Genome editing method based on bacterial endogenous homologous recombination system

文档序号：1609399 发布日期：2020-01-10 浏览：19次中文

阅读说明：本技术 一种基于细菌內源同源重组系统的基因组编辑方法 (Genome editing method based on bacterial endogenous homologous recombination system ) 是由霍毅欣黄潮勇于 2019-09-27 设计创作，主要内容包括：细菌是代谢工程中的主要宿主,基因组编辑是细菌工程改造的主要途径,而现有的针对细菌的基因组编辑方法都需要过表达外源的同源重组系统或非同源末端连接系统。本发明公开了一种基于细菌內源同源重组系统的基因组编辑方法。本发明所提供的基因组编辑方法是按照包括以下步骤进行：构建一个双质粒基因组编辑系统,通过转化将该系统导入待编辑的菌株。将含有基因组编辑系统的菌株在液体培养基中培养使其进行繁殖,待细胞数目足够时加入诱导剂诱导CRISPR/Cas9系统表达,随后发生DNA双链切割和修复,实现基因组编辑。本发明先通过细胞繁殖增加细胞的数量和活性再启动基因组编辑,显著提高了基因组编辑效率,并成功在大肠杆菌中实现了基因敲除和大片段删除。(Bacteria are the main host in metabolic engineering, genome editing is the main path of bacterial engineering, and the existing genome editing methods aiming at bacteria all need to over-express exogenous homologous recombination systems or non-homologous end connection systems. The invention discloses a genome editing method based on a bacterial endogenous homologous recombination system. The genome editing method provided by the invention comprises the following steps: a two-plasmid genome editing system was constructed, which was introduced into the strain to be edited by transformation. Culturing the strain containing the genome editing system in a liquid culture medium to propagate, adding an inducer to induce the expression of the CRISPR/Cas9 system when the cell number is enough, and then carrying out DNA double-strand cutting and repair to realize genome editing. According to the invention, the number and activity of cells are increased through cell propagation, then genome editing is started, the genome editing efficiency is obviously improved, and gene knockout and large fragment deletion are successfully realized in escherichia coli.)

1. A genome editing method based on a bacterial endogenous homologous recombination system is characterized in that after plasmids loaded with the genome editing system are transformed into host cells, the genome editing system is not started, the number and activity of the cells are increased through cell propagation, the genome editing system is started after the cells are propagated for a period of time, and the bacterial genome is efficiently edited by cutting DNA through a CRISPR/Cas9 system and repairing the DNA through the endogenous homologous recombination system.

2. The genome editing method based on the bacterial endogenous homologous recombination system according to claim 1, characterized by comprising the following steps:

A. a genome editing system taking a plasmid as a vector is constructed, the system consists of a Cas9 encoding gene, a sgRNA encoding gene and a DNA repair template, and the Cas9 encoding gene and the sgRNA encoding gene are controlled by an inducible promoter.

B. The plasmid loaded with the genome editing system is transformed into a host strain to be edited, and the obtained transformant is cultured in a liquid medium to proliferate the cells.

C. When the cells proliferate to exponential phase, an inducer is added to induce Cas9 protein and sgRNA expression.

D. After 2-3 hours of induction, the bacterial suspension was applied to plates containing the inducer at various dilutions.

E. And D, selecting a single colony from the plate in the step D for PCR verification to obtain a correct editing strain.

3. The genome editing method based on the bacterial endogenous homologous recombination system according to claim 2, wherein in the step A, the plasmid vector is a single plasmid vector or a double plasmid vector, and the inducible promoter includes but is not limited to P_BADAnd all the inducible promoters with strong stringency.

4. The genome editing method based on the bacterial endogenous homologous recombination system as claimed in claim 2, wherein in step A, only the Cas9 encoding gene and the sgRNA encoding gene are used for constructing the genome editing system, and the encoding genes of any foreign repair proteins such as lambda-Red are not used, and the used Cas9 comprises SpCas9 and all mutants of SpCas 9.

5. The genome editing method based on the bacterial endogenous homologous recombination system according to claim 2, wherein in step B, the host strains include but are not limited to all bacterial strains such as Escherichia coli which can adopt CRISPR/Cas9 technology.

6. The method for genome editing based on a bacterial endogenous homologous recombination system according to claim 2, wherein in the step C, the induction of the inducer occurs when the cells proliferate to the exponential phase.

Technical Field

The invention relates to a genome editing method based on a bacterial endogenous homologous recombination system, and belongs to the technical field of bioengineering.

Background

Synthetic biology is the most important biotechnology platform in the 21 st century, and has wide application in the fields of biological energy, biological materials, biomedicine, bioremediation and the like. The genome editing technology, the DNA assembly technology and the directed evolution technology form three leading edge technologies of synthetic biology.

Bacteria are the main host organisms in synthetic biology, metabolic engineering of microorganisms is an essential link in synthetic biology, and genome editing is the main means of bacterial metabolic engineering.

