Plasmid for mutation of purine base in blue-green algae as well as preparation method and application of plasmid

Abstract

The invention provides a plasmid for purine base mutation in blue-green algae and a preparation method and application thereof, and belongs to the field of bioengineering, the plasmid provided by the invention is named as pCyABE, the plasmid comprises a TadA-nCas9 fusion protein expression cassette and an sgRNA expression cassette, the nucleotide sequence of the TadA-nCas9 fusion protein expression cassette is shown as SEQ ID No.2, the nucleotide sequence of the sgRNA expression cassette is shown as SEQ ID No.2, and the nucleotide sequence of the sgRNA expression cassette is shown as SEQ ID No.2. The nucleotide sequence of the sgRNA expression cassette is as shown in SEQ ID No. 3. The plasmid provided by the invention is particularly suitable for mutation of adenine at any site on a blue-green algae genome into guanine, realizes fixed-point editing of the gene, has the advantages of high editing efficiency and low off-target rate, and has important significance on physiological property research and industrial application of blue-green algae.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a plasmid for purine base mutation in cyanobacteria, and a preparation method and application thereof. Background Art

[0002] Cyanobacteria are a class of photosynthetic autotrophic prokaryotes with efficient photosynthesis. They can be designed as microbial chassis for directly converting carbon dioxide into value-added biofuels and chemicals, such as biofuels, sugars and pigments, terpenoids, bioplastics, etc. In the past few decades, people have tried to apply synthetic biology tools and methods to the development of cyanobacterial cell factories to create biological chassis that can efficiently produce various high-value-added active substances. Large-scale synthetic biology transformation of cyanobacteria requires efficient and simple genetic manipulation tools. However, traditional cyanobacterial genome editing methods are mainly based on homologous recombination, with complex experimental steps, long time required, and antibiotic marks left at the editing site, which is not conducive to subsequent multiple editing. Therefore, new gene editing tools that can achieve precise, label-free, rapid and multiple editing in cyanobacteria are urgently needed to meet the needs of industrial production of cyanobacteria.

[0003] Currently, Cas9 and Cpf1 have been applied to genome editing of a variety of cyanobacteria, including Synechocystis, Synechococcus, and Anabaena, and can achieve efficient and traceless gene editing. However, these gene editing technologies still rely on homologous recombination repair mechanisms, so exogenous homologous recombination templates are required, and the transformation efficiency is low. In practical applications, especially in the process of multi-site gene editing, complex molecular cloning steps and experimental operations are required, which limits the promotion and application of this technology. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to improve the efficiency of gene editing in cyanobacteria.

[0005] To solve the above technical problems, the first aspect of the present invention provides a plasmid for purine base mutation in cyanobacteria, the plasmid is named pCyABE, the plasmid comprises a TadA-nCas9 fusion protein expression cassette and a sgRNA expression cassette, the nucleotide sequence of the TadA-nCas9 fusion protein expression cassette is shown in SEQ ID No.2, and the nucleotide sequence of the sgRNA expression cassette is shown in SEQ ID No.3.

[0006] The function of the TadA-nCas9 fusion protein expression cassette is to express the codon-optimized TadA (adenine deaminase) and nCas9 (Cas9D10A) fusion genes, driving the efficient expression of the editing protein in cyanobacteria.

[0007] Preferably, the plasmid further comprises a targeting sequence spacer fragment, and the targeting sequence spacer fragment is connected to the sgRNA via the BsaⅠ site on the sgRNA expression cassette.

[0008] In the present invention, the sgRNA backbone containing the BsaI site supports the rapid insertion of a targeting sequence spacer fragment, wherein the targeting sequence spacer fragment includes a selected cyanobacterial whole genome mutation site, the spacer fragment includes at least 20 bases, and the selection of the spacer fragment should follow the following rules: a DNA fragment of at least 20 base sequences before any NGG (N is an arbitrary base) sequence in the cyanobacterial whole genome is selected as a targeting sequence spacer fragment, and the NGG sequence is not within the targeting sequence spacer fragment, wherein positions 4 to 10 of the targeting sequence spacer fragment should have at least one adenine base as the target base for base editing.

