CN110408642A - A method for efficient deletion of large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application. The technical points include: constructing a plasmid containing an artificial CRISPR expression unit; determining a large fragment editing target site, selecting a guideRNA sequence for the editing target site, and designing primers; constructing a guideRNA primer sequence on a plasmid containing an artificial CRISPR expression unit, Construct the vector; construct the donor DNA sequence on the vector to obtain the editing plasmid; transform the editing plasmid into competent cells for editing. The invention utilizes a high-throughput gene editing platform based on the endogenous CRISPR-Cas system of Zymomonas mobilis to realize the editing purpose of deleting a large segment of the genome of the strain, and promote the development of metabolic engineering, systems biology and synthetic biology.

Description

Genome large fragment height based on endogenous CRISPR-Cas system of Zymomonas mobilis Efficient deletion method and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application.

Background technique

In recent years, the use of microorganisms for metabolic engineering, systems biology, and synthetic biology has made good progress. For the rational design and construction of microbial cell factories, living cells or enzymes are used to treat renewable biomass, such as It provides an important theoretical basis for the transformation of cellulose and other substances, the production of bioenergy, and the industrialization of biosmelting. Bioenergy regeneration is one of the effective means to solve the problems of resource and energy shortage and serious environmental pollution that human beings are currently facing.

Because Zymomonas mobilis can produce alcohol naturally, it has high tolerance to alcohol, and can use corn stalk hydrolyzate to produce high-concentration cellulose alcohol of 10.7% (v/v); Microorganisms that metabolize glucose or fructose to produce ethanol through the Entner-Doudoroff (ED) pathway produce only one molecule of ATP per molecule of glucose or fructose metabolized, and the production capacity is low, so most of the carbon source (>95%) is converted to For the product ethanol, only about 2-2.6% of the carbon source is used for cell growth, and the target product yield is very high; Zymomonas mobilis can grow in a wide range of temperature (24-45°C) and pH range (3.5-7.5), It is a recognized GRAS (Generally Recognized As Safe) strain and other characteristics, and is considered to be an ideal microbial cell factory. Moreover, its genome size is only about 2Mbp, which has great advantages in carrying out genome streamlining work and constructing an optimal genome cell factory.

The construction of a minimal genome is usually to delete non-essential DNA sequences in the genome, and the most effective method is to knock out large fragments. However, this need cannot be met by current conventional genetic manipulation methods. Although the commonly used CRISPR-Cas9-based gene editing tool can achieve high-throughput genome editing, the cytotoxicity of exogenously expressed Cas9 to Zymomonas mobilis limits the application of this tool.

Conventional genetic methods knock out genes through homologous recombination, but are currently limited to the knockout of a single small gene. Since these methods directly use the DNA recombination repair system in the host, they can be eliminated without any external DNA damage pressure. , the recombination efficiency is extremely low, which is particularly prominent in the deletion of large fragments of the genome. Moreover, for the knockout of each target gene, a specific selectable marker needs to be introduced, and there will be some problems such as limited available selectable markers, biosafety hazards caused by the introduction of antibiotic markers, and the like. In addition, there are also defects such as long time consumption and low efficiency.

The currently commonly used gene editing technology based on the CRISPR-Cas9 system is widely used, including in human cells, because it can introduce DNA damage at specific sites in the genome, prompting the DNA recombination repair system in the host to perform genome repair, thereby introducing mutations and improving recombination efficiency. In all kinds of organisms (cells) inside. However, with the deepening of research, more and more results show that heterologous expression of these nucleases can cause different degrees of cytotoxicity to the host. This may be one of the main reasons why related applications are difficult to carry out in Zymomonas mobilis so far.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application. The purpose of the present invention is to use the gene editing platform based on the I-F type CRISPR-Cas system encoded by the Zymomonas mobilis genome to knock out large fragments (>10kb, about 5‰ of the genome) on its own genome, for Research work such as the construction of minimal genome cell factories in this strain and similar cells provides efficient tools to promote the development of metabolic engineering, systems biology and synthetic biology.

