WO2021017200A1 - 一种基于运动发酵单胞菌的CRISPR-Cas系统、基因组编辑体系及其应用 - Google Patents

一种基于运动发酵单胞菌的CRISPR-Cas系统、基因组编辑体系及其应用 Download PDF

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WO2021017200A1
WO2021017200A1 PCT/CN2019/112556 CN2019112556W WO2021017200A1 WO 2021017200 A1 WO2021017200 A1 WO 2021017200A1 CN 2019112556 W CN2019112556 W CN 2019112556W WO 2021017200 A1 WO2021017200 A1 WO 2021017200A1
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crispr
sequence
gene
mobilis
cas12a
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杨世辉
彭文舫
沈威
郑艳丽
易犁
马立新
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湖北大学
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Priority claimed from CN201910692120.5A external-priority patent/CN110408642B/zh
Priority claimed from CN201910692118.8A external-priority patent/CN110331158B/zh
Priority claimed from CN201910692143.6A external-priority patent/CN110358768B/zh
Priority claimed from CN201910692112.0A external-priority patent/CN110358767B/zh
Application filed by 湖北大学 filed Critical 湖北大学
Priority to US17/295,045 priority Critical patent/US20220348939A1/en
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Definitions

  • the invention belongs to the technical field of genetic engineering, and particularly relates to a CRISPR-Cas system based on Zymomonas mobilis, a genome editing system and applications thereof.
  • Bio-energy regeneration is one of the effective means to solve the problems of human resources, energy shortages and serious environmental pollution.
  • Zymomonas mobilis has the relevant characteristics of an ideal microbial cell factory: (1) It can produce alcohol naturally, has high tolerance to alcohol, and can produce 10.7% (v/v) from the hydrolysate of corn stalks. ) High-concentration cellulose alcohol; (2) is the only known microorganism that can metabolize glucose or fructose to produce ethanol under anaerobic conditions through the Entner-Doudoroff (ED) pathway. It only metabolizes one molecule of glucose or fructose.
  • ED Entner-Doudoroff
  • Genome The size is only about 2Mbp, which has great advantages in carrying out genome streamlining work and constructing the most suitable genome cell factory.
  • Zymomonas mobilis can grow in a wide range of temperature (24-45°C) and pH range (3.5-7.5), and is a generally recognized GRAS (Generally Recognized As Safe) strain.
  • Zymomonas mobilis can be used as a model production strain for biomass renewable energy, which has attracted wide attention of researchers; analyze its physiological and genetic characteristics, rationally design and construct engineering strains, analyze its metabolic network and regulatory mechanism And so on, has also become a research focus.
  • ZFNs zinc finger nucleases
  • TALENs transcriptional activator-like effector nucleases
  • CRISPR-Cas9 and CRISPR-Cpf1 systems have been widely used in various organisms (cells) including human cells. Compared with ZFN and TALEN technologies, CRISPR technology has an obvious advantage. Advantages: Targeting of different target DNAs does not require protein modification, only a simple change of a single mediating RNA (gRNA) sequence, which is easy to operate. However, as of now, no related applications have been implemented in Z. mobilis. The main reasons may be: 1) Both Cas9 and Cpf1 are large nucleic acid proteins (greater than 1000 amino acids) with multiple domains, which are found in prokaryotic cells.
  • the effective transfer in the media has greater limitations; 2)
  • the exogenously expressed nucleases such as Cas9 or Cpf1 may be inactive, or active but have greater cytotoxicity to the host. With the deepening of research, more and more results show that heterologous expression of these nucleases will produce varying degrees of cytotoxicity to the host.
  • the present invention provides a CRISPR-Cas system based on Zymomonas mobilis, a genome editing system and applications thereof.
  • the purpose of the present invention is to take Z. mobilis 4 as a model strain and use its own genome-encoded I-F CRISPR-Cas system to build a genome editing platform.
  • the present invention is realized in this way, based on the CRISPR-Cas system of Zymomonas mobilis.
  • the CRISPR-Cas system includes 4 CRISPR structural sequences and a cas gene cluster.
  • the cas gene cluster includes cas1, cas3, csy1, and csy2. , Csy3 and csy4 genes, where cas3 gene is a fusion form of cas2 and cas3 genes.
  • Genome editing system based on exogenous Zymomonas mobilis CRISPR-Cas12a system, including editing plasmids carrying the targeting sequence guideRNA primer sequence, artificial CRISPR expression unit and donor DNA sequence, and containing the inducible expression of the nuclease Cas12a Recombinant Zymomonas mobilis.
  • the recombinant Z. mobilis containing the inducible expression of the nuclease Cas12a is obtained by integrating the exogenous nuclease Cas12a into the Z. mobilis containing the CRISPR-Cas system as claimed in claim 1,
  • the expression of the nuclease Cas12a is controlled by a tetracycline inducible promoter.
  • the artificial CRISPR expression unit includes a constitutive promoter PJ23119, a repetitive sequence, and two restriction sites.
  • the PAM sequence of the editing system is TTTN.
  • a genome editing system based on the endogenous Zymomonas mobilis CRISPR-Cas system including an editing plasmid carrying a targeting sequence guideRNA primer sequence, an artificial CRISPR expression unit, and a donor DNA sequence, and the motility fermenter described in claim 1 CRISPR-Cas system for spores.
  • the artificial CRISPR expression unit includes a leader sequence, a CRISPR cluster and a terminator, and the CRISPR cluster includes two restriction sites inserted between two repeat sequences.
  • leader sequence CRISPR cluster and terminator sequence are shown in SEQ ID NO: 34-SEQ ID NO: 36 respectively.
  • the PAM sequence of the editing system is NCC.
  • the CRISPR-Cas system based on Z. mobilis or the genome editing system based on the exogenous Z. mobilis CRISPR-Cas12a system as described above, or the endogenous Z. mobilis CRISPR-Cas system Genome editing system is used in gene knockout, gene insertion, or site-directed mutation DNA sequence, or simultaneous editing of multiple gene sites, or deletion of large fragments of the genome, or elimination of endogenous plasmids, or detection of protein active sites.
  • the present invention uses Zymomonas mobilis model strains as materials, develops its endogenous CRISPR-Cas system as a gene editing tool, and performs a series of efficient genetic operations.
  • the technology has the following beneficial effects: (1) It can effectively avoid the cytotoxicity of foreign Cas nucleic acid protein to the host; (2) The host's own CRISPR-Cas system has complete functions and can process mature crRNA to mediate the Cas effect The target DNA sequence is sheared by the complex; (3) there is no problem of transporting large proteins; (4) the editing purpose of deleting large fragments of the genome of the strain can be achieved. Therefore, cloning different mediator sequences into the same CRISPR cluster can achieve simultaneous editing of multiple target sites.
  • the invention breaks the limitation of low efficiency of exogenous CRISPR-Cas9 genome editing in such strains, realizes rapid and efficient knockout of multiple genes in the strain, and promotes the development of metabolic engineering, systems biology and synthetic biology.
  • the present invention has also developed a genome editing system based on the exogenous Zymomonas mobilis CRISPR-Cas12a system, and explored its applications in endogenous plasmid elimination, gene knockout, gene mutation and gene insertion.
  • the system has the following technical advantages: (1) Easy to operate, because CRISPR-Cas12a can process crRNA by itself, it only needs to assemble the target site sequence and provide an appropriate repair template to perform gene editing. (2) High positive rate and no trace editing. The CRISPR-Cas12a system can continuously cut the target sequence, has positive selection pressure, does not require additional resistance screening markers, and avoids safety hazards caused by the introduction of resistance genes. (3) The scope of application is wide.
  • the CRISPR-Cas12a system can be used in a variety of gene editing methods such as gene knockout, gene knock-in and site-directed mutation. (4) The process is simple and the time period is short, which greatly reduces the workload of prokaryotic genome editing.
  • Figure 1 is the CRISPR-Cas system encoded by Zymomonas mobilis
  • Figure 2 is the sequence of C2S7 and C3S4;
  • Figure 3 shows the results of in vivo cleavage activity detection of CRISPR-Cas system
  • Figure 4 is an artificial CRISPR expression unit
  • Figure 5 is a schematic diagram of the construction of guideRNA to vector in gene knockout
  • Figure 6 shows the results of PCR cloning in gene knockout
  • Figure 7 shows the sequencing results of PCR products in gene knockout
  • Figure 8 is a schematic diagram of the principle of site-specific insertion of DNA sequences
  • Figure 9 is the PCR cloning result of the site-specific insertion of the DNA sequence
  • Figure 10 is the sequencing result of PCR product of DNA sequence inserted at a specific point
  • Figure 11 is a schematic diagram of the principle of site-directed mutation DNA sequence
  • Figure 12 is the PCR cloning result of site-directed mutation DNA sequence
  • Figure 13 is the sequencing result of PCR product of site-directed mutation DNA sequence
  • Figure 14 is a multi-point simultaneous editing process mediated by endogenous CRISPR-Cas
  • Figure 15 is a schematic diagram of the process of cloning an artificial CRISPR cluster into an editing plasmid
  • Figure 16 is the result of electrophoresis of PCR product of transformant colony
  • Figure 17 is a schematic diagram of statistical analysis of transformant editing results
  • Figure 18 is the result of transformant sequencing
  • Figure 19 is a schematic diagram of the principle of large fragment sequence deletion
  • Figure 20 is the result of colony PCR positive cloning
  • Figure 21 is the result of transformant sequencing
  • Figure 22 is a product electrophoresis diagram of the elimination experiment of endogenous plasmid
  • Figure 23 is an electropherogram of the product of a point mutation experiment
  • Figure 24 is the product sequencing result of the point mutation experiment
  • Figure 25 is a schematic diagram of the principle of gene knockout editing
  • Figure 26 is an electropherogram of the product of a gene knockout experiment
  • Figure 27 is the sequencing result of the gene knockout experiment
  • Figure 28 is a schematic diagram of the principle of gene insertion editing
  • Figure 29 is an electropherogram of the product of a gene insertion experiment
  • Figure 30 is the product sequencing result of the gene insertion experiment
  • Figure 31 shows the results of flow cytometry in the gene insertion experiment.
