CRISPR-Cas9 gene editing system suitable for paracoccus and application of CRISPR-Cas9 gene editing system

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a CRISPR-Cas9 gene editing system suitable for paracoccus and application of the CRISPR-Cas9 gene editing system. The invention constructs a CRISPR-Cas9 gene editing system composed of a recombinant plasmid 1 with upstream and downstream homologous arms of a target gene to be knocked out and a plasmid 2 with a sgRNA DNA sequence of the target gene to be knocked out, and provides application of the gene editing system in the field of paracoccus gene knockout (including but not limited to a light sensitive protein PROKKA01468 gene). The system can directionally knock out the target gene of the KDSPL-02 strain, the gene knockout efficiency is not lower than 44.4%, the efficiency is far higher than that of a traditional homologous recombination gene knockout method, and the system has sufficient application prospects and practical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a CRISPR-Cas9 gene editing system suitable for Paracoccus and an application thereof. Background Art

[0002] The CRISPR-Cas system is an adaptive immune system acquired by bacteria and archaea through long-term evolution. After being invaded by a virus, if the bacteria do not die, they will store a small piece of DNA from the viral gene in a DNA sequence called CRISPR. If the virus invades again in the future, the bacteria can recognize it based on the spacer sequence stored in the CRISPR array, and then initiate the transcription of related genes to shear the exogenous DNA and render it ineffective. There are many types of CRISPR-Cas systems, among which CRISPR-Cas9 (CRISPR CasⅡ) technology is currently the most widely used one. It is developed from the bacterial CRISPR-Cas system and can be used to target or knock in almost any gene. The system mainly uses the Cas9 nuclease and directly uses sgRNA to guide the gene editing system in eukaryotes and prokaryotes.

[0003] An important limitation of using CRISPR-Cas RNA-guided nucleases is that they can generate extra DNA mutations at sites other than the intended target. A number of studies have directly demonstrated that CRISPR / Cas9 has serious off-target effects, meaning that the technology can cause non-specific cutting, causing mutations at non-targeted sites in the genome. The consequences of off-target effects may be incorrect phenotypes, or even more troublesome, pseudophenotypes, where the wrong gene is deleted but the same phenotype as deleting the target gene is produced. This can cause uncertainty in research results and a significant increase in research work, which severely limits the application of Cas9.

[0004] Therefore, how to design appropriate sgRNA and upper and lower homologous arm primers for specific genomes and target genes is the key to achieving gene knockout. Currently, there are methods for targeted gene knockout using the CRISPR-Cas9 gene editing system, but it is still very difficult to design appropriate sgRNA and upper and lower homologous arm primers for different genomes and target genes to ensure a high knockout rate. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a CRISPR-Cas9 gene editing system suitable for Paracoccus.

[0006] To solve the above problems, the technical solution adopted by the present invention is: The present invention provides a CRISPR-Cas9 gene editing system suitable for Paracoccus, which consists of a recombinant plasmid 1 with upstream and downstream homologous arms of a target gene to be knocked out and a plasmid 2 with a sgRNA DNA sequence of the target gene to be knocked out.

[0007] As a further improvement of the embodiment of the present invention, the PAM site nucleic acid sequence of the target gene to be knocked out is NGG.

[0008] As a further improvement of the embodiment of the present invention, the target gene to be knocked out is the light-sensing protein PROKKA_01468 gene.

[0009] As a further improvement of the embodiment of the present invention, the 5' end PCR primer sequence of the sgRNA DNA sequence of the target gene to be knocked out is shown as SEQ ID NO.1; the 3' end PCR primer sequence of the sgRNA DNA sequence of the target gene to be knocked out is shown as SEQ ID NO.2.

[0010] As a further improvement of the embodiment of the present invention, the 5'-end overlapping PCR primer sequence of the upstream homologous arm of the target gene to be knocked out is shown as SEQ ID NO.3; the 3'-end overlapping PCR primer sequence of the upstream homologous arm of the target gene to be knocked out is shown as SEQ ID NO.4; the sequence of the upstream homologous arm of the target gene to be knocked out is shown as SEQ ID NO.5.

