CN112980891B - Coli genome editing tool based on CRISPR-Cas - Google Patents

Abstract

The application discloses a CRISPR-Cas-based escherichia coli genome editing tool, which comprises a plasmid pEcCas for expressing endonuclease Cas9, wherein the plasmid pEcCas comprises Cas9 nuclease genes, lambda-RED recombinase genes, araC protein genes, sacB genes, replicon pSC101, rhamnose regulatory protein RhaS/RhaR genes and sgRNA sequences for guiding Cas9 to auxiliary plasmid DNA sequences. The genome editing tool can realize genome editing in B-series and K-12-series escherichia coli, shortens the editing period and has wider host adaptability.

Description

Coli genome editing tool based on CRISPR-Cas

Technical Field

The application belongs to the field of genetic engineering, and relates to a CRISPR-Cas-based escherichia coli genome editing tool and application thereof in escherichia coli.

Background

Coli is a common host for molecular biology as a model microbial strain. It is extremely important to establish an efficient genome editing method in E.coli. Several genome editing tools based on the type II CRISPR-Cas system have been reported to be established in e.coli, pCas using pTargetF or ptarget in combination being the most widely used set of plasmids at present. We originally established pCas/pTargetF or pTargetT dual plasmid systems (ECCRISPR 1.0) that enable iterative genome editing in E.coli MG1655, already shared on Addgene. However, some researchers have shown that the pCas/pTargetF or pTargetT double plasmid system does not work in E.coli Bl21 (DE 3) after using pCas. Thus, we did not obtain transformants after transformation of pTargetT-. DELTA.cadA or pTargetT-. DELTA.maeA plasmids (Jiang Y, chen B, et al appl Environ Microbiol, 2015) into pCas-containing Bl21 (DE 3) (see Table 2), indicating that existing genome editing tools do not work well in some E.coli such as E.coli BL 21B (DE 3). We speculate that the sgRNA anchoring the helper plasmid pTargetF or pTargetT replicon on the pCas plasmid had leaky transcription in BL21 (DE 3) such that the helper plasmid was cut so that we could not obtain transformants on dual-antibody screening plates containing kanamycin (50. Mu.g/mL) and spectinomycin (50. Mu.g/mL).

Disclosure of Invention

In order to overcome the defects of the double-plasmid CRISPR-Cas9 gene editing system in the prior art, a new thought is designed, and the new thought comprises the following strategies: 1. substitution of the lacI/trc promoter on the pCas plasmid with a rhamnose-inducible promoter (rhaS/rhaR) to control the gRNA of the cleavage helper plasmid DNA sequence, avoiding leaky transcription of the cleavage helper plasmid in certain E.coli, such as BL21 (DE 3), and thus enabling genome editing in more types of E.coli; 2. the temperature-sensitive replicon pSC101ts on the pCas plasmid is replaced by pSC101, so that the escherichia coli can grow rapidly at 37 ℃, the limitation of low-temperature (ECCRISPR 1.0 is 30 ℃) culture is avoided, and the experimental period is shortened; 3. the sacB gene is added to the pCas plasmid, and can be used for carrying out reverse screening on a sucrose flat plate when the plasmid is lost, so as to replace the original low-temperature reverse screening. Finally, an upgrade version plasmid pEcCas (ecrspr 2.0) was obtained.

Accordingly, a first object of the present application is to provide a universal E.coli genome editing tool. The method comprises the following technical scheme:

a CRISPR-Cas based escherichia coli genome editing tool comprising a plasmid pEcCas for expressing endonuclease Cas9, the plasmid pEcCas comprising Cas9 nuclease gene, lambda-RED recombination system (or weighing group enzyme) gene for improving editing efficiency, araC protein gene, sacB gene, replicon pSC101, rhamnose regulatory protein RhaS/RhaR gene, sgRNA sequence for guiding Cas9 to helper plasmid DNA sequence.

The lambda-RED recombination system comprises Exo protein, beta protein and Gam protein. The expression of the lambda-RED recombination system can be induced by arabinose and is regulated by an arabinose promoter.

The addition of the RhaS/RhaR gene described above allows the rhamnose-inducible promoter to replace the lacI/trc promoter to avoid leaky transcription of the sgRAN which cleaves helper plasmids.

Preferably, the above plasmid pEcCas further comprises the kanamycin resistance gene kanR.

In one embodiment, the cas9 nuclease gene is a cas9 nuclease gene derived from streptococcus pyogenes, and may be a cas nuclease gene derived from another microorganism.

To achieve the gene editing function, the above-described E.coli genome editing tool further includes another helper plasmid (selected from pTargetF or pTargetT) used in cooperation with the plasmid pEcCas, so that the above-described E.coli genome editing tool is presented as a pEcCas/pTargetF or pEcCas/pTargetT double plasmid system.

The above plasmid pEcCas also includes an sgRNA sequence for directing Cas9 to a helper plasmid DNA sequence, the transcription of which is induced by L-rhamnose.

