knockout and gene suppression) have been achieved, simultaneous manipulation of multiple sites on the genome has been difficult to succeed.

Disclosure of Invention

Aiming at the problems, the invention provides a dual-function system for simultaneously knocking out genes and inhibiting gene expression of corynebacterium glutamicum, which has higher gene knocking-out and gene inhibiting efficiency. Therefore, the invention adopts the following technical scheme:

a dual-function system for simultaneously knocking out genes and inhibiting gene expression of corynebacterium glutamicum is characterized in that:

the bifunctional system comprises a cas12a expression vector and a crRNA expression vector;

the cas12a expression vector comprises a cas12a sequence and a promoter sequence, wherein the cas12a sequence is shown as SEQ ID No.1, the promoter sequence is shown as SEQ ID No.2, and the promoter sequence is connected in front of an initiation codon ATG of a cas12a sequence;

the crRNA expression vector comprises a homologous repair sequence, a crRNA1 sequence and a crRNA2 sequence, wherein the crRNA1 sequence is shown as SEQ ID No.3, the crRNA2 sequence is shown as SEQ ID No.4, the crRNA1 sequence is a 18-24 bp guide sequence which is identical to the N18-N24 sequences of the target gene, and the crRNA2 sequence is a 13-16 bp guide sequence which is identical to the N13-N16 sequences of the target gene.

Further, the nucleotide sequence of the cas12a expression vector is shown as SEQ ID No. 5.

Further, the cas12a expression vector is prepared by the following method: a. synthesizing a cas12a gene sequence; PCR amplification promoter P with Corynebacterium glutamicum genome as template_tufA sequence; amplification of kanamycin resistance Gene Kan Using pEC-XK99E plasmid as template^RC.glutamicum replicon and e.coli replicon elements and terminator rrnB; b. ligation of P by homologous recombination using homologous recombination technique_tufPromoter, cas12a Gene, rrnB terminator element, Kan^RResistance gene, Corynebacterium glutamicum and Escherichia coli replicon elements, to construct the cas12a expression vector.

Furthermore, the homologous repair sequence contained in the crRNA expression vector is formed by connecting an upstream sequence of a knocked-out target gene and a downstream sequence of the target gene, and the lengths of the upstream sequence and the downstream sequence of the target gene are 300 bp-1500 bp.

Further, in the above-mentioned case,a. synthesizing crRNA1 sequence, using pXMJ19 plasmid as template, amplifying Cm^RResistance genes, Corynebacterium glutamicum and E.coli replicon elements; constructing a homologous repair sequence, and introducing XhoI, PstI, BamHI, XbaI, EcoRI and HindIII enzyme cutting sites at the 5' end of the homologous repair sequence; b. homologous recombination using homologous recombination techniques to join the crRNA1 sequence, Cm^RThe resistance gene, the replicon element of the corynebacterium glutamicum and the escherichia coli and the homologous repair sequence are obtained to obtain psg-A1-HR vector; c. synthesizing a crRNA2 sequence, and introducing two enzyme digestion recognition sequences of XhoI, PstI, BamHI, XbaI, EcoRI and HindIII at two ends of crRNA 2; d. and (3) digesting psg-A1-HR plasmid and crRNA2 sequence by using two selected restriction enzymes, purifying and then connecting to obtain the crRNA expression vector for gene knockout and gene expression inhibition simultaneously.

Further, the application of the above bifunctional system for simultaneous gene knockout and gene expression inhibition in corynebacterium glutamicum specifically operates as follows: a. transferring the cas12a expression vector into corynebacterium glutamicum, culturing and screening to obtain a transformant; b. the crRNA expression plasmid is transferred into corynebacterium glutamicum containing cas12a expression vector, cultured and screened to obtain transformant.

