A eukaryotic gene editing method based on the gene cas7-3 in the type I CRISPR-Cas system

Abstract

The present invention discloses for the first time the gene knockout and gene insertion of two Cas proteins (Cas7 and Cas3) in the class I CRISPR-Cas system in the genome of eukaryotic organisms; the invention of this method breaks the traditional practice of relying on a single gene cas 9 and cpf 1, providing a new vision for multi-gene gene editing in eukaryotic organisms. The system can be used to conveniently, quickly and effectively edit the genome of the eukaryotic yeast Saccharomyces cerevisiae. The optimized tool is expected to be widely used in gene editing of other eukaryotic organisms.

Description

[0001] The present invention relates to genetic engineering technology in the field of biotechnology, and more specifically to a eukaryotic gene editing method based on gene cas7-3 in a type I CRISPR-Cas system. Background Art

[0002] Genetic engineering, or DNA recombination technology, is a revolutionary technology based on restriction endonucleases and ligases. It usually refers to the technology of integrating genes from different sources into vectors (such as plasmids or viruses) in vitro according to a pre-designed blueprint, and then introducing them into living cells to change the original genetic characteristics of the organism, obtain new varieties or increase product yields. Its characteristic is that the transformation and function of the DNA are mainly achieved through engineered plasmids and engineered viruses that can replicate autonomously. Genome editing technology (referred to as gene editing technology) is the same as the commonly said genetic engineering in that it changes the genetic characteristics of cells, but the main difference is that gene editing technology directly targets the chromosomes of cells, making its function far greater than traditional genetic engineering technology that relies on vectors.

[0003] The CRISPR / Cas system is widely present in bacteria and archaea. It is an RNA-guided adaptive immune system that degrades invading viral or phage DNA formed by the long-term evolution of prokaryotes. The modification of the type II CRISPR / Cas system has made it a new and efficient genome editing technology after zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). Compared with ZFNs and TALENs, the CRISPR / Cas gene editing system has the advantages of being simpler and more efficient. CRISPR consists of a series of short, highly conserved direct repeat sequences (repeats) and spacers of similar length. The number of CRISPR sites varies between species, and the number of repeat sequences in CRISPR also varies with different species. CRISPR sites are usually located on prokaryotic chromosomes, and some are located in plasmids (Horvath P, Barrangou R (January 2010). CRISPR / CAS, the immune system of bacteria and archaea. Science. 327(5962): 167-70.)

[0004] To date, the only two type II CRISPR-Cas systems with a single Cas effector, Cas9, and two type V Cpf1s that have been reported to be useful for gene editing (Mohanraju, P., Makarova, KS, Zetsche, B., Zhang, F., Koonin, EV, and van der Oost, J. (2016). Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 353, aad5147). Although we have reported on the use of a class I CRISPR-Cas system containing multiple Cas effectors for prokaryotic gene editing (Tong Wangyu, Xu Xin, Zhang Yan, Sun Yan, Cao Suli, A virginia IBL14type IB-sv14 CAS gene editing system, CN201611113137.3; Tong Wangyu, Qiu Caihua, Yang Xingwang, Wang Anjing, A gene editing method based on cas7-5-3 of the Streptomyces virginia IBL14 gene, CN201611089333.1), there have been no reports on its application in eukaryotic gene editing.

[0005] The present invention discloses for the first time a gene editing system consisting of a Cas7-3 protein expression vector (vector-cas7-3) and a gene editing vector (vector-t / g-gene abbreviation) designed based on the dual-gene cas7 and cas3 in the type I Cas system in the chromosome of Streptomyces virginianus IBL14. Since eukaryotic organisms have nuclear membranes and are only translated with monocistronics, the two cas genes after base optimization in the present invention are respectively provided with a transcription promoter, a terminator and a nuclear localization sequence. The emergence of this gene editing tool breaks the limitations of cas9 in gene editing and provides a new basis for discovering other gene editing tools. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a eukaryotic gene editing tool constructed based on the dual genes cas7 and cas3 of the Class 1 Type I CRISPR-Cas system.

[0007] To achieve this purpose, the technical solution adopted by the present invention is:

[0008] A eukaryotic gene editing method based on the gene cas7-3 in the type I CRISPR-Cas system is characterized by: using a protein expression plasmid and / or a gene editing plasmid constructed by the cas7 and cas3 genes in the genome of Streptomyces virginica IBL14 to effectively edit the genome of a eukaryotic organism.