[0009] Preferably, the plasmid further comprises sacB Reverse screening of genes.

[0010] sacB The counter-selection gene has no effect on the host under normal culture conditions, but produces a lethal toxin in the presence of sucrose for plasmid elimination.

[0011] Preferably, the plasmid further comprises the broad host replicon RSF1010.

[0012] The broad-host replicon RSF1010 supports stable plasmid replication in Escherichia coli and cyanobacteria.

[0013] Preferably, the plasmid further comprises a construction screening gene, and the construction screening gene is selected from at least one of kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, tetracycline resistance gene, erythromycin resistance gene, gentamicin resistance gene and bleomycin resistance gene.

[0014] The pCyABE plasmid provided by the present invention can be used to mutate adenine into guanine at any site on the cyanobacterial genome to achieve site-specific editing of genes. The implementation principle is: under the guidance of sgRNA, the TadA-nCas9 fusion protein specifically recognizes and targets a specific site of the genome through the principle of base complementary pairing. Then the TadA protein can catalyze the adenine (A) at the site to convert it into hypoxanthine (I), and at the same time nCas9 cuts the non-edited chain, allowing the cell to perform DNA repair using the edited chain as a template. During the repair process, hypoxanthine (I) is recognized as guanine (G) in DNA replication, and finally adenine is accurately mutated to guanine at a specific site in the genome.

[0015] Furthermore, the second aspect of the present invention also provides a method for preparing the aforementioned plasmid, comprising the following steps: S1: In vitro synthesis of TadA-nCas9 fusion protein expression cassette, sgRNA expression cassette, sacB Reverse screening genes, construct screening genes; S2: Using pCpf1b-Sp plasmid as template, amplify plasmid replicon RSF1010; S3: Assemble the fragments in steps S1 and S2 into a circular plasmid using seamless cloning technology; S4: Transform the assembled product into E. coli DH5α competent cells, screen the corresponding resistant strains and verify the plasmid sequence.

[0016] Furthermore, the third aspect of the present invention provides an application of the aforementioned plasmid, wherein the application is to use the plasmid for purine base mutation in cyanobacteria to direct mutation of adenine to guanine at any site on the cyanobacterial genome.

[0017] Preferably, the active window for directional mutation of adenine to guanine at any site on the cyanobacterial genome is located at positions 4 to 10 of the spacer fragment of the targeting sequence, measured from 5' to 3'.

[0018] Furthermore, the fourth aspect of the present invention provides an expression cell strain, which contains the aforementioned plasmid, and the expression cell strain is selected from any one or more of bacteria, fungi, and algae.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The plasmid for purine base mutation in cyanobacteria provided by the present invention is designed based on the CRISPR-Cas9 system and TadA adenine deaminase. The plasmid provided by the present invention can directly mutate adenine at any site in cyanobacteria to guanine, realize base editing, and then realize amino acid directed mutation or gene inactivation in cyanobacteria. The plasmid provided by the present invention realizes multi-target editing by replacing the sgRNA sequence. sacB Reverse screening of genes can eliminate the plasmid of the present invention through sucrose screening to avoid residual marker genes. The plasmid provided by the present invention has the advantages of high editing efficiency and low off-target rate, and is of great significance to the research on the physiological characteristics of cyanobacteria and industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The plasmid map of the pCyABE plasmid provided for the specific embodiment of the present invention; Figure 2 A schematic diagram of the principle of targeted mutation of adenine to guanine in the pCyABE plasmid provided in a specific embodiment of the present invention; Figure 3 is the sequencing result in Example 2 of the present invention; Figure 4 The results of the plasmid elimination experiment in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0022] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0023] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0024] Base editing technology can achieve the directed transformation of DNA bases without relying on double-strand breaks of genomic DNA and homologous recombination. However, currently, due to the genomic characteristics of cyanobacteria, base editors cannot be directly applied to gene editing of cyanobacteria.