The present invention is achieved in this way. The method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis comprises the following steps:

Step 1: constructing a plasmid containing an artificial CRISPR expression unit;

Step 2: Determine the large fragment editing target site, select the guideRNA sequence for the editing target site, and design primers;

Step 3: Construct the guideRNA primer sequence on the plasmid containing the artificial CRISPR expression unit to construct the vector;

Step 4: Construct the donor DNA sequence onto the vector to obtain the editing plasmid;

Step 5: Transform the editing plasmid into competent cells for editing.

Further, the length of the large fragment sequence is above 10kb.

Further, the plasmid containing the artificial CRISPR expression unit is an artificial CRISPR expression unit constructed on the Z.mobilis-E.coli shuttle vector pEZ15Asp.

Further, the artificial CRISPR expression unit includes a promoter leader sequence, a CRISPR cluster and a terminator.

Further, the artificial CRISPR cluster includes two repeats, and two BsaI restriction sites are inserted between the two repeats.

Further, the competent cells described in step 5 are Zymomonas mobilis ZM4 competent cells.

The application of the method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis in the deletion of large gene fragments according to any one of claims 1-6.

In summary, the advantages and positive effects of the present invention are:

The invention utilizes a high-throughput gene editing platform based on the endogenous CRISPR-Cas system of Zymomonas mobilis to realize the editing purpose of deleting a large segment of the genome of the strain, and promote the development of metabolic engineering, systems biology and synthetic biology. In addition, the present invention uses Zymomonas mobilis as a model strain and uses its endogenous CRISPR-Cas system for gene editing, which can effectively avoid the cytotoxicity of exogenous Cas nucleic acid proteins on the host and at the same time realize efficient deletion of large genome fragments.

Description of drawings

Figure 1 is the CRISPR-Cas system encoded by Zymomonas mobilis;

Figure 2 is the sequence of C2S7 and C3S4;

Figure 3 is the detection results of the in vivo shearing activity of the CRISPR-Cas system;

Figure 4 is an artificial CRISPR expression unit;

Figure 5 is a schematic diagram of the principle of deletion of large fragment sequences;

Fig. 6 is colony PCR positive clone result;

Fig. 7 is the sequencing result of transformants.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The present invention discloses a method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis and its application, as shown in the following examples.

Example 1 Construction of Zymomonas mobilis Endogenous CRISPR-Cas Genome Editing System

(1) CRISPR-Cas phylogenetic analysis of the genome encoding of Zymomonas mobilis

The premise of the present invention is that the research strain needs to encode the CRISPR-Cas system itself and have DNA splicing activity, which requires the host genome to encode the CRISPR cluster and the complete Cas protein system.

Taking Zymomonas mobilis Z.mobilis 4 as the model strain, the sequencing data of Z.mobilis 4 were analyzed. The results showed that the genome of the strain encoded four CRISPR structural sequences. According to their sequence in the genome, we sequenced them Named CRISPR1-CRISPR4, see Figure 1. CRISPR1 occupies the 113,783-114,170 region of the genome and contains 7 repeat sequences; CRISPR2 occupies the 1,244,355-1,245,866 region and contains 9 repeat sequences; CRISPR3 occupies the 1,598,754-1,599,144 region and contains 7 repeat sequences. CRISPR4 consists of 2 repeats and 1 spacer, occupying the 1,595,315-1,599,403 region. CRISPR2-4 are on the same strand, while CRISPR1 is on the complementary strand. The repeats in these CRISPR structures are all conserved 28bp sequences, and 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 an operon. In the above results, the cas3 gene is a fusion form of the cas2 and cas3 genes, which is a hallmark feature of the I-F type CRISPR-Cas system.

