CN109811010B - Method for enhancing gene editing efficiency of actinomycetes and application thereof - Google Patents

Abstract

The invention provides a method for enhancing gene editing efficiency of actinomycetes and application thereof, which take deletion of actII-ORF4 in streptomyces coelicolor as an example, and control the activity of Cas9 by using an inducible promoter tipA, a ribosome switch and split Cas9, wherein the transformation efficiency is improved by about 260 times compared with the traditional constitutive Cas9 expression in the transformation process; the editing process adds an inducer and increases the ATP concentration to achieve an editing efficiency of 80%. The method establishes a CRISPR/Cas9 system which can be induced by chemical micromolecules and can be reversibly regulated and controlled by blue light, thereby ensuring that the accurate and efficient gene editing based on double-strand break is realized under the condition that the transformation efficiency of plasmids and the normal growth and metabolism of a target host are not influenced to the maximum extent.

Description

Method for enhancing gene editing efficiency of actinomycetes and application thereof

Technical Field

The invention belongs to the field of genetic engineering and genetic modification, and particularly relates to a method for enhancing gene editing efficiency by controlling Cas9 activity and ATP concentration in actinomycetes and application thereof.

Background

The CRISPR (clustered regulated short palindromic repeats)/Cas (CRISPR-associated) system is an immune system unique to prokaryotes and is accompanied by RNA-mediated recognition and degradation of specific sequences to foreign DNA, causing it to be inactivated by loss of function. In recent years, the CRISPR/Cas9 system, as a gene-directed editing technology for specific sites, has the advantages of simple and convenient operation, low investment, high efficiency, good specificity and universality, and the like, is considered as a genome editing kit with strongest molecular mechanism dissection and gene expression control in recent two years, shows huge potential in cell reprogramming of synthetic biology of mammalian protein engineering, therapeutic intervention and drug discovery, and is also used for microbial metabolic engineering, giant chromosome construction and even manipulation of genetically intractable microorganisms.

The CRISPR/Cas9 system can accurately edit a target gene. And (3) directing exogenous DNA to a specific site of a host cell chromosome to realize positioning and directional gene editing. The system firstly utilizes designed guide RNA (single-guide RNA, sgRNA) to mediate specific combination of Cas9 protein and a target site, completes specific cutting of DNA, and self-repairs in a mode of homologous recombination or non-homologous end connection, thereby realizing gene knockout, knock-in and the like.

However, off-target effects and toxic activity of the CRISPR/Cas9 system would greatly impede its application. The visible consequence of Cas9 toxicity in microbial manipulation is a dramatic reduction in transformants, such as in the superior genetically engineered hosts escherichia coli, yeast and streptomyces, most often leading to failure of genetic modification. Wherein the streptomyces has special industrial value in the production of clinical use medicines. Therefore, how to improve the gene editing efficiency of the CRISPR/Cas technology and improve the targeting property of the CRISPR/Cas technology is a big problem to be faced by the technology at present.

The current research shows that thiostrepton inducible promoter tipAp is found in streptomyces and can be used as a regulatory element for inhibiting Cas overexpression in a CRISPR/Cas9 editing system. And through artificially adding thiostrepton, open transcription of a tipAp promoter is induced, Cas9 activity reconstruction is realized in the editing process, and the aim of controlling the activity of Cas9 at the transcription level is fulfilled.

On the other hand, related researches report that theophylline-dependent ribosome switches with the length of 85bp have a high-level structure in non-coding RNA transcribed under a non-induction condition, mRNA expressed by downstream coding genes can be prevented from being combined with ribosome, and polypeptide chain synthesis is inhibited, so that protein expression is at an extremely low level, and the theophylline-dependent ribosome switches can be used for controlling transformation efficiency. But can unlock higher-order structure under the induction of theophylline, so that hundreds of times of induced expression can be generated after theophylline is added, and the genetic tool can realize the control of Cas9 activity at the translation level.

In addition, a blue-light inducible Cas9 protein reconstitution technology for achieving Cas9 activity control in human cells has been researched and developed at present. The fungal light receptor Vivid is designed to be divided into two proteins, namely a positive protein (pMag) and a negative protein High1(nMAGHIGH1), which are heterodimerized under the blue light induction condition to reconstruct the structure and activity of Cas 9. This photoswitchable advantage has been exploited to develop photoactivatable cleaved Cas9(N713-pMag and nMagHigh1-C714), enabling precise control of Cas9 activity and gene editing at the protein level.

In addition, the double-strand break caused by Cas9 needs to be repaired precisely by Homologous Directed Recombination (HDR) imposed by an ATP-dependent DNA repair system, including Rad51/Rad52 in eukaryotic cells and RecA-triggered SOS pathway in e. Where RecA is an ATP-dependent recombinase that forms filaments on single-stranded DNA after DSB, and subsequently facilitates homology search and crossover for homologous recombination, thereby ensuring genome integrity and stability. ATP plays an important role during HDR, since ATP is associated with Rad51/RecA filaments on DNA, and hydrolysis of ATP plays an important role at least in RecA-ssDNA, dynamic interactions between RecA sliding along DNA, prevention of pairing between long repeats, and conformational changes during disassembly of Rad51 filaments. Therefore, the ATP concentration is increased in the editing process, so that Homologous Directed Recombination (HDR) is facilitated, and the gene editing efficiency is further improved.