[0025] Based on this, the present invention specifically implements a plasmid for purine base mutation in cyanobacteria, which is named pCyABE. The plasmid includes a TadA-nCas9 fusion protein expression cassette and a sgRNA expression cassette. The nucleotide sequence of the TadA-nCas9 fusion protein expression cassette is shown in SEQ ID No. 2, and the nucleotide sequence of the sgRNA expression cassette is shown in SEQ ID No. 3.

[0026] In some embodiments, the nucleotide sequence of the pCyABE plasmid is shown in SEQ ID No. 1, and the plasmid map of the pCyABE plasmid is shown in Figure 1 shown.

[0027] like Figure 1 As shown, the pCyABE plasmid provided by the present invention further includes a targeting sequence spacer fragment, and the targeting sequence spacer fragment is connected to the sgRNA through the BsaⅠ site on the sgRNA expression cassette. The role of the targeting sequence spacer fragment is to locate the base site that needs to be edited.

[0028] like Figure 1 As shown, the pCyABE plasmid provided by the present invention also includes sacB Reverse screening of genes. Used for subsequent plasmid removal.

[0029] like Figure 1 As shown, the pCyABE plasmid provided by the present invention also includes a broad host replicon RSF1010.

[0030] In some specific embodiments, the pCyABE plasmid also includes a construction screening gene, and the construction screening gene is selected from at least one of kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, tetracycline resistance gene, erythromycin resistance gene, gentamicin resistance gene, and bleomycin resistance gene.

[0031] The pCyABE plasmid provided in the embodiment of the present invention is particularly suitable for mutating adenine into guanine at any site on the cyanobacterial genome to achieve site-specific editing of genes. The implementation principle is as follows: Figure 2 As shown in the figure, specifically, under the guidance of sgRNA, TadA-nCas9 fusion protein specifically recognizes and targets a specific site in the genome through the principle of base complementary pairing. Then TadA protein can catalyze adenine (A) at this site, causing the adenine at this site to eventually mutate to guanine (G). Among them, the adenine at positions 4-10 in the spacer sequence has the highest efficiency in mutation.

[0032] The technical solution of the present invention is further illustrated by specific examples. In the following, unless otherwise specified, the cyanobacteria strains used in this article are all Synechocystis PCC 6803 strains (purchased from ATCC, USA, catalog number: 27184).

[0033] Example 1

[0034] pCyABE plasmid construction The pCyABE plasmid provided in this example includes the following fragments: (1) a TadA-nCas9 fusion protein expression cassette (as shown in SEQ ID No. 2); (2) a sgRNA expression cassette containing two BsaI restriction sites for insertion of a targeting sequence (as shown in SEQ ID No. 3); (3) a kanamycin resistance gene fragment; (4) sacB Reverse screening of gene fragments; (5) The replicon RSF1010 with a wide range of hosts.

[0035] The construction method of pCyABE plasmid includes the following steps: S1: In vitro synthesis of TadA-nCas9 fusion protein expression cassette, sgRNA expression cassette, sacB Reverse screening gene, kanamycin resistance gene fragment; S2: Using pCpf1b-Sp plasmid as template, amplify plasmid replicon RSF1010. Use 2×Phanta MaxMaster Mix for PCR amplification, the reaction system is: 22μl ddH 2 O, 1 μl 5' Primer (10 μM), 1 μl 3' Primer (10 μM), 1 μl template DNA (100 ng / μl), 25 μl 2× Phanta Max Master Mix. After the system was prepared, polymerase chain reaction (PCR) was performed with the following cycles: 98 o C 2min; then 98 o C 20s, 55 o C 25s, 72 o C 4min, 30 cycles in total; the final 72 o C for 5 min. Use a DNA product purification kit to extract the amplified DNA fragment; S3: Use seamless cloning technology to assemble the fragments in steps S1 and S2 into a circular plasmid. The specific reaction system is: 2 μl 2×CE Mix, 100 ng of each of the five DNA fragments, and finally add appropriate amount of ddH 2 O to a total volume of 10 μl. The 2×CE Mix used in the reaction was produced by Vazyme. The reaction was carried out in a PCR instrument: 50 o C 15min; then drop to 4 o C or immediately store on ice; S4: The assembled product was transformed into E. coli DH5α competent cells and plated on LB solid medium containing 50 μg / ml kanamycin. After the transformation solution was absorbed by the solid medium, incubate at 37 oIncubate the cells upside down in a C incubator overnight. The transformed strains grown on the culture medium were transferred and stored, and the pCyABE plasmid was extracted for subsequent experiments. The plasmid was also sequenced and confirmed.