(2) Detection of in vivo cleavage activity of the endogenous I-F type CRISPR-Cas system of Zymomonas mobilis

In order to test whether the type I-F CRISPR-Cas system of Z. mobilis 4 strain can cleave DNA in vivo under the mediation of crRNA, according to the results of transcriptome analysis, we selected spacer7 (C2S7) in CRISPR2 and spacer4 (C3S4) in CRISPR3, Add 5'-CCC-3'PAM at the 5' end of the sequence, as shown in Figure 2. The specific operation is as follows: the Z.mobilis-E.col shuttle vector pEZ15Asp was double-digested with XbaI and EcoRI, and the restriction system is shown in Table 1. After annealing with the primers of C2S7 and C3S4 (Table 2), they were ligated with T4 DNA ligase (Table 3). Plasmid amplification was performed by transforming into E.coli DH5α, and then the transformants were verified by colony PCR (Table 4), and the constructed plasmids were verified by sequencing. At the same time, insert the spacer with 5' plus 5'-AAA-3' sequence into the vector to obtain the corresponding reference plasmid. The shuttle vector pEZ15Asp contains the spectinomycin resistance coding gene.

Table 1: Vector double enzyme digestion system

Carrier double enzyme digestion program: 37 ℃ incubation for 3-4h.

C2S7 and C3S4 primer sequences:

C2S7(CCC)-F: AATTCCCGATCGCGGGCAACGGTTTATTCAGCTATCCGCGC, see SEQ ID NO: 1;

C2S7(CCC)-R: CTAGGCGCGGATAGCTGAATAAACCGTTGCCCGCGATCGGG, see SEQ ID NO: 2;

C2S7(AAA)-F: AATTAAAGATCGCGGGCAACGGTTTTATTCAGCTATCCGCG, see SEQ ID NO: 3;

C2S7(AAA)-R: CTAGGCGCGGATAGCTGAATAAACCGTTGCCCGCGATCTT, see SEQ ID NO:4;

C3S4(CCC)-F: AATTCCCGTCTGGCTGAAATGAGGTCCGACGATTTGCAT, see SEQ ID NO: 5;

C3S4(CCC)-R: GTTAATGCAAATCGTCGGACCTCATTTCAGCCAGACGGG, see SEQ ID NO: 6;

C3S4(AAA)-F: AATTAAAGTCTGGCTGAAATGAGGTCCGACGATTTGCAT, see SEQ ID NO: 7;

C3S4(AAA)-R: GTTAATGCAAATCGTCGGACCTCATTTCAGCCAGACTTT, see SEQ ID NO:8.

Table 2: Primer Annealing System

Primer annealing procedure: keep at 95°C for 5 minutes, then anneal at room temperature.

Table 3: T4 DNA Ligase Ligation System

T4DNA ligase ligation program: keep warm at 22°C for 2h.

Transformation method of Escherichia coli DH5α: Take 50 μL of competent cells on ice, add 5 μL of the enzyme-linked product, let stand on ice for 10 minutes, heat shock at 42°C for 35 seconds, add 450 μL of liquid LB, and recover at 37°C for 1 hour. Take 100 μL and spread it on a 100 μg/mL spectinomycin-resistant plate, and incubate at 37°C for 16 hours.

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 cycles 25 times; step 5, 72°C , 2min.

Table 4: Colony PCR system

ZM4 was electrotransformed with the extracted plasmid. Take ZM4 competent cells on ice, after the competent cells are thawed, take 50 μL and add them to the electroporation cup, and add 1 μg plasmid into the electroporation cup. Electroporation conditions were 1600V, 25μF, 200Ω. Resuscitate in RMG liquid medium at 30°C after electroporation. The cultures recovered for 6-12 hours were centrifuged at 6000 rpm for 1 min, and the supernatant was removed. Add 200 μL of fresh RMG medium, take 100 μL and spread it on a 100 μg/mL spectinomycin-resistant plate, and incubate at 30°C for 2 days.

