A method for constructing a plasmid that identifies and deletes large fragment non-essential gene regions and its application in gene editing

Abstract

The present application relates to a kind of plasmid construction method for identifying and deleting large fragment non-essential gene region and its application in gene editing.The plasmid is the gene editing plasmid pMHAGE containing mini-CRISPR cluster and multiple donor DNA fragments.The plasmid carries artificial CRISPR cluster and multiple donor DNA simultaneously, and multiple donor DNA can carry out multiple forms of gene editing to prokaryote using CRISPR-Cas system through homologous recombination, the identification and deletion of large fragment non-essential gene region, realize the simplification of prokaryote genome.The plasmid application host range is wide, and all bacteria and archaea using CRISPR-Cas system can be operated;It can be used to identify large fragment non-essential gene region and editable non-essential gene region combination on genome;With the ability of screening growth phenotype and the optimal non-essential gene region large fragment deletion of genome simplification chassis cell resistance;Simple process, short time cycle, greatly reduce the workload when prokaryote genome simplification.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genomics, genetic engineering and biotechnology, and particularly relates to a construction method of a plasmid for recognizing and deleting a large fragment of a non-essential gene region and application thereof in gene editing. BACKGROUND

[0002] The method of synthetic biology usually needs to perform genome simplification to construct a chassis cell from a wild-type strain, however, single gene iterative knockout is time-consuming and laborious, and a large fragment gene deletion is the most effective.

[0003] In the past decade, an effective genetic manipulation system and tool have been established in the archaeal model species Sulfolobus islandicus REY 15A (Yingjun Li et al., 2016). That is, a genetic manipulation technology for gene editing using an endogenous CRISPR-Cas system. The current gene editing method has been proved to be applicable to the deletion of ≧10K gene fragments, and this technology is applied to genome simplification.

[0004] Genome simplification optimization is not blind knockout of non-essential genes, but to modify a microorganism containing only beneficial genes as much as possible. Determining the essential genes in the genome is the first step of simplification. By comparing the genomes of closely related strains, the positions of essential genes can be predicted, but this does not represent the accuracy of essential gene prediction and the combination of non-essential genes.

[0005] Using the endogenous CRISPR-Cas system to edit the predicted large fragment of non-essential genes often shows that it is not editable. This indicates that there are essential genes in the predicted large fragment of non-essential gene region or multiple non-essential genes combined to perform essential gene functions.

[0006] Therefore, in order to realize the construction of a genome-simplified chassis cell, it is urgent to develop a gene editing method capable of recognizing and deleting a large fragment of a non-essential gene region in the genome. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides a construction method of a plasmid for recognizing and deleting a large fragment of a non-essential gene region and application thereof in gene editing. The plasmid simultaneously carries an artificial CRISPR cluster and multiple donor DNAs, and through homologous recombination of the multiple donor DNAs, multiple forms of gene editing can be performed on prokaryotes using the CRISPR-Cas system, the large fragment of the non-essential gene region is recognized and deleted, and the genome of the prokaryote is simplified.

[0008] The technical scheme of the present application is as follows:

[0009] In a first aspect of the present application, a plasmid for identifying and deleting a large fragment of a non-essential gene region is provided, wherein the plasmid is a gene editing plasmid pMHAGE containing a mini-CRISPR cluster and a multi-donor DNA fragment.

[0010] The gene editing plasmid pMHAGE is obtained by selecting a protospacer sequence from a target site in a region to be edited in a prokaryotic genome, designing a corresponding primer to anneal to obtain a desired spacer fragment, ligating the spacer fragment with a pSe-Rp plasmid to form a mini-CRISPR cluster, obtaining an artificial CRISPR plasmid (pAC) capable of expressing crRNA, and then inserting a multi-donor DNA fragment required for identifying and deleting a target gene region into the pAC plasmid.

[0011] In a second aspect of the present application, a construction method of the plasmid for identifying and deleting a large fragment of a non-essential gene region is provided, and the specific steps are as follows:

[0012] A protospacer sequence is selected as a target site in a region to be edited in a prokaryotic genome, two reverse complementary primers are designed according to the protospacer sequence, the sequences of the primers are forward primer: 5'-AAG-Nn-3' and reverse primer: 5'-AGC-N'n-3', wherein Nn and N'n are reverse complementary sequences, N and N' represent bases A, T, G or C, and n represents the number of bases of the protospacer sequence; the two primers are annealed to form double-stranded DNA with sticky ends, i.e., a spacer fragment; an artificial CRISPR vector pSe-Rp is treated with a restriction endonuclease Lgu I, and then ligated with the spacer fragment with sticky ends by T4 ligase to obtain an artificial CRISPR plasmid (pAC) capable of producing mature crRNA; and a multi-donor DNA fragment required for identifying and deleting a large fragment of a non-essential target region is inserted into the pAC plasmid to obtain a gene editing plasmid (pMHAGE) containing the multi-donor DNA fragment;

[0013] The multi-donor DNA fragment is obtained by SOE-PCR concatenation of DNA fragments homologous to the target site in the region to be edited in the genome of a host cell.

[0014] According to a preferred embodiment of the present application, the method for obtaining the multi-donor DNA fragment comprises the following steps:

[0015] 1) According to the homologous DNA fragments on both sides of the target site of the editing region of the host cell genome, the amplification primers of each donor DNA fragment with adjacent donor DNA adapters are designed by SOE-PCR, and each donor DNA fragment can be but not limited to be labeled as HA1, HA2,..., HAn;

[0016] 2) The same molar concentration of each donor DNA fragment is mixed in equal volume, and the fusion fragment is obtained by amplifying 15-20 cycles by the overlap-PCR method without adding any primer;

[0017] 3) The first donor DNA fragment amplification forward primer F1 and the last donor DNA fragment amplification reverse primer Rn are used as amplification primers, and the fusion fragment obtained in step 2) is used as a template for PCR amplification, and after enrichment and purification, a multi-donor DNA fragment is obtained, which is a tandem fusion product of each donor DNA HA1-HA2-...-HAn;

[0018] Among them, the multi-donor DNA is greater than or equal to 3, that is, n of HAn is greater than or equal to 3, and the specific number can be adjusted according to experimental requirements; the length of each donor DNA fragment in the multi-donor DNA is 250bp-600bp, that is, 250bp≤HAn length≤600bp; the interval of each adjacent donor DNA fragment in the multi-donor DNA on the host cell genome is 4kb-40kb.