[0009] The eukaryotic gene editing method based on the gene cas7-3 in the type I CRISPR-Cas system includes the construction of a protein expression plasmid, the construction of a gene editing plasmid, and the acquisition and testing of recombinants, and the specific steps are as follows:

[0010] (1) Construction of protein expression vector

[0011] According to the gene cas7 and cas3 sequences and vector sequences in Streptomyces virginiana IBL14, the codon-optimized genes cas7 and cas3 and related primers were synthesized, and the optimized genes cas7 and cas3 were connected to the vector to obtain a protein expression vector vector-cas7-3;

[0012] (2) Construction of gene editing vector

[0013] Primers are designed according to the DNA sequence information of the eukaryotic target gene, and the eukaryotic genome is used as a template. The upstream and downstream homologous arm fragments of the target gene with restriction endonuclease cutting sites and overlap PCR complementary sequences at the ends are respectively amplified by PCR reaction, and the upstream and downstream homologous arms are connected to the gene editing template t-DNA by overlap PCR. At the same time, the target gene fragment g-DNA containing a galactose promoter and an RNA terminator at the head and tail, respectively, is designed and directly synthesized according to the sequence information of the eukaryotic target gene, and the gene editing template t-DNA and the target gene fragment g-DNA are connected to the vector according to the sticky ends on the restriction enzyme cutting site to obtain the gene editing vector vector-t / g-geneabbreviation;

[0014] (3) Acquisition and testing of recombinants

[0015] Prepare eukaryotic cell competent cells, transform or transfect the protein expression vector vector-cas7-3 obtained in step (1) and the gene editing vector vector-t / g-DNA obtained in step (2) into target competent cells respectively to obtain candidate gene editing recombinants; perform PCR and gene sequencing or functional analysis on the recombinant chromosomes to obtain correct gene editing recombinants.

[0016] The eukaryotic organisms are microorganisms such as Saccharomyces cerevisiae or eukaryotic cells of animals and plants.

[0017] The gene editing refers to the insertion, knockout, traceless point mutation and any combination of the chromosomal genes of eukaryotic cells.

[0018] The present invention discloses for the first time the gene knockout and gene insertion of eukaryotic genomes by two Cas proteins (Cas7 and Cas3) in a type I CRISPR-Cas system; the invention of this method breaks the limitation of relying on single genes cas9 and cpf1, and provides a new vision for gene editing of eukaryotic organisms with multiple genes. The system can be used to conveniently, quickly and effectively edit the genome of eukaryotic yeast Saccharomyces cerevisiae. The optimized tool is expected to be widely used in gene editing of other eukaryotic organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of gene editing tool construction. (A) Protein expression plasmid pRS415-cas7-3: Ori / origin: DNA replication origin, f1ori: replication origin of f1 phage, CEN / ARS: centriole / autonomous replication, gal1promoter: galactose promoter, binds to RNA polymerase to initiate DNA transcription, LEU2promoter: leucine promoter, AmpR / ampicillin resistance: ampicillin resistance; (B) Gene editing plasmid pYES2 / NTA-t / g-ΔcrtI: Ori / origin: DNA replication origin, f1ori: phage replication origin, produces single-stranded DNA, 2μori: replication origin, URA3promoter: uracil promoter, T7promoter: T7 promoter, initiates DNA transcription, AmpR / ampicillin resistance: ampicillin resistance.

[0020] Figure 2 Knockout results of crtI gene in SCBY4741 genome. (A) Red and white spot screening (black is the original strain, white is the recombinant strain); (B) PCR electrophoresis (lane M: 5000 bp DNA ladder, lane 1: plasmid pRS415-cas7-3 and pYES2 / NTA-t / g-ΔcrtI transformants, lane 2: pRS415-cas7-3 and pYES2 / NTA-t-ΔcrtI transformants, lane 3: pRS415-cas7-3-t / g-ΔcrtI transformants, lane 4: plasmid pRS415-cas7-3-t-ΔcrtI transformants, lane 5: host SCBY4741). DETAILED DESCRIPTION

[0021] In order to more fully understand the technical content of the present invention, the technical solution of the present invention is further introduced and illustrated in conjunction with specific embodiments below, in order to better explain the content of the present invention, and the following embodiments do not limit the protection scope of the present invention. In addition, in the examples listed, the following materials are used unless otherwise specified:

[0022] 1) Strains, cells and plasmids

[0023] Escherichia coli DH5α / EC DH5α, human embryonic kidney cells human embryonic kidney 293 cells / HEK293, Saccharomyces cerevisiae BY4741 / SC BY4741; plasmids pRS415, pYES2 / NTA, pUC19, pSp-2A-Puro

[0024] 2) Solutions and culture media

[0025] 1.0 mol / L LiAc solution:

[0026] Weigh 6.6 g of LiAc solid powder, add 90 ml of double distilled water to dissolve, and make up to 100 ml.

[0027] 100mmol / L LiAc solution:

[0028] Weigh 0.66 g of LiAc solid powder, add 90 ml of double distilled water to dissolve, and make up to 100 ml.

[0029] HEPES Buffer:

[0030] Weigh 0.0135g Na2HPO4﹒H2O, 0.8g NaCl, 0.037g KCl, 0.5g glucose, and 0.5g HEPES, dissolve in 90ml double distilled water, adjust pH to 7.5 with NaOH, and make up to 100ml.

[0031] 50mg / ml ampicillin:

[0032] Weigh 0.5 g of ampicillin powder and place it in a 10 ml centrifuge tube. Add 4 ml of double distilled water, mix well, make up to 10 ml, filter with a 0.2 μm sterile water filter membrane, divide into 1.5 ml EP tubes, and store in a -20°C refrigerator.