[0011] As a further improvement of the embodiment of the present invention, the 5'-end overlapping PCR primer sequence of the downstream homologous arm of the target gene to be knocked out is shown as SEQ ID NO.6; the 3'-end overlapping PCR primer sequence of the downstream homologous arm of the target gene to be knocked out is shown as SEQ ID NO.7; the sequence of the downstream homologous arm of the target gene to be knocked out is shown as SEQ ID NO.8.

[0012] As a further improvement of the embodiment of the present invention, the Paracoccus is selected from Paracoccus sp. KDSPL-02 Paracoccus sp.

[0013] As a further improvement of the embodiment of the present invention, the plasmid 1 is a PUC57-sgRNA recombinant plasmid; the plasmid 2 is a pCas9 plasmid.

[0014] The present invention further provides an application of the CRISPR-Cas9 gene editing system in the field of Paracoccus gene knockout.

[0015] As a further improvement of the embodiment of the present invention, the application method is: the plasmid 2 inserted with the sgRNA DNA sequence of the target gene to be knocked out is transformed into Paracoccus; the plasmid 1 inserted with the upstream and downstream homologous arms of the target gene to be knocked out is also transformed and immersed in the above-mentioned Paracoccus to perform gene knockout.

[0016] The principle and beneficial effects of adopting the above technical solution are: 1. A recombinant plasmid with the target gene sgRNA DNA sequence inserted was constructed. Since the DNA sequence of the sgRNA is located at the CRISPR site of the pCas9 plasmid, as long as the recombinant plasmid is expressed in Paracoccus, the target gene will be recognized by the sgRNA and cut off under the action of the Cas9 nuclease; at the same time, the fusion homology arm carried on the homology arm plasmid will act as a repair donor to induce homology repair in bacteria, so that the target gene is deleted in the bacterial genome.

[0017] 2. The CRISPR-Cas9 gene editing system provided by the present invention obtains a CRISPR-Cas9 gene editing system suitable for Paracoccus by designing appropriate sgRNA and upstream and downstream homologous arm primers. Its gene knockout efficiency can reach 55.6%, not less than 44.44%, which is much higher than the traditional homologous recombination method. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a map of the pCas9 recombinant plasmid used in Example 1 of the present invention; Figure 2 This is a map of the PUC57-sgRNA recombinant plasmid used in Example 1 of the present invention; Figure 3 This is a graph showing the results of electrophoresis detection of E. coli clones transformed with pCas9 that are positive for colony PCR obtained in Example 1 of the present invention, wherein lane M is a 2000 bp marker and lane 1 is the result of electrophoresis verification of clone sequencing; Figure 4 The electrophoresis verification result of the PCR product of the overlapping upstream and downstream homology arms of the target gene obtained in Example 1 of the present invention, wherein lane M is a 2000 bp marker, and lane 1 is the electrophoresis result of the PCR product of the upstream and downstream homology arm connection fragments; Figure 5 The electrophoresis verification result of the PCR product of the Paracoccus colony after transformation with the pCas9 plasmid obtained in Example 2 of the present invention, wherein lane M is a 2000 bp marker, and lane Cas9 is a KDSPL-02 strain transformed with pCas9; Figure 6The figure is a graph showing the electrophoresis verification result of the PCR product of the Paracoccus colony after the PROKKA_01468 gene was knocked out obtained in Example 2 of the present invention, wherein lane M is a 2000 bp marker, lane 1 is a wild-type strain KDSPL-02, and lanes 2-10 are the electrophoresis results for the knockout strain verification; Figure 7 This is the sequencing map (partial) of the PROKKA_01468 gene of the gene knockout strain in Example 2 of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention clearer, the invention is described clearly and completely in conjunction with specific embodiments below, which is only for the convenience of describing the present invention and cannot be understood as limiting the present invention. The test methods and operations in this embodiment are conventional methods and conventional operations unless otherwise specified; the raw materials, reagents, etc. used are commercially available products unless otherwise specified.