  • CRISPR1-CRISPR4 CRISPR1-CRISPR4 in turn, as shown in Figure 1.
  • CRISPR1 occupies 113,783-114,170 regions of the genome and contains 7 repeat sequences
  • CRISPR2 occupies 1,244,355-1,245,866 regions and contains 9 repeat sequences
  • CRISPR3 occupies 1,598,754-1,599,144 regions and contains 7 repeat sequences.
  • CRISPR4 is composed of 2 repeats and 1 spacer, occupying 1,595,315-1,599,403 regions.
  • CRISPR2-4 are on the same chain, while CRISPR1 is on the complementary chain.
  • the repeat in these CRISPR structures is a conservative 28bp sequence, the spacer length is 32 or 33bp, of which 32bp accounts for 70%.
  • the genome also encodes a cas gene cluster, including cas1, cas3, csy1, csy2, csy3 and csy4 genes, where cas1, cas3 form an operon, and all csy genes are arranged in the form of an operon.
  • the cas3 gene is a fusion of cas2 and cas3 genes, which is a hallmark feature of the I-F CRISPR-Cas system.
  • the digestion system is system 1: pEZ15Asp, 2-3 ⁇ g; Xba I, and EcoR I each 1 ⁇ L; Buffer, 2 ⁇ L ; Make up H 2 O to 20 ⁇ L. Carrier double digestion conditions: 37°C for 3-4 hours. After annealing with C2S7 and C3S4 primers, they were ligated with T4 DNA ligase.
  • the annealing system is system 2: Buffer, 1 ⁇ L; forward primer and reverse primer each 1 ⁇ L; H 2 O to 10 ⁇ L. Annealing procedure: hold at 95°C for 5 minutes, then anneal at room temperature.
  • the T4 DNA ligase ligation system is system 3: linearized vector 100-200ng; primer 1 ⁇ L after annealing; T4 DNA ligase 1 ⁇ L; Buffer 2 ⁇ L; H 2 O to 20 ⁇ L. Connection procedure: heat preservation at 22°C for 2h. Transform into E.coli DH5 ⁇ by the standard heat shock transformation method at 42°C for plasmid amplification, and then verify the colony PCR on the transformants.
  • the PCR system is system 4: PCRmix, 5 ⁇ L; forward primer and reverse primer each 0.5 ⁇ L; Template 1 ⁇ L; supplement H 2 O to 10 ⁇ L.
  • the PCR program is: Step 1, 98°C, 3min; Step 2, 98°C, 10s; Step 3, 55°C, 15s; Step 4, 72°C, 30s; Step 2-Step 4 cycle 25 times; Step 5, 72°C , 2min.
  • the constructed plasmids are verified by sequencing. At the same time, insert the spacer of 5'plus 5'-AAA-3' sequence into the vector to obtain the corresponding reference plasmid.
  • the shuttle vector pEZ15Asp contains a gene encoding spectinomycin resistance.
  • ZM4 was electrotransformed with the extracted plasmid.
  • the electro-transformation method is a common standard method in the field, and will not be repeated here.
  • the results of the experiment are shown in Figure 3.
  • the efficiency of transforming the interference plasmid is 103 times lower than that of the reference plasmid, which indicates that the DNA activity of the IF-type system mediated by crRNA expressed in CRISPR on the genome has been cut to the protospacer in the interference plasmid Cutting, thus confirming that the system can be used for site-specific targeting and cutting of DNA sequences.
  • An artificial CRISPR expression unit was constructed on the plasmid pEZ15Asp, as shown in Figure 4, consisting of a promoter leader sequence, a CRISPR cluster and a terminator.
  • the artificial CRISPR expression unit was artificially synthesized by Ssweeping Gene Synthesis Company.
  • the artificial CRISPR cluster includes two Bsa I restriction sites inserted between two repeats. The second is the provision of donor DNA. According to different genome editing methods, the design of donor DNA is also different, but the donor DNA is amplified and connected by fusion PCR technology.
  • the promoter Leader sequence, RgR module sequence, and T7 terminator sequence are shown in SEQ ID NO: 34-SEQ ID NO: 36, respectively.
  • the sequence of about 300bp in the upstream and downstream of the target gene is amplified and connected.
  • the mutation site is introduced into the primer and the mutation is introduced into the donor DNA.
  • the purified donor DNA is cloned into the corresponding editing plasmid for transformation.
  • the nuclease Cas12a from Francisella novicida was integrated into the ZMO0038 site in the Z.mobilis ZM4 genome by homologous recombination, and an inducible promoter was used to control the expression of the nuclease Quantity, the recombinant strain ZM-Cas12a was constructed.
  • PCR amplification program is set as follows: 98°C pre-denaturation 2min; 98°C denaturation 10s, 55°C annealing 10s, 72°C extension (set according to the fragment length according to 10s/kb), a total of 30 cycles; after the cycle reaction is completed, 72°C is maintained 5min; the product is purified and stored at -20°C.
  • the PCR amplification condition system is system 5: 10 ⁇ M forward and reverse primers each 0.5 ⁇ L; PrimerSTAR DNA Polymerase (Takara), 10 ⁇ L; Template (5-10ng), X ⁇ L; supplement H 2 O to 20 ⁇ L.
  • the templates used to amplify Cas12a and inducible promoter fragments are all synthetic sequences
  • the Cas12a gene sequence is shown in SEQ ID NO: 69
  • the promoter Ptet sequence is shown in SEQ ID NO: 70.
  • the spectinomycin resistance gene was amplified from the well-known vector pEZ15a
  • the upstream and downstream gene sequence template was from the Zymomonas mobilis ZM4 genome
  • the template for reverse amplification of pUC57 was the pUC57 vector.
  • For the primer sequence see SEQ ID NO: 71-SEQ ID NO: 82.
  • the obtained fragment and the vector were mixed in a ratio of 3:1, according to System 6 (DNA fragment, 0.12pM; Vector, 0.04pM; 10 ⁇ Buffer 4 (Thermo), 0.5 ⁇ L; T5 Exonuclease, 0.5U; H 2 0 to 5 ⁇ L) After the preparation is completed, let it stand on ice for 5 minutes, and then add chemically competent state for chemical transformation.
  • System 6 DNA fragment, 0.12pM; Vector, 0.04pM; 10 ⁇ Buffer 4 (Thermo), 0.5 ⁇ L; T5 Exonuclease, 0.5U; H 2 0 to 5 ⁇ L
  • spectinomycin resistant plates for screening, pick single colonies, and use universal M13 primers to verify by colony PCR (the PCR amplification program is set to: 98°C pre-denaturation 3min; 98°C denaturation 10s, 55°C annealing 10s, 72 Extend for 80s at °C, 30 cycles in total), and the band size is consistent with the expectation and verified by sequencing.
  • the PCR amplification system is System 7: 10 ⁇ M forward and reverse primers 0.3 ⁇ L each; 2 ⁇ T5 Super PCR Mix (Tsingke), 5 ⁇ L; Template, X ⁇ L; H 2 O to 10 ⁇ L.
  • the PCR amplification procedure is the same as the above procedure when constructing the recombinant plasmid. Strains with the same band size as expected are verified by sequencing, and the correct strain is stored for use.
  • the editing plasmid uses pEZ15a as the vector backbone to construct the artificial expression unit of crRNA. It consists of a 19-nt repeat sequence and a 23-nt guide sequence for expression under the control of a constitutive promoter PJ23119, in which two inserts are inserted after the repeat sequence. A Bsa I restriction site to facilitate the insertion of the guide sequence.
  • the specific construction process is to assemble PJ23119 (TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC), 19-nt repeat sequence (AATTTCTACTCTTGTAGAT) and two Bsa I restriction sites (GGAGACCGAGGTCTCA) in series and then assemble them into pEZ15a vector. The process is completed by the tripartite company.
  • the annealed product was ligated with the linearized vector with T4 DNA ligase (Annealed oligonucleotide, 0.5 ⁇ L; vector, 10ng; 10 ⁇ T4 DNA Polymerase Buffer, 1 ⁇ L; T4 DNA Polymerase, 0.2 ⁇ L; H 2 O to 10 ⁇ L), and then Transformed into E. coli cloning strain DH5 ⁇ by chemical transformation methods commonly used in the art for plasmid construction, colony PCR is used to screen recombinants and finally verified by sequencing.
  • T4 DNA ligase Annealed oligonucleotide, 0.5 ⁇ L; vector, 10ng; 10 ⁇ T4 DNA Polymerase Buffer, 1 ⁇ L; T4 DNA Polymerase, 0.2 ⁇ L; H 2 O to 10 ⁇ L
  • the sequences of about 700 bp in the upstream and downstream of the target gene were selected, and the DNA fragments were amplified by PCR.
  • the PCR system and procedures were the same as those in the part (1) of Example 2.
  • the target vector constructed in the previous step is amplified with primers by reverse PCR.