[0033] 50mg / ml G418:

[0034] Weigh 0.5 g of G418 and place it in a 10 ml centrifuge tube. Add 4 ml of HEPES buffer, mix well, make up to 10 ml, filter with a 0.2 μm sterile water filter membrane, dispense into 1.5 ml EP tubes, and store in a -20°C refrigerator.

[0035] SD-LEU induction medium (100 ml):

[0036] 0.17g YNB, 0.5g ammonium sulfate, 2g galactose, 1g raffinose, add appropriate amount of double distilled water to dissolve, dilute to 99.6ml, package and sterilize at 115℃ / 30min. Add 400μl amino acids (methionine 0.5g / 100ml, histidine 0.5g / 100ml, uracil 0.5g / 100ml) that have been sterilized by filtration.

[0037] LB medium:

[0038] Yeast powder 5g, peptone 10g, NaCl 10g, add appropriate amount of tap water to dissolve, make up to 1L, adjust pH to 7.0-7.2, package and seal, sterilize at 121℃ / 20min, add 20g agar if making plates.

[0039] SD-LEU / URA selection medium (100 mL)

[0040] 0.17g YNB, 0.5g ammonium sulfate, 2g glucose, add appropriate amount of double distilled water to dissolve, dilute to 99.6ml, package and sterilize at 115℃ / 30min. Add 400μl amino acids (methionine 0.5g / 100ml, histidine 0.5g / 100ml) that have been sterilized by filtration, and add 2g agar and 18.2g sorbitol if making plates.

[0041] YNB medium:

[0042] Yeast extract 10g, peptone 20g, glucose 20g, add appropriate amount of tap water to dissolve, make up to 1L, divide into packages, sterilize at 115℃ / 30min, add 20g agar if making plates.

[0043] SD-LEU selection medium (100 mL):

[0044] 0.17g YNB, 0.5g ammonium sulfate, 2g glucose, add appropriate amount of double distilled water to dissolve, dilute to 99.6ml, package and sterilize at 115℃ / 30min. Add 400μl amino acids (methionine 0.5g / 100ml, histidine 0.5g / 100ml, uracil 0.5g / 100ml) that have been sterilized by filtration, and add 2g agar and 18.2g sorbitol if making a plate.

[0045] High glucose DMEM medium:

[0046] Anhydrous calcium chloride.2H2O265.00mg / L, ferric nitrate.9H2O 0.10mg / L, potassium chloride 400.0mg / L, anhydrous magnesium sulfate 97.67mg / L, sodium chloride 6400.0mg / L, anhydrous sodium dihydrogen phosphate 109.0mg / L, succinic acid 75.0mg / L, sodium succinate 100.0mg / L, L-arginine hydrochloride 84.0mg / L, L-cystine hydrochloride 63.0mg / L, glycine 30.0mg / L, L-histidine hydrochloride 42.0mg / L, L-isoleucine 105.0mg / L, L-leucine 105.0mg / L, L-lysine hydrochloride 146.0mg / L, L-methionine 30.0mg / L, L-phenylalanine 66.0mg / L, L-serine 42.0mg / L, L-threonine 95.0mg / L, L-tryptophan 16.0mg / L, L-tyrosine 72.0mg / L, L-valine 94.0mg / L, D-calcium pantothenate 4.0mg / L, choline bitartrate 7.20mg / L, folic acid 4.0mg / L, inositol 7.2mg / L, niacinamide 4.0mg / L, riboflavin 0.40mg / L, thiamine hydrochloride 4.0mg / L, pyridoxine hydrochloride 4.0mg / L, glucose 4500.0mg / L, sodium pyruvate 110.0mg / L, phenol red 9.3mg / L.

[0047] All reagents used were commercially available.

[0048] Example 1 pRS415-cas7-3 and pYES2 / NTA-t / g-ΔcrtI were used to transform and knock out the crtI gene in SC BY4741

[0049] (1) Construction of protein expression plasmid pRS415-cas7-3

[0050] (A) Construction of protein expression plasmid pRS415-cas3

[0051] According to the sequence information of plasmid pRS415, specific primers cas3-F / cas3-R and pRS415-cas3-F / pRS415-cas3-R were designed, and the complementary sequences of cas3 gene and plasmid pRS415-cas9 were carried out at both ends of the primers. Cas3 gene PCR amplification was performed using TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd., and the reaction conditions were: 95±1℃5±1min, 95±1℃30s, 62±3℃30s, 72℃1±1min (50μl reaction system), 30 cycles, 72℃10min. The PCR product was detected by 1% agarose electrophoresis, and the kit was recovered to obtain a purified cas3 gene fragment. The same PCR amplification was performed again to obtain a purified pRS415 plasmid skeleton. Rapid cloning was performed using the one-step cloning seamless cloning kit produced by Quanshijin Biotechnology Co., Ltd., and the ligation product was transformed into EC DH5α competence. After a single clone grows out, colony PCR is performed using primers cas3-F / cas3-R. The remaining PCR-positive colonies are inoculated into LB medium containing Amp and cultured overnight. The plasmid is extracted and identified to obtain the protein expression plasmid pRS415-cas3.