[0036] After testing, the pCyABE plasmid is characterized in that it is a shuttle plasmid that can replicate and propagate in Escherichia coli and cyanobacteria; that the plasmid is kanamycin-resistant in both Escherichia coli and cyanobacteria and can be used for strain screening; that the plasmid can perform genome base editing in cyanobacteria to achieve a mutation of a specific base from adenine to guanine; and that sacB Reverse screening of genes, which can achieve plasmid elimination in the strain by culturing under sucrose conditions; it contains two BsaI sites that can be used to insert spacer targeting fragments.

[0037] Example 2

[0038] pCyABE plasmid achieves efficient base editing in the genome of Synechocystis PCC 6803 (1) First, select the target gene in the Synechocystis PCC 6803 strain, and select the 20 base DNA sequence (called spacer) before the specific PAM site (NGG, N represents any base) on the target gene. It is worth noting that on the spacer selected in this step, the adenine base A that needs to be mutated for editing is generally located at the 4th to 10th position of the spacer (5'→3' direction). For example, the selected slr1609 The DNA sequence of the gene spacer is: 5'-gaaattAcagcttttgccgc-3' (SEQ ID No. 4), in which the adenine A that needs to undergo base editing is at position 7.

[0039] In order to insert the spacer fragment into the BsaI restriction site of the pCyABE plasmid, it is necessary to add TAGT to the 5' end of the selected spacer sequence fragment and add AAAC to the 5' end of the complementary strand sequence. Therefore, the specific primer sequences designed are as follows: slr1609spF: 5'-TAGTgaaattacagcttttgccgc-3' (SEQ ID No. 5); slr1609spR: 5'-AAACgcggcaaaagctgtaatttc-3' (SEQ ID No. 6).

[0040] (2) Synthesize the two designed primers mentioned above, and anneal the synthesized spacer forward and reverse single-stranded primers to synthesize a double-stranded DNA. The specific reaction system of this step is as follows: 1) 5 μl 10×T4 DNA ligase Buffer (NEB); 2) 10μl slr1609spF (10μM); 3) 10μl slr1609spR (10μM); 4) 25 μl ddHO 2 O.

[0041] The reaction solution was heated at 95°C for 5 min and then naturally cooled to room temperature, allowing the two single-stranded primers to form double-stranded DNA through base pairing.

[0042] (3) The double-stranded DNA obtained by the above reaction is inserted into the BsaI restriction site of pCyABE. The specific reaction system of this step is as follows: 1) 1μl 10×T4 DNA ligase Buffer; 2) 1 μl of 20-fold diluted double-stranded DNA; 3) 200 ng pCyABE plasmid; 4) 0.5μl T4 DNA ligase (400 units / μl); 5) 0.5μl BsaI-HF (20 units / μl); 6) Add ddH 2 O to make the total system up to 10 μl.

[0043] (The buffer and enzyme used in this reaction are produced by NEB) Then the reaction was carried out in a PCR instrument, and the reaction cycle was as follows: 37 o C 2min; 16 o C for 3 min, (25 cycles); then 50 o C 5min,80 o C 15min.

[0044] (4) Transform 10 μl of the reaction product into the competent E. coli DH5α. Specific steps: Add 10 μl of the reaction product to 100 μl of the competent E. coli DH5α, let it stand on ice for half an hour, then heat it in a 42°C water bath for 90 seconds, and then continue to stand on ice for 2 minutes. Add 900 μl of fresh LB culture medium to the competent medium and culture it in a shaker (37°C, 220 rpm) for 1 hour. Centrifuge the bacterial solution, pour off the supernatant, take 100 μl of the bacterial solution and spread it on an LB solid culture plate containing 50 μg / ml kanamycin, and invert and culture it in a 37°C incubator overnight. Only successfully constructed bacteria can grow on kanamycin-resistant LB solid culture plates.