  • the PCR amplification system and procedures are the same as those in Part (1) of Example 2, and the primers are as follows.
  • the fragment and the vector were connected by the standard general Gibson assembly method, and then transferred into the E. coli cloning strain DH5 ⁇ for plasmid construction, and the recombinant was screened by colony PCR and finally verified by sequencing. See SEQ ID NO: 104-SEQ ID NO: 105 for reverse amplification sequence.
  • SEQ ID NO: 106-SEQ ID NO: 109 for the sequence of the upstream and downstream primers for amplification.
  • primers are used to perform colony PCR detection on the recombinant strains.
  • the system and conditions are the same as those in Example 2 for the detection of recombinant bacteria.
  • the detection primer sequence is shown in SEQ ID NO: 110-SEQ ID NO: 111.
  • the CRISPR-Cas system in Example 1 can also be selected.
  • the guideRNA primer sequence was constructed on the vector, as shown in Figure 5:
  • the above-mentioned plasmid containing the artificial CRISPR expression unit was linearized with Bsa I, annealed with the guideRNA primer and then ligated with T4 DNA ligase, and then transformed into E. coli DH5 ⁇ for plasmid expansion. Then the transformants were verified by colony PCR, and the constructed plasmids were all verified by sequencing.
  • the experimental conditions are the same as above.
  • For colony PCR verification primer sequences see SEQ ID NO: 11 and SEQ ID NO: 10.
  • the donor DNA sequence is about 300bp upstream and downstream of the target gene, and the PCR technology (PCRmix, 10 ⁇ L; forward and reverse primers are 1 ⁇ L each; templates 1 and 2 are each 1 ⁇ L; H 2 O to 20 ⁇ L.
  • the PCR program is : 98°C, 3min; 98°C, 10s; 55°C, 15s; 72°C, 30s; cycle 25 times; 72°C, 2min.) Amplify and connect them.
  • the vector constructed in the previous step was digested with Xba I and EcoR I (same as in Example 1), and then transformed with the donor DNA sequence into E.coli DH5 ⁇ by Gibson assembly method, and then the transformants were verified by colony PCR (same as In Example 1), the constructed editing plasmids were all verified by sequencing.
  • the donor DNA primer sequences are shown in SEQ ID NO: 12 and SEQ ID NO: 15.
  • the editing plasmid was transformed into ZM4 competent cells by the general electroporation method, and then primer 0038-check-F, see SEQ ID NO: 16; 0038-check-R, see SEQ ID NO: 17; Perform colony PCR screening (the conditions are the same as above ) Positive clones.
  • Example 2 Connect the guide RNA primer sequence to the plasmid containing the CRISPR expression unit prepared in Example 2: First, linearize the vector with restriction endonuclease Bsa I, then anneal the guide RNA primer pair, and the annealed product The linearized vector is ligated using T4 DNA ligase, and then transferred to the E. coli cloning strain DH5 ⁇ for plasmid construction. The recombinants are screened by colony PCR and finally verified by sequencing. The specific experimental operation is the same as in Example 3.
  • the donor DNA sequence was constructed on the target vector: the sequence of about 700 bp in the upstream and downstream of the target gene and the mCherry expression element were respectively amplified by PCR for their DNA fragments.
  • the PCR system and procedures were the same as in Example 2.
  • the target vector constructed in the previous step was amplified by reverse PCR using primers, and then the fragment and vector were connected by Gibson assembly method, where the mCherry expression element was sandwiched by the upstream and downstream homology arm fragments, and then transferred to the large intestine
  • the plasmid was constructed in the bacillus clone strain DH5 ⁇ , and the recombinants were screened by colony PCR and finally verified by sequencing.
  • the results are shown in Figure 29 and Figure 30.
  • the results show that the CRISPR-Cas12a system can insert genes in a targeted manner, and the sequencing results show that the knocked-out genes are consistent with the design.
  • the flow cytometer was used for detection.
  • the results are shown in Figure 31.
  • the results indicate that the reporter gene can be expressed normally at the insertion site. It shows that this method is a precise method of gene insertion.
  • the CRISPR-Cas system in Example 1 can also be selected.
  • the following is an example of inserting a His-Tag tag after the start code ATG of the ZMO0038 gene, so that the encoded protein can be purified by a nickel column. This method can be extended to any protein to be purified. The principle is shown in Figure 8.
  • the 32bp sequence immediately downstream of 5'-TCC-3' was cut from the target gene sequence ZMO0038 near the start codon ATG as the guideRNA, which can be located on any strand of the genome.
  • ZMO0038(His-Tag)-guideRNA primer see SEQ ID NO: 18-SEQ ID NO: 19.
  • the guideRNA primer is constructed on the vector: the plasmid containing the artificial CRISPR expression unit is linearized with Bsa I, annealed with the guideRNA primer, and then ligated with T4 DNA ligase, transformed into E. coli DH5 ⁇ for plasmid amplification, and then the transformant Perform colony PCR verification.
  • the experimental conditions are the same as in Example 3.
  • the verification primers are: pEZ15A-F and 0038His-gRNA-R. The sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 19, respectively.
  • the constructed plasmids are all verified by sequencing.
  • the donor DNA sequence of the His-Tag tag is inserted into the His-Tag by using the primer design.
  • the upstream arm and the downstream arm of the donor DNA are selected to insert the His-Tag tag at the upstream and downstream positions of about 300bp each.
  • the sequence is amplified and connected by fusion PCR technology.
  • the vector constructed in the previous step was digested with Xba I and EcoR I, and then transformed with the donor DNA sequence into E. coli DH5 ⁇ by Gibson assembly method, and then the transformants were verified by colony PCR.
  • the constructed editing plasmids all passed Sequencing verification.
  • the experimental conditions are the same as in Example 3.
  • the donor DNA primer sequence is shown in SEQ ID NO: 21-SEQ ID NO: 24.
  • the editing plasmid was electro-transformed into ZM4 competent cells, and positive clones were screened.
  • the method was the same as that in Example 3.
  • the primer sequence is shown in SEQ ID NO: 25-SEQ ID NO: 26.
  • ssDNA single-stranded nucleotide
  • Pst I enzyme Cut bit The length of ssDNA is 59-nt, which is complementary to the lagging strand of the coding DNA.
  • Transformation of editing plasmid about 200ng editing plasmid and 1 ⁇ g ssDNA are transformed into competent cells of recombinant bacteria containing Cas12a and cultured.
  • the experimental method and conditions are the same as in Example 3.
  • Colony PCR detection conditions are the same as in Example 2, and the detection primer sequence is shown in SEQ ID NO: 100-SEQ ID NO: 101.
  • the DNA fragment was recovered, and the DNA was digested with restriction endonuclease Pst I, and the digestion system (DNA fragment, 200 ng; 10 ⁇ buffer, 1 ⁇ L; Pst I, 0.2 ⁇ L; supplemented with H 2 O to 10 ⁇ L.
  • the conditions are the same as in the example 3).
  • the ability to cut the DNA fragment correctly indicates that it was edited correctly, and the recombinant strain was further verified by sequencing.
  • Example 1 the CRISPR-Cas system in Example 1 can also be selected.
  • the ZMO0038 gene with a His-Tag tag inserted in Example 4 is taken as the target, and a few base sequences are site-directed mutations in the coding region of the gene, thereby introducing the stop code 5'-AAA-3' to advance the encoded protein termination.
  • This method can also be extended to the study of protein active sites. The principle is shown in Figure 11.
  • the 32bp sequence immediately downstream from the 5'-CCC-3' of the target gene sequence His-ZMO0038 coding region is used as the guideRNA, and this sequence can only be located on the coding strand.
  • ZMO0038(PM)-guideRNA primer see SEQ ID NO: 27-SEQ ID NO: 28.
  • the plasmid containing the artificial CRISPR expression unit was linearized with Bsa I, annealed with the guideRNA primer, and then ligated with T4 DNA ligase.
  • the plasmid was transformed into E. coli DH5 ⁇ for plasmid amplification, and then the transformants were subjected to colony PCR verification.
  • the experimental method is the same as in Example 3.
  • the verification primers are: pEZ15A-F and 0038PM-gRNA-R.
  • the constructed plasmids are all verified by sequencing.
  • the donor DNA sequence for site-directed mutagenesis is the base sequence with the mutation introduced by the primer design.
  • the upstream and downstream arms of the donor DNA select the sequence of about 300bp upstream and downstream of the site-directed mutagenesis position, and expand it by fusion PCR technology. Increase and connect.
  • the vector constructed in the previous step was digested with Xba I and EcoR I, and then transformed with the donor DNA sequence into E. coli DH5 ⁇ by Gibson assembly method, and then the transformants were verified by colony PCR.
  • the constructed editing plasmids all passed Sequencing verification.
  • the experimental method is the same as in Example 3.
  • For the donor DNA primer sequence see SEQ ID NO: 29-SEQ ID NO: 32.
  • the editing plasmid was electro-transformed into ZM4 competent cells, and colony PCR and sequencing were performed. The conditions were the same as in Example 3. For primer sequence, see SEQ ID NO: 16-SEQ ID NO: 33.
  • the target of multi-site gene editing in this embodiment is genome-encoded CRISPR genes 1-4 (CRISPR1-4), where CRISPR3 and 4 are located in close proximity on the genome, and they are used as an editing target for knockout.
  • CRISPR1-4 genome-encoded CRISPR genes 1-4
  • the 32bp sequence immediately downstream of 5'-NCC-3' was cut from the 3'end of the target gene sequence CRISPR1-4 as gRNA, and the sequence could be located on any strand of the genome.