In view of the above considerations and background studies, the present invention discloses a method for enhancing gene editing efficiency by controlling Cas9 activity and ATP concentration in actinomycetes and applications thereof. The method is clear, efficient and easy to operate, and provides a novel method for effectively improving the target gene editing efficiency in a microbial host.

Disclosure of Invention

The invention aims to provide a method for enhancing gene editing efficiency of actinomycetes and application thereof, and provides a novel CRISPR/Cas9 system for improving target gene editing efficiency by controlling Cas9 activity and ATP concentration in actinomycetes. The technology is a novel technology for efficiently regulating and controlling the transformation efficiency and the editing efficiency based on the addition of an element for controlling the activity of Cas9 and the concentration of ATP. The method is realized by the following steps:

(1) selecting proper inducible promoters, strong promoters and skeleton plasmids according to the physiological characteristics of a target host;

(2) inserting the inducible promoter screened in the step (1), a ribosome switch, a split Cas9 and a strong promoter-atpD high-energy gene cassette into the skeleton plasmid selected in the step (1);

(3) designing a spacer sequence (left arm and right arm) of a target editing site, respectively inserting the spacer sequence into the plasmids constructed in the step (2), and guiding the Cas9 protein to move to a target sequence;

(4) selecting a proper screening mode for screening antibiotics or auxotrophs and the like of the transformant according to the physiological properties of the host and the plasmid elements, and additionally adding related genes for expressing the antibiotics, the auxotrophs and the like into the plasmid constructed in the step (3);

(5) under a non-induction condition, transforming the recombinant plasmid in the step (4) (or performing genetic operation suitable for a target host such as conjugation transduction) into the target host, and screening by adding a culture medium of a screening condition to obtain a transformant carrying the recombinant plasmid;

(6) inoculating the transformant in the step (5) into an enrichment medium with proper screening conditions, and fermenting at constant temperature;

(7) taking the fermentation liquor fermented for 24-48 h in the step (6), centrifuging to collect thalli, washing twice with sterile water, centrifuging to remove supernatant, replacing a culture medium added with promoter induction conditions with proper concentration and theophylline, and then carrying out shake constant-temperature culture under blue light induction conditions;

(8) and (4) centrifuging the fermentation liquor obtained in the step (7), discarding the supernatant, and collecting the precipitate to obtain the strain for completing target gene editing.

The invention aims to provide application of the method in effectively improving the gene editing efficiency of a target host.

1. The method realizes the suppression of Csa9 protein overexpression in the transformation process by screening a proper inducible promoter (such as thiostrepton inducible promoter tipAp), and realizes the accurate control of the Cas9 activity at the transcription level by inducing and reconstructing the Cas9 activity by subsequently adding a promoter induction condition (such as thiostrepton).

2. According to the method, the synthesis of Cas9 protein polypeptide chain is inhibited in the conversion process of the theophylline-dependent ribosome switch, and the subsequent addition of theophylline induces the ribosome switch to start the synthesis of the polypeptide chain, so that the precise control of the Cas9 activity at the translation level is realized.

3. According to the method, the blue light-induced split Cas9 protein is split into two proteins in the conversion process to cause function loss, the two proteins are induced to be heterodimerized by adding blue light subsequently, the structure and activity of Cas9 are reconstructed, and the purpose of accurately controlling the activity of Cas9 at the protein level is achieved.

4. The method of the invention provides ATP to facilitate Homologous Directed Recombination (HDR) and improve editing efficiency by expressing atpD gene of beta-subunit of ATP synthase under a strong constitutive promoter (such as ermEp).

5. The invention provides a novel CRISPR/Cas9 system for enhancing gene editing efficiency by controlling Cas9 activity and ATP concentration in actinomycetes, wherein a proper inducible promoter (such as thiostrepton inducible promoter tipAp), a ribosome switch and a split Cas9 are added to realize triple control on Cas9 activity, so that cytotoxicity and off-target probability of the CRISPR/Cas9 system are inhibited to the maximum extent when the CRISPR/Cas9 system is not induced; the atpD gene of the constructed beta-subunit of the ATP synthase ensures the effective supply of ATP, further improves the gene editing efficiency in a target host, and can be suitable for gene engineering and efficient genetic modification of other modes including streptomycete or industrial actinomycetes.