[0052] (B) Preparation of cas7 gene fragment with promoter, terminator and nuclear localization sequence

[0053] Plasmid pMD-18T-crtI sequence information, design specific primers cas7-F (1) / cas7-R (1) and pMD18T-cas7-F / pMD18T-cas7-R with complementary sequences of plasmid pMD-18T-crtI and cas7 gene, respectively. Cas7 gene PCR amplification was performed using TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd., and the amplification conditions were the same as those in Example 1 step (1A). The PCR product was detected by 1% agarose electrophoresis, and the kit was recovered to obtain a purified cas7 gene fragment. The same PCR amplification was performed again to obtain a purified pMD-18T plasmid backbone containing CDC19p and CDC19t. Then, through one-step cloning and seamless connection, the reaction system and transformation steps were the same as those for the construction of the pRS415-cas3 plasmid, and the successfully constructed pMD-18T-cas7 plasmid was obtained; then, PCR amplification was performed using TransStart FastPfu DNA Polymerase and primers pRS415-cas7-F / pRS415-cas7-R to obtain a purified cas7 gene fragment containing CDC19p, CDC19t and SV40NLS.

[0054] (C) Construction of protein expression plasmid pRS415-cas7-3

[0055] According to the constructed pRS415-cas3 sequence information, specific primers pRS415-cas7-3-F / pRS415-cas7-3-R were designed, and the complementary sequence of cas7 was carried out at both ends of the primer. PCR amplification was performed using TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd., and the amplification conditions were the same as those in Example 1 step (1A) to obtain the plasmid backbone of purified pRS415-cas3. Rapid cloning was performed by the one-step cloning seamless cloning kit produced by Quanshijin Biotechnology Co., Ltd., and the cas7 gene fragment containing CDC19p, CDC19t and SV40NLS obtained in the previous step was connected to the plasmid backbone of pRS415-cas3. The connection product was transformed into EC DH5α competence, and colony PCR was performed by primers cas7-F / cas7-R after monoclonal growth, and the remaining colonies of PCR positive colonies were inoculated into LB culture medium containing Amp and cultured overnight, and plasmids were extracted and identified to obtain protein expression plasmid pRS415-cas7-3.

[0056] (2) Construction of gene editing plasmid pYES2 / NTA-t / g-ΔcrtI

[0057] (A) Preparation of upstream and downstream homology arms

[0058] According to the full sequence of crtI gene, crtI-UF and crtI-UR, crtI-DF and crtI-DR (the boldface is the overlap PCR complementary sequence) were designed for the upstream homology arm primers of crtI gene, and the upstream primer of the upper homology arm contained a HindIII restriction endonuclease cleavage site, and the downstream primer of the lower homology arm contained a BamHI restriction endonuclease cleavage site. Using the purified crtI gene DNA as a template, the upstream and downstream homology arms were amplified respectively, and the amplification conditions were the same as those in step (1) of Example 1. The PCR products were detected by 1.5% agarose electrophoresis, and the kit was recovered to obtain the purified upstream and downstream homology arm DNA fragments for standby use.

[0059] (B) Construction of gene editing template vector pYES2 / NTA-t-ΔcrtI

[0060] 0.5 μl of the purified product of the upper homology arm and the purified product of the lower homology arm were mixed as templates, and overlap PCR was performed in a 25 μl reaction system. The reaction conditions were: 94°C 5 min, 94°C 1 min, 61°C 30 s, 72°C 1 min. After one cycle, 1 μl of primers crtI-UF and crtI-DR were added, and PCR was continued. The reaction conditions were: 94°C 5 min, 94°C 30 s, 61°C 30 s, 72°C 1 min, and 30 cycles were performed, 72°C 10 min. The amplified product was detected by 1.5% agarose gel electrophoresis and purified to obtain the gene editing template t-ΔcrtI; the obtained t-ΔcrtI was cut out of the sticky end by HindIII restriction endonuclease and BamHI restriction endonuclease, and then connected to the pYES2 / NTA plasmid by T4 ligase produced by Quanshijin Biotechnology Co., Ltd. to obtain the gene editing template vector pYES2 / NTA-t-ΔcrtI.

[0061] (C) Construction of gene editing plasmid pYES2 / NTA-t / g-ΔcrtI

[0062] The targeted gene fragment containing the galactose promoter and the g-crtI ligation product was directly synthesized by Chuzhou General Biotechnology Co., Ltd., with EcoRI and XhoI restriction sites added at the beginning and end, respectively, and the promoter, repeat sequence, spacer, repeat sequence and terminator in the middle; the synthetic targeted gene fragment was cut out of the sticky end by EcoRI and XhoI restriction endonucleases, and then ligated to the gene editing template vector pYES2 / NTA-t-ΔcrtI by T4 ligase to obtain the gene editing plasmid pYES2 / NTA-t / g-ΔcrtI ( Figure 1 (B)).