  • the cloning process is shown in Figure 15.
  • the gRNA of the above target gene was connected in series by artificial synthesis, and then assembled with Gibson with the plasmid containing the artificial CRISPR expression unit prepared in Example 1, and then the transformants were verified by colony PCR.
  • the constructed plasmids were all verified by sequencing .
  • the experimental method is the same as in Example 1.
  • the sequence of the colony PCR verification primers used is shown in SEQ ID NO: 40-SEQ ID NO: 41.
  • the sequences of about 300 bp in the upstream and downstream of the target gene were selected as the homologous recombination donor template DNA, which was amplified and ligated by fusion PCR technology, and then connected to the editing plasmid by the Gibson assembly method. Transform E.coli DH5 ⁇ , and then perform colony PCR verification on the transformants. The constructed editing plasmids are all verified by sequencing.
  • the experimental conditions are the same as in Example 3.
  • the donor DNA primer sequence is shown in SEQ ID NO: 42-SEQ ID NO: 53.
  • a bioinformatics method is used to determine the essential genes that need to be retained and the non-essential genes that can be deleted, and a non-essential gene with a length of 10 kb is selected as the target knockout large fragment. Then design the guide RNA and donor DNA sequences. Finally, load the artificial CRISPR cluster expression module and the donor DNA sequence on the plasmid, and electrotransform the plasmid into the Zymomonas mobilis cell to complete editing.
  • the CRISPR-Cas system described in Example 1 is used. The schematic diagram of the principle is shown in Figure 19, and the specific experimental scheme is as follows:
  • the plasmid containing the artificial CRISPR expression unit prepared in Example 1 was linearized with Bsa I, annealed with guide RNA primers, and then ligated with T4 DNA ligase.
  • the plasmid was amplified by transforming into E.coli DH5 ⁇ , and then the transformants were amplified. Colony PCR verification was performed, and the constructed plasmids were all verified by sequencing.
  • the experimental conditions are the same as in Example 3. See SEQ ID NO: 62-SEQ ID NO: 10 for colony PCR verification primer sequence.
  • the donor DNA sequence is about 1Kb upstream and downstream of the target gene, and amplified and connected by fusion PCR technology.
  • the vector constructed in the previous step was digested with Xma I and Sac I (except for the different types of enzymes used in the digestion system, the other conditions and conditions are the same as in Example 1), and then transformed with the donor DNA sequence by Gibson assembly method E.coli DH5 ⁇ , and then the transformants were verified by colony PCR, and the constructed editing plasmids were all verified by sequencing.
  • the experimental conditions are the same as in Example 3.
  • the donor DNA primer sequence is shown in SEQ ID NO: 63-SEQ ID NO: 66.
  • Z.mobilis ZM4 contains 4 endogenous plasmids and named pZM32 (32,791bp), pZM33 (33,006bp), pZM36 (36,494bp) and pZM39 (39,266bp) according to the size of the sequence. . Sequence analysis showed that the four endogenous plasmid bacteria edit the replicase. If the replicase is inactivated, the endogenous plasmid will lose the ability to replicate and the endogenous plasmid will be eliminated from the strain.
  • a sequence of 23 bp downstream of the PAM site TTTN was selected from the replicase gene of the endogenous plasmid as the targeting guide sequence for constructing the guide RNA in the target plasmid to guide the cleavage of the target site by the nuclease.
  • the forward primer is 5'-AGAT+ (target sequence)-3'
  • the reverse primer is 5'-TGAC+ (target sequence complementary sequence)-3'.
  • the CRISPR-Cas12a system described in Example 2 is used.
  • the guide RNA primer sequences of the four endogenous plasmids are shown in SEQ ID NO: 83-SEQ ID NO: 90.
  • primers are used to perform colony PCR detection on the recombinant strains.
  • the PCR system and procedures are the same as those in Example 2, and the detection primers are shown in SEQ ID NO: 91-SEQ ID NO: 96.

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Abstract

一种基于运动发酵单胞菌的CRISPR-Cas系统，包括4个CRISPR结构序列和一个cas基因簇，所述cas基因簇包括cas1，cas3，csy1，csy2，csy3和csy4基因，其中cas3基因是一个cas2和cas3基因的融合形式。还提供了以运动发酵单胞菌为模式菌，是利用其自身基因组编码的CRISPR-Cas系统及外源Cas12a搭建的基因组编辑系统。

Description

一种基于运动发酵单胞菌的CRISPR-Cas系统、基因组编辑体系及其应用 技术领域
本发明属于基因工程技术领域，尤其涉及一种基于运动发酵单胞菌的CRISPR-Cas系统、基因组编辑体系及其应用。