Compared with the prior art, the gain effect and obvious advantages of the invention are as follows: 1) the invention provides a method tool for artificially inducing and regulating Cas9 activity and ATP concentration and effectively improving editing efficiency for genetic engineering and genetic modification of model microorganisms such as actinomycetes and the like. 2) The CRISPR/Cas9 system added with a proper inducible promoter (such as thiostrepton inducible promoter tipAp), a ribosome switch and a split Cas9 triple control is constructed, and the transformation efficiency is obviously increased under a non-inducible condition. 3) According to the invention, the expression of the Cas9 is restored by adding an inducible promoter inducing condition (such as thiostrepton), theophylline and blue light, and the structure and activity of the Cas9 are recombined. 4) The atpD gene of the beta-subunit of the ATP synthase is expressed under a strong constitutive promoter (such as ermEp), the ATP concentration in actinomycetes is increased, and the gene editing efficiency is further remarkably increased. 5) The invention has wide application in the genetic engineering of microorganisms such as actinomycetes and the like, and all modes capable of carrying out genetic operation or industrial actinomycetes can be used for carrying out high-efficiency genetic modification, thereby improving the economic benefit of the microorganism production industry.

Drawings

FIG. 1 is a schematic diagram of the construction process of the universal editing plasmid pKC1139-trMD of Streptomyces coelicolor.

FIG. 2 is a schematic diagram of the knock-out actII-ORF4 gene in Streptomyces coelicolor A3 (2).

FIG. 3 shows the efficiency of conjugation transduction of pKC1139-trMD-cas9-actII-orf4 and each control plasmid against Streptomyces coelicolor A3 (2). And (4) after conjugation transduction, obtaining a transformant which can be seen by naked eyes and realizes counting through antibiotic screening. Plate count can see: gradually adding each control element until the final plasmid pKC1139-trMD-Cas9-actII-orf4, gradually increasing the number of transformants, and improving the final transformation efficiency by about 260 times compared with the traditional constitutive Cas9 expression.

FIG. 4 shows PCR knock-out verification of strains into which pKC1139-trMD-cas9-actII-orf4 plasmid was successfully introduced under non-inducible conditions. Successful transformants were verified by colony PCR. The results show that under non-induced conditions, only 19# amplified a 1.35kb band with both pairs of primers, i.e., the knock-out efficiency of the Cas9 system was only 5%

FIG. 5 shows PCR knock-out verification of strains into which pKC1139-trMD-cas9-actII-orf4 plasmid was successfully introduced under triple induction conditions. Successful transformants were verified by colony PCR. The results show that under the condition that all three induction conditions are available and autonomously replicate, the knockout efficiency of the optimized Cas9 system can reach 80 percent

Detailed Description

The following describes the method of the present invention in further detail with reference to the accompanying drawings and specific examples.

Example 1

The strain selected in example 1 is Streptomyces coelicolor A3(2) (Bentley SD et al. Nature,2002) (accession number: CGMCC 4.7168), and the whole genome sequence (sequence number: GenBank: NC-003888.3) thereof. The present invention will be described in detail by taking the case of knocking out the gene actII-ORF4 in Streptomyces coelicolor A3(2) as a model bacterium by using the method. The method comprises the following specific steps:

(1) according to the literature report on the regulation of related streptomyces promoters (He Huang et al, acta Biochim Biophys Sin,2015), thiostrepton-induced tipAp promoter and ermEp promoter were selected, and a constitutive promoter and a strong promoter for atpD gene expression, which can be used as a target strain streptomyces coelicolor A3(2), were obtained. (details are shown in Table 1 and attached figure 1)

(2) According to the streptomyces coelicolor A3(2) plasmid library, the size of a determined element, a suitable enzyme cutting site in the position construction process and an antibiotic resistance gene suitable for transformant screening, a pKC1139 plasmid is screened out and used as a skeleton plasmid of an editing plasmid.

(3) Constructing an editing plasmid according to the promoter and the skeleton plasmid determined in the step (1) and the step (2)

In order to ensure that Csa9 is expressed under the control of tipAp (SEQ NO.1 for sequence details) and riboswitch (SEQ NO.2 for sequence details), plasmids pWHU2653 and pET28a (Novagene) are digested with EcoRI/XbaI, and a digested fragment with the length of 4.7kb and containing cas9-gRNA is connected to a vector skeleton with the length of 5.2kb obtained after digestion of pET28a vector, so that plasmid pET28a-cas9-gRNA can be obtained. Then, a tsr-to-tipAp fragment was PCR-amplified from pIJ8600(2) plasmid using primer pair 1+2, and inserted into SacI/NotI digested pET28a-cas9-gRNA by seamless cloning (Vazyme, China) to obtain pET28a-tipAp-cas 9-gRNA. Subsequently, pET28a-tipAp-cas9-gRNA was further digested with EcoRI/BglII to obtain a 6.3kb fragment, and this fragment was ligated to a 9.4kb plasmid backbone obtained by digesting pWHU2653 plasmid with EcoRI/BamHI to construct pWHU2653-tipAp plasmid. Meanwhile, a fragment with the length of 114bp and containing a ribosome switch tibosiltch is amplified in vitro by a primer pair 3+4+5 and is inserted into an NdeI enzyme cutting site of pET28a-tipAp-cas9-gRNA, and the construction of pET28a-tipAp-ribo is completed. Further, the pET28a-tipAp-ribo plasmid was digested with EcoRI/BglII, and the digested fragment was ligated to a 9.4kb plasmid backbone obtained by digestion of pWHU2653, to finally obtain pWHU2653-tipAp-ribo plasmid.

