CN111676237A

CN111676237A - Vector for expressing CRISPR/dCas9 system in acetobacter xylinum

Info

Publication number: CN111676237A
Application number: CN202010534620.9A
Authority: CN
Inventors: 钟成; 李雪晶; 孙雪文
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2020-09-18

Abstract

The invention discloses a vector construction method for weakening gene expression in acetobacter xylinum by using a CRSPR/dCas9 vector. Experiment the vector for weakening the expression of the bcsA gene in acetobacter xylinum is constructed by the weakening principle of CRSPR/dCas9, so that the attenuation of the bcsA gene in acetobacter xylinum is realized. In the research, the yield of the bacterial cellulose is reduced by 80% after the expression of the bcsA gene of the acetobacter xylinum is weakened, and the expression quantity of the bcsA is reduced by 70% through RT-PCR (reverse transcription-polymerase chain reaction) calculation, which shows that the vector can effectively weaken the expression of the gene in the acetobacter xylinum. The research provides a new method for researching gene expression and regulation of acetobacter xylinum in future.

Description

Vector for expressing CRISPR/dCas9 system in acetobacter xylinum

Technical Field

The invention relates to a construction of a CRSPR/dCas9 vector and application of the vector in the technical fields of genetic engineering, metabolic engineering and synthetic biology. The invention also relates to a host cell for expressing the CRSPR/dCas9 vector and application of the host cell in production.

Background

CRISPR systems are the adaptive immune system of bacteria, and have been modified for genome engineering today. The engineered CRISPR system comprises two components: guide RNAs (grnas or sgrnas) and CRISPR-associated endonucleases (Cas proteins). grnas are short synthetic RNAs consisting of a backbone sequence required for Cas binding and 20-specific nucleotides, which function to determine the site at which the Cas protein modifies the genome. Thus, the genomic target of the Cas protein can be altered by simply altering the target sequence present in the gRNA. In addition, since grnas are very easy to edit, this makes CRISPR one of the most extensive genome editing technologies.

CRISPR is a very flexible tool for genome manipulation, as Cas proteins bind to target DNA independent of the ability to cleave the target DNA. Specifically, RuvC and HNH nuclease domains can be inactivated by point mutations (D10A and H840A in SpCas 9), resulting in the death of nuclease Cas9(dCas9) molecules that cannot cleave target DNA. The dCas9 molecule retained the ability to bind to the target DNA based on gRNA targeting sequences. Early experiments showed that targeting dCas9 to the transcription initiation site was sufficient to inhibit transcription by blocking initiation. dCas9 can also be labeled with transcriptional repressors or activators, and targeting these dCas9 fusion proteins to the promoter region results in strong transcriptional repression or activation of downstream target genes. The simplest dCas 9-based activators and repressors consist of dCas9 fused directly to a single transcriptional activator or transcriptional repressor. In addition, due to the differences in Cas9 or Cas9 nickase-induced genome modification, dCas 9-mediated gene activation or suppression is reversible, as it does not permanently alter genomic DNA.

Disclosure of Invention

The invention constructs a dCas9 vector capable of being expressed in acetobacter xylinum. The vector uses a replicon and a resistance gene of pSEVA331 as a vector framework, uses Pj23100 as a promoter to express a dCas9 gene, and uses a Pj2119 promoter to express specific gRNA. The vector has the capacity of specifically weakening gene expression in the genome of acetobacter xylinum.

Has the advantages that:

this patent constructed a vector that was able to efficiently express dCas9 in acetobacter xylinum. The vector expresses both gRNA and dCas9 so that genes in acetobacter xylinum can be specifically attenuated. The vector can help to regulate and control gene expression in acetobacter xylinum, and provides a new tool for researching the influence of different gene expression amounts in acetobacter xylinum on the yield of bacterial cellulose.

Description of the drawings:

FIG. 1: plasmid map of pSEVA331-dCas9 vector.

FIG. 2: change in transcript levels before and after attenuation of bcsA

FIG. 3: change in BC production before and after attenuation of bcsA

Detailed Description

Construction of pdCas9 vector

(1) Amplification of target Gene

And designing a primer amplification vector framework according to the plasmid sequence of the plasmid pSEVA 331. RuvC and HNH domains of Cas9 were inactivated by point mutations. Promoters J23100 and J23119 were obtained by ordinary PCR. The sequences of J23100 and ribosome binding site as well as cleavage sites HpaI and PstI were added to the 5 ' end of the upstream primer of dCas9, and table 1 is the specific primer sequence (5 ' -3 '):

TABLE 1 primer sequences

(2) Design of gRNA

The gRNA fragment was designed from CRISPR RGEN (http:// www.rgenome.net) and was synthesized by GeneCo. The synthetic gRNA gene fragment was inserted into pSEVA331-dcas9 (the only BasI restriction site) by the Golden Gate method.