[0020] It should be noted that the PUC57-sgRNA plasmid used in this embodiment was purchased from Miaoling Biotechnology Co., Ltd.; the pCas9 plasmid was purchased from Miaoling Biotechnology Co., Ltd.; the 2×Taq mix reagent was purchased from Shanghai Sangon Biotechnology Co., Ltd.; and the PCR gel recovery reagent was purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0021] Example 1 S1. Construction of plasmid 1 (pCas9 recombinant plasmid): (1) Based on the principle of complementary base pairing, the appropriate PAM site was determined based on the DNA sequence of the target gene PROKKA_01468, and about 20 bases upstream of the site were used as templates to design overlapping PCR primers for the sgRNA DNA fragment of the target gene to be knocked out; (2) Add 1 μL of the 5' end primer of the target gene sgRNA DNA fragment, 1 μL of the 3' end primer of the target gene sgRNA DNA fragment, 1 μL of the target gene DNA, 12.5 μL of 2×Taq mix, and 9.5 μL of ddH2O to the EP tube; pre-denature at 94°C for 10 min, denature at 98°C for 10 s, anneal at 58°C for 5 s, then extend at 72°C for 1 min, and finally extend at -72°C for 10 min to obtain the PCR product; (3) Add 1 μL of BsaI, 1 μL of pCas9 plasmid, 5 μL of NEB buffer, and 13 μL of ddH2O to the EP tube and react at 37°C for 30 min for single enzyme digestion; after single enzyme digestion, add 2 μL of oligo I, 2 μL of oligo II, 5 μL of enzyme digestion buffer, 1 μL of T4 polynucleotide kinase, and 40 μL of ddH2O and perform enzyme digestion again at 37°C for 1 h; (4) After the above reaction is completed, add 2.5 μL of 1 M NaCl solution to the EP tube, incubate at 95°C for 3 min on a PCR instrument, and then slowly cool to room temperature. After obtaining the annealed oligonucleotide chain product, add ddH2O to dilute it 20 times and store it in a 4°C refrigerator for a short term. (5) Add 1 μL T4 DNA ligase, 2 μL T4 DNA ligase buffer, and 4 μL ddH2O to 3 μL pCas9 plasmid after enzyme digestion and 10 μL target gene sgRNA DNA fragment, and react at 16°C for 12 h to allow the plasmid and target gene sgRNA DNA fragment to fully combine; (6) Add the obtained pCas9 plasmid containing the target gene sgRNA DNA sequence to 50 μL of E. coli DH5α competent cells, flick a few times, incubate on ice for 30 minutes, heat shock in a 42°C water bath for 45 seconds, and then quickly put it on ice for 5 minutes; add 500 μL of LB liquid culture medium, culture at 37°C and 180 rpm for 30 minutes, centrifuge at 4000 rpm for 3 minutes to collect the bacteria, and evenly spread 200 μL of the bacterial solution on a plate containing chloramphenicol; pick a single colony for colony PCR and electrophoresis detection, and then perform plasmid sequencing and electrophoresis detection on the E. coli with positive colony PCR results; inoculate the positive clones with correct sequencing results into LB liquid culture medium and culture at 37°C and 180 rpm for shaking until OD 600 The value was 0.8, and the strain was preserved and the plasmid pCas9 was extracted using a plasmid extraction kit. The extracted plasmid was stored in a -20°C refrigerator; S2, construction of plasmid 2 (PUC57-sgRNA-updown plasmid): The primer at the 5' end of the upstream homology arm is F1, and the primer at the 3' end of the upstream homology arm is R1; the primer at the 5' end of the downstream homology arm is F2, and the primer at the 3' end of the downstream homology arm is R2; a site for connecting with the vector plasmid PUC57-sgRNA and an overlapping fragment for overlapping PCR are added to the 5' end of primer F2 / R2, and the overlapping length is about 20bp; (1) Add 1 μL of the 5' primers of the upstream and downstream homology arms, 1 μL of the 3' primers of the upstream and downstream homology arms, 1 μL of the target gene DNA, 12.5 μL of 2×Taq mix, and 9.5 μL of ddH2O to an EP tube; perform pre-denaturation at 94°C for 10 min, denature at 98°C for 10 s, anneal at 58°C for 5 s, then extend at 72°C for 1 min, and finally extend at -72°C for 10 min to obtain the PCR product, and detect by electrophoresis; (2) Using the upstream and downstream homology arms as templates, overlap extension PCR was performed to obtain a DNA sequence in which the upstream and downstream homology arms were connected and did not contain the fragment to be knocked out. The overlapping PCR products were detected by electrophoresis, purified by gel extraction, and stored in a -20°C refrigerator; (3) Add 2.5 μL NEB buffer, 0.5 μL BamHI, and 0.5 μL NH4Cl to 5 μL PUC57-sgRNA recombinant plasmid and 1 μL target gene upstream and downstream homology arm connection fragments, respectively. Eco RI and 11.5 μL ddH2O, incubate in a 37°C water bath overnight to double-digest the plasmid PUC57-sgRNA and the upstream and downstream homology arm DNA of the target gene PROKKA_01468; purify and recover the digestion products, and detect the digestion effect by electrophoresis; (4) Add 1 μL T4 DNA ligase, 2 μL T4 DNA ligase buffer and 4 μL ddH2O to 3 μL of PUC57-sgRNA recombinant plasmid after restriction digestion and 10 μL of upstream and downstream homology arm connection fragments of the target gene after restriction digestion, react at 16°C for 12 h to allow the plasmid and upstream and downstream homology arm DNA sequences to fully combine, and detect by electrophoresis; (5) Add 5 μL of the PUC57-sgRNA-updown recombinant plasmid with upstream and downstream homologous arms of the target gene to 50 μL of E. coli DH5α competent cells, flick a few times, incubate on ice for 30 min, heat shock in a 42°C water bath for 45 s, and quickly put on ice for 5 min; add 500 μL of LB liquid culture medium and culture on a shaker at 37°C, 180 rpm for 30 min; collect the bacteria by centrifugation at 4000 rpm for 3 min, and evenly spread 200 μL of the bacterial solution on a plate containing kanamycin; pick a single colony, and add 1 μL of the upstream verification primer (sequence as shown in SEQ ID NO.9), 1 μL of the downstream verification primer (sequence as shown in SEQ ID NO.10), 12.5 μL of 2×Taq mix and ddH2O to 1 μL of the picked E. coli colony. 9.5μL, perform colony PCR, and detect by electrophoresis whether the plasmid has been successfully transferred into E. coli; inoculate the positive bacteria into LB liquid culture medium containing kanamycin resistance, and culture on a shaking table for a period of time until OD 600 The value is 0.8, and the strain is preserved; (6) Use a plasmid extraction kit to extract the PUC57-sgRNA-updown plasmid and store the extracted plasmid in a -20°C refrigerator.