[0019] The third aspect of the present application provides the application of the above-mentioned plasmid for recognizing and deleting a large fragment of a non-essential gene region in gene editing.

[0020] According to the present application, the gene editing includes gene deletion and genome simplification.

[0021] The fourth aspect of the present application provides a method for simplifying the genome of a prokaryote by using the above-mentioned plasmid for recognizing and deleting a large fragment of a non-essential gene region, comprising the following steps:

[0022] (1) Obtaining of mutant strain: after the pMHAGE plasmid is electroporated into the host prokaryote competent cells, the artificial CRISPR cluster on the plasmid transcribes pre-crRNA, which is processed into mature crRNA in the cell; the crRNA forms a target effect complex with the CRISPR-Cas protein, and then the crRNA pairs with the target DNA strand on the host cell genome to recognize and cut the target site; subsequently, the multiple different donor DNA fragments on both sides of the target site on the pMHAGE plasmid are homologous recombined with each other and the host genome sequence, and then the genome editing mutant strain after homologous recombination of different donor DNA is obtained;

[0023] (2) Verification of mutant strains: design verification primers outside each donor DNA fragment according to the genomic sequence of the host prokaryote, verify the genomic editing by PCR according to the possible two-by-two combination of the multi-donor DNA located on both sides of the target site, determine the region of large fragment of non-essential genes that can be deleted, and purify the strain containing the region of large fragment of non-essential genes that is deleted;

[0024] Wherein, the PCR verification of the genomic editing is: a, in the transformants with editing bands, the two-by-two combination of the multi-donor DNA located on both sides of the target site is homologously recombined, which reflects that the region of the two donor DNAs is the region of large fragment of non-essential genes that can be deleted; b, in the transformants without editing bands, the two-by-two combination of the multi-donor DNA located on both sides of the target site is homologously recombined, which reflects that there can be essential genes or non-essential genes that cannot be deleted in the region of the two donor DNAs;

[0025] (3) Purification of the strain with maximum deletion of the region of non-essential genes: according to the strain with the genomic editing band of large fragment of non-essential genes deletion obtained in step (2), the single strain with deletion of large fragment of genes is further purified by dilution separation method; or according to the region of large fragment of non-essential genes that can be deleted obtained in step (2), in order to improve the editing and purification efficiency, a gene editing plasmid pGE with mini CRISPR cluster and donor DNA at both ends of the deletion region is constructed; then the genomic editing plasmid pGE is transformed into the host prokaryote cells, a large number of transformants are obtained on the screening medium, and after purification, the strain with maximum deletion of the region of non-essential genes is obtained, thereby completing the simplification of the prokaryote genome.

[0026] The specific construction method of the gene editing plasmid pGE can be referred to in "A method for editing prokaryote genome by using endogenous CRISPR-Cas system" (CN 105331627B). The screening medium is SCV medium.

[0027] According to the present application, the prokaryote is preferably bacteria or archaea containing endogenous type I and / or type III CRISPR-Cas system, or bacteria or archaea into which endogenous type I and / or type III CRISPR-Cas system is introduced.

[0028] Further preferably, the prokaryote includes, but is not limited to, S. islandicus (preferably S. islandicus REY15A), Escherichia coli, Staphylococcus epidermidis, Shewanella putrefaciens, Thermus thermophilus, Streptococcus thermophilus, Pyrococcus furiosus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Methanothermobacter thermautotrophicus, Haloferax volcanii, Pectobacterium atrosepticum, Corynebacterium diphtheriae, and the like.

[0029] The present application has the following advantages:

[0030] 1. The present application provides a plasmid for identifying and deleting large fragment non-essential gene regions, which carries an artificial CRISPR cluster and multiple donor DNAs, and can perform multiple forms of gene editing on prokaryotes containing a CRISPR-Cas system through homologous recombination of the multiple donor DNAs, and identify and delete large fragment non-essential gene regions.

[0031] 2. The present application provides a method for simplifying a prokaryote genome, which only needs to construct an editing plasmid carrying an artificial CRISPR cluster and multiple donor DNAs, and can achieve multiple forms of gene editing on the genome through homologous recombination of different donor DNA combinations on both sides of the target site after DNA interference on the genome by the CRISPR system, thereby simplifying the prokaryote genome. The biggest advantage of this method is that the application host range is wide, and all bacteria and archaea that can utilize the CRISPR-Cas system can be operated; it can be used to identify large fragment non-essential gene regions and combinations of editable non-essential gene regions on the genome; it has the ability to screen for genome simplification chassis cells with large fragment deletion of non-essential gene regions with optimal growth phenotypes and stress resistance; the process is simple, the time period is short, and the workload of prokaryote genome simplification is greatly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Schematic diagram of the principle of the method for reducing the genome of a prokaryote using plasmids that recognize and delete large non-essential gene regions.

[0033] Figure 2 Schematic diagram of the selection of multiple donor DNA fragment positions and two Spacer positions in the N1 region of S. islandicus REY15A in Example 1.

[0034] Figure 3 Nucleic acid electrophoresis diagram of PCR detection of each gene editing condition of 24 transformants of plasmid N1-pMHAGE-1 in Example 1, WT is the wild type bacterial solution as the template of the negative control, and H2O is the blank control without template.