背景技术
近年来，利用微生物进行代谢工程、系统生物学及合成生物学等方面的研究取得了良好的进展，为理性化设计、构建微生物细胞工厂，利用生物体活细胞或酶对可再生生物质，如纤维素等进行物质转化，生产生物能源，实现生物冶炼的工业化提供了重要的理论基础。生物能源再生化是解决人类目前面临的资源、能源短缺及环境污染严重等问题的有效手段之一。
运动发酵单胞菌(Zymomonas mobilis，Z.mobilis)具有理想微生物细胞工厂的相关特性：(1)能天然产酒精，对酒精耐受性高，可以利用玉米秸秆水解物生产10.7％(v/v)的高浓度纤维素酒精；(2)是目前已知的唯一可以在厌氧条件下通过Entner-Doudoroff(ED)途径代谢葡萄糖或果糖生产乙醇的微生物，其每代谢一分子的葡萄糖或者果糖只产生一分子的ATP，产能低，因而大部分碳源(>95％)被转化为产物乙醇，只有约2-2.6％的碳源用于细胞生长，目标产物产率非常高；(3)基因组大小仅为约2Mbp，在开展基因组精简工作，构建最适基因组细胞工厂方面具有很大优势。此外，运动发酵单胞菌可在广泛的温度(24-45℃)及pH范围(3.5-7.5)生长，是公认的GRAS(Generally Recognized As Safe)菌株。因此，运动发酵单胞菌可作为生物质再生能源的模式生产菌株，已引起了研究者们的广泛关注；分析其生理和遗传等特性、理性设计与构建工程菌株、解析其代谢网络和调控机制等，也成为了研究的热点。
开展细胞生理和遗传特性分析、工程菌株理性设计与构建、代谢网络和调控机制解析等工作，需要在细胞内对基因组DNA序列进行可控改变。对于具有优良的微生物细胞工厂特性的运动发酵单胞菌，亟需一个高效且精确的基因组编辑系统来实现对其的充分研究和利用。
常规的遗传学方法直接利用宿主体内DNA重组修复系统，也能对基因进行突变，但通常只能单个单个地完成，耗时较长，效率太低，显然无法满足诸如构建复杂的代谢通路等研究的要求。而且，对于每一个目标基因的突变，均需要引入特定的选择标记，会存在可用选择标记有限、引入抗生素标记而产生生物安全隐患等等一些问题。此外，为了提高细胞体内DNA重组效率，可利用序列特异性核酸酶对目的基因进行定点剪切，促进基因组与供体DNA的重组，定点引入突变，实现基因组的精准编辑。例如，锌指核酸酶(Zinc finger nucleases，ZFNs)和转录激活样效应物核酸酶(Transcription activator-like effector nucleases，TALENs)被成功用于对基因组进行定点剪切。但是，对每一个靶标位点的剪切，均需要对上述蛋白进行一次改造，实验步骤较为繁琐。
自2013年以来，基于CRISPR-Cas9与CRISPR-Cpf1系统的基因组编辑技术被广泛应用于包括人体细胞在内的各类生物(细胞)中，相比于ZFN和TALEN技术，CRISPR技术具有一个明显的优 势：对不同目的DNA的靶定不需要进行蛋白改造，只需要简单改变单个介导RNA(gRNA)序列，易于操作。然而，截止目前为止，尚无相关应用在运动发酵单胞菌中实现，主要原因可能是：1)Cas9和Cpf1均为具有多个结构域的大核酸蛋白(大于1000氨基酸)，其在原核细胞中的有效转移存在较大的局限性；2)外源表达的Cas9或Cpf1等核酸酶可能无活性，或者有活性但对宿主产生了较大的细胞毒性。随着研究的深入，越来越多的结果显示，异源表达这些核酸酶会对宿主产生不同程度的细胞毒性。
发明内容
针对现有技术存在的问题，本发明提供了涉及一种基于运动发酵单胞菌的CRISPR-Cas系统、基因组编辑体系及其应用。本发明的目的在于以Z.mobilis 4为模式菌株，利用其自身基因组编码的I-F型CRISPR-Cas系统搭建一套基因组编辑平台。为在该菌株及类似细胞中开展基础研究与应用研究提供一套强大的工具，促进代谢工程、系统生物学及合成生物学的发展。
本发明是这样实现的，基于运动发酵单胞菌的CRISPR-Cas系统，所述CRISPR-Cas系统包括4个CRISPR结构序列和一个cas基因簇，所述cas基因簇包括cas1，cas3，csy1，csy2，csy3和csy4基因，其中cas3基因是一个cas2和cas3基因的融合形式。
基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系，包括携带靶向序列guideRNA引物序列、人工CRISPR表达单元以及供体DNA序列的编辑质粒，及含有诱导型表达的核酸酶Cas12a的重组运动发酵单胞菌。
进一步，所述含有诱导型表达的核酸酶Cas12a的重组运动发酵单胞菌通过将外源的核酸酶Cas12a整合至含有如权利要求1所述的CRISPR-Cas系统的运动发酵单胞菌中获得，且所述核酸酶Cas12a通过四环素诱导型启动子控制表达。
进一步，所述人工CRISPR表达单元包括组成型启动子PJ23119、重复序列以及两个酶切位点。
进一步，所述编辑体系的PAM序列为TTTN。
基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系，包括携带靶向序列guideRNA引物序列、人工CRISPR表达单元以及供体DNA序列的编辑质粒，及权利要求1所述的运动发酵单胞菌的CRISPR-Cas系统。
进一步，所述人工CRISPR表达单元包括leader序列，CRISPR簇及终止子，所述CRISPR簇包括在两个repeat序列中间插入了两个酶切位点。
进一步，所述leader序列，CRISPR簇及终止子序列分别见SEQ ID NO:34-SEQ ID NO:36。
进一步，所述编辑体系的PAM序列为NCC。
如上所述的基于运动发酵单胞菌的CRISPR-Cas系统、或基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系、或基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系在基因敲除、或基因插入、或定点突变DNA序列、或多基因位点同时编辑、或基因组大片段删除、或内源质粒消除、或蛋白活性位点检测中的应用。
综上所述，本发明的优点及积极效果为：
本发明以运动发酵单胞菌模式菌株为材料，开发其內源CRISPR-Cas系统作为基因编辑工具，进行一系列高效的遗传学操作。该技术有以下有益效果：(1)可有效避免外源Cas核酸蛋白对宿主产生的细胞毒性；(2)宿主自身CRISPR-Cas系统具有完整的功能，能加工成熟的crRNA用于介导Cas效应复合体对目标DNA序列的剪切；(3)不存在对大蛋白的转运问题；(4)能够实现对该菌株基因组大片段删除的编辑目的。因此，将不同的介导序列克隆到同一个CRISPR簇中，即能实现对多个靶位点的同时编辑。而相对于传统遗传学操作方法，利用该CRISPR-Cas系统，(1)能对预编辑的目标序列进行持续剪切，具有很强的正选压力，不需要额外使用选择标记，避免了传统操作方法中常遇到的可用选择标记有限、引入抗生素标记产生生物安全隐患；(2)极大程度上缩短了基因编辑的周期。以单个位点编辑为例，常规遗传操作方法至少需要15天才能得到纯化的目的菌株，而利用该CRISPR-Cas系统仅需要3天；对于多位点的编辑则差异更大，常规遗传操作方法只能在单个位点编辑的基础上进行累加，即2位点需至少30天，3位点则需至少45天，以此类推，利用CRISPR-Cas系统则仍然只需要3天(一个编辑周期)。本发明已实现了至少3位点的同时编辑。
本发明打破外源CRISPR-Cas9基因组编辑在该类菌株中效率低下的限制，在该菌株中实现多基因的同时快速、高效敲除，促进代谢工程、系统生物学及合成生物学的发展。
另外，本发明还开发了基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系，并探索了其在内源质粒消除、基因敲除、基因突变及基因插入中的应用。该体系有以下的技术优势：(1)操作简便，由于CRISPR-Cas12a可自己加工形成crRNA，仅需要装配完成靶位点序列，提供适当的修复模板，即可进行基因编辑。(2)阳性率高，无痕编辑，CRISPR-Cas12a系统能对靶序列持续进行剪切，具有正选压力，无需额外的抗性筛选标记，避免引入抗性基因造成的安全隐患。(3)适用范围广泛，CRISPR-Cas12a系统可用于多种基因编辑方式如：基因的敲除、基因敲入和定点突变等。(4)流程简单，时间周期短，大大减轻原核生物基因组编辑的工作量。
附图说明
图1是运动发酵单胞菌编码的CRISPR-Cas系统；
图2是C2S7和C3S4序列；
图3是CRISPR-Cas系统体内剪切活性检测结果；
图4是人工CRISPR表达单元；
图5是基因敲除中guideRNA构建到载体原理示意图；
图6是基因敲除中PCR克隆结果；
图7是基因敲除中PCR产物测序结果；
图8是定点插入DNA序列原理示意图；
图9是定点插入DNA序列PCR克隆结果；
图10是定点插入DNA序列PCR产物测序结果；
图11是定点突变DNA序列原理示意图；
图12是定点突变DNA序列PCR克隆结果；
图13是定点突变DNA序列PCR产物测序结果；
图14是內源CRISPR-Cas介导的多点同时编辑流程；
图15是将人工CRISPR簇克隆到编辑质粒的流程示意图；
图16是转化子菌落PCR产物电泳结果；
图17是转化子编辑结果统计分析示意图；
图18是转化子测序结果；
图19是大片段序列删除原理示意图；
图20是菌落PCR阳性克隆结果；
图21是转化子测序结果；
图22是内源质粒的消除实验的产物电泳图；
图23是点突变实验的产物电泳图；
图24是点突变实验的产物测序结果；
图25是基因敲除编辑的原理示意图；
图26是基因敲除实验的产物电泳图；
图27是基因敲除实验的产物测序结果；
图28是基因插入编辑的原理示意图；
图29是基因插入实验的产物电泳图；
图30是基因插入实验的产物测序结果；
图31是基因插入实验的流式细胞仪检测结果。
具体实施方式
以下结合实施例对本发明进行进一步详细说明，各实施例及试验例中所用的设备和试剂如无特殊说明，均可从商业途径得到。
实施例1运动发酵单胞菌內源CRISPR-Cas基因组编辑系统的构建
(1)运动发酵单胞菌基因组编码的CRISPR-Cas系统组学分析