To achieve split expression of Cas9, the literature references teach the construction of pMag (SEQ No.3 for sequence details) and nMagHigh1(nMag) (SEQ No.4 for sequence details) by chemical synthesis by cloning a GSGGSSGSGG linker at the carboxy terminus of pMag and a GGSGSSGGSG linker at the amino terminus of nMag into pTA2(Toyobo, Japan), respectively, to obtain pTA2-nMag-Cas9C and pTA2-Cas 9N-pMag. Then, the primer pair 6+7 was selected, and a to-tipAp fragment containing XbaI/NdeI cleavage sites was PCR-amplified using the plasmid pIJ8600 as a template and inserted into the XbaI site of pTA2 to obtain pTA 2-tipAp. Then, by using pTA2-nMAG and pWHU2653 as templates, nMAG and Ccas9 (1965 bp at the carboxyl terminal of cas 9) fragments are amplified by primer pairs 8+9 and 10+11 respectively, and the two fragments are assembled together by Gibson and inserted into the NdeI enzyme cutting site of pTA2-tipAP plasmid to assemble the plasmid pTA2-tipAP-nMAG-Ccas 9. Similarly, the plasmid pET28 a-tip-Ncas 9-pMag was assembled by amplifying Ncas9 (2136 bp of the amino terminus of cas 9) and pMag fragment with pWHU2653 and pTA2-pMag as templates in primer pairs 12+13 and 14+15, respectively, and inserting the amplified fragments into pET28a-tipAp-cas9-gRNA plasmid by Gibson assembly, and cutting the resulting vector with NdeI/NheI. pET28a-tipAp-Ncas9-pMag was further digested with XbaI/BglII and ligated to the plasmid backbone of tipAp-nMAG-Ccas9 which was obtained by digestion with XbaI/BglII of pTA2-tipAp-nMAG-Ccas9, to obtain pET28a-tipAp-Mag-cas9 plasmid. Finally, the plasmid pET28a-tipAp-Mag-cas9 was digested with EcoRI/BglII and inserted into the 9.4kb plasmid backbone obtained by EcoRI/BamHI digestion of pWHU2653, resulting in plasmid pWHU2653-tipAp-Mag-cas 9.

To ensure that the cleaved Csa9 could be further controlled by ribosome switching, the above 114bp fragment was inserted into NdeI-digested pET28a-tipAp-Ncas9-pMag and pTA2-tipAp-nMAG-Ccas9 vectors using Gibson assembly, respectively, to give pET28a-tipAp-ribo-Ncas9-pMag and pTA2-tipAp-ribo-nMAG-Ccas9 plasmids, respectively. Then TA2-tipAp-ribo-nMAG-Ccas9 was digested with XbaI/BglII to give a tipAp-ribo-nMAG-Ccas9 fragment, which was ligated to pET28a-tipAp-ribo-Ncas9-pMag plasmid into the vector backbone after digestion with XbaI/BglII to give pET28a-tipAp-ribo-Mag-cas9 plasmid. Finally, pET28a-tipAp-ribo-Mag-cas9 was digested with EcoRI/BglII to obtain an 8.6kb fragment, which was then ligated to the 9.4kb vector backbone of pWHU2653, which was digested with EcoRI/BamHI, to obtain pWHU2653-trM-cas9 plasmid.

In order to realize ectopic binding expression of recA and aptD, a primer pair 16+17 and a primer pair 18+19 are respectively selected, genomes of pLM1(4) and S.coelicolor A3(2) are used as templates to amplify constitutive promoters ermEp and recA, and the constitutive promoters ermEp and recA are inserted into NdeI sites of pWHU2653-trM-cas9 to obtain pWHU2653-trM-cas9-recA plasmids. In addition, constitutive promoters ermEp and atpD were amplified by using genomes of pLM1(4) and S.coelicolor A3(2) as templates through primer pairs 20+21 and 22+23, respectively, and inserted into EcoRI sites of pWHU2653-trM-cas9 to obtain pWHU2653-trMD-cas9 plasmid, and inserted into EcoRI sites of pWHU2653-trM-cas9-recA to obtain pWHU2653-trMD-cas9-recA plasmid.

In order to replace pIJ101ori on pWHU2653 with pSG5ori on pWHC 1139, pWHU2653-trM-cas9 was digested with EcoRI/BglII to obtain an 8.0kb fragment, which was inserted into the 6.0kb plasmid backbone digested with EcoRI/BglII on pKC1139, yielding pKC1139-trM-cas9 plasmid. Then, ermEp and atpD were amplified by using the genome of pLM1(4) and S.coelicolor A3(2) as templates, respectively, and finally, the two plasmids are Gibson assembled and inserted into the EcoRI site of pKC1139-trM-cas9, so that pKC1139-trMD-cas9 was obtained.