[0022] Example 2 1. Cultivation of Paracoccus and preparation of competent cells: (1) Cultivation and preservation of strains: The LB solid medium consists of 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder and 20 g / L agar. The pH of the medium is adjusted to 7.0 and sterilized at 121°C for 20 min. The LB liquid culture medium contains: peptone 10 g / L, NaCl 10 g / L and yeast powder 5 g / L. The pH of the culture medium is adjusted to 7.0 and sterilized at 121°C for 20 min. Strain culture: Paracoccus can be cultured in LB liquid medium at 30°C; Strain preservation: Add 2% Paracoccus bacteria solution to LB liquid medium and culture at 30°C with horizontal shaking at 200 rpm for a period of time until OD 600 The value is 0.8, then 800 μL of bacterial solution and 200 μL of glycerol are added to the centrifuge tube and stored in a -80℃ refrigerator; (2) Preparation of competent cells: Take 200 μL of Paracoccus KDSPL-02 bacterial solution and inoculate it into 5 mL of LB liquid medium, and culture it at 28°C and 200 rpm until it becomes turbid; then take 1 mL of the above bacterial solution and inoculate it into 50 mL of LB liquid medium, and culture it at 30°C and 200 rpm until the OD 600 After reaching 1.0, transfer the bacterial solution to a 50mL centrifuge tube and place it in an ice bath for 15-20 minutes, gently shake it 2-3 times; then centrifuge the bacterial solution at 4°C and 6000rpm for 10 minutes, discard the supernatant, add 2-3mL of pre-cooled and sterilized 10% glycerol solution to the bacteria obtained by centrifugation, mix carefully, and add 10% glycerol solution to a total system of 25mL, then continue to centrifuge the bacterial solution at 4°C and 6000rpm for 10 minutes, discard the supernatant, and obtain the bacteria; repeatedly wash the bacteria with 10% glycerol until the culture medium components are completely washed, then add 1mL of pre-cooled 10% glycerol to the bacteria, blow and pipette to mix, divide into pre-cooled 1.5mL EP tubes, and store in a -80°C refrigerator.