When genome editing is completed, the sgRNA sequence is used for targeting a helper plasmid DNA sequence, guiding Cas9 to cleave the helper plasmid, and achieving the effect of eliminating the helper plasmid.

Preferably, the sgRNA sequence is a 20nt N20 sequence located downstream of the rhamnose promoter Prha, complementary to the target sequence, which is: 5'-CTATCGTCTTGAGTCCAACC-3' (SEQ ID NO: 2).

The N20 sequence is identical to part of the sequences of the replicon pMB1 and P15A genes, which N20 sequence is followed by the PAM sequence 5'-CGG-3' on the pMB1 and P15A genes.

The nucleotide sequence of the plasmid pEcCas described above can be SEQ ID NO. 1, which includes 14605 bases.

The helper plasmid pTargetF or pTargetT described above is used in combination with a plasmid pEcCas comprising sgRNA, pMB1 and/or P15A replicons for targeting predetermined sites of the E.coli genome.

The length of the sgRNA on the helper plasmid may be 18-25nt, preferably 19-22nt, more preferably 20nt.

Preferably, the helper plasmid pTargetF or ptarget further comprises the spectinomycin resistance gene aadA.

In one embodiment, transcription of sgRNA on the helper plasmid described above is controlled by a Pj23119 constitutive promoter.

The above-described E.coli genome editing tool is in the form of a kit comprising a plasmid pEcCas, helper plasmid pTargetF or pTargetT, various PCR primers for performing genome editing, and the like.

A second object of the present application is to provide a method for constructing the pEcCas described above, comprising the steps of:

obtaining pSC101 replicon fragments by PCR amplification; PCR amplification of kanamycin resistance gene kanR fragment using pCas plasmid as template; PCR amplification of cas9 and lambda-RED recombinase gene fragments by using pCas plasmid as template; amplifying sacB gene fragments from bacillus subtilis genome DNA; the sgRNA fragment was amplified from the pTargetF-cadA plasmid reported in the literature Jiang Y, chen B, et al Appl Environ Microbiol, 2015; the rhaRS fragment was amplified from the genomic DNA of E.coli MG1655, and the six fragments were assembled to obtain the pEcCas plasmid.

Preferably, the assembly may be a ClonExpress MultiS One Step Cloning Kit (Vazyme) assembly or a Gibson assembly.

A third object of the present application is to provide the use of the novel E.coli genome editing tool described above in B-and K-12-series E.coli for genome editing. The E.coli genome editing tool of the present application has wide applicability, and can be applied to genome editing of B-series, K-12-series and W-series E.coli, for example, to genome editing of E.coli BL21 and derivatives thereof such as BL21 (DE 3) or E.coli MG 1655.

In a preferred embodiment, the above application comprises the steps of:

1) Transforming plasmid pEcCas into competent cells of escherichia coli, and screening on an LB solid plate containing kanamycin at 37 ℃;

2) Selecting positive clones, preparing electrotransformation competent cells, and adding arabinose to induce RED recombinase expression 1 hour before centrifugal collection;

3) Transferring helper plasmids pTargetF or pTargetT into competent cells obtained in the step 2) through electrotransformation, coating bacterial liquid on a dual-antibody LB solid plate containing kanamycin and spectinomycin, culturing overnight at 37 ℃, and then verifying and screening clones;

4) Selecting positive clones in LB culture solution containing kanamycin, adding rhamnose, inducing the sgRNA sequence of the DNA sequence of the guide auxiliary plasmid to transcribe, and verifying the elimination of the auxiliary plasmid on LB solid plates containing spectinomycin;

5) Picking auxiliary plasmid to eliminate positive clones, directly culturing overnight at 37 ℃ in an LB liquid culture medium containing glucose, sucking bacterial liquid, streaking and dividing single bacteria on an LB solid plate containing glucose and sucrose, identifying single bacteria on an LB plate containing kanamycin resistance, and verifying pEcCas plasmid elimination to obtain an editing strain.

The escherichia coli genome editing tool pEcCas/pTargetF or pTargetT is a universal double-plasmid system, can effectively realize genome editing in K-12, B and W escherichia coli, shortens editing period, and has wider host adaptability.

Drawings

FIG. 1 is a schematic representation of the structure of plasmid pEcCas plasmid.

FIG. 2 is a schematic representation of the structure of the sgRNA sequence in plasmid pEcCas. The gene fragment was used to anchor the pMB1 and/or P15A replicon of the helper plasmid DNA sequence, wherein the N20 sequence was identical to part of the sequences of the replicon pMB1 and P15A genes, which N20 sequence was followed by the PAM sequence 5'-CGG-3' on the pMB1 and P15A genes.