To obtain a plasmid editing the pSG5 site, the spacer sequence of actII-ORF4 was inserted into the BaeI site of pKC1139-trM-cas9 and pKC1139-trMD-cas9 plasmids, respectively. The left arm of actII-ORF4 was amplified with primer pair 26+28, while the right arm of both actII-ORFs 4 was amplified with primer pair 27+29 and primer pair 27+30, respectively. The insertion of the left arm with the first right arm into pKC1139-trM-cas9-spacer yielded pKC1139-trM-cas9-actII-orf4 plasmid, while the insertion of the left arm with the second right arm into KC1139-trMD-cas9-spacer yielded pKC1139-trMD-cas9-actII-orf4 plasmid. (details are shown in Table 1 and attached figure 1)

(4) Ampicillin is selected as an appropriate antibiotic for screening transformants according to the physiological properties of the streptomyces coelicolor A3(2) and the elements of the skeleton plasmid pKC1139, and is used for screening the transformant screening conditions for successfully introducing the plasmid in the subsequent steps;

(5) under non-induction conditions, the recombinant plasmid in the step (4) is conjugately transduced into streptomyces coelicolor A3(2), and the specific steps are as follows: introducing a target plasmid into E.coli ET12567/pUZ8002(Kieser T, 2000), picking a monoclonal to an LB test tube containing ampicillin for overnight culture, then transferring the E.coli ET12567/pUZ8002 containing the target plasmid after the overnight culture into 25mL of LB culture medium containing corresponding antibiotics according to 1% inoculation amount, culturing until A260 is about 0.4, and centrifuging at 4000rpm for 5 minutes to collect thalli; adding 1mL of LB liquid culture medium to resuspend the thalli, centrifuging at 4000rpm for 5 minutes, discarding the supernatant, and finally resuspending the thalli by using 0.5mL of LB liquid culture medium; taking a proper amount of streptomycete spores collected in advance, subpackaging the streptomycete spores into each centrifuge tube, washing the spores for 2 times similarly to the steps, suspending the bacteria by using 2 XYT culture medium, placing the centrifuge tube filled with the spores into a water bath kettle for germination for 10 minutes, and adjusting the specific temperature according to different types of streptomycetes; mixing 0.5mL of coli bacteria liquid and 0.5mL of streptomyces spore suspension; centrifuging the mixed solution for 5 minutes at 5000rpm, sucking part of supernatant, resuspending thalli and spores in the rest 200 microliters of supernatant, and uniformly coating the mixed solution on an MS (Mass spectrometer) flat plate; and (3) inversely placing the MS plate in a 30-degree incubator for culture, coating corresponding antibiotics and nalidixic acid after 16 hours, and culturing at 30 ℃ for about 5 days to grow a transformant. The transformants obtained at this time were successfully edited;

(6) inoculating the monoclonal transformant in the step (5) into an enrichment medium (the liquid loading amount is 35mL/250mL) added with ampicillin, and fermenting for 24-48 h (30 ℃,220rpm) at constant temperature by a shaking table;

(7) taking the fermentation liquor obtained in the step (6), centrifuging to collect thalli, washing twice with sterile water, centrifuging to remove supernatant, replacing a culture medium added with thiostrepton with the final concentration of 5 mu g/mL and theophylline with the final concentration of 4mM, and then carrying out shake table constant-temperature culture for 24-48 h under the blue light induction condition;

(8) and (4) centrifuging the fermentation liquor obtained in the step (7) (4000rpm for 5min), and discarding the supernatant to obtain a strain precipitate which is edited by the target.

TABLE 1 construction of (related) primers for editing plasmids

Sequence listing

<110> Zhejiang university

<120> method for enhancing gene editing efficiency of actinomycetes and application thereof