It is predicted that after the plasmids are respectively transformed into the host, if the I-F type CRISPR-Cas system is active, the inserted protospacer will be sheared and screened on the RMG medium plate containing 100 μg/mL spectinomycin, resulting in Few colonies formed; plates transformed with the reference plasmid produced a large number of colonies.

The experimental results are shown in Figure 3. The efficiency of transforming the interference plasmid is 10 ³ times lower than that of the reference plasmid, which shows that the crRNA expressed in the CRISPR on the genome mediates the DNA activity of the IF-type system to cut the protospacer in the interference plasmid. cleavage, thus confirming that the system can be used for site-specific targeting and cleavage of DNA sequences.

(3) Composition of genome editing plasmids

An artificial CRISPR expression unit was constructed on the plasmid pEZ15Asp, as shown in Figure 4, which consists of a promoter leader sequence, a CRISPR cluster and a terminator. The artificial CRISPR expression unit was artificially synthesized by Sanfang Gene Synthesis Company. The artificial CRISPR cluster includes two BsaI restriction sites inserted in the middle of two repeats. The function of these two BsaI restriction sites is to facilitate the insertion of the selected guideRNA sequence on the target gene. In this example, the promoter Leader sequence, RgR module sequence and T7 terminator sequence are respectively shown in SEQ ID NO: 18-SEQ ID NO: 20.

Example 2 Application of the method for efficient deletion of large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis

In the present invention, bioinformatics methods are firstly used to determine the essential genes that need to be retained and the non-essential genes that can be deleted, and select a non-essential gene with a length of 10 kb as the target knockout large fragment. Then design guide RNA and donor DNA sequences. Finally, the artificial CRISPR cluster expression module and donor DNA sequence were loaded on the plasmid, and the plasmid was electrotransformed into Zymomonas mobilis cells to complete the editing. The schematic diagram of the principle is shown in Figure 5, and the specific experimental scheme is as follows:

(1) Selection of target knockout large fragments

Through bioinformatics analysis, a non-essential gene ZMO1815-ZMO1822 (10,021bp) was found on the genome of the bacteria, which can be used as the target sequence for large fragment knockout.

(2) Target sequence guideRNA sequence selection

From the target gene sequence ZMO1815-ZMO1822, the 32bp sequence immediately downstream of any 5'-CCC-3' was intercepted as a guide RNA, and the sequence could be located on any strand of the genome. Design primers.

10K-guideRNA primer sequence:

10K-gRNA1-F: gaaagacccttatgcctatgtcgatacaaccacgaat, see SEQ ID NO: 9;

10K-gRNA1-R: gaacattcgtggttgtatcgacataggcataaggtc, see SEQ ID NO:10.

(3) Loading of guide RNA on the gene editing plasmid

The plasmid containing the artificial CRISPR expression unit prepared in Example 1 was linearized with BsaI (Table 5), annealed with the guide RNA primer and ligated with T4 DNA ligase (same as above, Table 3), and transformed into E.coli DH5α The plasmid was amplified (the method was the same as in Example 1), and then the transformants were verified by colony PCR (same as above, Table 4), and the constructed plasmids were verified by sequencing.

Table 5: Vector single enzyme digestion system

Carrier single enzyme digestion procedure: 37°C incubation for 3-4h.

Colony PCR verification primer sequences:

pEZ15A-F: ggcaaagccaccctatttttag, see SEQ ID NO: 11;

10K-gRNA1-R: gaacattcgtggttgtatcgacataggcataaggtc, see SEQ ID NO:10.

(4) Acquisition of donor DNA sequence and its loading on the editing plasmid

For the donor DNA sequence, select the upstream and downstream sequences of about 1 Kb each of the target gene, and amplify and connect them by fusion PCR technology (Table 6). The vector constructed in the previous step was digested with XmaI and SacI (Table 7), and then transformed into E.coli DH5α with the donor DNA sequence by Gibson assembly method, and then the transformant was verified by colony PCR to construct the edited plasmid All were verified by sequencing.