以运动发酵单胞菌Z.mobilis 4为模式菌株，对Z.mobilis 4测序数据进行分析，结果显示，该菌株基因组编码4个CRISPR结构序列，根据其在基因组的排列顺序，本发明中将其依次命名为CRISPR1-CRISPR4，见图1。CRISPR1占据基因组113,783-114,170区域，包含7个repeat序列；CRISPR2占据1,244,355-1,245,866区域，包含9个repeat序列；CRISPR3占据1,598,754-1,599,144区域，包含7个repeat序列。CRISPR4由2个repeat和1个spacer组成，占据1,595,315-1,599,403区域。CRISPR2-4位于同一条链，而CRISPR1位于互补链上。这些CRISPR结构中repeat均为保守的28bp序列，spacer长度为32或33bp，其中32bp占70％。该基因组还编码一个cas基因簇，包括cas1，cas3，csy1，csy2， csy3和csy4基因，其中cas1，cas3形成一个操纵子，所有csy基因以一个操纵子形式排列。上述结果中cas3基因是一个cas2和cas3基因的融合形式，是I-F型CRISPR-Cas系统的一个标志性特征。
(2)运动发酵单胞菌内源I-F型CRISPR-Cas系统体内剪切活性检测
为了检测Z.mobilis 4菌株I-F型CRISPR-Cas系统是否可在crRNA的介导下体内剪切DNA，根据转录组分析结果，分别选取CRISPR2中spacer7(C2S7)和CRISPR3中spacer4(C3S4)，在其序列5’端加上5’-CCC-3’PAM，见图2。具体操作为：将Z.mobilis-E.col穿梭载体pEZ15Asp用Xba Ⅰ和EcoR Ⅰ双酶切，酶切体系为体系1：pEZ15Asp，2-3μg；Xba Ⅰ，和EcoR Ⅰ各1μL；Buffer，2μL；补H 2O至20μL。载体双酶切条件：37℃保温3-4h。与C2S7和C3S4的引物退火后用T4 DNA连接酶连接。退火体系为体系2：Buffer，1μL；正向引物和反向引物各1μL；补H 2O至10μL。退火程序：95℃保温5min，然后常温退火。T4 DNA连接酶连接体系为体系3：线性化载体100-200ng；退火后引物1μL；T4 DNA连接酶1μL；Buffer 2μL；补H 2O至20μL。连接程序：22℃保温2h。通过42℃标准热激转化法转化到E.coli DH5α中进行质粒扩增，然后对转化子进行菌落PCR验证，PCR体系为体系4：PCRmix，5μL；正向引物和反向引物各0.5μL；模板1μL；补H 2O至10μL。PCR程序为：步骤1，98℃，3min；步骤2，98℃，10s；步骤3，55℃，15s；步骤4，72℃，30s；步骤2-步骤4循环25次；步骤5，72℃，2min。构建好的质粒均通过测序验证。同时，将5’加上5’-AAA-3’序列的spacer插入到载体，得到对应的参照质粒。穿梭载体pEZ15Asp上含有壮观霉素抗性编码基因。
C2S7和C3S4引物序列见SEQ ID NO:1-SEQ ID NO:8。
用提取的质粒对ZM4进行电转化。电转化方法为本领域通用标准方法，在此不做赘述。
预测，将质粒分别转化到宿主后，如果I-F型CRISPR-Cas系统有活性，插入的protospacer将被剪切，在含有100μg/mL壮观霉素的RM培养基平板上进行筛选，则会造成平板上形成少数几个菌落；而转化参照质粒的平板上则会形成大量菌落。
实验结果如图3，转化干涉质粒的效率相比于转化参照质粒要低10 3倍，由此说明，基因组上CRISPR中表达的crRNA介导I-F型系统的DNA活性对干涉质粒中protospacer进行了剪切，从而确定该系统可被用于DNA序列的定点靶向和切割。
(3)基因组编辑质粒的组成
在质粒pEZ15Asp上构建了一个人工CRISPR表达单元，见图4，由启动子leader序列，CRISPR簇及终止子组成。该人工CRISPR表达单元由三方基因合成公司人工合成。人工CRISPR簇包括两个repeat中间插入了两个Bsa Ⅰ的酶切位点。其次是供体DNA的提供，根据不同的基因组编辑形式，供体DNA的设计也不同，但都是通过融合PCR技术对供体DNA进行扩增和连接。本实施例中启动子Leader序列、RgR模块序列以及T7终止子序列分别见SEQ ID NO:34-SEQ ID NO:36。
在基因敲除的应用中，将目标基因上、下游各300bp左右的序列进行扩增和连接。在基因定点突变的应用中，则是将突变位点引入到引物上，在供体DNA中引入突变。纯化后的供体DNA克隆到对应的编辑质粒上进行转化。
实施例2运动发酵单胞菌CRISPR-Cas12a基因组编辑系统的构建
本实施例在实施例1的基础上，将来源于Francisella novicida的核酸酶Cas12a通过同源重组的方法整合到Z.mobilis ZM4基因组中的ZMO0038位点，并采用诱导型启动子来控制核酸酶表达量，构建重组菌株ZM-Cas12a。
具体构建过程如下：
(1)重组质粒的构建
利用PCR分别扩增Cas12a基因序列，抗性筛选标记(壮观霉素)，诱导型启动子基因序列(四环素诱导启动子)，插入位点上下游的基因序列及反向扩增用于整合的pUC57的载体序列。PCR扩增程序设置为：98℃预变性2min；98℃变性10s，55℃退火10s，72℃延伸(根据片段长度按照10s/kb进行设置)，共30个循环；循环反应结束后72℃保持5min；产物经纯化后-20℃保存。PCR扩增条件体系为体系5：10μM正、反向引物各0.5μL；PrimerSTAR DNA Polymerase(Takara)，10μL；Template(5-10ng)，XμL；补H 2O至20μL。
其中，用于扩增Cas12a和诱导型启动子片段的模板均为合成的序列，Cas12a基因序列见SEQ ID NO:69，启动子Ptet序列见SEQ ID NO:70。扩增壮观霉素抗性基因的来自于公知载体pEZ15a，用于扩增上下游的基因序列模板来自于发酵单胞菌Zymomonas mobilis ZM4的基因组，反向扩增pUC57的模板为pUC57载体，扩增引物序列见SEQ ID NO:71-SEQ ID NO:82。
将获取的片段和载体按照3:1的比例进行混合，按照体系6(DNA fragment，0.12pM；Vector，0.04pM；10×Buffer 4(Thermo)，0.5μL；T5 Exonuclease，0.5U；补H 2O至5μL)配制完成后，在冰上静置5分钟，然后添加化学感受态，进行化学转化。利用壮观霉素抗性平板进行筛选，挑取单菌落，分别用通用M13引物通过colony PCR进行验证(PCR扩增程序设置为：98℃预变性3min；98℃变性10s，55℃退火10s，72℃延伸80s，共30个循环)，条带大小与预期一致的通过测序进行验证。
之后按照通用电转化方法进行转化、培养。待有菌落生长出来之后，对重组菌株进行菌落PCR检测，PCR扩增体系为体系7：10μM正、反向引物各0.3μL；2×T5 Super PCR Mix(Tsingke)，5μL；Template，XμL；补H 2O至10μL。PCR扩增程序同上述构建重组质粒时的程序。条带大小与预期一致的菌株通过测序进行验证，正确的菌株保存待用。
(2)构建编辑质粒
编辑质粒以pEZ15a为载体骨架，进行crRNA的人工表达单元的构建，其由19-nt的重复序列和23-nt的引导序列在组成型启动子PJ23119控制下进行表达，其中重复序列后面插入了两个Bsa Ⅰ的酶切位点以方便引导序列插入。其具体构建过程为，将PJ23119(TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC)，19-nt重复序列(AATTTCTACTCTTGTAGAT)以及两个Bsa Ⅰ的酶切位点(GGAGACCGAGGTCTCA)串联后组装到pEZ15a载体上。该过程由三方公司完成。
(3)在获得含有人工CRISPR表达单元的质粒后，根据具体的编辑需要设计供体DNA、构建靶 向性质粒，并将供体DNA整合到靶向性质粒，之后将靶向性质粒转入感受态细胞进行编辑。最终通过菌落PCR及测序验证编辑效果。
实施例3运动发酵单胞菌CRISPR基因组编辑系统在基因敲除中的应用
1.根据靶基因及其编辑类型选择基因组编辑系统，本实施例中将Z.mobilis ZM4基因组中ZMO0028作为靶位点，对其进行敲除，选用实施例2所述的CRISPR-Cas12a系统。原理如图25所示。从靶基因中选择PAM位点TTTN下游23bp的序列作为构建靶质粒中向导RNA的靶向引导序列，引导核酸酶对靶位点的切割。其中正向引物为5′-AGAT+(靶序列)-3′，反向引物为5′-TGAC+(靶序列互补序列)-3′。引物序列见SEQ ID NO:102和SEQ ID NO:103。
2.构建靶质粒
将向导RNA引物序列连接到实施例2中制备的含有CRISPR表达单元的编辑质粒上：首先利用限制性内切酶Bsa Ⅰ将载体进行线性化处理(质粒2μg；10×buffer，5μL；酶1μL；补H 2O至50μL。37℃保温3-4h)，然后将向导RNA引物对进行退火(10μM的引物各取1μL加水补足至10μL，95℃变性5min，然后冷却至室温备用)。退火的产物与线性化载体使用T4 DNA连接酶进行连接(Annealed oligonucleotide，0.5μL；载体，10ng；10×T4 DNA Polymerase Buffer，1μL；T4 DNA Polymerase，0.2μL；补H 2O至10μL)，然后通过本领域通用化学转化法转入到大肠杆菌克隆菌株DH5α中进行质粒构建，通过菌落PCR对重组子进行筛选最后通过测序验证。
3.构建编辑质粒
分别选取靶基因上、下游各700bp左右的序列，通过PCR分别扩增其DNA片段，PCR体系及程序同实施例2第(1)部分。将上一步构建好的靶载体利用引物进行反向PCR扩增，PCR扩增体系及程序均同实施例2第(1)部分，引物见下述。然后通过标准通用的Gibson装配的方法将片段和载体进行连接，然后转入到大肠杆菌克隆菌株DH5α中进行质粒构建，通过菌落PCR对重组子进行筛选最后通过测序验证。反向扩增序列见SEQ ID NO:104-SEQ ID NO:105。扩增上下游引物序列见SEQ ID NO:106-SEQ ID NO:109。
4.编辑质粒的转化
取步骤3构建的编辑质粒500ng左右，采用通用电转化方法转化至实施例2中制备的含Cas12a的重组菌的感受态细胞中。电转程序完成后，将其转入到1mL RM培养基中在30℃条件下静置培养4-6h，然后取200μL左右涂布于50μg/mL氯霉素抗性平板上，30℃静置培养2-3天。
5.重组菌的筛选
待有菌落生长出来之后，分别用引物对重组菌株进行菌落PCR检测，体系及条件同实施例2中重组菌检测，检测引物序列见SEQ ID NO:110-SEQ ID NO:111。
PCR电泳结果及测序结果见图26和图27，说明CRISPR-Cas12a系统可以定向敲除基因，且测序结果表明敲除的基因与设计的一致，说明该方法是一种精确的基因敲除的方法。