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 183

<212> DNA

<213> Streptomyces coelicolor

<400> 1

tccgctccct tcttctctga cgccgtccac gctgcctcct cacgtgacgt gaggtgcaag 60

cccggacgtt ccgcgtgcca cgccgtgagc cgccgcgtgc cgtcggctcc ctcagcccag 120

atccccgatc cggtccagta atgacctcag aactccatct ggatttgttc agaacgctcg 180

gtt 183

<210> 2

<211> 60

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 2

catcttgttg cctccttagc agggtgctgc caagggcatc aagacgatgc tggtatcacc 60

<210> 3

<211> 2136

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 3

gacctgggcc ttctggatgt cctccttgaa ggtcaggctg tcgtcgtgga tgagctgcat 60

gaagttgcgg ttggcgaagc cgtcggactt gaggaagtcc aggatggtct tgccgctctg 120

cttgtcccgg atgccgttga tcagcttccg ggagaggcgg ccccagccgg tgtaccggcg 180

ccgcttcagc tgcttcatca ccttgtcgtc gaacaggtgg gcgtaggtct tgagccgctc 240

ctcgatcatc tcgcggtcct cgaagagggt cagggtgagg acgatgtcct ccaggatgtc 300

ctcgttctcc tcgttgtcga ggaagtcctt gtccttgatg atcttgagca ggtcgtggta 360

ggtgcccagg gaggcgttga agcggtcctc cacgccgctg atctcgacgg agtcgaagca 420

ctcgatcttc ttgaagtagt cctccttcag ctgcttcacg gtgaccttgc ggttggtctt 480

gaagagcagg tcgacgatcg ccttcttctg ctcgcccgac aggaaggccg gcttccgcat 540

gccctcggtc acgtacttga ccttggtcag ctcgttgtac acggtgaagt actcgtagag 600

cagggagtgc ttgggcagga ccttctcgtt cgggaggttc ttgtcgaagt tggtcatgcg 660

ctcgatgaac gactgggcgg aggcgccctt gtccacgacc tcctcgaagt tccacggggt 720

gatggtctcc tccgacttcc gggtcatcca cgcgaaccgg gagttgccgc gggccagggg 780

gccgacgtag tacgggatgc ggaaggtcag gatcttctcg atcttctcgc ggttgtcctt 840

caggaagggg tagaagtcct cctggcgccg gaggatggcg tgcagctcgc ccaggtggat 900

ctggtgcggg atggagccgt tgtcgaaggt ccgctgcttg cggagcaggt cctcgcggtt 960

cagcttgacg agcagctcct cggtgccgtc catcttctcc aggatcggct tgatgaactt 1020

gtagaactcc tcctgcgacg cgccgccgtc gatgtagccg gcgtagccgt tcttggactg 1080

gtcgaagaag atttccttgt acttctcggg cagctgctgg cgcacgaggg ccttgagcag 1140

ggtcaggtcc tggtggtgct cgtcgtaccg cttgatcatg ctcgccgaca gcggggcctt 1200

ggtgatctcg gtgttgaccc gcaggatgtc gctgagcagg atggcgtccg agaggttctt 1260

cgcggccagg aagaggtccg cgtactggtc gccgatctgg gcgagcaggt tgtccaggtc 1320

gtcgtcgtag gtgtccttgg acagctggag cttcgcgtcc tcggccaggt cgaagttgct 1380

cttgaagttg ggggtcaggc cgagcgacag cgcgatcagg ttgccgaaga ggccgttctt 1440

cttctcgccc ggcagctggg cgatgaggtt ctccaggcgc cgggacttgc tcaggcgcgc 1500

ggagaggatc gccttggcgt cgacgccgct ggcgttgatg gggttctcct cgaacagctg 1560

gttgtaggtc tgcaccagct ggatgaagag cttgtcgacg tcggagttgt ccgggttcag 1620

gtcgccctcg atgaggaagt ggccgcggaa cttgatcatg tgcgcgaggg ccaggtagat 1680

gagccgcagg tccgccttgt cggtcgagtc gaccagcttc ttgcggaggt ggtagatggt 1740

ggggtacttc tcgtggtagg ccacctcgtc gacgatgttg ccgaagatcg ggtggcgctc 1800

gtgcttcttg tcctcctcca ccaggaagct ctcctcgagc cggtggaaga acgagtcgtc 1860

gaccttggcc atctcgttgg agaagatttc ctggaggtag cagatgcggt tcttgcgccg 1920

ggtgtagcgc cggcgcgcgg tccgcttcag gcgggtcgcc tcggcggtct cgccgctgtc 1980

gaagagcagg gcgccgatca ggttcttctt gatcgagtgc cggtcggtgt tgcccaggac 2040

cttgaacttc ttggagggga ccttgtactc gtcggtgatg accgcccagc ccacgctgtt 2100

ggtgccgatg tccaggccga tgctgtactt cttgtc 2136

<210> 4

<211> 1965

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 4

gtcgccgccc agctggctca ggtcgatgcg ggtctcgtac aggccggtga tgctctggtg 60

gatcagggtc gcgtcgagga cctccttggt ggaggtgtac cgcttgcggt cgatggtggt 120

gtcgaagtac ttgaaggcgg ccggggcacc caggttggtg agggtgaaca ggtggatgat 180

gttctccgcc tgctcccgga tcggcttgtc gcggtgcttg ttgtaggcgg acagcacctt 240

gtcgaggttc gcgtcggcca ggatgacgcg cttgctgaac tcgctgatct gctcgatgat 300

ctcgtccagg tagtgcttgt gctgctccac gaagagctgc ttctgctcgt tgtcctcggg 360

cgagcccttc agcttctcgt agtgggacgc gaggtacagg aagttgacgt acttcgacgg 420

gagggccagc tcgttgccct tttgcagctc gcccgcggag gcgagcatcc gcttgcggcc 480

gttctccagc tcgaacaggc tgtacttggg cagcttgatg atgaggtcct tcttgacctc 540

cttgtagccc ttggcctcca ggaagtcgat cgggttcttc tcgaacgagg agcgctccat 600

gatggtgatg ccgagcagct ccttcacgga cttcagcttc ttgctcttgc ccttctcgac 660

cttcgccacg accagcacgg agtaggcgac ggtggggctg tcgaagccgc cgtacttctt 720

cgggtcccag tccttcttcc gggcgatcag cttgtcgctg ttgcgcttgg ggaggatgga 780

ctccttgctg aagccgccgg tctgcacctc ggtcttcttc acgatgttga cctgcggcat 840

cgacagcacc ttccggacgg tggcgaagtc gcggcccttg tcccagacga tctcgccggt 900

ctcgccgttg gtctcgatca ggggccgctt gcggatctcg ccgttggcca gggtgatctc 960

ggtcttgaag aagttcatga tgttggagta gaagaagtac ttcgcggtgg ccttgccgat 1020

ctcctgctcg ctcttggcga tcatcttgcg cacgtcgtag accttgtagt cgccgtagac 1080