[0035] Figure 4 Nucleic acid electrophoresis diagram of PCR detection of each gene editing condition of 24 transformants of plasmid N1-pMHAGE-2 in Example 1, WT is the wild type bacterial solution as the template of the negative control, and H2O is the blank control without template.

[0036] Figure 5 Nucleic acid electrophoresis verification diagram of the N1 region HA2-HA4 gene region deletion editing strain obtained by purifying the N1-2 / 4HA-pGE transformant in Example 1; wherein DH1 is the N1 region HA2-HA4 gene region deletion editing strain, WT is the wild type bacterial solution as the template of the negative control, and H2O is the blank control without template.

[0037] Figure 6 Schematic diagram of the selection of multiple donor DNA fragment positions and two Spacer positions in the N6 region of S. islandicus REY15A in Example 2.

[0038] Figure 7 Nucleic acid electrophoresis diagram of multiplex primer PCR verification of gene editing conditions of 16 transformants of plasmid N6-pMHAGE-1 in Example 2, WT is the wild type bacterial solution as the template of the negative control, and H2O is the blank control without template.

[0039] Figure 8 Nucleic acid electrophoresis diagram of multiplex primer PCR verification of gene editing conditions of 16 transformants of plasmid N6-pMHAGE-2 in Example 2, WT is the wild type bacterial solution as the template of the negative control, and H2O is the blank control without template.

[0040] Figure 9The nucleic acid electrophoresis verification chart of the N6 region HA3-HA4 intergenic region deletion edited strain purified from the N6-pMHAGE-2 transformant in Example 2, DH4 is the obtained N6 region HA3-HA4 intergenic region deletion edited strain, WT is the wild type bacterial solution as a negative control, and H2O is a blank control without template. DETAILED DESCRIPTION

[0041] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically stated, the experimental operations in the examples are all carried out according to the conventional experimental conditions or the conditions suggested by the manufacturers of the materials and reagents.

[0042] In one specific embodiment of the present application, a gene editing plasmid for recognizing and deleting a large fragment of an unnecessary gene region is provided, and the specific method is as follows:

[0043] A protospacer sequence is selected as a target site in the region to be edited in the genome of a prokaryote, and two reverse complementary primers are designed according to the protospacer sequence, and the sequences are forward primer: 5'-AAG-Nn-3' and reverse primer: 5'-AGC-N'n-3', wherein Nn and N'n are reverse complementary sequences, N and N' represent bases A, T, G or C, and n represents the number of bases of the protospacer sequence; the above two primers are annealed to form double-stranded DNA with sticky ends, i.e. a spacer fragment; an artificial CRISPR vector pSe-Rp is treated by restriction enzyme Lgu I, and then connected with the spacer fragment with sticky ends by T4 ligase to obtain an artificial CRISPR plasmid (pAC) capable of producing mature crRNA; and a multi-donor DNA fragment required for recognizing and deleting a large fragment of an unnecessary target region is inserted into the above pAC plasmid to obtain a gene editing plasmid (pMHAGE) containing a multi-donor DNA fragment;

[0044] The multi-donor DNA fragment is obtained by SOE-PCR concatenation of DNA fragments homologous to multiple target sites on both sides of the region to be edited in the genome of a host cell. That is, the DNA fragments homologous to the target sites on both sides of the region to be edited in the genome of a host cell are donor DNA fragments.

[0045] According to the present application, preferably, the method for obtaining the multi-donor DNA fragment comprises the following steps:

[0046] 1) According to the homologous DNA fragments on both sides of the target site of the editing region of the host cell genome, the amplification primers of each donor DNA fragment with adjacent donor DNA adapters are designed by SOE-PCR, and each donor DNA fragment can be but not limited to be labeled as HA1, HA2,..., HAn;

[0047] 2) The donor DNA fragments are mixed in equal volume at the same molar concentration, and the fusion fragment is obtained by amplifying 15-20 cycles by overlap-PCR without adding any primers;

[0048] 3) The first donor DNA fragment amplification forward primer F1 and the last donor DNA fragment amplification reverse primer Rn are used as amplification primers, and the fusion fragment obtained in step 2) is used as a template for PCR amplification, and after enrichment and purification, a multi-donor DNA fragment is obtained, which is a tandem fusion product of each donor DNA HA1-HA2-...-HAn;

[0049] Among them, the multi-donor DNA is greater than or equal to 3, that is, n of HAn is greater than or equal to 3, and the specific number can be adjusted according to experimental requirements; the length of each donor DNA fragment in the multi-donor DNA is 250bp-600bp, that is, 250bp≤HAn length≤600bp; the interval of each adjacent donor DNA fragment in the multi-donor DNA on the host cell genome is 4kb-40kb.

[0050] In another specific embodiment of the application, a method for simplifying the genome of a prokaryote by using the above-mentioned plasmid for recognizing and deleting a large fragment of an unnecessary gene region is provided, which comprises the following steps:

[0051] (1) Obtaining of the mutant strain: after the pMHAGE plasmid is electroporated into the host prokaryote competent cells, the artificial CRISPR cluster on the plasmid transcribes pre-crRNA, which is processed into mature crRNA in the cells; the crRNA forms a target effect complex with the CRISPR-Cas protein, and then the crRNA pairs with the target DNA strand on the host cell genome to recognize and cut the target site; then the multiple different donor DNA fragments on both sides of the target site on the pMHAGE plasmid are homologous recombined with each other and the host genome sequence, and then the genome editing mutant strain after homologous recombination of different donor DNA is obtained;

[0052] (2) Verification of the mutant: design verification primers outside each donor DNA fragment according to the genomic sequence of the host prokaryote, verify the genome editing by PCR according to the possible two-by-two combination of the multi-donor DNA located on both sides of the target site, determine the large fragment of the non-essential gene region that can be deleted, and purify the strain containing the large fragment of the non-essential gene region that can be deleted;