The CRISPR/Cas9 technology has been widely applied to genome editing of bacteria, and various methods for genome editing of bacteria based on the CRISPR/Cas9 technology have been reported. The principles of these methods can be briefly summarized as: the Cas9-sgRNA complex specifically recognizes the target DNA and cleaves to generate a DNA double strand break, and the broken DNA is subsequently repaired by a repair system, completing genome editing during repair. However, these genome editing methods for bacteria all rely on exogenous homologous recombination systems or non-homologous end joining systems to repair genomic DNA cleaved by Cas9 protein, and this limitation has not been broken up to date. The reason for this dependence is that most of the homologous recombination systems and non-homologous end joining systems in bacteria are very weak in repairing DNA double strand breaks, unlike eukaryotes such as yeast.

In order to break this limitation on bacterial genome editing, the present invention proposes a new approach. The expression of Cas9 and sgRNA is controlled using a highly stringent inducible promoter, so that the CRISPR/Cas9 system is initiated only when an inducer is added, before which the cell can grow normally without dying due to cleavage of Cas 9. Prior to induction of Cas9 and sgRNA expression, the cells were allowed to proliferate by liquid culture for a period of time during which the number and activity of the cells increased. After the inducer is added, the CRISPR/Cas9 system is initiated and DNA double strand breaks are generated, and then the DNA repair system endogenous to the cell repairs the broken DNA, thereby realizing genome editing. Most cells die due to failure of DNA repair, but because the cell population is large enough, a large number of cells with successful repair survive.

Disclosure of Invention

The invention aims to provide a simple and efficient bacterial genome editing method, which removes the dependence of the existing method on an exogenous repair system and accelerates the process of bacterial metabolic engineering.

According to the technical scheme provided by the invention, the genome editing method based on the bacterial endogenous homologous recombination system adopts the following steps:

1. a genome editing system taking a plasmid as a vector is constructed, the system consists of a Cas9 encoding gene, a sgRNA encoding gene and a DNA repair template, and the Cas9 encoding gene and the sgRNA encoding gene are controlled by an inducible promoter.

2. The plasmid loaded with the genome editing system is transformed into a host strain to be edited, and the obtained transformant is cultured in a liquid medium to proliferate the cells.

3. When the cells proliferate to exponential phase, an inducer is added to induce Cas9 protein and sgRNA expression.

4. After 2-3 hours of induction, the bacterial suspension was applied to plates containing the inducer at various dilutions.

5. And (4) selecting a single colony from the plate in the step (4) for PCR verification to obtain a correct editing strain.

Drawings

FIG. 1 is a diagram of a two-plasmid genome editing system constructed for E.coli. (a) Plasmid P15A-P_BADMap of Cas 9. This plasmid was used to express Cas9 protein. (b, c) plasmid pSC101-P_BADMap of sgRNA-Donor. The plasmid is used for expressing sgRNA and providing a DNA repair template, and is divided into pSC101-P only expressing one sgRNA_BAD-sgRNA-Donor plasmid (see FIG. 1b) and pSC101-P for simultaneous expression of two sgRNAs_BADThe sgRNA-Donor plasmid (FIG. 1 c).

FIG. 2 shows pSC101-P_BADConstruction flow chart of (E) -sgRNA-Donor plasmid

FIG. 3 is a flowchart of gene insertion in E.coli

FIG. 4 is a flow chart of genome large fragment deletion in E.coli

FIG. 5 is a graph showing the results of gene insertion and deletion of a large fragment. (a, b) inserting purple protein gene. (c, d) deletion of the 100kb genomic fragment.

Sequence listing

The sequence 1 is a plasmid pSC101-P_BAD-sgRNA-Donor-T1

The sequence 2 is a plasmid pSC101-P_BAD-sgRNA-Donor-T2

The sequence 3 is a plasmid pSC101-P_BAD-sgRNA-Donor-T3

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

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

The following examples are further illustrative of the present invention and are not to be construed as limiting the spirit of the present invention.

Example 1 was carried out: insertion of purple protein Gene into lacZ Gene of E.coli MG1655 Strain

Construction of plasmid P15A-P_BADCas9, as in fig. 1 a. The plasmid takes P15A as a replicon, takes a kanamycin resistance gene as a selection marker, and carries P_BADThe Cas9 under the control of the promoter encodes the gene. The plasmid only needs to be constructed once, and does not need to be repeatedly constructed when other genes are edited.

Construction of plasmid pSC101-P expressing sgRNA_BADsgRNA-Donor as in FIG. 1 b. The plasmid takes pSC101 as a replicon, takes an ampicillin resistance gene as a selection marker, and is provided with a P_BADsgRNA coding gene controlled by promoter and a Donor sequence (repair template). The plasmid is specially used for inserting a purple protein gene into lacZ, and the expressed sgRNA specifically targets the open reading frame of the lacZ gene.