[0023] , Plasmid transformation: (1) Electrochemical transformation of pCas9 recombinant plasmid containing target gene sgRNA DNA sequence: Add 3µL of recombinant plasmid pCas9 connected with the target gene sgRNA DNA sequence to 50μL Paracoccus competent cells, let it stand on ice for 20 minutes, then electroporate at 1.8kV, ice bath for 3 minutes, add 700μL LB liquid culture medium, and culture at 30℃, 180rpm for 4h; centrifuge the cultured bacterial solution at 4000rpm for 3min to collect the bacteria; spread 200μL of the collected bacterial solution on LB solid culture medium containing chloramphenicol resistance, invert and culture at 30℃ for 72~84h, and after a single colony grows on the culture dish, pick the colony and use pCas9 universal primers for colony PCR, and perform electrophoresis on the PCR product. If a band is visible, the transformation is successful; (2) Electrochemical transformation of the PUC57-1468 plasmid carrying the upstream and downstream homology arms of the target gene: After the pCas9 recombinant plasmid carrying the sgRNA DNA sequence and the Cas9 nuclease was transformed into Paracoccus, 3 µL of the PUC57-1468 plasmid carrying the upstream and downstream homologous arm DNA sequences of the light-sensing protein was added to 50 µL of Paracoccus competent cells. After standing on ice for 20 minutes, the cells were electroporated at 1.8 kV, and then ice-bathed for 3 minutes. 700 µL of LB liquid culture medium was added and cultured at 30°C and 180 rpm for 4 hours. The cultured bacterial solution was centrifuged at 4000 rpm for 3 minutes to collect the bacteria. 200 µL of the collected bacterial solution was spread on the LB solid culture medium containing chloramphenicol resistance and kanamycin resistance, and inverted cultured at 30°C for 72 hours.