[0053] wherein the PCR verification of the genome editing is: a. in the transformants with editing bands, the two-by-two combination of the multi-donor DNA located on both sides of the target site is homologously recombined, which reflects that the region of the two donor DNAs is a possible large fragment of the non-essential gene deletion region; b. in the transformants without editing bands, the two-by-two combination of the multi-donor DNA located on both sides of the target site is homologously recombined, which reflects that there may be essential genes or non-essential genes that cannot be deleted in the region of the two donor DNAs;

[0054] (3) Purification of the non-essential gene region maximized deletion strain: according to the strain with a large fragment of the non-essential gene deletion gene editing band verified in step (2), the single large fragment of the gene fragment deletion strain is further purified by dilution separation method; or according to the large fragment of the non-essential gene region that can be deleted verified in step (2), a gene editing plasmid pGE with a mini CRISPR cluster and donor DNA at both ends of the deletion region is constructed to improve the editing and purification efficiency; then the genome editing plasmid pGE is transformed into the host prokaryote cells, a large number of transformants are obtained on the screening medium (the screening medium used in the embodiment of the present application is SCV medium), and after purification, the non-essential gene region maximized deletion strain is obtained, thereby completing the simplification of the prokaryote genome.

[0055] The prokaryote is a bacterium or archaea containing an endogenous type I and / or type III CRISPR-Cas system, or a bacterium or archaea into which an endogenous type I and / or type III CRISPR-Cas system is introduced.

[0056] The application scope of the method includes, but is not limited to, *S. islandicus* (preferably prokaryotic *S. islandicus* REY15A), *Escherichia coli*, *Staphylococcus epidermidis*, *Shewanella putrefaciens*, thermophilic bacteria *Thermus thermophilus*, *Streptococcus thermophilus*, *Pyrococcus furiosus*, acidophilic thermosulfuric bacteria *Sulfolobus acidocaldarius*, *Sulfolobus solfataricus*, thermoautotrophic methanothermobacter thermautotrophicus*, *Haloferax volcanii*, *Pectobacterium atrosepticum*, and *Corynebacterium diphtheriae*.

[0057] Example 1

[0058] like Figure 1 The diagram illustrates the principle of prokaryotic genome simplification using the pMHAGE gene-editing plasmid to identify and delete large non-essential gene regions. Figure 1 It is known that the gene editing plasmid pMHAGE, which contains mini-CRISPR clusters and multiple donor DNA fragments, can target different donor DNA fragments on both sides of the cleavage site after DNA interference in the genome by the endogenous CRISPR system. Through homologous recombination, it can edit gene fragments of different lengths in the genome, thus realizing the identification and deletion of large non-essential genomic regions.

[0059] This embodiment uses *S. islandicus* REY15A as an example, which contains an endogenous type IA CRISPR system. The N1 region (800, 630 bp - 877, 876 bp) of this strain was selected as the target editing region for non-essential gene region exploration. The specific process of identifying and deleting large non-essential gene fragments is as follows:

[0060] 1. Construction of multi-homologous arm editing plasmids

[0061] (1) In the region of S. islandicus REY15A SiRe_0285, 40 bases from +134 to +173 were selected as N1-protospacer 1, and in the region of SiRe_0344, 40 bases from +508 to +547 were selected as N1-protospacer 2. Both target sites are adjacent to CCA-PAM (Protospacer Adjacent Motif), so they can be targeted by type I-A CRISPR system; at the same time, because the 5' end sequence of crRNA is mismatched with the sequence of the corresponding target site, they can also be targeted by type III-B CRISPR system; based on N1-protospacer 1, two primers N1-Sp1-F / N1-Sp1-R (Table 1) were designed, and the two primers were annealed to generate N1-Spacer1 fragment containing sticky ends at both ends; based on N1-protospacer 2, two primers N1-Sp2-F / N1-Sp2-R (Table 1) were designed, and the two primers were annealed to generate N1-Spacer2 fragment containing sticky ends at both ends;

[0062] Table 1, primer sequences used in the application

[0063]

[0064]

[0065]

[0066] The black underlined in the above table is the homologous region of the enzyme-digested plasmid used for Jibson recombination. The three bases forming the sticky end of the spacer are shown in black and bold.

[0067] (2) The artificial CRISPR vector pSe-Rp was treated with LguⅠ enzyme, and the enzyme digestion products were respectively ligated with N1-spacer1 fragment and N1-spacer2 fragment prepared in step (1) to obtain artificial CRISPR plasmids N1-PAC1 and N1-PAC2;

[0068] (3) According to the sequence of the N1 region and the two target sites, homologous arm fragments 1-5 are selected as donor DNA fragments, which are labeled as HA1, HA2, HA3, HA4 and HA5, and then 10 SOE PCR primers N1-HA1-F-Jibson / N1-HA1-R, N1-HA2-F / N1-HA2-R, N1-HA3-F / N1-HA3-R, N1-HA4-F / N1-HA4-R, N1-HA5-F / N1-HA5-R-Jibson (Table 1) are designed to amplify the homologous arm fragments 1-5, and then the donor DNA fragments are obtained by PCR amplification, and then the donor DNA fragments are fused in series by SOE-PCR to obtain a multi-donor DNA fragment N1-5HA containing a homologous region with the Sph I and Xho I enzyme digestion sites of the Pse-Rp plasmid;

[0069] Among them, the positions of N1-protospacer 1 and N1-protospacer 2 in the N1 region and the positions of each donor DNA fragment (HA1-5) in the genomic N1 are as shown in Figure 2

[0070] (4) The multi-donor DNA fragment N1-5HA obtained in step (3) is connected to the artificial CRISPR plasmids N1-PAC1 and N1-PAC2 digested by Sph I and Xho I respectively using Jibson recombination to obtain a multi-homologous arm editing plasmid N1-pMHAGE-1 and a multi-homologous arm plasmid N1-pMHAGE-2.