Detailed Description

In this context, for ease of description, a protein such as Cas9 is sometimes used in combination with its coding gene (DNA) name, and those skilled in the art will appreciate that they represent different substances in different descriptive contexts. Those skilled in the art will readily understand their meaning depending on the context and context. For example, for Cas9, when used to describe nuclease function or class, refers to a protein; when described as a gene, it refers to the gene encoding the Cas 9. Similarly, RNA such as sgRNA is sometimes used in combination with the name of its encoding gene (DNA), and those skilled in the art will appreciate that they represent different substances in different descriptive contexts.

The E.coli genome editing tool of the present application may be a double plasmid combination of plasmid pEcCas and helper plasmid, wherein the helper plasmid may be pTargetF or pTargetT, pTargetF and pTargetT, the difference being that the pTargetF plasmid does not contain the homology arm sequences required for homologous recombination, the homology arms being provided separately in fragment form; and pTargetT is a plasmid in which the homology arm sequence is cloned.

In this two-plasmid system, the nickase Cas9 expressed by the pEcCas plasmid is to function in two places: firstly, sgRNA on an auxiliary plasmid guides Cas9 to cut an escherichia coli genome target point, so that genome transformation is realized; second, after genome engineering is completed, to obtain a plasmid-free host bacterium for iterative editing, pMB1 and/or P15A replicons of helper plasmids need to be cleaved using sgrnas on the pEcCas plasmid to guide Cas9 to eliminate the helper plasmids.

In view of the convenience of experimental operation, the escherichia coli genome editing tool of the application can be a kit, and the required materials are integrated in one kit. In a preferred embodiment, the above-described kit may include at least one of the following items, in addition to the plasmid pEcCas, pTargetF or ptarget plasmid, various PCR primers for performing genome editing, and the like: a carrying means, the space of which is divided into a defined space which can house one or more containers, such as kits, vials, test tubes, and the like, each of which contains a separate component for use in the method of the application; the instructions, which can be written on bottles, test tubes and the like, or on a single piece of paper, or outside or inside the container, for example paper with a video APP download window for the operation demonstration, such as a two-dimensional code, can also be in the form of multimedia, such as a CD, a U-disc, a netdisk, etc.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are for illustrative purposes only and are not limiting of the application. It is also to be understood that various changes and modifications may be made by those skilled in the art after reading the inventive concept, and that such equivalents are intended to fall within the scope of the application as defined by the appended claims.

The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.

In the examples, to simplify the expression of pEcCas in a two-plasmid system with a helper plasmid (pTargetF or pTargetT), pEcCas is referred to as plasmid A and helper plasmids such as pTargetT-. DELTA.cadA and pTargetT-. DELTA.maeA are referred to as plasmid B.

Examples

Materials and methods

Primer synthesis herein was done by Shanghai Bai Lige Biotechnology Co., ltd, and sequencing was done by Shanghai Biotechnology Co., ltd.

Molecular biology experiments herein include plasmid construction, digestion, ligation, competent cell preparation, transformation, medium preparation, etc., and are mainly described in "molecular cloning Experimental guidelines (third edition), J.Sam Broker, D.W. Lassel (America) code, huang Peitang, et al, scientific Press, beijing, 2002). The specific experimental conditions can be determined by simple experiments, if necessary.

The PCR amplification experiments were performed according to the reaction conditions or instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.

Example 1 construction of plasmid pEcCas

The primers required for construction of the pEcCas plasmid are shown in table 1.

TABLE 1 PCR primer sequences

Note that: the number 1 of the suffix of the primer name in the table represents the forward primer, i.e., -F, and the number 2 represents the reverse primer, i.e., -R.

1.1 construction of pSC101 replicon fragments

pSC101 replicon fragments were obtained by PCR amplification using repA1/repA2 as primers and pMW119 vector (Nippon Gene) as a template.

PCR reaction System (50. Mu.L):

PCR reaction conditions:

KOD Plus kit was purchased from Toyobo (Shanghai) Biotechnology Co.

1.2 construction of kanamycin resistance Gene kanR fragment

The kanamycin resistance gene kanR fragment was amplified by PCR using kan1/kan2 as a primer and pCas plasmid (Addgene: 62225) as a template.

The PCR reaction conditions and the required reagents are the same as in step 1.1.

1.3 amplification of cas9 and lambda-RED recombinase Gene fragments

PCR was performed using cas9 and lambda-RED recombinase gene fragments, respectively, as primers, pCas plasmid (Addgene: 62225) as a template.

The PCR reaction conditions and the required reagents are the same as in step 1.1.

1.4 amplification of sacB Gene fragments

The sacB gene fragment was amplified from the genomic DNA of Bacillus subtilis (ATCC 23857) using sac1/sac2 as a primer.

The PCR reaction conditions and the required reagents are the same as in step 1.1.

1.5 construction of sgRNA fragments

The sgRNA fragment was amplified from the pTargetF-cadA plasmid (Jiang Y, chen B, et al appl Environ Microbiol, 2015) using primers sgnna 1/sgnna 2.