[0045] (5) Pick a single clone colony on a kanamycin-resistant LB solid culture plate and perform PCR verification. Use gel electrophoresis to identify whether the PCR is successful. Select a colony that has successfully been verified by PCR, inoculate it into 5 ml of LB culture medium containing 50 μg / ml kanamycin, and place it in a 37°C shaker (220 rpm) for overnight culture for plasmid extraction. The plasmid is sequenced and verified to obtain the successfully constructed editing plasmid pCyABE-slr1609.

[0046] (6) The successfully constructed editing plasmid pCyABE-slr1609 was transformed into the conjugation transfer DH5α competent cell. The specific transformation steps are as follows: 1 μl of the editing plasmid pCyABE-slr1609, 20 μl of 5xKCM solution and 79 μl of ddHO 2 O was added to the competent state for conjugation transfer DH5α, and after standing on ice for half an hour, it was heated in a 42°C water bath for 90 seconds, and then continued to stand on ice for 2 minutes. 900μl of fresh LB culture medium was added to the competent state, and cultured in a shaker (37°C, 220 rpm) for 1 hour. Centrifuge the bacterial solution, pour out 900μl of supernatant, and take the remaining 100μl of bacterial solution and spread it on an LB solid culture plate containing 100μg / ml ampicillin, 50μg / ml chloramphenicol, and 50μg / ml kanamycin. The LB solid culture plate with bacterial solution was placed in a 37°C incubator and inverted for overnight culture. Only bacteria that have successfully transferred plasmids can grow on the LB solid culture plate.

[0047] (7) Pick a single clone colony from the LB solid culture plate with three antibodies and inoculate it into 5 mL LB liquid culture medium containing 100 μg / ml ampicillin, 50 μg / ml chloramphenicol, and 50 μg / ml kanamycin for overnight culture. Centrifuge the overnight bacterial solution for 5 min (5000 rpm, 25°C), discard the supernatant, add 4 ml of non-antibiotic LB to wash twice, and finally add 200 μl of non-antibiotic LB to resuspend. Take 50 ml of logarithmic phase Synechocystis PCC6803, centrifuge and wash twice with 30 ml BG11 liquid culture medium, and finally resuspend with 1 ml BG11 liquid culture medium. Mix 180 μl of the above bacterial solution and 20 μl of the above algae solution, and dilute 100 times with non-antibiotic LB liquid culture medium. Take 200 μl of the bacterial and algae mixture and apply it on the non-antibiotic BG11 solid culture plate with a filter membrane. Two days later, the filter was transferred to a new BG11 solid culture plate with 30 μg / mL kanamycin antibiotic. -2 S -1 Leave it under the right conditions for 10 to 15 days.

[0048] (8) After a single colony of algae grows on the plate, pick a single colony for PCR verification. The reaction system is as follows: 1) Upstream primer slr1609VF: 0.75 μL; 2) Downstream primer slr1609VR: 0.75 μL; 3) 2×Phanta Max Master Mix: 12.5μL; 4) ddH 2 O: 11 μL; The reaction was carried out in a PCR instrument with the following reaction cycles: 98°C for 3 min; then 98°C for 20 s, 55°C for 25 s, and 72°C for 30 s (34 cycles); 72°C for 2 min. The PCR primer sequences used were: slr1609VF: 5'-cattccatccattgcctggc-3' (SEQ ID No. 7); slr1609VR: 5'-ggtttgccgattttctgcga-3' (SEQ ID No. 8).

[0049] The PCR products were sequenced and verified. The sequencing results showed that in the selected single algae colony, adenine A at position 7 was successfully mutated to guanine G (see attached Figure 3 as shown).