[0071] 2. Verification of multi-homologous arm editing plasmid transformants

[0072] 500 ng of the multi-homologous arm editing plasmid N1-pMHAGE-1 and the multi-homologous arm plasmid N1-pMHAGE-2 are respectively electroporated into the competent cells of S. islandicus REY15A, and are plated on SCV (0.2% sucrose, 0.2% casaminoacids plus 1% vitamin solution) solid medium and cultured at 76°C for 7 days. 24 transformants of each multi-homologous arm plasmid are picked, and bacterial liquid PCR verification is performed according to the corresponding editing method of the plasmid N1-pMHAGE-1 and the plasmid N1-pMHAGE-2 using the corresponding multi-homologous arm editing verification primers.

[0073] The possible gene editing results of the N1-pMHAGE-1 transformant and the N1-pMHAGE-2 transformant are as shown in Figure 2 ​Figure 1 shows the schematic diagram of the N1-pMHAGE-1 and N1-pMHAGE-2 plasmids. The HA is the homologous arm, representing the position of homologous recombination on the genome, the Spacer represents the targeted cleavage site on the genome, the arrow represents the position and direction of the verification primer. The white rectangle represents the possible deleted gene fragment.

[0074] By Figure 2 It can be seen that there are four possible gene editing results of N1-pMHAGE-1 transformants, which are ① deleting HA1-HA2, verified by primers N1-HA1-FP-F / N1-HA2-FP-R; ② deleting HA1-HA3, verified by primers N1-HA1-FP-F / N1-HA3-FP-R; ③ deleting HA1-HA4, verified by primers N1-HA1-FP-F / N1-HA4-FP-R; ④ deleting HA1-HA5, verified by primers N1-HA1-FP-F / N1-HA5-FP-R. There are six possible gene editing results of N1-pMHAGE-2 transformants, which are ① deleting HA3-HA4, verified by primers N1-HA3-FP-F / N1-HA4-FP-R; ② deleting HA2-HA4, verified by primers N1-HA2-FP-F / N1-HA4-FP-R; ③ deleting HA1-HA4, verified by primers N1-HA1-FP-F / N1-HA4-FP-R; ④ deleting HA3-HA5, verified by primers N1-HA3-FP-F / N1-HA5-FP-R; ⑤ deleting HA2-HA5, verified by primers N1-HA2-FP-F / N1-HA5-FP-R; ⑥ deleting HA1-HA5, verified by primers N1-HA1-FP-F / N1-HA5-FP-R. Among them, the editing of the repeated N1 region HA1-HA4 region is verified only once for the transformants of the two plasmids, that is, the primers N1-HA1-FP-F / N1-HA4-FP-R are used to verify the N1-pMHAGE-1 transformant. The above primers are listed in Table 1.

[0075] The PCR verification results of the bacterial liquid of the 24 N1-pMHAGE-1 transformants are shown in Figure 3 The PCR verification results of the bacterial liquid of the 24 N1-pMHAGE-2 transformants are shown in Figure 4 The statistics of the possible gene editing results of the N1-pMHAGE-1 transformants and the N1-pMHAGE-2 transformants are shown in Table 2.

[0076] Table 2

[0077]

[0078] By Figure 3 , 4As shown in Table 2, different gene editing occurred among different individuals within the same transformant colony. The results indicated that gene editing, i.e., gene deletion, occurred in the HA2-HA4, HA3-HA4, and HA3-HA5 homologous arm regions within the N1 region of the N1 transformant. The HA2-HA4 gene fragment was the longest, at 47.1 kb. However, no corresponding editing band was observed in N1-pMHAGE-1. This result suggests that the gene fragment between HA1 and HA2 within the N1 region of N1-Spacer1 contains either an essential gene or a combination of non-essential genes that cannot be deleted.

[0079] 3. Purification of strains with maximum deletion of non-essential gene regions

[0080] Depend on Figure 4 The verification results showed that the largest deleteable non-essential gene region obtained from the N1-pMHAGE-2 transformant editing was a 47.1 kb gene fragment from HA2 to HA4 in the N1 region. To improve editing and purification efficiency, a gene editing plasmid N1-2 / 4HA-pGE containing a mini CRISPR cluster and donor DNA at both ends of the deletion region was constructed. Then, the genome editing plasmid N1-2 / 4HA-pGE was transformed into host prokaryotic cells, and a large number of transformants were obtained on the selection medium (SCV medium used in this embodiment of the invention). After purification, strains with maximum deletion of non-essential gene regions were obtained, thus completing the simplification of the prokaryotic genome.

[0081] The construction method of the gene editing plasmid N1-2 / 4HA-pGE is as follows: the artificial CRISPR vector pSe-Rp is treated by Lgu I enzyme, and the enzyme product is ligated with the N1-spacer2 fragment prepared in step 1(1) in the case 1 to obtain the artificial CRISPR plasmid N1-2 / 4HA-PAC; the left homologous arm is amplified by using the SOE PCR primer N1-HA2-F-Jibson / N1-HA2-R-SOE-HA4, and the right homologous arm is amplified by using the SOE PCR primer N1-HA4-F-SOE-HA2 / N1-HA4-R-Jibson; the left and right arm DNA fragments are fused by SOE-PCR to obtain the double-donor DNA fragment N1-2+4HA containing the homologous region with the Sph I and Xho I enzyme cutting sites of the Pse-Rp plasmid; the obtained double-donor DNA fragment N1-2+4HA is connected to the artificial CRISPR plasmid N1-2 / 4HA-PAC treated by Sph I and Xho I enzyme by using the Jibson recombination method to obtain the double-homologous arm editing plasmid N1-2 / 4HA-pGE. The double-homologous arm editing plasmid N1-2 / 4HA-pGE is transformed according to step 2, and the obtained transformant is verified by using the primers N1-HA2-FP-F / N1-HA4-FP-R (Table 1) to verify the editing band.