The PCR reaction conditions and the required reagents are the same as in step 1.1.

1.6 amplification of rhaR, rhaS fragments

The rhaRS fragment was amplified from E.coli MG1655 (ATCC 700926) genomic DNA using primers rha1/rha 2.

The PCR reaction conditions and the required reagents are the same as in step 1.1.

1.7 fragment Assembly

Six fragments obtained in the above steps were assembled using ClonExpress MultiS One Step Cloning Kit (Vazyme) to obtain pEcCas plasmid. Sequencing was verified to be correct.

The PCR reaction conditions and the required reagents are the same as in step 1.1.

EXAMPLE 2 E.coli genome editing

2.1 transformation of pEcCas plasmid into E.coli MG1655 and BL21 (DE 3) chemocompetent cells, respectively, screening on LB solid plates containing kanamycin (50. Mu.g/mL) at 37 ℃; preparation of chemically transformed competent cells reference the guidelines for molecular cloning experiments (third edition);

2.2 picking positive clones, preparing electrotransformation competent cells, and adding 10mM arabinose to induce RED recombinase expression 1 hour before centrifugation collection;

2.3 electrotransformation into helper plasmid pTargetT-DeltacadA or pTargetT-DeltamaeA plasmid (electrotransformation voltage is 2.5 kV), recovering for 1 hour at 37 ℃ and 200rpm, then coating bacterial liquid on double-anti-LB solid plate containing kanamycin (50 mug/mL) and spectinomycin (50 mug/mL), and verifying and screening clone after overnight culture at 37 ℃; electrotransformation competent cell preparation methods are described in molecular cloning Experimental guidelines (third edition);

2.4 helper plasmid elimination: selecting positive clones in LB liquid (containing 50 mug/mL kanamycin), adding 10mM rhamnose, inducing transcription of sgRNA sequence leading to helper plasmid DNA sequence, culturing overnight, streaking out single bacteria, and verifying elimination of helper plasmid on LB solid plate containing spectinomycin (50 mug/mL);

2.5pEcCas plasmid elimination: picking auxiliary plasmids to eliminate positive clones, directly performing shake culture in a liquid LB medium containing 5g/L glucose at 37 ℃ overnight, and sucking a small amount of bacterial liquid to score and divide single bacteria on a solid LB plate containing 5g/L glucose and 10g/L sucrose. Single colonies were identified on LB plates containing kanamycin (50. Mu.g/mL) resistance and pEcCas plasmid elimination was verified.

We successfully obtained the editing strains after transformation of helper plasmid pTargetT-. DELTA.cadA or pTargetT-. DELTA.maeA plasmids into pEcCas-containing E.coli Bl21 (DE 3) and MG61655, respectively, with editing efficiency and plasmid elimination rates as shown in Table 2.

Table 2, comparison of Gene editing by pEcCas in E.coli MG1655 and Bl21 (DE 3)

a. Number of correctly edited clones/total number of clones selected

The comparison results in Table 2 show that, in E.coli MG1655, the editing efficiency using pEcCas is comparable to pCas; however, in E.coli Bl21 (DE 3), pEcCas has high editing efficiency, but pCas has no editing effect. The novel E.coli genome editing tool pEcCas is shown to have wider host adaptability.

The above experiments verify the function and characteristics of the E.coli genome editing tool of the present application using pEcCas/pTargetF or pTargetT double plasmid system as examples, and those skilled in the art will understand that various changes or modifications can be made on the basis of the ideas of the present application, and equivalents of the various changes or modifications can be made, which shall also fall within the scope of the present application.

Sequence listing

<110> Shanghai life science institute of China academy of sciences

<120> an E.coli genome editing tool based on CRISPR-Cas

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catgggtatg gacagttttc cctttgatat gtaacggtga acagttgttc tacttttgtt 60

tgttagtctt gatgcttcac tgatagatac aagagccata agaacctcag atccttccgt 120

atttagccag tatgttctct agtgtggttc gttgtttttg cgtgagccat gagaacgaac 180

cattgagatc atacttactt tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct 240

gaatttttgc agttaaagca tcgtgtagtg tttttcttag tccgttacgt aggtaggaat 300

ctgatgtaat ggttgttggt attttgtcac cattcatttt tatctggttg ttctcaagtt 360

cggttacgag atccatttgt ctatctagtt caacttggaa aatcaacgta tcagtcgggc 420

ggcctcgctt atcaaccacc aatttcatat tgctgtaagt gtttaaatct ttacttattg 480

gtttcaaaac ccattggtta agccttttaa actcatggta gttattttca agcattaaca 540

tgaacttaaa ttcatcaagg ctaatctcta tatttgcctt gtgagttttc ttttgtgtta 600

gttcttttaa taaccactca taaatcctca tagagtattt gttttcaaaa gacttaacat 660

gttccagatt atattttatg aattttttta actggaaaag ataaggcaat atctcttcac 720

taaaaactaa ttctaatttt tcgcttgaga acttggcata gtttgtccac tggaaaatct 780

caaagccttt aaccaaagga ttcctgattt ccacagttct cgtcatcagc tctctggttg 840

ctttagctaa tacaccataa gcattttccc tactgatgtt catcatctga gcgtattggt 900

tataagtgaa cgataccgtc cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta 960