[0063] Synthetic g-crtI gene sequence (PAM: ttc):

[0064] The capital letters are restriction sites, the wavy lines are protection bases, the single underline is the promoter, the italic is the spacer, the bold is the repeat, and the double underline is the terminator.

[0065] (3) Acquisition and testing of recombinants

[0066] (A) Preparation of SCBY4741 competent cells and transformation with pRS415-cas7-3

[0067] In the clean bench, pick a single colony from a fresh plate into 5 ml YPD medium, and shake culture at 30°C overnight; inoculate 1 ml of bacterial solution into 50 ml YPD, shake culture at 30°C until OD600 is 1.0-1.3; centrifuge at 4000 rpm for 5 min in a 50 ml sterile centrifuge tube to precipitate the cells; discard the culture medium, resuspend the cells in 25 ml sterile water, and centrifuge at 4000 rpm for 5 min; discard the water, suspend the cells in 1 ml of 100 mmol / L lithium acetate (LiAc), and transfer the suspension to a 1.5 ml centrifuge tube; centrifuge at high speed for 5 s to precipitate the cells, and use a micropipette to absorb all the lithium acetate; add 400 μl 100mmol / L lithium acetate; boil 1ml ssDNA for 5min, and quickly cool in ice water; shake the cell suspension, take 50μl sample and add it to the marked centrifuge tube, centrifuge to precipitate the cells, and remove the lithium acetate with a micropipette; carefully add the basic transformation mixture 240μl PEG (50% m / V), 36μl 1.0mol / L lithium acetate, 25μl ssDNA (2.0mg / ml), 50μl water or plasmid DNA or PCR purified product in sequence; shake each reaction tube vigorously until the cells are completely mixed, which usually takes about 1min; keep warm at 30℃ for 30min; heat shock in a 42℃ water bath for 60min; centrifuge at high speed for 15s, and remove the transformation mixture with a micropipette; aspirate 1ml YPD medium was added to each centrifuge tube, and the tube was placed at 30°C for 4 hours of shaking; centrifugation was performed, the upper medium was discarded, 1 ml of sterile water was added, and the bacteria were suspended; 100 μl was applied to the SD-LEU defect selection plate, and cultured at 30°C for 3 days; a red single clone was selected as a template, and PCR amplification reaction was performed with the cas7-3 gene verification primers cas3-F / cas3-R, and the amplification conditions were the same as those in step (1) of Example 1. The PCR product was detected by 1% agarose electrophoresis, and the cas3 peripheral amplification band in the recombinant SC BY4741-pRS415-cas7-3 was observed to be consistent with the expected result, proving that the pRS415-cas7-3 plasmid had been successfully transferred into yeast.

[0068] (B) Preparation of SC BY4741-pRS415-cas7-3 competent cells and transformation with pYES2 / NTA-t / g-ΔcrtI

[0069] Except that the single clone of SCBY4741-pRS415-cas7-3 successfully transferred into the pRS415-cas7-3 plasmid was selected and transformed in 50 ml SC-LEU induction medium, and the obtained recombinant was a white single clone, the rest was the same as step (3A) of Example 1. The knockout results of crtI gene in SCBY4741 genome are shown in Figure 2 .

[0070] The primers and their sequences involved in each step are shown in Table 1. Capital letters represent restriction sites, and boldface letters represent complementary regions.

[0071] Table 1 Primers and their sequences

[0072]

[0073] Example 2 pRS415-cas7-3 and pYES2 / NTA-t-ΔcrtI were used to transform and knock out the crtI gene in SC BY4741

[0074] (1) Construction of protein expression plasmid pRS415-cas7-3

[0075] Same as step (1) of Example 1

[0076] (2) Construction of gene editing template vector pYES2 / NTA-t-ΔcrtI

[0077] (A) Preparation of upstream and downstream homology arms

[0078] Same as step (2A) of Example 1

[0079] (B) Construction of gene editing template vector pYES2 / NTA-t-ΔcrtI

[0080] Same as step (2B) of Example 1

[0081] (3) Acquisition and testing of recombinants

[0082] (A) Preparation of SCBY4741 competent cells and transformation with pRS415-cas7-3

[0083] Same as step (3-A) of Example 1

[0084] (B) Preparation of SC BY4741-pRS415-cas7-3 competent cells and transformation with pYES2 / NTA-t-ΔcrtI

[0085] Except for the introduction of plasmid pYES2 / NTA-t-ΔcrtI, the rest was the same as step (3B) of Example 1 (see Figure 2 ).