According to the CRISPR/Cas12a system for simultaneously performing gene knockout and gene suppression in the genome, two artificially designed crRNAs are combined with Cas12a protein to perform gene knockout and gene suppression on the gene target position respectively; wherein, the DNA double strand at the target site for gene knockout is cut to form a double strand break gap and provide a homologous repair sequence to achieve the purpose of gene knockout; wherein, the crRNA and cas12a protein nucleic acid complex is combined on the target site DNA to inhibit gene expression, which does not cause the cutting of DNA double strand, but prevents the transcription of gene; wherein P is_tufThe promoter enables the Cas12a protein to be expressed continuously in corynebacterium glutamicum.

Experiments prove that the CRISPR/Cas12a system for simultaneously knocking out genes and inhibiting genes, which is constructed by the invention, can simultaneously knock out genes and inhibit gene expression of corynebacterium glutamicum genomes, and has the advantages of short period, high efficiency and the like.

Drawings

FIG. 1 is a plasmid map of pEC-XK 99E.

FIG. 2 is a plasmid map of cas12a expression vector.

FIG. 3 is a plasmid map of pXMJ 19.

FIG. 4 is a plasmid map of psg-A1-HR.

FIG. 5 is a plasmid map of the crRNA expression vector.

FIG. 6 is a schematic diagram of the design of simultaneous gene knockout and gene expression suppression in a genome.

FIG. 7 shows the genotype validation result (A) after aecD gene knockout and the aecD knockout mcbR gene expression inhibition result (B). M represents Marker, 1-20 represents single colony selected at random; cg- Δ aecD-Control represents aecD gene knockdown bacteria as a Control; cg-delta aecD-mcbRi represents that a gene suppression plasmid is added into aecD gene knockout bacteria to serve as a control; Cg-aecDd-mcbti represent simultaneous aecD knockout and mcbR expression inhibiting strains constructed using the CRISPR/Cas12a system of the invention.

FIG. 8 shows the aecD gene knockout and the mcbR gene suppression strain cysteine production. WT represents wild type c.glutamicum; cg- Δ aecD stands for aecD gene knock-out; cg-delta aecD-mcbRi represents that a gene suppression plasmid is added into aecD gene knockout bacteria to serve as a control; Cg-aecDd-mcbti represent simultaneous aecD knockout and mcbR expression inhibiting strains constructed using the CRISPR/Cas12a system of the invention.

Detailed Description

The experimental methods in the following examples or application examples are conventional methods unless otherwise specified.

Materials, reagents, gene synthesis and the like used in the following examples or application examples are commercially available unless otherwise specified.

The vector backbones used in the examples or applications described below were pEC-XK99E and pXMJ19, both purchased from Addgene.

Coli MC1061 used in the following examples or applications was obtained from Tokyo.

Corynebacterium glutamicum C.glutamicum B253 used in the examples or applications described below was purchased from the China center for Industrial culture Collection of microorganisms (CICC).

PrimeSTAR Max DNA polymerase, restriction enzymes and T4ligase used in the following examples or application examples were purchased from TaKaRa.

pEASY-Uni Senamless Cloning and Assembly Kit used in the following examples or application examples was purchased from TransGen corporation.

In the following examples or application examples, LB medium was used as the medium for Escherichia coli, and the medium formulation was: 10g of peptone, 5g of yeast extract, 10g of NaCl and 1L of deionized water.

In the following examples or application examples, the culture medium of Corynebacterium glutamicum used was BHISG medium, and the medium formulation was: 37g of brain-heart infusion, 91g of D-sorbitol, 10g of glucose and 1L of deionized water.

The following examples or application examples use MC medium for the cysteine synthesis medium of Corynebacterium glutamicum, the medium formulation being: 50 g glucose, 25g corn steep liquor, 15g (NH)₄)₂SO₄,1g K₂HPO₄,1g KH₂PO₄,1g MgSO₄·7H₂O,5gNa₂S₂O₃,10mg FeSO₄·7H₂O,10mg MnSO₄·H₂O,1 mg ZnSO₄·7H₂O,0.2mg CuSO₄,0.02mgNiCl₂0.2mg biotin, 0.2mg thiamine and 20g CaCO3, 1L deionized water.