gtgccacaca gcataaaatt agcttggttt catgctccgt taagtcatag cgactaatcg 1020

ctagttcatt tgctttgaaa acaactaatt cagacataca tctcaattgg tctaggtgat 1080

tttaatcact ataccaattg agatgggcta gtcaatgata attactagtc cttttccttt 1140

gagttgtggg tatctgtaaa ttctgctaga cctttgctgg aaaacttgta aattctgcta 1200

gaccctctgt aaattccgct agacctttgt gtgttttttt tgtttatatt caagtggtta 1260

taatttatag aataaagaaa gaataaaaaa agataaaaag aatagatccc agccctgtgt 1320

ataactcact actttagtca gttccgcagt attacaaaag gatgtcgcaa acgctgtttg 1380

ctcctctaca aaacagacct taaaacccta aaggcttaag tagcaccctc gcaagctcgg 1440

ttgcggccgc aatcgggcaa atcgctgaat attccttttg tctccgacca tcaggcacct 1500

gagtcgctgt ctttttcgtg acattcagtt cgctgcgctc acggctctgg cagtgaatgg 1560

gggtaaatgg cactacaggc gccttttatg gattcatgca aggaaactac ccataataca 1620

agaaaagccc gtcacgggct tctcagggcg ttttatggcg ggtctgctat gtggtgctat 1680

ctgacttttt gctgttcagc agttcctgcc ctctgatttt ccagtctgac cacttcggat 1740

tatcccgtga caggtcattc agactggcta atgcacccag taaggcagcg gtatcatcaa 1800

cccactgcag caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa 1860

actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 1920

atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 1980

cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 2040

tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat 2100

gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 2160

catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 2220

tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg 2280

catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 2340

cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 2400

tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat 2460

tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 2520

atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 2580

aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat 2640

ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg 2700

atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttccc 2760

tgcagctgaa caatacttaa tactatagaa tgataacaaa ataaactact ttttaaaaga 2820

attttgtgtt ataatctatt tattattaag tattgggtaa tattttttga agagatattt 2880

tgaaaaagaa aaattaaagc atattaaact aatttcggag gtcattaaaa ctattattga 2940

aatcatcaaa ctcattatgg atttaattta aactttttat tttaggaggc aaaaatggat 3000

aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc ggtgatcact 3060

gatgattata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt 3120

atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact 3180

cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta 3240

caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa 3300

gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata 3360

gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg 3420

gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt 3480

aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac 3540

aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac 3600

gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta 3660

gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt 3720

gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct 3780

aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt 3840

ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt 3900

tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt 3960

aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa 4020

cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat 4080

attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa 4140

atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa 4200

cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt 4260

ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa 4320

atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt 4380

gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc 4440

gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt 4500

ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac 4560

gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt 4620

gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag 4680

caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga 4740

gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa 4800

gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca 4860

ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc 4920

tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg 4980

tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt 5040

ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg 5100

acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa 5160

catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa 5220

gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa 5280

atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa 5340

cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa 5400

aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg 5460

tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tcacattgtt 5520

ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa 5580

aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat 5640

tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa 5700

gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt 5760

gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa 5820

tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta 5880

gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat 5940

catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca 6000

aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt 6060

gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc 6120

atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta 6180

atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca 6240

gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca 6300

ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt 6360

aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca 6420

gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag 6480

ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta 6540

gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt 6600

ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa 6660

ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat 6720

gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat 6780

aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta 6840

gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata 6900

cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct 6960

gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt 7020

ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg 7080

agtcagctag gaggtgactg aagtatattt tagatgaaga ttatttctta atctagacat 7140

gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 7200

tccccgaaaa gtgccacctg catcgattta ttatgacaac ttgacggcta catcattcac 7260

tttttcttca caaccggcac ggaactcgct cgggctggcc ccggtgcatt ttttaaatac 7320

ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga ccgacggtgg cgataggcat 7380

ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa 7440

gacgctaatc cctaactgct ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac 7500

atgctgtgcg acgctggcga tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg 7560

acaagcctcg cgtacccgat tatccatcgg tggatggagc gactcgttaa tcgcttccat 7620

gcgccgcagt aacaattgct caagcagatt tatcgccagc agctccgaat agcgcccttc 7680

cccttgcccg gcgttaatga tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc 7740

atccgggcga aagaaccccg tattggcaaa tattgacggc cagttaagcc attcatgcca 7800

gtaggcgcgc ggacgaaagt aaacccactg gtgataccat tcgcgagcct ccggatgacg 7860

accgtagtga tgaatctctc ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa 7920

ttctcgtccc tgatttttca ccaccccctg accgcgaatg gtgagattga gaatataacc 7980

tttcattccc agcggtcggt cgataaaaaa atcgagataa ccgttggcct caatcggcgt 8040

taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat cattttgcgc 8100

ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc catattgcat 8160

cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac cggtaacccc 8220

gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa 8280

aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg tcacactttg 8340

ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct ttttatcgca 8400

actctctact gtttctccat acccgttttt ttgggaattc gagctctaag gaggttataa 8460

aaaatggata ttaatactga aactgagatc aagcaaaagc attcactaac cccctttcct 8520

gttttcctaa tcagcccggc atttcgcggg cgatattttc acagctattt caggagttca 8580

gccatgaacg cttattacat tcaggatcgt cttgaggctc agagctgggc gcgtcactac 8640

cagcagctcg cccgtgaaga gaaagaggca gaactggcag acgacatgga aaaaggcctg 8700

ccccagcacc tgtttgaatc gctatgcatc gatcatttgc aacgccacgg ggccagcaaa 8760

aaatccatta cccgtgcgtt tgatgacgat gttgagtttc aggagcgcat ggcagaacac 8820

atccggtaca tggttgaaac cattgctcac caccaggttg atattgattc agaggtataa 8880

aacgaatgag tactgcactc gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg 8940

attctgtcga cccacaggaa ctgatcacca ctcttcgcca gacggcattt aaaggtgatg 9000

ccagcgatgc gcagttcatc gcattactga tcgttgccaa ccagtacggc cttaatccgt 9060

ggacgaaaga aatttacgcc tttcctgata agcagaatgg catcgttccg gtggtgggcg 9120

ttgatggctg gtcccgcatc atcaatgaaa accagcagtt tgatggcatg gactttgagc 9180

aggacaatga atcctgtaca tgccggattt accgcaagga ccgtaatcat ccgatctgcg 9240

ttaccgaatg gatggatgaa tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa 9300

tcacggggcc gtggcagtcg catcccaaac ggatgttacg tcataaagcc atgattcagt 9360

gtgcccgtct ggccttcgga tttgctggta tctatgacaa ggatgaagcc gagcgcattg 9420

tcgaaaatac tgcatacact gcagaacgtc agccggaacg cgacatcact ccggttaacg 9480

atgaaaccat gcaggagatt aacactctgc tgatcgccct ggataaaaca tgggatgacg 9540

acttattgcc gctctgttcc cagatatttc gccgcgacat tcgtgcatcg tcagaactga 9600

cacaggccga agcagtaaaa gctcttggat tcctgaaaca gaaagccgca gagcagaagg 9660

tggcagcatg acaccggaca ttatcctgca gcgtaccggg atcgatgtga gagctgtcga 9720

acagggggat gatgcgtggc acaaattacg gctcggcgtc atcaccgctt cagaagttca 9780

caacgtgata gcaaaacccc gctccggaaa gaagtggcct gacatgaaaa tgtcctactt 9840

ccacaccctg cttgctgagg tttgcaccgg tgtggctccg gaagttaacg ctaaagcact 9900

ggcctgggga aaacagtacg agaacgacgc cagaaccctg tttgaattca cttccggcgt 9960

gaatgttact gaatccccga tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc 10020

cgatggttta tgcagtgacg gcaacggcct tgaactgaaa tgcccgttta cctcccggga 10080

tttcatgaag ttccggctcg gtggtttcga ggccataaag tcagcttaca tggcccaggt 10140

gcagtacagc atgtgggtga cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg 10200