[0086] Example 3 Single plasmid pRS415-cas7-3-t / g-ΔcrtI transformation to knock out the crtI gene in SC BY4741

[0087] (1) Construction of protein expression plasmid pRS415-cas7-3

[0088] Same as step (1) of Example 1

[0089] (2) Protein expression and construction of the single plasmid pRS415-cas7-3-t / g-ΔcrtI for gene editing

[0090] (A) Preparation of upstream and downstream homology arms

[0091] Except that the upstream homology arm primer crtI-UF2 and the downstream homology arm primer crtI-DR2 of the crtI gene both contain XhoI restriction enzyme cleavage sites, the rest is the same as step (2A) of Example 1, and the sequence of the upstream homology arm primer crtI-UF2 used is ccgCTCGAGatcgtttgcgtcgggtgttctcct, and the sequence of the downstream homology arm primer crtI-DR2 is ccgCTCGAGaccaactgaacgagcaataacgg.

[0092] (B) Protein expression and construction of the single plasmid pRS415-cas7-3-t / g-ΔcrtI for gene editing

[0093] Except that the g-crtI-DNA and t-ΔcrtI-DNA fragments were ligated to the pRS415-cas7-3 plasmid, the remaining steps were the same as step (2B) of Example 1.

[0094] Synthetic g-crtI sequence (PAM: ttc):

[0095] The capital letters represent restriction sites, the single underline represents the promoter, the italic represents the spacer, the bold represents the repeat, and the double underline represents the terminator.

[0096] (3) Acquisition and testing of recombinants

[0097] (A) Preparation of SCBY4741 competent cells and one-step transformation of plasmid pRS415-cas7-3-t / g-ΔcrtI

[0098] Same as step (3B) of Example 1, the test results are shown in Figure 2 .

[0099] Example 4 Single plasmid pRS415-cas7-3-t-ΔcrtI transformation to knock out the crtI gene in SC BY4741

[0100] (1) Construction of protein expression plasmid pRS415-cas7-3

[0101] Same as step (1) of Example 1

[0102] (2) Construction of protein expression and gene editing template single plasmid pRS415-cas7-3-t-ΔcrtI

[0103] (A) Preparation of upstream and downstream homology arms

[0104] Same as step (2A) of Example 3

[0105] (B) Construction of the gene editing targeting vector single plasmid pRS415-cas7-3-t-ΔcrtI

[0106] Except that the t-ΔcrtI-DNA fragment was directly connected to the pRS415-cas7-3 plasmid, the remaining steps were the same as in Example 1.

[0107] 1 step (2B).

[0108] (3) Acquisition and testing of recombinants

[0109] Except for using plasmid pRS415-cas7-3-t-ΔcrtI for transformation, the rest is the same as step (3B) of Example 1. The detection results are shown in Figure 2 .

[0110] Example 5 One-step transformation of pRS415-cas7-3 and pYES2 / NTA-t / g-ΔcrtYB::G418 to knock out the crtYB gene in SCBY4741

[0111] (1) Construction of protein expression plasmid pRS415-cas7-3

[0112] Same as step (1) of Example 1

[0113] (2) Construction of gene editing plasmid pYES2 / NTA-t / g-ΔcrtYB::G418

[0114] (A) Preparation of LoxP-G418-LoxP fragment and crtYB upstream and downstream homology arms

[0115] According to the whole genome sequencing information of SC BY4741, the LoxP-G418-LoxP fragment-specific primers crtYB-G418-F / crtYB-G418-R and crtYB-UF / crtYB-UR and crtYB-DF / crtYB-DR were designed to amplify and detect the purified LoxP-G418-LoxP fragment and the upstream and downstream homology arms of crtYB, respectively. The amplification conditions were the same as those in Example 1 (2-A).

[0116] (B) Construction of gene editing template vector pYES2 / NTA-t-ΔcrtYB::G418

[0117] The upper homology arm purified product and LoxP-G418-LoxP purified product were mixed with 0.5 μl each as templates, and overlap PCR was performed in 25 μl reaction system. The reaction conditions were: 94°C 5 min, 94°C 30 s, 65°C 30 s, 72°C 3 min. After one cycle, 1 μl each of primers crtYB-UF and crtYB-G418-R were added, and PCR was continued. The reaction conditions were: 94°C 5 min, 94°C 30 s, 65°C 30 s, 72°C 3 min, and 30 cycles were performed. The amplification product was detected by 1.5% agarose gel electrophoresis and purified. The purified product crtYB-UHA-LoxP-G418-LoxP was mixed with 0.5 μl each of the lower homology arm purified product as templates, and overlap PCR was performed in 25 μl reaction system. PCR, the reaction conditions are: 94°C 5 min, 94°C 30 s, 62°C 30 s, 72°C 4 min. After one cycle, 1 μl of primers crtYB-UF and crtYB-DR were added, and PCR was continued. The reaction conditions are: 94°C 5 min, 94°C 30 s, 59°C 30 s, 72°C 4 min. The amplified product was detected by 1.5% agarose gel electrophoresis and purified to obtain the gene editing template t-ΔcrtYB::G418; the obtained t-ΔcrtYB::G418 was cut out of the sticky ends by HindIII restriction endonuclease and BamHI restriction endonuclease, and then it was ligated to the pYES2 / NTA plasmid by T4 ligase produced by Quanshijin Biotechnology Co., Ltd. to obtain the gene editing template vector pYES2 / NTA-t-ΔcrtYB::G418.