[0024] , Gene knockout and verification: The Paracoccus colonies containing two recombinant plasmids after successful electroporation were incubated at 30°C for 4 h to allow them to fully react and knock out the target gene; Prepare a dual-resistance LB solid medium containing chloramphenicol and kanamycin, spread the incubated Paracoccus strain on the dual-resistance solid medium, place it in a 37°C constant temperature incubator for 72-84 hours, pick a single colony for colony PCR, and perform electrophoresis verification to screen the target colony that has successfully knocked out the PROKKA_01468 gene; 4. Elimination of the dual plasmid system: The strain with the PROKKA_01468 gene successfully knocked out was placed in an LB liquid culture medium without antibiotics and cultured at 37°C for 12 hours. The bacteria will metabolize and eliminate the plasmid system due to their own metabolic pressure. The bacterial liquid was transferred to an LB liquid culture medium containing kanamycin and chloramphenicol, and an LB liquid culture medium containing only chloramphenicol and only kanamycin. If the bacteria did not grow, it proved that the elimination was complete and the knockout strain of the PROKKA_01468 gene was obtained; Effect Example 1 A number of strains obtained in Example 1 were randomly selected for colony PCR, and their colony PCR products were verified by electrophoresis. The results were as follows: Figure 6 As shown, sequencing verification was performed and the knockout rate of the gene editing system was calculated; Knockout rate = (number of knockout strains / total number of strains) × 100%; Experimental results: Figure 6 As shown, the knockout results of about 500bp of each left and right homologous arms, lane 1 is the verification result of the wild-type strain of Paracoccus, the sequence length of PROKKA_01468 gene is 984bp, so the result of the wild-type strain after colony PCR should be the fusion length of the PROKKA_01468 gene sequence length and the left and right homologous arms (~2000bp); lanes 4, 6, 7, 8, 10 are consistent with the colony PCR band size of the wild-type strain, so these transformants are not PROKKA_01468 deletion strains, and the colony PCR bands of lanes 2, 3, 5, and 9 are compared with the wild-type strain of Paracoccus. The size of the bands is significantly reduced and the size is consistent with the length of the bands obtained by Overlap PCR fusion. Sequencing verification confirmed that the knockout of the PROKKA_01468 gene was achieved, and the knockout rate was 44.44%.

[0025] Effect Example 2 A plurality of strains obtained in Example 1 were randomly selected and subjected to colony PCR. The colony PCR products were verified by electrophoresis and sequencing, and the knockout rate of the gene editing system was calculated to be 50%.

[0026] Effect Example 3 A plurality of strains obtained in Example 1 were randomly selected and subjected to colony PCR. The colony PCR products were verified by electrophoresis and sequencing, and the knockout rate of the gene editing system was calculated to be 55.6%.

[0027] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CRISPR-Cas9 gene editing system suitable for Paracoccus, characterized in that: It consists of a recombinant plasmid 1 with upstream and downstream homology arms of the target gene to be knocked out and a plasmid 2 with the sgRNA DNA sequence of the target gene to be knocked out.

2. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The PAM site nucleic acid sequence of the target gene to be knocked out is NGG.

3. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The target gene to be knocked out is the light-sensing protein PROKKA_01468 gene.

4. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The 5' end PCR primer sequence of the target gene to be knocked out sgRNA DNA sequence is shown as SEQ ID NO.1; the 3' end PCR primer sequence of the target gene to be knocked out sgRNA DNA sequence is shown as SEQ ID NO.

2.

5. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The 5' overlapping PCR primer sequence of the upstream homology arm of the target gene to be knocked out is shown in SEQ ID NO.3; The 3'-end overlapping PCR primer sequence of the upstream homology arm of the target gene to be knocked out is shown in SEQ ID NO.4; the sequence of the upstream homology arm of the target gene to be knocked out is shown in SEQ ID NO.

5.

6. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The 5' overlapping PCR primer sequence of the downstream homology arm of the target gene to be knocked out is shown in SEQ ID NO.6; The 3'-end overlapping PCR primer sequence of the downstream homology arm of the target gene to be knocked out is shown in SEQ ID NO.7; the sequence of the downstream homology arm of the target gene to be knocked out is shown in SEQ ID NO.

8.

7. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The Paracoccus is selected from Paracoccus sp. KDSPL-02 Paracoccus sp.

8. A CRISPR-Cas9 gene editing system suitable for Paracoccus according to claim 1, characterized in that: The plasmid 1 is a PUC57-sgRNA recombinant plasmid; the plasmid 2 is a pCas9 plasmid.

9. An application of the CRISPR-Cas9 gene editing system as described in claims 1-8 in the field of Paracoccus gene knockout.

10. The use according to claim 9, characterized in that: The application method is: the plasmid 2 inserted with the sgRNA DNA sequence of the target gene to be knocked out is transformed into Paracoccus; the plasmid 1 inserted with the upstream and downstream homologous arms of the target gene to be knocked out is also transformed and immersed in the above Paracoccus to perform gene knockout.

Images