The fusion 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 cycles 30 times; step 5, 72 ℃, 2min.

Donor DNA primer sequences:

10K-up-F: tcgagcgtcccatagatctcgagctcggttgtgataagcggcagat, see SEQ ID NO: 12;

10K-up-R: aaacccgttacagaaatgggatgcaccctttagggagtcg, see SEQ ID NO: 13;

10K-down-F: cgactccctaaagggtgcatcccatttctgtaacgggttt, see SEQ ID NO: 14;

10K-down-R: gggtcaaataaagggtcaaacccgggcgagttcatattcacggtcg, see SEQ ID NO:15.

Table 6: Fusion PCR reaction system

Table 7: Vector double enzyme digestion system

Carrier double enzyme digestion program: 37 ℃ incubation for 3-4h.

(5) Electrotransform ZM4 Competent Cells with Editing Plasmid

Take 50 μL of competent cells on ice, add 1 μg of edited plasmid, and transfer the competent cells into a 0.1 cm electroporation cuvette after they are thawed. Electroporation conditions were 1600V, 25μF, 200Ω. Resuscitate in RM liquid medium at 30°C after electroporation. The cultures recovered for 6-12 hours were centrifuged at 6000 rpm for 1 min, and the supernatant was removed. Add 200 μL of fresh RM medium, take 100 μL and spread it on a 200 μg/mL spectinomycin-resistant plate, and incubate at 30°C for 2 days. Colony PCR (same as above, Table 4) was used to screen positive clones with primers 10K-check-F and 10K-check-R.

10K-check-F: gacaagagcggaatccgcgt, see SEQ ID NO: 16;

10K-check-R: ggatggtgcctttccctgaa, see SEQ ID NO:17.

(6) Results and analysis

The results of positive clones screened by colony PCR are shown in Figure 6. The results show that a large genome fragment of 10kb was successfully knocked out, and the editing efficiency reached 40%, thus illustrating that the invention can be used to efficiently delete large genome fragments. The above PCR products were sequenced and analyzed, and the results are shown in Figure 7. In the obtained edited strain, the deletion of gene clusters was completely consistent with the experimental design, which demonstrated the accuracy of the invention in gene editing.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

sequence listing

<110> Hubei University

<120> Method for efficient deletion of large genome fragments based on endogenous CRISPR-Cas system of Zymomonas mobilis and its application

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 41

<212>DNA

<213> Artificial sequence (C2S7CCC-F)

<400> 1

aattcccgat cgcgggcaac ggtttatca gctatccgcg c 41

<210> 2

<211> 41

<212>DNA

<213> Artificial sequence (C2S7CCC-R)

<400> 2

ctaggcgcgg atagctgaat aaaccgttgc ccgcgatcgg g 41

<210> 3

<211> 40

<212>DNA

<213> Artificial sequence (C2S7AAA-F)

<400> 3

aattaaagat cgcgggcaac ggtttattca gctatccgcg 40

<210> 4

<211> 40

<212>DNA

<213> Artificial sequence (C2S7AAA-R)

<400> 4

ctaggcgcgg atagctgaat aaaccgttgc ccgcgatctt 40

<210> 5

<211> 39

<212>DNA

<213> Artificial sequence (C3S4CCC-F)

<400> 5

aattcccgtc tggctgaaat gaggtccgac gatttgcat 39

<210> 6

<211> 39

<212>DNA

<213> Artificial sequence (C3S4CCC-R)

<400> 6

gttaatgcaa atcgtcggac ctcatttcag ccagacggg 39

<210> 7

<211> 39

<212>DNA

<213> Artificial sequence (C3S4AAA-F)

<400> 7

aattaaagtc tggctgaaat gaggtccgac gatttgcat 39

<210> 8

<211> 39

<212>DNA

<213> Artificial sequence (C3S4AAA-R)

<400> 8

gttaatgcaa atcgtcggac ctcatttcag ccagacttt 39

<210> 9

<211> 36

<212>DNA

<213> artificial sequence (10K-gRNA1-F)