同理，对于其他类型靶基因的敲除也可以选择实施例1中的CRISPR-Cas系统。以下以运动发酵 单胞菌基因组编码的ZMO0038基因作为靶基因进行叙述。
从目标基因序列ZMO0038中截取任意5’-CCC-3’下游紧邻的32bp序列作为guideRNA，该序列可位于基因组任一条链上，并设计引物序列见SEQ ID NO:9-SEQ ID NO:10。
guideRNA引物序列构建到载体上，见图5：将上述含有人工CRISPR表达单元的质粒用Bsa Ⅰ线性化，与guideRNA引物退火后用T4 DNA连接酶连接，通过转化到E.coli DH5α中进行质粒扩增，然后对转化子进行菌落PCR验证，构建好的质粒均通过测序验证。实验条件同上。菌落PCR验证引物序列见SEQ ID NO:11和SEQ ID NO:10。
供体DNA序列选取目标基因上、下游各300bp左右的序列，通过融合PCR技术(PCRmix，10μL；正、反向引物各1μL；模板1和2各1μL；补H 2O至20μL。PCR程序为：98℃，3min；98℃，10s；55℃，15s；72℃，30s；循环25次；72℃，2min。)对其进行扩增和连接。将上一步构建好的载体用Xba Ⅰ和EcoR Ⅰ双酶切(同实施例1)，再与供体DNA序列通过Gibson装配的方法转化E.coli DH5α，然后对转化子进行菌落PCR验证(同实施例1)，构建好的编辑质粒均通过测序验证。供体DNA引物序列见SEQ ID NO:12和SEQ ID NO:15。
编辑质粒采用通用电转化法转化至ZM4感受态细胞，再用引物0038-check-F,见SEQ ID NO:16；0038-check-R，见SEQ ID NO:17；进行菌落PCR筛选(条件同上)阳性克隆。
阳性克隆结果见图6，ZMO0038基因敲除的效率达到了100％，充分的说明了该发明是一种高效的基因编辑方法。利用上述PCR产物进行测序分析，结果如图7，通过测序结果与野生型菌株基因组序列进行比对，发现编辑方式完全按照实验设计的方案，进一步说明了该发明是一种精确的基因编辑方法。
实施例4运动发酵单胞菌CRISPR基因组编辑系统在基因插入中的应用
1.根据靶基因及其编辑类型选择基因组编辑系统，本实施例中将ZMO0028作为靶位点，将报告基因mCherry插入基因组中，对ZMO0028进行替换，选用实施例2所述的CRISPR-Cas12a系统。原理如图28所示。从靶基因中选择PAM位点TTTN位点下游23bp的序列作为构建靶质粒中向导RNA的靶向引导序列，引导核酸酶对靶位点的切割。其中正向引物为5′-AGAT+(靶序列)-3′，反向引物为5′-TGAC+(靶序列互补序列)-3′。向导RNA引物序列见SEQ ID NO:112-SEQ ID NO:113。
2.构建靶质粒
将向导RNA引物序列连接到实施例2中制备的含有CRISPR表达单元的质粒上：首先利用限制性内切酶Bsa Ⅰ将载体进行线性化处理，然后将向导RNA引物对进行退火，退火的产物与线性化载体使用T4 DNA连接酶进行连接，然后转入到大肠杆菌克隆菌株DH5α中进行质粒构建，通过菌落PCR对重组子进行筛选最后通过测序验证。具体实验操作同实施例3。
3.构建编辑质粒
将供体DNA序列构建到靶载体上：靶基因上、下游各700bp左右的序列及mCherry表达元件分别通过PCR扩增其DNA片段，PCR体系及程序同实施例2。将上一步构建好的靶载体利用引物进行 反向PCR扩增，然后通过Gibson装配的方法将片段和载体进行连接，其中mCherry表达元件被上下游同源臂片段夹在当中，然后转入到大肠杆菌克隆菌株DH5α中进行质粒构建，通过菌落PCR对重组子进行筛选最后通过测序验证。
反向扩增的引物序列见SEQ ID NO:114-SEQ ID NO:115。
扩增上下游引物序列见SEQ ID NO:116-SEQ ID NO:119。
扩增报告基因表达元件引物序列见SEQ ID NO:120-SEQ ID NO:121。
4.将编辑质粒电转化及培养，实验方法同实施例3。
5.重组菌株的筛选，实验方法同实施例3，检测引物序列见SEQ ID NO:122-SEQ ID NO:123。
结果如图29和图30所示，结果表明，CRISPR-Cas12a系统可以定向插入基因，且测序结果表明敲除的基因与设计的一致。此外，在插入报告基因后利用流式细胞仪进行检测，结果如图31，结果说明报告基因可以在插入位点正常表达。说明该方法是一种精确的基因插入的方法。
同理，对于其他类型的基因插入也可以选择实施例1中的CRISPR-Cas系统。以下以在ZMO0038基因起始密码ATG后面定点插入一个His-Tag标签为例进行叙述，使其编码的蛋白可以通过镍柱纯化出来，此方法可以延伸到任意一个拟纯化的蛋白。原理见图8。
从目标基因序列ZMO0038起始密码ATG附近截取5’-TCC-3’下游紧邻的32bp序列作为guideRNA，该序列可位于基因组任一条链上。ZMO0038(His-Tag)-guideRNA引物序列见SEQ ID NO:18-SEQ ID NO:19。
guideRNA引物构建到载体上：将含有人工CRISPR表达单元的质粒用Bsa Ⅰ线性化，与guideRNA引物退火后用T4 DNA连接酶连接，通过转化到E.coli DH5α中进行质粒扩增，然后对转化子进行菌落PCR验证。实验条件同实施例3。验证引物为：pEZ15A-F和0038His-gRNA-R，序列分别见SEQ ID NO:11和SEQ ID NO:19，构建好的质粒均通过测序验证。
定点插入His-Tag标签的供体DNA序列是利用引物设计带入了His-Tag的DNA序列，供体DNA的上游臂和下游臂选取了定点插入His-Tag标签的位置上下游各300bp左右的序列，通过融合PCR技术对其进行扩增和连接。将上一步构建好的载体用Xba Ⅰ和EcoR Ⅰ双酶切，再与供体DNA序列通过Gibson装配的方法转化E.coli DH5α，然后对转化子进行菌落PCR验证，构建好的编辑质粒均通过测序验证。实验条件同实施例3。His-Tag标签序列：catcatcatcatcatcac，见SEQ ID NO:20。供体DNA引物序列见SEQ ID NO:21-SEQ ID NO:24。
编辑质粒电转化至ZM4感受态细胞，并筛选阳性克隆，方法同实施例3，引物序列见SEQ ID NO:25-SEQ ID NO:26。
菌落PCR筛选阳性克隆结果见图9，结果显示定点插入的效率达到了100％，又从另外一个方面证实了该发明是一种高效的基因编辑方法。利用上述PCR产物进行测序分析，结果如图10，通过测序结果与野生型菌株基因组序列进行比对，发现His-Tag标签的插入方式完全按照实验设计的方案，进一步说明了该发明的准确性。
实施例5运动发酵单胞菌CRISPR基因组编辑系统在点突变中的应用
1.根据靶基因及其编辑类型选择基因组编辑系统，本实施例中将ZMO1237作为靶位点，对其进行点突变，从靶基因中选择PAM位点TTTN位点下游23bp的序列作为构建靶质粒中向导RNA的靶向引导序列。其中正向引物为5′-AGAT+(靶序列)-3′，反向引物为5′-TGAC+(靶序列互补序列)-3′。选用实施例2所述的CRISPR-Cas12a系统。向导RNA引物序列见SEQ ID NO:97-SEQ ID NO:98。
2.构建靶质粒
将向导RNA引物序列连接到实施例2中制备的含有CRISPR表达单元的编辑质粒上：首先利用限制性内切酶Bsa Ⅰ将载体进行线性化处理，然后将向导RNA引物对进行退火，退火的产物与线性化载体使用T4 DNA连接酶进行连接，然后转入到大肠杆菌DH5α中进行质粒构建，通过菌落PCR对重组子进行筛选最后通过测序验证，具体操作过程同实施例3。
3.供体DNA的设计
通过CRISPR-Cas12a系统切割运动发酵单胞菌的基因组，以单链核苷酸(ssDNA)为修复模板对断裂的部分进行修改，同时在靶位点附近改变两个碱基以引入Pst Ⅰ的酶切位。ssDNA的长度为59-nt，其互补于编码DNA的后随链。ssDNA的序列见SEQ ID NO:99。
4.编辑质粒的转化，将200ng左右编辑质粒和1μg ssDNA转化至含Cas12a的重组菌的感受态细胞中，并培养。实验方法及条件同实施例3。
5.重组菌株的筛选
菌落PCR检测条件同实施例2，检测引物序列见SEQ ID NO:100-SEQ ID NO:101。
回收DNA片段，用限制性内切酶Pst Ⅰ对DNA进行酶切，酶切体系(DNA fragment，200ng；10×buffer，1μL；Pst Ⅰ，0.2μL；补H 2O至10μL。条件同实施例3)。能够对DNA片段进行正确切割的说明其被正确编辑，并通过测序对重组菌株进行进一步的验证。
酶切产物电泳及测序检测结果见图23和图24，结果表明，CRISPR-Cas12a在ssDNA的协助下，可以高效的对基因组中的碱基进行替换，且与设计的一致，其效率可以高达100％。
同理，对于其他类型的点突变中也可以选择实施例1中的CRISPR-Cas系统。以下以实施例4中插入一个His-Tag标签的ZMO0038基因为目标，在其基因的编码区定点突变几个碱基序列，从而引入终止密码5’-AAA-3’，使其编码的蛋白提前终止。此方法也可以延伸应用到蛋白的活性位点的研究中。原理见图11。
从目标基因序列His-ZMO0038编码区5’-CCC-3’下游紧邻的32bp序列作为guideRNA，该序列只能位于编码链上。ZMO0038(PM)-guideRNA引物序列见SEQ ID NO:27-SEQ ID NO:28。
将含有人工CRISPR表达单元的质粒用Bsa Ⅰ线性化，与guideRNA引物退火后用T4 DNA连接酶连接，通过转化到E.coli DH5α中进行质粒扩增，然后对转化子进行菌落PCR验证。实验方法同实施例3。验证引物为：pEZ15A-F和0038PM-gRNA-R，构建好的质粒均通过测序验证。
定点突变的供体DNA序列是利用引物设计带入了突变的碱基序列，供体DNA的上游臂和下游 臂选取了定点突变位置上下游各300bp左右的序列，通过融合PCR技术对其进行扩增和连接。将上一步构建好的载体用Xba Ⅰ和EcoR Ⅰ双酶切，再与供体DNA序列通过Gibson装配的方法转化E.coli DH5α，然后对转化子进行菌落PCR验证，构建好的编辑质粒均通过测序验证。实验方法同实施例3。供体DNA引物序列见SEQ ID NO:29-SEQ ID NO:32。
编辑质粒电转化至ZM4感受态细胞，并进行菌落PCR及测序，条件同实施例3。引物序列见SEQ ID NO:16-SEQ ID NO:33。