Construction of pSC101-P_BADBefore the-sgRNA-Donor plasmid, a proper targeting site needs to be selected, the targeting site is positioned in an open reading frame of a gene to be lacZ, and the selection principle of the targeting site is (1) high in efficiency and (2) low in off-target rate. The selection of the target site can be accomplished by means of a dedicated website or software, or the like, which utilizes a websitehttps:// chopchop.cbu.uib.no/#. The target site sequence selected was 5'-gatgaaagctggctacaggaagg-3', consisting of protospacer (20bp) and PAM (3 bp). In pSC101-P_BADIn the sgRNA-Donor plasmid, the spacer sequence is the protospacer sequence in the targeting site, i.e., gatgaaagctggctacagga.

Construction of pSC101-P_BADThe sgRNA-Donor plasmid,as shown in FIG. 2a, the plasmid pSC101-P_BADThe sgRNA-Donor-T1 (see sequence 1) is a starting plasmid, and a Donor sequence and a spacer sequence are sequentially inserted on the starting plasmid through two plasmid constructions.

For genome editing, as shown in FIG. 3, plasmid P15A-P was used first_BADCas9 was transformed into MG1655 competent cells, 1mL of SOC medium was added to the transformed product, and after 40 minutes of recovery at 37 degrees, the cells were transferred to a shake tube containing 4mL of LB medium (containing kanamycin) and cultured at 37 degrees. When cultured to the exponential phase of cell growth (OD)₆₀₀0.4-0.6), competent cells were prepared, and the plasmid pSC101-P was introduced_BADThe sgRNA-Donor was transformed into competent cells, 1mLSOC medium was added to the transformed product, and after 40 minutes of recovery at 30 degrees, the cells were transferred to a shake tube containing 4mL of LB medium (containing kanamycin, ampicillin and glucose) and cultured overnight at 30 degrees. Transferring 1mL of overnight culture into a shake tube containing 4mL of LB culture medium (containing kanamycin and ampicillin), adding L-arabinose after culturing for 1 hour at 30 degrees to induce Cas9 and sgRNA expression, taking bacterial liquid after culturing for 2-3 hours at 30 degrees, coating LB plates (containing kanamycin, ampicillin and L-arabinose) at different dilutions, and picking out single colonies from the plates after culturing overnight at 30 degrees for verification. The colony PCR results and sequencing results are shown in fig. 5.

After the colonies in which the purple protein gene was successfully inserted were obtained, if a new round of gene editing was to be performed, the colonies were inoculated into LB medium (containing kanamycin), cultured at 42 ℃ for 8 hours to remove the temperature-sensitive plasmid pSC101-P_BAD-sgRNA-Donor followed by transfer of new pSC101-P_BAD-sgRNA-Donor plasmid; if no new round of gene editing is performed, the colony is inoculated into LB medium (containing no antibiotics), cultured at 40 ℃ for 20 hours, and the plasmid P15A-P is removed_BADCas9 and pSC101-P_BAD-sgRNA-Donor。

The working concentrations of kanamycin, ampicillin and L-arabinose used in the above procedures were 0.05g/L, 0.1g/L and 3g/L, respectively. The LB culture medium has the formula: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L (solid medium: agar 20 g/L). The formula of the SOC culture medium is as follows: tryptone 20g/L, yeast extract 5g/L, sodium chloride 0.5g/L, potassium chloride 2.5mM, magnesium chloride 10mM, magnesium sulfate 10mM, glucose 20 mM.

Example 2 was carried out: a100 kb genomic fragment was knocked out in E.coli MG1655 strain.

Construction of plasmid P15A-P_BADCas9, same as [0025 ]]。

Construction of plasmid pSC101-P expressing two sgRNAs_BADsgRNA-Donor as in FIG. 1 c. The plasmid takes pSC101 as a replicon, takes an ampicillin resistance gene as a selection marker, and is provided with two P_BADsgRNA coding gene and Donor sequence controlled by promoter. Two sgrnas expressed by the plasmid specifically target both ends of a 100kb genomic fragment.

Construction of pSC101-P_BADPlasmid pSC101-P in the case of the-sgRNA-Donor plasmid, as shown in FIG. 2b_BAD-sgRNA-Donor-T2 (see sequence 2) is a starting plasmid, and a Donor sequence is inserted into the starting plasmid to form an intermediate plasmid; using the intermediate plasmid as template, performing PCR amplification to obtain skeleton, and using plasmid pSC101-P_BADAn insert is obtained by PCR amplification by taking sgRNA-Donor-T3 (see sequence 3) as a template; the skeleton and the insert are assembled by DNA to obtain pSC101-P with Donor sequence and two spacer sequences_BAD-sgRNA-Donor plasmid.

Genome editing was performed as described in [0029], and is shown in FIG. 4. The colony PCR results and sequencing results are shown in fig. 5.

Sequence listing

<110> Beijing university of science and technology

<120> genome editing method based on bacterial endogenous homologous recombination system

<141>2019-09-20

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