tatgaagcgt gaaggcctgc attatgtcgt gattgagcgg gatgaaaagt acatggcgag 10260

ttttgacgag atcgtgccgg agttcatcga aaaaatggac gaggcactgg ctgaaattgg 10320

ttttgtattt ggggagcaat ggcgatgacg catcctcacg ataatatccg ggtaggcgca 10380

atcactttcg tctactccgt tacaaagcga ggctgggtat ttcccggcct ttctgttatc 10440

cgaaatccac tgaaagcaca gcggctggct gaggagataa ataataaacg aggggctgta 10500

tgcacaaagc atcttctgtt gagttaagaa cgagtatcga gatggcacat agccttgctc 10560

aaattggaat caggtttgtg ccaataccag tagaaacaga cgaagaatcc atgggtatgg 10620

acagatcctc tttaggcccg tagtctgcaa atccttttat gattttctat caaacaaaag 10680

aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc gaaaagtaaa 10740

tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt 10800

atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt aactaacttg 10860

ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat aaaaaaggag 10920

acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc 10980

gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca aaagccatat 11040

aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat ccctgaacag 11100

caaaaaaatg aaaaatatca agttcctgaa ttcgattcgt ccacaattaa aaatatctct 11160

tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacgctga cggcactgtc 11220

gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa aaatgcggat 11280

gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa 11340

aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc tatcctaaaa 11400

gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa aatccgttta 11460

ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac tgcacaagtt 11520

aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta taaatcaatc 11580

tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga aggcaactac 11640

agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa aggccacaaa 11700

tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga agaatcttta 11760

tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt 11820

ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg tatgattgag 11880

ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc taacacagta 11940

acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta cctgttcact 12000

gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat ttacatgctt 12060

ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac tggccttgtg 12120

ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt cgctgtacct 12180

caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg attctacgca 12240

gacaaacaat caacgtttgc gccaagcttc ctgctgaaca tcaaaggcaa gaaaacatct 12300

gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata aaaacgcaaa 12360

agaaaatgcc gatatcctat tggcattttc ttttatttct tatcaacata aaggtgaatc 12420

ccatatgaac tatataaaag caggcaaatg gctaaccgta ttcctcaaaa aaagcaccga 12480

ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatttctag 12540

ctctaaaacg gttggactca agacgatagt tacgaccagt ctaaaaagcg cctgaattcg 12600

cgaccttctc gttactgaca ggaaaatggg ccattggcaa ccagggaaag atgaacgtga 12660

tgatgttcac aatttgctga attgtggtga tgtgatgctc accgcatttc ctgaaaattc 12720

acgctgtatc ttgaaaaatc gacgtttttt acgtggtttt ccgtcgaaaa tttaaggtaa 12780

gaacctgacc tcgtgattac tatttcgccg tgttgacgac atcaggaggc cagtatgacc 12840

gtattacata gtgtggattt ttttccgtct ggtaacgcgt ccgtggcgat agaaccccgg 12900

ctcccgcagg cggattttcc tgaacatcat catgattttc atgaaattgt gattgtcgaa 12960

catggcacgg gtattcatgt gtttaatggg cagccctata ccatcaccgg tggcacggtc 13020

tgtttcgtac gcgatcatga tcggcatctg tatgaacata ccgataatct gtgtctgacc 13080

aatgtgctgt atcgctcgcc ggatcgattt cagtttctcg ccgggctgaa tcagttgctg 13140

ccacaagagc tggatgggca gtatccgtct cactggcgcg ttaaccacag cgtattgcag 13200

caggtgcgac agctggttgc acagatggaa cagcaggaag gggaaaatga tttaccctcg 13260

accgccagtc gcgagatctt gtttatgcaa ttactgctct tgctgcgtaa aagcagtttg 13320

caggagaacc tggaaaacag cgcatcacgt ctcaacttgc ttctggcctg gctggaggac 13380

cattttgccg atgaggtgaa ttgggatgcc gtggcggatc aattttctct ttcactgcgt 13440

acgctacatc ggcagcttaa gcagcaaacg ggactgacgc ctcagcgata cctgaaccgc 13500

ctgcgactga tgaaagcccg acatctgcta cgccacagcg aggccagcgt tactgacatc 13560

gcctatcgct gtggattcag cgacagtaac cacttttcga cgctttttcg ccgagagttt 13620

aactggtcac cgcgtgatat tcgccaggga cgggatggct ttctgcaata acgcgaatct 13680

tctcaacgta tttgtacgcc atattgcgaa taatcaactt cgttctctgg ccgaggtagc 13740

cacggtggcg catcagttaa aacttctcaa agatgatttt tttgccagcg accagcaggc 13800

agtcgctgtg gctgaccgtt atccgcaaga tgtctttgct gaacatacac atgatttttg 13860

tgagctggtg attgtctggc gcggtaatgg cctgcatgta ctcaacgatc gcccttatcg 13920

cattacccgt ggcgatctct tttacattca tgctgacgat aaacactcct acgcttccgt 13980

taacgatctg gttttgcaga atattattta ttgcccggag cgtctgaagc tgaatcttga 14040

ctggcagggg gcgattccgg gatttaacgc cagcgcaggg caaccacact ggcgcttagg 14100

tagcatgggg atggcgcagg cgcggcaggt tatcggtcag cttgagcatg aaagtagtca 14160

gcatgtgccg tttgctaacg aaatggctga gttgctgttc gggcagttgg tgatgttgct 14220

gaatcgccat cgttacacca gtgattcgtt gccgccaaca tccagcgaaa cgttgctgga 14280

taagctgatt acccggctgg cggctagcct gaaaagtccc tttgcgctgg ataaattttg 14340

tgatgaggca tcgtgcagtg agcgcgtttt gcgtcagcaa tttcgccagc agactggaat 14400

gaccatcaat caatatctgc gacaggtcag agtgtgtcat gcgcaatatc ttctccagca 14460

tagccgcctg ttaatcagtg atatttcgac cgaatgtggc tttgaagata gtaactattt 14520

ttcggtggtg tttacccggg aaaccgggat gacgcccagc cagtggcgtc atctcaattc 14580

gcagaaagat taattcgcca tgccc 14605

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 2

ctatcgtctt gagtccaacc 20

Claims (16)