<400> 9

gaaagacctt atgcctatgt cgatacaacc acgaat 36

<210> 10

<211> 36

<212>DNA

<213> artificial sequence (10K-gRNA1-R)

<400> 10

gaacattcgt ggttgtatcg acataggcat aaggtc 36

<210> 11

<211> 22

<212>DNA

<213> Artificial sequence (pEZ15A-F)

<400> 11

ggcaaagcca ccctattttt ag 22

<210> 12

<211> 46

<212>DNA

<213> Artificial sequence (10K-up-F)

<400> 12

tcgagcgtcc catagatctc gagctcggtt gtgataagcg gcagat 46

<210> 13

<211> 40

<212>DNA

<213> Artificial sequence (10K-up-R)

<400> 13

aaacccgtta cagaaatggg atgcaccctt tagggagtcg 40

<210> 14

<211> 40

<212>DNA

<213> Artificial sequence (10K-down-F)

<400> 14

cgactcccta aagggtgcat cccatttctg taacgggttt 40

<210> 15

<211> 46

<212>DNA

<213> artificial sequence (10K-down-R)

<400> 15

gggtcaaata aagggtcaaa cccgggcgag ttcatattca cggtcg 46

<210> 16

<211> 20

<212>DNA

<213> Artificial sequence (10K-check-F)

<400> 16

gacaagagcg gaatccgcgt 20

<210> 17

<211> 20

<212>DNA

<213> Artificial sequence (10K-check-R)

<400> 17

ggatggtgcc tttccctgaa 20

<210> 18

<211> 148

<212>DNA

<213> promoter leader (promoter leader)

<400> 18

tttgaccctt tatttgaccc tctttttttg gcatgtaaaa aaatccttta aaatcaatag 60

gttaaaaata ggctctattt ttagggttat ttggctattt ttgcccgata ttcctttcat 120

ttagggggat ttttaattat ttactcta 148

<210> 19

<211> 72

<212>DNA

<213> RgR(RgR)

<400> 19

gttcactgcc gcacaggcag cttagaaagg agaccgaggt ctcagttcac tgccgcacag 60

gcagcttaga aa 72

<210> 20

<211> 28

<212>DNA

<213> T7 terminator (T7 terminator)

<400> 20

ggctcacctt cgggtgggcc tttctgcg 28

Claims (7)

1. A method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis, comprising the following steps: Step 1: constructing a plasmid containing an artificial CRISPR expression unit; Step 2: Determine the large fragment editing target site, select the guideRNA sequence for the editing target site, and design primers; Step 3: Construct the guideRNA primer sequence on the plasmid containing the artificial CRISPR expression unit to construct the vector; Step 4: Construct the donor DNA sequence onto the vector to obtain the editing plasmid; Step 5: Transform the editing plasmid into competent cells for editing. 2. The method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis according to claim 1, characterized in that: the length of the large fragment sequence is more than 10 kb. 3. The method for efficiently deleting large genome fragments based on Zymomonas mobilis endogenous CRISPR-Cas system according to claim 1, characterized in that: the plasmid containing the artificial CRISPR expression unit is in Z.mobilis-E. The artificial CRISPR expression unit was constructed on the coli shuttle vector pEZ15Asp. 4. The method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis according to claim 3, characterized in that: the artificial CRISPR expression unit comprises a promoter leader sequence, a CRISPR cluster and a terminator . 5. The method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis according to claim 4, characterized in that: the artificial CRISPR cluster includes two repeats, and two repeats are inserted in the middle Two restriction sites for BsaI. 6. The method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis according to claim 1, characterized in that: the competent cell described in step 5 is Zymomonas mobilis ZM4 competent cell. 7. The application of the method for efficiently deleting large genome fragments based on the endogenous CRISPR-Cas system of Zymomonas mobilis as described in any one of claims 1-6 in deleting large gene fragments.