[0082] The transformant (N1-pMHAGE-2 transformant) colony with the gene editing band is resuspended with 50 μl of water, and then diluted 10 3 times and coated on the SCV solid medium containing 20 mg / L uracil and 6-12 mg / L 5-FOA, and then cultured at 76°C under humid conditions for 7 days, and then the obtained single colony is verified by PCR to be a pure single gene editing strain.

[0083] The specific verification method is as follows: the editing band is verified by using the primers N1-HA2-FP-F / N1-HA4-FP-R (Table 1), and the wild type band is verified by using the primers N1-HA2-CP-F / N1-HA4-CP-R (Table 1). The pure N1 region HA2-HA4 large fragment gene deletion strain with the editing band and without the wild type band is named as DH1.

[0084] The specific verification result is shown in Figure 5 It can be known from Figure 5 the above that the strain in which the N1 region HA2-HA4 gene fragment is deleted, that is, the non-essential gene fragment in the N1 region can be maximally deleted, can be purified from the double-homologous arm editing plasmid N1-2 / 4HA-pGE transformant. Therefore, it is verified that the multi-donor DNA gene editing plasmid can recognize the largest non-essential gene fragment that can be deleted in a certain gene region.

[0085] In summary, the plasmid for recognizing and deleting large fragment of non-essential gene region provided by the application can perform various forms of gene editing on prokaryotes containing CRISPR-Cas system through homologous recombination, and the essential genes are skipped to recognize and delete the largest length non-essential genes, so as to realize the simplification of prokaryote genome.

[0086] Example 2

[0087] This embodiment is described by taking S. islandicus REY15A as an example, and the strain contains an endogenous I-A type CRISPR system. The N6 region (2,298,485bp-2,382,752bp) on the strain is taken as the editing region to explore the non-essential gene region, and the process of specifically recognizing and deleting large fragment of non-essential gene region is as follows:

[0088] 1. Construction of multi-homologous arm editing plasmid

[0089] (1) 40 bases from +344 to +383 in the SiRe_2466 region of S. islandicus REY15A are selected as N6-protospacer 1, and 40 bases from +297 to +336 in the SiRe_2507 region are selected as N6-protospacer 2, both of which are adjacent to CCA-PAM (Protospacer Adjacent Motif), so they can be targeted by I-A type CRISPR system; at the same time, because the 5' end sequence of crRNA is mismatched with the sequence of the corresponding target site, they can also be targeted by III-B type CRISPR system; two primers N6-Sp1-F / N6-Sp1-R (Table 1) are designed based on N6-protospacer 1, and the two primers generate N6-spacer 1 fragment containing sticky ends at both ends by annealing; two primers N6-Sp2-F / N6-Sp2-R (Table 1) are designed based on N6-protospacer 2, and the two primers generate N6-spacer 2 fragment containing sticky ends at both ends by annealing;

[0090] (2) The artificial CRISPR vector pSe-Rp is treated by LguⅠ enzyme, and the enzyme cutting products are respectively connected with the N6-spacer 1 fragment and the N6-spacer 2 fragment prepared in step (1) to obtain artificial CRISPR plasmids N6-PAC1 and N6-PAC2;

[0091] (3) According to the sequence of the N6 region and the two target sites, homologous arm fragments 1-5 are selected as donor DNA fragments, labeled as HA1, HA2, HA3, HA4 and HA5, then 10 SOE PCR primers N6-HA1-F-Jibson / N6-HA1-R, N6-HA2-F / N6-HA2-R, N6-HA3-F / N6-HA3-R, N6-HA4-F / N6-HA4-R and N6-HA5-F / N6-HA5-R-Jibson (Table 1) are designed to amplify the homologous arm fragments 1-5, and after PCR amplification, the donor DNA fragments are obtained, then the donor DNA fragments are fused in series by SOE-PCR to obtain a multi-donor DNA fragment N6-5HA containing a homologous region with the Sph I and Xho I enzyme digestion sites of the Pse-Rp plasmid;

[0092] Among them, the positions of N6-protospacer 1 and N6-protospacer 2 in the N6 region and the positions of each donor DNA fragment (HA1-5) in the genomic N6 are as shown in Figure 6 ;

[0093] (4) The multi-donor DNA fragment N6-5HA obtained in step (3) is connected to the artificial CRISPR plasmids N6-PAC1 and N6-PAC2 digested by Sph I and Xho I respectively using Jibson recombination to obtain a multi-homologous arm editing plasmid N6-pMHAGE-1 and a multi-homologous arm plasmid N6-pMHAGE-2.

[0094] 2. Verification of multi-homologous arm editing plasmid transformants

[0095] 500 ng of the multi-homologous arm editing plasmid N6-pMHAGE-1 and the multi-homologous arm plasmid N6-pMHAGE-2 are respectively electroporated into the competent cells of S. islandicus REY15A, and are plated on SCV (0.2% sucrose, 0.2% casaminoacids plus 1% vitamin solution) solid medium and cultured at 76°C for 7 days. 16 transformants of each multi-homologous arm plasmid are picked, and multiplex primer PCR is performed according to the corresponding editing method of the plasmid N6-pMHAGE-1 and the plasmid N6-pMHAGE-2 using the corresponding multi-homologous arm editing verification primers (Table 1).