1. A CRISPR-Cas based escherichia coli genome editing tool comprising a plasmid pEcCas for expressing an endonuclease Cas9, the plasmid pEcCas comprising Cas9 nuclease gene, lambda-RED recombination system gene, araC protein gene, sacB gene, replicon pSC101, rhamnose regulatory protein RhaS/RhaR gene, sgRNA sequence for directing Cas9 to helper plasmid DNA sequences, the escherichia coli genome editing tool for genome editing of B-and K-12-line escherichia coli, wherein

Transcription of the sgRNA sequence of the targeting helper plasmid DNA sequence, which is the N20 sequence located 20nt downstream of the rhamnose promoter Prha complementary to the target sequence, is induced by L-rhamnose.

2. The escherichia coli genome editing tool of claim 1 wherein the N20 sequence is SEQ ID No. 2.

3. The escherichia coli genome editing tool of claim 1 or 2, wherein the plasmid pEcCas plasmid further comprises a kanamycin resistance gene kanR.

4. The escherichia coli genome editing tool of claim 1, wherein the cas9 nuclease gene is a streptococcus pyogenes-derived cas9 nuclease gene.

5. The escherichia coli genome editing tool of claim 4 wherein the nucleotide sequence of the plasmid pEcCas is SEQ ID No. 1.

6. The escherichia coli genome editing tool of claim 1, further comprising a helper plasmid, thereby making the escherichia coli genome editing tool a two-plasmid system.

7. The escherichia coli genome editing tool of claim 6 wherein the helper plasmid is selected from pTargetF or ptarget comprising: (a) An sgRNA sequence for targeting the genome of escherichia coli, (b) a pMB1 replicon or a P15A replicon.

8. The escherichia coli genome editing tool of claim 7 wherein the helper plasmid pTargetF or ptarget comprises a spectinomycin resistance gene aadA.

9. The escherichia coli genome editing tool of claim 7, wherein transcription of the sgRNA for targeting the escherichia coli genome is controlled by a Pj23119 constitutive promoter.

10. The escherichia coli genome editing tool of claim 7, wherein the sgRNA for targeting the escherichia coli genome is 18-25nt in length.

11. The escherichia coli genome editing tool of claim 1 wherein the N20 sequence is identical to a partial sequence of replicons pMB1 and P15A, the N20 sequence on the pMB1 and P15A replicons being followed by the PAM sequence 5'-CGG-3'.

12. The escherichia coli genome editing tool of claim 7, wherein the tool comprises a plasmid pEcCas, a helper plasmid pTargetF or ptarget, and PCR primers for performing genome editing in the form of a kit.

13. The escherichia coli genome editing tool of claim 1, wherein the plasmid pEcCas is constructed by a method comprising: obtaining pSC101 replicon fragments by PCR amplification; PCR amplification of kanamycin resistance gene kanR fragment using pCas plasmid as template; PCR amplification of cas9 and lambda-RED recombinase gene fragments by using pCas plasmid as template; amplifying sacB gene fragments from bacillus subtilis genome DNA; amplifying sgRNA fragments directed to pMB1 and P15A replicons; the rhaRS fragment was amplified from the genomic DNA of E.coli MG1655, and the six fragments were assembled to obtain the pEcCas plasmid.

14. Use of the escherichia coli genome editing tool of any one of claims 1-13 in escherichia coli genome editing of B-line and K-12-line.

15. Use according to claim 14 for genome editing of e.coli BL21 (DE 3) or e.coli MG 1655.

16. The use according to claim 14, comprising the steps of:

1) Transforming plasmid pEcCas into competent cells of escherichia coli, and screening on an LB solid plate containing kanamycin at 37 ℃;

2) Selecting positive clones, preparing electrotransformation competent cells, and adding arabinose to induce RED recombinase expression 1 hour before centrifugal collection;

3) Transferring helper plasmids pTargetF or pTargetT into competent cells obtained in the step 2) through electrotransformation, coating bacterial liquid on a dual-antibody LB solid plate containing kanamycin and spectinomycin, and verifying clones after overnight culture at 37 ℃;

4) Selecting positive clones in LB culture solution containing kanamycin, adding rhamnose, inducing the sgRNA sequence of the DNA sequence of the guide auxiliary plasmid to transcribe, and verifying the elimination of the auxiliary plasmid on LB solid plates containing spectinomycin;

5) Picking auxiliary plasmid to eliminate positive clones, directly culturing overnight at 37 ℃ in an LB liquid culture medium containing glucose, sucking bacterial liquid, streaking and dividing single bacteria on an LB solid plate containing glucose and sucrose, identifying single bacteria on an LB plate containing kanamycin resistance, and verifying pEcCas plasmid elimination to obtain an editing strain.