[0118] (C) Construction of gene editing plasmid pYES2 / NTA-t / g-ΔcrtYB::G418

[0119] Except for the different targeted gene fragments of the g-crtYB ligation product, the rest was the same as step (2C) of Example 1 to obtain the gene editing plasmid pYES2 / NTA-t / g-ΔcrtYB::G418.

[0120] Synthetic g-crtYB gene sequence (PAM: ttc):

[0121] The capital letters are restriction sites, the wavy lines are protection bases, the single underline is the promoter, the italic is the spacer, the bold is the repeat, and the double underline is the terminator.

[0122] (3) Acquisition and testing of recombinants

[0123] The procedure was the same as step (3) of Example 4 except that the plasmid pRS415-cas7-3 and the plasmid pYES2 / NTA-t / g-ΔcrtYB::G418 were simultaneously transformed into SCBY4741 competent cells.

[0124] The primers and their sequences involved in each step are shown in Table 2, in which the capital letters represent restriction sites and the bold letters represent complementary regions.

[0125] Table 2 Primers and their sequences

[0126]

[0127] Example 6 Knockout of HEK293 gene drosha using pSp-cas3-7 and pUC19-t / g-Δdrosha

[0128] (1) Construction of protein expression plasmid pSp-cas3-cas7

[0129] (A) Construction of protein expression plasmid pSp-cas3

[0130] According to the sequence information of the constructed pRS415-cas7-3 plasmid, the specific primers cas3R (1) / cas3F (1) were designed, and the TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd. was used for PCR amplification, and the reaction conditions were the same as those in step (1A) of Example 1. The PCR product was detected by 1% agarose electrophoresis, and the kit was recovered to obtain the purified cas3 fragment. According to the sequence information of the plasmid pSp-cas9, the specific primers VectorR (2) / VectorF (2) were designed, and the TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd. was used for PCR amplification. The remaining steps were the same as step (2B) of Example 1, and the reaction conditions were the same as those in step (1A) of Example 1. The PCR product was detected by 1% agarose electrophoresis, and the kit was recovered to obtain the purified pSp plasmid skeleton. The one-step cloning enzyme produced by Quanshijin Biotechnology Co., Ltd. was used to connect the cas3 fragment and the pSp plasmid skeleton to obtain the pSp-cas3 plasmid.

[0131] (B) Construction of protein expression plasmid pSp-cas3-7

[0132] According to the sequence information of the constructed pRS415-cas7-3 plasmid, specific primers cas7R / cas7F were designed, and TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd. was used for PCR amplification, and the reaction conditions were the same as those in Example 1 step (1A). The PCR product was detected by 1% agarose electrophoresis, and the kit was recovered to obtain the purified cas7 fragment. According to the sequence information of the plasmid pSp-cas3, specific primers VectorR(1) / VectorF(1) were designed, and TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd. was used for PCR amplification, and the reaction conditions were the same as those in Example 1 step (1A). The PCR product was detected by 1% agarose electrophoresis, and the kit was recovered to obtain the purified pSp-cas3 plasmid skeleton. The one-step cloning enzyme produced by Quanshijin Biotechnology Co., Ltd. was used to connect the cas7 fragment and the pSp-cas3 plasmid skeleton to obtain the pSp-cas3-7 protein expression plasmid.

[0133] (2) Construction of gene editing plasmid pUC19-t / g-Δdrosha

[0134] (A) Preparation of upstream and downstream homology arms

[0135] The remaining steps are the same as step (2A) of Example 1, except that the primers used are drosha-UF / drosha-UR and drosha-DR / drosha-DF, and the upstream primer of the upper homology arm contains a HindIII restriction endonuclease cleavage site, and the downstream primer of the lower homology arm contains a SphI restriction endonuclease cleavage site.

[0136] (B) Construction of gene editing template vector pUC19-t-Δdrosha

[0137] In addition to the different restriction endonuclease cutting sites HindIII and SphI,

[0138] (C) Construction of gene editing plasmid pUC19-t / g-Δdrosha

[0139] Except for the restriction endonuclease cleavage sites XbaI and BamHI added at the beginning and end of the synthetic g-drosha, the remaining steps are the same as step (2C) of Example 1.

[0140] Synthetic g-drosha sequence (PAM:ttc):

[0141] The capital letters are restriction sites, the wavy lines are protection bases, the single underline is the promoter, the italic is the spacer, the bold is the repeat, and the double underline is the terminator.