(3) Construction of recombinant plasmids

The vector skeleton and the target gene obtained in (1) and the gRNA obtained in (2) are connected into a pSEVA331-dCas9 vector by a homologous recombination method. Reaction conditions are as follows: 30min at 37 ℃. The reaction system is as follows

TABLE 2 homologous recombination System

Remarking: and calculating the vector dosage and the insert dosage according to a formula.

The formula: the optimum amount of cloning vector used was [0.02 Xcloning vector base number ng ] (0.03pmol)

The optimum amount of the insert used was [0.04 Xthe base number ng of the insert ] (0.06pmol)

After the construction is successful, the pSEVA331-dCas9 plasmid sequentially comprises an Ori V replicon, a Pj23119 promoter, a Pj23100 promoter, an sg RNA sequence, a dCas9 sequence and a Cm resistance gene.

(4) Transformation and identification of recombinant plasmids

With CaCl₂The method transforms the recombinant plasmid into escherichia coli DH5 α competent cells, adopts an alkaline lysis method to extract plasmid DNA, and performs enzyme digestion identification and PCR identification on the recombinant plasmid.

(5) Sequencing of dCas9, gRNA, Pj23100 and Pj23119 fragment genes on recombinant plasmid

And (4) carrying out overnight culture on the positive colonies identified correctly in the step (4), preserving bacterial liquid by using a glycerol tube, extracting plasmid DNA, and sending to a gene company for sequencing.

(5) Homologous alignment of gene sequences

The result of sequencing the overlapping genes of dCas9 with SnapGene software was aligned to the sequence in GenBank (Gene ID: 3344282). The alignment result is consistent with the sequence of the constructed overlapping fragment.

The sequencing of the gRNA overlapping Gene was aligned to the sequence in GenBank (Gene ID: 3342760) using SnapGene software. The alignment result is consistent with the sequence of the constructed overlapping fragment.

The Pj23119 promoter overlap Gene sequencing results were aligned with the GenBank sequence (Gene ID: 1021246) using SnapGene software. The alignment result is consistent with the sequence of the constructed overlapping fragment.

The Pj23100 promoter overlap Gene sequencing results were aligned with the GenBank sequence (Gene ID: 1021001) using SnapGene software. The alignment result is consistent with the sequence of the constructed overlapping fragment.

Claims

1. Construction of a vector for expressing a CRISPR/dCas9 system in Acetobacter xylinum, wherein the dCas9 plasmid sequentially comprises an OriV replicon, a Pj23119 promoter, a Pj23100 promoter, a gRNA sequence, a dCas9 sequence and a Cm resistance gene and is named as dCas9 plasmid; the dCas9 gene is located at the downstream of Pj23110 promoter of dCas9 expression vector; the gRNA is positioned at the downstream of Pj23119 resistance gene of a dCas9 expression vector and at the upstream of replication origin (OriV) of a dCas9 expression vector, and the gRNA sequence is a targeting sequence which can specifically target a target sequence; the target sequence refers to a nucleotide sequence of a gene needing editing.

2. The construction method according to claim 1, characterized by comprising the following specific steps:

(1) obtaining the target Gene

And designing a primer amplification vector framework according to the plasmid sequence of the plasmid pSEVA 331. The promoter Pj23100 and Pj23119 fragments were obtained by ordinary PCR. Inactivating RuvC and HNH domains of Cas9 by point mutation to obtain dCas9 fragment; gRNA fragments were synthesized by Gene Synthesis;

(2) construction of recombinant plasmids

Connecting the vector skeleton obtained in the step (1) and the target gene by a homologous recombination method to obtain a dCas9 vector;

(3) transformation and identification of recombinant plasmids

With CaCl₂Transforming the recombinant plasmid into escherichia coli DH5 α competent cells, extracting plasmid DNA by an alkaline lysis method, and performing enzyme digestion identification and PCR identification on the recombinant plasmid;

(4) sequencing of dCas9, gRNA, Pj23100 and Pj23119 fragment genes on recombinant plasmid

And (4) performing overnight culture on the positive colonies identified correctly in the step (3), preserving bacterial liquid by using a glycerol tube, extracting plasmid DNA, and sending to a gene company for sequencing.

3. The method of claim 1-2, wherein the gRNA is synthesized by GeneCo; it is an RNA consisting of a backbone sequence required for Cas binding and 20bp specific nucleotides, and the genome of the Cas protein can be altered by simply changing the target sequence present in the gRNA.

4. The method according to claims 1-2, characterized in that dCas9 expression vector is constructed in acetobacter xylinum, so that the vector can change the specific 20bp in gRNA through BasI enzyme cutting site, and can regulate the expression of acetobacter xylinum gene.