[0050] Example 3

[0051] Plasmid elimination Since the pCyABE plasmid contains the sacB gene expression cassette, toxic substances will be produced in the presence of sucrose, causing the death of cyanobacteria. Therefore, for the successfully edited mutant strain of Synechocystis PCC6803, the strain can be cultured on a BG11 plate containing sucrose and the plasmid can be eliminated through reverse screening, thereby avoiding the influence of the edited plasmid on the physiological state of the mutant strain. The specific steps of this experiment are: (1) The successfully edited PCC6803 algae strain was inoculated into 50 ml of antibiotic-free BG11 liquid culture medium at 30°C and a light density of 30 μmol m -2 S -1 Cultivate to logarithmic growth phase under the same conditions; (2) Take 100 μl of algal liquid, dilute it with BG11 liquid medium, and spread it on a BG11 solid culture plate containing 5% w / v sucrose. o C, light density is 30 μmol m -2 S -1 Cultivated under conditions; (3) Pick the monoclonal algae colonies grown on the BG11 plate containing sucrose and inoculate them on the BG11 solid culture plate without antibiotics and the BG11 solid culture plate containing 50μg / ml kanamycin. If they can grow on the BG11 solid culture plate without antibiotics but cannot grow on the BG11 solid culture plate containing 50μg / ml kanamycin, it means that the pCyABE plasmid has been successfully eliminated. The final result is as follows: Figure 4 As shown in the figure, after successful gene editing, the plate verification experiment of the elimination of the editing plasmid. The wild-type Synechocystis PCC 6803 strain (WT), the Synechocystis PCC 6803 strain containing the pCyABE plasmid (P), and the six mutant strains of Synechocystis PCC 6803 (1-6) were spread on BG11 plates without antibiotics and containing antibiotics, respectively. It can be seen that all strains on the antibiotic-free plate can grow normally, while all six mutant strains on the antibiotic-containing plate cannot grow, indicating that the pCyABE editing plasmid in the mutant strains has been eliminated.

[0052] (4) The mutant algae strains in which the plasmid has been successfully eliminated are cultured and subcultured, and then preserved and frozen.

[0053] It can be seen from the experimental results of the above examples that the pCyABE plasmid provided by the present invention has extremely high efficiency and accuracy in gene editing and has great application potential.

[0054] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A plasmid for purine base mutation in cyanobacteria, characterized in that: The plasmid is named pCyABE, and the plasmid includes a TadA-nCas9 fusion protein expression cassette and a sgRNA expression cassette. The nucleotide sequence of the TadA-nCas9 fusion protein expression cassette is shown in SEQ ID No.2, and the nucleotide sequence of the sgRNA expression cassette is shown in SEQ ID No.

3.

2. The plasmid according to claim 1, characterized in that The plasmid also includes a targeting sequence spacer fragment, and the targeting sequence spacer fragment is connected to the sgRNA through the BsaⅠ site on the sgRNA expression cassette.

3. The plasmid according to claim 1, characterized in that The plasmid also includes sacB Reverse screening of genes.

4. The plasmid according to claim 1, characterized in that The plasmid also includes the broad host replicon RSF1010.

5. The plasmid according to claim 1, characterized in that The plasmid also includes a construction screening gene, which is selected from at least one of kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, tetracycline resistance gene, erythromycin resistance gene, gentamicin resistance gene and bleomycin resistance gene.

6. A method for preparing the plasmid according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: In vitro synthesis of TadA-nCas9 fusion protein expression cassette, sgRNA expression cassette, sacB Reverse screening genes, construct screening genes; S2: Using pCpf1b-Sp plasmid as template, amplify plasmid replicon RSF1010; S3: Assemble the fragments in steps S1 and S2 into a circular plasmid using seamless cloning technology; S4: Transform the assembled product into E. coli DH5α competent cells, screen the corresponding resistant strains and verify the plasmid sequence.

7. A use of the plasmid according to any one of claims 1 to 5, characterized in that: The application is to use the plasmid for purine base mutation in cyanobacteria to direct mutation of adenine to guanine at any site on the cyanobacteria genome.

8. The use according to claim 7, characterized in that The active window for directional mutation of adenine to guanine at any site on the cyanobacterial genome is located at positions 4 to 10 of the spacer fragment of the targeting sequence, measured from 5' to 3'.

9. An expression cell line, characterized in that The expression cell strain contains the plasmid according to any one of claims 1 to 5, and the expression cell strain is selected from any one or more of bacteria, fungi, and algae.

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