[0096] There are four possible gene editing outcomes for the N6-pMHAGE-1 transformant: ① deletion of HA1-HA2, verified using primers N6-HA1-FP-F / N6-HA2-FP-R; ② deletion of HA1-HA3, verified using primers N6-HA1-FP-F / N6-HA3-FP-R; ③ deletion of HA1-HA4, verified using primers N6-HA1-FP-F / N6-HA4-FP-R; ④ deletion of HA1-HA5, verified using primers N6-HA1-FP-F / N1-HA5-FP-R. There are six possible gene editing outcomes for the N6-pMHAGE-2 transformant: ① deletion of HA3-HA4, verified using primer N6-HA3-FP-F / N1-HA4-FP-R; ② deletion of HA2-HA4, verified using primer N6-HA2-FP-F / N1-HA4-FP-R; ③ deletion of HA1-HA4, verified using primer N6-HA1-FP-F / N1-HA4-FP-R; ④ deletion of HA3-HA5, verified using primer N6-HA3-FP-F / N1-HA5-FP-R; ⑤ deletion of HA2-HA5, verified using primer N6-HA2-FP-F / N1-HA5-FP-R; ⑥ deletion of HA1-HA5, verified using primer N6-HA1-FP-F / N1-HA5-FP-R. All primers are listed in Table 1.

[0097] The multiplex primer PCR validation results of the 16 N6-pMHAGE-1 transformants are as follows: Figure 7 As shown. The primers used in the multiplex PCR system were combinations of N6-HA1-FP-F, N6-HA2-FP-R, N6-HA3-FP-R, N6-HA4-FP-R, and N6-HA5-FP-R (Table 1). The molar ratio of each primer was 4:1:1:1:1. Figure 7 The abbreviation is N6-1F / 2345R-FP.

[0098] The multiplex primer PCR validation results of the 16 N6-pMHAGE-2 transformants are as follows: Figure 8 As shown. In Figure 8 In the multiplex PCR system 1, the primers used were a combination of N6-HA4-FP-R, N6-HA1-FP-F, N6-HA2-FP-F, and N6-HA3-FP-F (Table 1), with a molar ratio of 3:1:1:1. Figure 8 The abbreviation is N6-123F / 4R-FP; the primers used in multiplex PCR system 2 are a combination of N6-HA5-FP-R, N6-HA1-FP-F, N6-HA2-FP-F, N6-HA3-FP-F, and N6-HA4-FP-F, with a molar ratio of 3:1:1:1. Figure 8N6-pMHAGE-1 is abbreviated as N6-123F / 5R-FP.

[0099] The statistics of possible gene editing results of the N6-pMHAGE-1 transformants and N1-pMHAGE-2 transformants are shown in Table 3.

[0100] Table 3

[0101]

[0102] From Figure 7 , 8 and Table 3, it can be seen that different gene editing occurred between different individual bacteria in the same transformant colony. The results show that gene editing, i.e. gene deletion, occurred in the region between HA2-HA4 and HA3-HA4 in the N6 region, and the 2 / 4 HA editing bands are relatively shallow. This result shows that HA3-HA4 between the N6 region can be knocked out, and HA2-HA4 between them can be knocked out. There is no corresponding editing band for N6-pMHAGE-1. This result shows that the gene fragment between HA1-HA2 in the N6 region where N6-Spacer1 is located contains "essential genes" or non-essential gene combinations that cannot be deleted.

[0103] 3. Purification of the strain with maximum deletion of non-essential gene region

[0104] The transformant (N6-pMHAGE-2 transformant) colony with gene editing bands was resuspended with 50 μl of water, and then diluted 10 3 times and spread on SCV solid medium containing 20 mg / L uracil and 6-12 mg / L 5-FOA, and incubated at 76°C under humid conditions for 7 days. Then the obtained single colony was verified by PCR to determine whether it was a pure single gene editing strain.

[0105] The specific verification method is as follows: when verifying the strain with deletion of the gene fragment between HA2-HA4 homologous arms in the N6 region, primers N6-HA2-FP-F and N6-HA4-FP-R (Table 1) are used to verify the editing band, and N6-HA2-CP-F and N6-HA4-CP-R (Table 1) are used to verify the wild type band. The single colony with editing band and no wild type band in the PCR result of the bacterial solution is the pure gene editing strain purified, but a pure strain with deletion of the gene region between HA2-HA4 in the N6 region is not obtained.

[0106] When verifying whether it is a strain for deleting the gene fragment between HA3-HA4 in the N6 region, then the primer N6-HA3-FP-F and N6HA4-FP-R (Table 1) are required to verify the editing band, and N6-HA3-CP-F and N6-HA4-CP-R (Table 1) are required to verify the wild type band, and the single colony with the editing band and without the wild type band in the PCR result of the bacterial liquid shows that the pure gene editing strain is purified, which is named as DH4, and the specific verification result is shown in Figure 9 .

[0107] It can be known from Figure 9 that the strain for deleting the gene fragment between HA3-HA4 in the N6 region can be purified from the N6-pMHAGE-2 transformant.

[0108] In conclusion, the plasmid for recognizing and deleting the large fragment of the non-essential gene region can perform various forms of gene editing on the prokaryote containing the CRISPR-Cas system through homologous recombination, skips the essential gene to recognize and delete the largest length non-essential gene, and realizes the simplification of the prokaryote genome.