gaactcggac tccagcttcg ggtacttctt gatcagcgcg gtgcccacga cggcgttcag 1140

gtacgcgtcg tgggcgtggt ggtagttgtt gatctcccgg accttgtaga actggaagtc 1200

cttgcggaag tcggagacca gcttgctctt gagggtgatc accttgacct cgcggatcag 1260

cttgtcgttc tcgtcgtact tggtgttcat ccgggagtcc aggatctggg ccacgtgctt 1320

ggtgatctgc cgggtctcga ccagctggcg cttgatgaag cccgccttgt ccagctcgct 1380

caggccgccc cgctcggcct tggtcaggtt gtcgaacttg cgctgggtga tgagcttggc 1440

gttgagcagc tggcgccagt agttcttcat cttcttcacg acctcctccg agggcacgtt 1500

gtcggacttg ccccggttct tgtccgagcg ggtcaggacc ttgttgtcga tcgagtcgtc 1560

cttcaggaag gactgcggca cgatgtggtc gacgtcgtag tcggacagcc ggttgatgtc 1620

cagctcctgg tccacgtaca tgtcgcggcc gttctggagg tagtagaggt acagcttctc 1680

gttctggagc tgggtgttct cgaccgggtg ctccttcagg atctggctgc ccagctcctt 1740

gatgccctcc tcgatccgct tcatccgctc gcgcgagttc ttctggccct tctgggtggt 1800

ctggttctcc cgggccatct cgatcacgat gttctcgggc ttgtggcggc ccatcacctt 1860

gaccagctcg tccacgacct tgacggtctg gaggatgccc ttcttgatcg ccggggagcc 1920

cgccaggttg gcgatgtgct cgtggaggct gtcgccctgg cccga 1965

<210> 5

<211> 46

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 5

gtggtggtgg tggtgctcga gtgaattcga tggggatcaa ggcgaa 46

<210> 6

<211> 45

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 6

ccgatgctgt acttcttgtc catatgtccg ctcccttctt ctctg 45

<210> 7

<211> 55

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 7

gaagaaggga gcggacataa tacgactcac tataggttcc ggtgatacca gcatc 55

<210> 8

<211> 58

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 8

cttgttgcct ccttagcagg gtgctgccaa gggcatcaag acgatgctgg tatcaccg 58

<210> 9

<211> 34

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 9

ctgtacttct tgtccatctt gttgcctcct tagc 34

<210> 10

<211> 42

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 10

tgtactgaga gtgcaccatc tagagattac tgtcgtttaa tg 42

<210> 11

<211> 41

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 11

caccgcggtg gcggccgcca tatgtccgct cccttcttct c 41

<210> 12

<211> 40

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 12

gagaagaagg gagcggacat atgcacaccc tgtacgcccc 40

<210> 13

<211> 18

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 13

gccgcccgag ccggagcc 18

<210> 14

<211> 40

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 14

ggctccggct cgggcggcga ttcgggccag ggcgacagcc 40

<210> 15

<211> 43

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 15

accgcggtgg cggccgcaga tctttagtcg ccgcccagct ggc 43

<210> 16

<211> 41

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 16

gagaagaagg gagcggacat atggacaaga agtacagcat c 41

<210> 17

<211> 36

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 17

gagccggagc cgccgatgac ctgggccttc tggatg 36

<210> 18

<211> 20

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 18

atcggcggct ccggctcgtc 20

<210> 19

<211> 39

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 19

tagacacgtc tgaagctagc ctcactcggt ctcgcactg 39

<210> 20

<211> 39

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 20

ttcagcgtga catcattcat aggcggcttg cgcccgatg 39

<210> 21

<211> 19

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 21

atgtccgcct cctttggtc 19

<210> 22

<211> 39

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 22

accaaaggag gcggacatat ggcaggaacc gaccgcgag 39

<210> 23

<211> 40

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 23

gatcaaggcg aatacttcat atgatccgtc tcgtacgggg 40

<210> 24

<211> 37

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 24

gaggcccttt cgtcttcaag gcggcttgcg cccgatg 37

<210> 25

<211> 40

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 25

ggtctcaaca gtggtggtca tatgtccgcc tcctttggtc 40

<210> 26

<211> 18

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 26

atgaccacca ctgttgag 18

<210> 27

<211> 36

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 27

ttcgccttga tccccatcgc gaccagcgcg acgtgc 36

<210> 28

<211> 37

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 28

acagctatga catgattacg gcggcttgcg cccgatg 37

<210> 29

<211> 41

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 29

ttcgccttga tccccatcga attcgcgacc agcgcgacgt g 41

<210> 30

<211> 18

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 30

ctgcgccccc gtcgagat 18

<210> 31

<211> 36

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 31

atctcgacgg gggcgcagga gaaggtgctc gtgtag 36

<210> 32

<211> 40

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 32