PCR电泳结果见图12，菌落PCR产物测序结果分析见图13，送测序的16个样品结果全部都为突变株，定点突变的效率达到了100％，证实了该发明是一种高效切精确的基因编辑方法。
实施例6运动发酵单胞菌CRISPR基因组编辑系统在多基因位点同时编辑中的应用
1.根据靶基因及其编辑类型选择基因组编辑系统，选用实施例1所述的CRISPR-Cas系统。本实施例的多位点基因编辑对象为基因组编码的CRISPR基因1-4(CRISPR1-4)，其中CRISPR3和4在基因组上的位置紧邻，把它们作为一个编辑靶标进行敲除。
从目标基因序列CRISPR1-4的3’末端截取5’-NCC-3’下游紧邻的32bp序列作为gRNA，该序列可位于基因组任一条链上。
CRISPR1-gRNA、CRISPR2-gRNA和CRISPR3&4-gRNA序列见SEQ ID NO:37-SEQ ID NO:39。
2.将人工CRISPR簇克隆到编辑质粒
克隆流程见图15。将上述目标基因的gRNA用人工合成的方法串联起来，再与实施例1中制备的含有人工CRISPR表达单元的质粒用Gibson装配，然后对转化子进行菌落PCR验证，构建好的质粒均通过测序验证。实验方法同实施例1。所用菌落PCR验证引物序列见SEQ ID NO:40-SEQ ID NO:41。
3.将供体DNA序列克隆到编辑质粒
分别选取目标基因上、下游各300bp左右的序列作为同源重组供体模板DNA，通过融合PCR技术对其进行扩增和连接，通过Gibson装配的方法连入编辑质粒。转化E.coli DH5α，然后对转化子进行菌落PCR验证，构建好的编辑质粒均通过测序验证。实验条件同实施例3。供体DNA引物序列见SEQ ID NO:42-SEQ ID NO:53。
4.将编辑质粒电转化到运动发酵单胞菌ZM4感受态细胞，再用引物CRISPR1-check-F和CRISPR1-check-R；CRISPR2-check-F和CRISPR2-check-R；CRISPR3&4-check-F和CRISP3&4-check-R分别进行菌落PCR筛选阳性克隆。方法同实施例3。菌落PCR筛选引物序列见SEQ ID NO:54-SEQ ID NO:59。
5.对得到的转化子进行菌落PCR验证，筛选阳性克隆，并进一步对PCR产物进行测序验证。方法同实施例3。
结果如图16-图18所示，检测的16个转化子中，至少发生一处靶位点编辑的占75％，而3号、4号和5号样品为成功同时敲除3个靶基因位点的菌株，测序分析结果说明，所有位点均按实验设计进行了准确编辑，说明了本发明在基因编辑应用中的高效性、精确性和稳定性。
实施例7运动发酵单胞菌CRISPR基因组编辑系统在基因组大片段高效删除中的应用
本实施例首先利用生物信息学方法确定需要保留的必需基因及可以删除的非必需基因，选择一段长度为10kb的非必须基因作为目标敲除大片段。然后设计guide RNA和供体DNA序列。最后在质粒上装载人工CRISPR簇表达模块和供体DNA序列，并将该质粒电转化到运动发酵单胞菌细胞内完成编辑。选用实施例1所述的CRISPR-Cas系统。原理示意图见图19所示，具体实验方案如下所示：
(1)目标敲除大片段的选取
通过生物信息学分析在该菌的基因组上找到了一段非必须基因ZMO1815-ZMO1822(10,021bp)可作为大片段敲除的目标序列。
(2)目标序列guideRNA序列选取
从目标基因序列ZMO1815-ZMO1822中截取任意5’-CCC-3’下游紧邻的32bp序列作为guide RNA，该序列可位于基因组任一条链上。设计引物。10K-guideRNA引物序列见SEQ ID NO:60-SEQ ID NO:61。
(3)guide RNA在基因编辑质粒上的装载
将实施例1中制备的含有人工CRISPR表达单元的质粒用Bsa Ⅰ线性化，与guide RNA引物退火后用T4 DNA连接酶连接，通过转化到E.coli DH5α中进行质粒扩增，然后对转化子进行菌落PCR验证，构建好的质粒均通过测序验证。实验条件同实施例3。菌落PCR验证引物序列见SEQ ID NO:62-SEQ ID NO:10。
(4)供体DNA序列的获取及其在编辑质粒上的装载
供体DNA序列选取目标基因上、下游各1Kb左右的序列，通过融合PCR技术对其进行扩增和连接。将上一步构建好的载体用Xma Ⅰ和Sac Ⅰ双酶切(酶切体系中除所用酶的种类不同外，其他及条件同实施例1)，再与供体DNA序列通过Gibson装配的方法转化E.coli DH5α，然后对转化子进行菌落PCR验证，构建好的编辑质粒均通过测序验证。实验条件同实施例3。供体DNA引物序列见SEQ ID NO:63-SEQ ID NO:66。
(5)编辑质粒电转化ZM4感受态细胞。再用引物10K-check-F，见SEQ ID NO:67；10K-check-R见SEQ ID NO:68，进行菌落PCR筛选阳性克隆。方法同实施例3。
(6)结果与分析
菌落PCR筛选阳性克隆结果见图20，结果显示，成功的敲除了10kb的基因组大片段，编辑效率达到40％，从而说明了该发明可用于高效删除基因组大片段。对上述PCR产物进行了测序分析，结果如图21所示，得到的编辑菌株中，基因簇的删除与实验设计完全吻合，说明了该发明在基因编辑上的精准性。
实施例8运动发酵单胞菌CRISPR基因组编辑系统在内源质粒消除中的应用
1.靶位点的选取
Z.mobilis ZM4的基因组数据已经被公布，其包含4个内源质粒，并且根据序列的大小分别命名为pZM32(32,791bp)，pZM33(33,006bp)，pZM36(36,494bp)和pZM39(39,266bp)。序列分析表明四 个内源质粒菌编辑复制酶，如果将复制酶失活，内源质粒将会失去复制的能力，内源质粒会被从菌株中消除。
本实施例分别从内源质粒的复制酶基因中选择PAM位点TTTN下游23bp的序列作为构建靶质粒中向导RNA的靶向引导序列，引导核酸酶对靶位点的切割。其中正向引物为5′-AGAT+(靶序列)-3′，反向引物为5′-TGAC+(靶序列互补序列)-3′。选用实施例2所述的CRISPR-Cas12a系统。
四个内源质粒的向导RNA引物序列见SEQ ID NO:83-SEQ ID NO:90。
2.构建靶质粒
将向导RNA引物序列连接到实施例2中制备的含有CRISPR表达单元的编辑质粒载体上：首先利用限制性内切酶Bsa Ⅰ将载体进行线性化处理，然后将向导RNA引物对进行退火，退火的产物与线性化载体使用T4 DNA连接酶进行连接，然后通过本领域通用化学转化法转入到大肠杆菌克隆菌株DH5α中进行质粒构建，通过菌落PCR对重组子进行筛选最后通过测序验证。实验条件同实施例3。
3.将靶质粒按照电转化转化至，含Cas12a的重组菌的感受态细胞，并培养。方法同实施例3。
4.重组菌株的筛选
待有菌落生长出来之后，分别用引物对重组菌株进行菌落PCR检测，PCR体系及程序同实施例2，检测引物见SEQ ID NO:91-SEQ ID NO:96。
PCR电泳检测结果见图22，结果表明，有三个内源质粒被成功消除，充分说明该方法是一种高效的消除内源质粒的方法，对用于分析质粒的功能以及进行基因组精简提供了一种有效的手段。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种基于运动发酵单胞菌的CRISPR-Cas系统，所述CRISPR-Cas系统包括4个CRISPR结构序列和一个cas基因簇，所述cas基因簇包括cas1，cas3，csy1，csy2，csy3和csy4基因，其中cas3基因是一个cas2和cas3基因的融合形式。
  2. 基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系，其特征在于：包括携带靶向序列guideRNA引物序列、人工CRISPR表达单元以及供体DNA序列的编辑质粒，及含有诱导型表达的核酸酶Cas12a的重组运动发酵单胞菌。
  3. 根据权利要求2所述的基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系，其特征在于：所述含有诱导型表达的核酸酶Cas12a的重组运动发酵单胞菌通过将外源的核酸酶Cas12a整合至含有如权利要求1所述的CRISPR-Cas系统的运动发酵单胞菌中获得，且所述核酸酶Cas12a通过四环素诱导型启动子控制表达。
  4. 根据权利要求2所述的基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系，其特征在于：所述人工CRISPR表达单元包括组成型启动子PJ23119、重复序列以及两个酶切位点。
  5. 根据权利要求2所述的基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系，其特征在于：所述编辑体系的PAM序列为TTTN。
  6. 基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系，其特征在于：包括携带靶向序列guideRNA引物序列、人工CRISPR表达单元以及供体DNA序列的编辑质粒，及权利要求1所述的运动发酵单胞菌的CRISPR-Cas系统。
  7. 根据权利要求6所述的基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系，其特征在于：所述人工CRISPR表达单元包括leader序列，CRISPR簇及终止子，所述CRISPR簇包括在两个repeat序列中间插入了两个酶切位点。
  8. 根据权利要求7所述的基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系，其特征在于：所述leader序列，CRISPR簇及终止子序列分别见SEQ ID NO:34-SEQ ID NO:36。
  9. 根据权利要求6所述的基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系，其特征在于：所述编辑体系的PAM序列为NCC。
  10. 如权利要求1所述的基于运动发酵单胞菌的CRISPR-Cas系统、或权利要求2-5任一所述的基于外源的运动发酵单胞菌CRISPR-Cas12a系统的基因组编辑体系、或权利要求6-9任一所述的基于内源的运动发酵单胞菌CRISPR-Cas系统的基因组编辑体系在基因敲除、或基因插入、或定点突变DNA序列、或多基因位点同时编辑、或基因组大片段删除、或内源质粒消除、或蛋白活性位点检测中的应用。
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