Claims

1. A plasmid which recognizes and deletes a large fragment of an unnecessary gene region, characterized by comprising a DNA sequence represented by SEQ ID NO:

1. The plasmid is a gene editing plasmid pMHAGE containing a mini-CRISPR cluster and a multi-donor DNA fragment; A protospacer sequence is selected as a target site in a quasi-editing region of a prokaryotic genome, and two reverse complementary primers are designed according to the protospacer sequence, and the sequences are forward primer: 5'-AAG-Nn-3' and reverse primer: 5'-AGC-N'n-3', wherein Nn and N'n are reverse complementary sequences, N and N' represent bases A, T, G or C, and n represents the number of bases of the protospacer sequence; the two primers are annealed to form double-stranded DNA with sticky ends, i.e. a spacer fragment; the artificial CRISPR vector pSe-Rp is treated by restriction enzyme Lgu I, and then the spacer fragment with sticky ends is connected by T4 ligase to obtain an artificial CRISPR plasmid pAC capable of producing mature crRNA; and a multi-donor DNA fragment required for recognizing a non-essential large fragment of a target region is inserted into the artificial CRISPR plasmid pAC capable of producing mature crRNA to obtain a gene editing plasmid pMHAGE containing a multi-donor DNA fragment; The prokaryote is bacteria or archaea containing an endogenous type I and / or type III CRISPR-Cas system, or bacteria or archaea into which an endogenous type I and / or type III CRISPR-Cas system is introduced. The multi-donor DNA fragment is obtained by SOE-PCR fusion of DNA fragments homologous to multiple target sites on both sides of the quasi-editing region of the host cell genome, and the obtaining method comprises the following steps: 1) According to the DNA fragments homologous to multiple target sites on both sides of the quasi-editing region of the host cell genome, the amplification primers of each donor DNA fragment with adjacent donor DNA adapters are designed by SOE-PCR, and each donor DNA fragment can be but is not limited to labeled as HA1, HA2,..., HAn; 2) The same molar concentration of each donor DNA fragment is mixed in equal volume, and the fusion fragment is obtained by amplifying 15-20 cycles by overlap-PCR without adding any primer; 3) The first donor DNA fragment amplification forward primer F1 and the last donor DNA fragment amplification reverse primer Rn are used as amplification primers, and the fusion fragment obtained in step 2) is used as a template for PCR amplification, and after enrichment and purification, a multi-donor DNA fragment is obtained, which is a tandem fusion product of each donor DNA HA1-HA2-...-HAn; The multi-donor DNA is greater than or equal to 3, i.e. n of HAn is greater than or equal to 3.

2. The plasmid of claim 1, wherein the large fragment of the non-essential gene region is identified and deleted. The length of each donor DNA fragment in the multi-donor DNA is 250-600 bp, i.e. 250 bp≤HAn length≤600 bp; and the interval of each adjacent donor DNA fragment in the multi-donor DNA on the host cell genome is 4-40 kb.

3. The use of the plasmid for identifying and deleting large fragment of non-essential gene region in gene editing according to claim 1.

4. Use according to claim 3, wherein the compound is ###0002### The gene editing is gene deletion.

5. The use according to claim 3, wherein the compound is ###0002### The gene editing is genome reduction.

6. A method for reducing the genome of a prokaryote using the plasmid of claim 1 which recognizes and deletes large nonessential gene regions, characterized in that, The method comprises the following steps: (1) Obtaining a mutant strain: after the pMHAGE plasmid is electroporated into a host prokaryotic cell, a pre-crRNA is transcribed from an artificial CRISPR cluster on the plasmid, which is processed into a mature crRNA in the cell; the crRNA forms a target effect complex with a CRISPR-Cas protein, and then the crRNA pairs with a target DNA strand on the host cell genome to identify and cut the target site; subsequently, a plurality of different donor DNA fragments on both sides of the target site on the pMHAGE plasmid are homologously recombined with each other and the host genome sequence, thereby obtaining a genome editing mutant strain after homologous recombination of different donor DNA combinations; The prokaryote is a bacterium or archaea containing an endogenous type I and / or type III CRISPR-Cas system, or a bacterium or archaea into which an endogenous type I and / or type III CRISPR-Cas system is introduced. (2) Verification of the mutant strain: verification primers outside each donor DNA fragment are designed according to the host prokaryotic genome sequence, and the genome editing is verified by PCR of the bacterial liquid according to the possible two-by-two combination of the plurality of donor DNA on both sides of the target site to determine the large fragment of non-essential gene region that can be deleted, and the strain containing the large fragment of non-essential gene region that is deleted is purified; The PCR verification of the genome editing is as follows: a, in the transformant with editing bands, the plurality of donor DNA on both sides of the target site are homologously recombined in a two-by-two combination, which reflects that the region of the two donor DNAs is a possible large fragment of non-essential gene deletion region; b, in the transformant without editing bands, the plurality of donor DNA on both sides of the target site are homologously recombined in a two-by-two combination, which reflects that the region of the two donor DNAs may contain essential genes or non-essential genes that cannot be deleted. (3) Purification of the strain with maximum deletion of the non-essential gene region: according to the strain with the large fragment of non-essential gene deletion gene editing bands verified in step (2), a single large fragment of gene fragment deletion strain is further purified by dilution separation; or according to the large fragment of non-essential gene region that can be deleted in step (2), a gene editing plasmid pGE with a mini CRISPR cluster and donor DNA at both ends of the deletion region is constructed to improve the editing and purification efficiency; then the genome editing plasmid pGE is transformed into the host prokaryotic cell, a large number of transformants are obtained on the selection medium, and after purification, the strain with maximum deletion of the non-essential gene region is obtained, thereby completing the genome reduction of the prokaryote.

7. The method of reducing a prokaryotic genome according to claim 6, wherein, The prokaryotes include S. icelandicus, E. coli Escherichia coli , S. epidermidis Staphylococcus epidermidis , S. putrefaciens Shewanella putrefaciens , S. thermophilus Thermus thermophilus , S. pyogenes Streptococcus thermophilus , S. acidocaldarius Pyrococcus furiosus , S. solfataricus Sulfolobus acidocaldarius , M. thermoautotrophicum Sulfolobus solfataricus , H. salinarum Methanothermobacter thermautotrophicus , P. melioides Haloferax volcanii , P. atrosepticum Pectobacterium atrosepticum , C. diphtheriae Corynebacterium diphtheriae .

Images