cgccttgatc cccatcgaat tcaccaagcc ggagtcggtg 40

<210> 33

<211> 40

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 33

cgacctgcag gcatgcaagc ttgtggtcgt cgtcatcgtc 40

<210> 34

<211> 41

<212> DNA

<213> Artificial Synthesis (Unknow)

<400> 34

gtgcttgcgg cagcgtgaag cttagcagcg agacgacgag g 41

Claims (6)

1. A method for enhancing the gene editing efficiency of streptomycete, which is characterized by comprising the following steps:

(1) selecting an inducible promoter tipA, a ribosome switch shown by SEQ ID number 2, a strong promoter and a skeleton plasmid according to the physiological characteristics of a target host;

(2) the inducible promoter tipA screened in the step (1), the ribosome switch shown as SEQ ID number 2, the split Cas9 and the strong promoter-atpDInserting a high-energy-supply gene cassette into the backbone plasmid selected in the step (1); to achieve split expression of Cas9, pMag and nMAG were constructed, where the sequence of pMag is as set forth in SEQ ID NO.3The sequence of nMAG is shown as SEQ ID NO.4, nMAG is sequentially connected with 1965bp coding protein at the carboxyl end of Cas9, 2136bp at the amino terminal of Cas9 is sequentially connected with pMag, two fragments are connected with a framework plasmid, and then two proteins are induced to be heterodimerized by adding blue light to rebuild Cas 9;

(3) designing a spacer sequence of a target editing site, respectively inserting the spacer sequence into the plasmids constructed in the step (2), and guiding the Cas9 protein to move to a target sequence;

(4) selecting a proper screening mode for screening antibiotics or auxotrophs of the transformants according to the physiological properties of the host and plasmid elements, and additionally adding related genes for expressing the antibiotics and the auxotrophs into the plasmid constructed in the step (3);

(5) under a non-induction condition, transforming the recombinant plasmid in the step (4) into a target host, and screening by adding a culture medium of a screening condition to obtain a transformant carrying the recombinant plasmid;

(6) inoculating the transformant in the step (5) into an enrichment medium with proper screening conditions, and fermenting at constant temperature;

(7) taking the fermentation liquor fermented for 24-48 h in the step (6), centrifuging to collect thalli, washing twice with sterile water, centrifuging to remove supernatant, replacing a culture medium added with thiostrepton and theophylline with proper concentration, and then performing shaking table constant-temperature culture under the blue light induction condition;

(8) and (4) centrifuging the fermentation liquor obtained in the step (7), discarding the supernatant, and collecting the precipitate to obtain the strain for completing target gene editing.

2. The method for enhancing the gene editing efficiency of streptomyces as claimed in claim 1, wherein step (5) is carried out by genetically manipulating the recombinant plasmid in step (4) into the target host through conjugation or transduction under non-inducing conditions.

3. Use of the method of claim 1 for increasing the efficiency of gene editing in streptomyces.

4. The use of claim 3, wherein the transformation process is achieved with triple control of Cas9 activity at the transcriptional, translational and protein levels by the addition of a suitable inducible promoter, ribosomal switch and split Cas9 to the editing plasmid.

5. The application of claim 3, wherein in the editing process, the aim of improving the editing efficiency of the target gene is fulfilled by recovering Cas9 protein expression and the reconstruction of the structure and activity thereof through thiostrepton induction, theophylline induction and blue light induction in a manual controlled manner.

6. The use according to claim 3, characterized in that the editing plasmid is inserted with a suitable strong promoteratpDThe highly energized gene cassette of (1), encoding the beta-subunit of ATP synthase during the editing processatpDThe gene is expressed under the screened strong constitutive promoter, ATP is provided to facilitate the completion of homologous directional recombination, which is a key step of gene editing, and the homologous recombination efficiency is enhanced, so that the editing efficiency is further improved.

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