[0142] (3) Acquisition and testing of recombinants

[0143] HEK293 cells were inoculated into a 6-well plate containing DMEM medium and cultured in a 37°C 5% CO2 incubator until 70%-90% confluence. The constructed pSp-cas3-7 (4 μl) and pUC19-t / g-Δdrosha (6 μl) were co-transfected into 90 μl HEK293 cells (Lipofectamine 2000 transfection reagent). Fresh cell culture medium was replaced after 6 hours. After 5-7 days, the transfected HEK293 cells were inoculated into a large cell culture dish (1×10 6 After about 10-15 days, the single clone visible to the naked eye in the cell culture dish was inoculated and subcultured into another 6-well plate. When the cell culture filled the plate, the genome was extracted for PCR and detected by 1% agarose electrophoresis to observe the change in the size of the recombinant chromosome DNA amplification band, and gene sequencing was used to prove the drosha gene knockout of HEK293 cells.

[0144] The primers and their sequences involved in each step are shown in Table 3, in which the capital letters represent restriction sites and the bold letters represent complementary regions.

[0145] Table 3 Primers and their sequences

[0146]

[0147] The above description is only used to further illustrate the technical content of the present invention by examples, so as to facilitate readers to understand more easily, but it does not mean that the implementation mode of the present invention is limited to this. Any technical extension or re-creation made according to the present invention is protected by the present invention. Sequence Listing <110> Anhui University <120> A eukaryotic gene editing method based on the gene cas7-3 in the type I CRISPR-Cas system <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1020 <212> DNA <213> S. virginiae IBL14 <400> 1 atggttgccg gtgcaccaaa caatggtgaa ggtgaagata atacaggtag agttaaaaaa 60 ttaagggttg gtagggaaga attccctac gtttccgcac aagcctttag acgttggttg 120 agggattcct tacctgccca agaacctagg tccgttgtta ctagatccgg ttcaggtgct 180 aaaaaag ctcatacagc aggtagacct gatttgcatt tggatgatga cctctttggt 240 tatatggttg ctgttaaagg taaaggtggt tcatatcaaa gagatacagt tttagcaact 300 ggtacattag tttccgttgt tcctcaaagg ccaactttgg attttggtac tatgtctagg 360 gattttccag ccggtgaaca tcctgttat catagtcatg aattgtatag tgccactttg 420 gccggcgatg ttttattgga cctccctagg gccggtgttt ttgaaactga tggtaatggt 480 ttaagagttg caatttcacc agccgttgct gaaagccg ctaaaaatgg tgccgaagtt 540 actactttga ggggttctgc tgctattaga ttaccattga cagaaagaca tcggcggatt 600 ggtacattat tgaggacttt agcatccgtt aggggtggtg ccaaacaagc cttgcattat 660 ggcgaccgag caccatcctt agttttatta gctccattaa aaggtggtgt taatccgttt 720 actagagttt tgggtgccag agatggtaaa cctgttttct tgtccgatgt ttgaggggaa 780 gaattggaag cctgggccga tgaattggat ggtccagttt tattgggttg ggcaccaggg 840 ttcttggggg accaaaggga acaagttaga agggaattaa aagaccttat tgatgaaggt 900 agagttgttt tgtcacatcc tagagtttta ttgactcaat tagcagatag gattgaacaa 960 ggtgaccatg atgcctggtt tgaagatagc gctgctccca agaagaagag gaaggtgtga 1020 <210> 2 <211> 2337 <212> DNA <213> S. virginiae IBL14 <400> 2 atgggtagat tggatgccgt tgaagatgtt tttggtggta gattttggcc agttgttgaa 60 ttggccggtt tgacacatga tgccggaaag attccagaag gttttcaaag aatgttagcc 120 ggttatagta gagcatgggg tgaaagacat gaagttgctt ccttggggtt cttacctgcc 180 ttaattgggg accctgatgt tttattgtgg gttgctacag ctgttgcaac acatcataga 240 ccattaactg gtcaaaatgg tagggacctc caaactttgt atagtggtgt tactattaca 300 gaattggctc ataggtttgg tccatttgat ccaagggccg ttcctgcctt ggaagcctgg 360 ttaagggctt cagcaattag agttggttta cctgccgccg ccgttcctga tgatggtact 420 ttaactgata caggtgttgt tgctggtgct catcaattat tggaagaaat tttggatagg 480 tgggccgata gagttagacc agaagttggt ttagcagcag ttttattaca aggtgccgtt 540 actttggccg atcatttgtc ttccgcccat caagccttac caacagttca accattgggt 600 gcaggttttc gtagtaggtt agaaaaagaa tttgcagaaa ggggtaggac attaagggca 660 catcaattgg aagccgccac tgttacaggt catttgttat tgagaggtcc tacaggttca 720 ggaaagacag aagcagcctt gttgtgggct gcttcacaag ttgaagcctt aaaagccgaa 780 ggtagaggtg ttcctagagt tttctttaca ttaccatacc ttgcttctat taatgctatg 840 gccactagat tgggagacac tttgggggat ggtgaagctg ttggtgttgc ccattcgcgc 900 gctgcttctt atcatttggc tcaagctatt gccccacaag atggcgacga agaagatgaa 960 catggtgcac catgtagagt tgatgcagct gctaaagcct tgtcacgggc agctgctact 1020 aaactcttta gggaatccgt tagagttgcc actccatatc aattattaag ggccgcctta 1080 gccggtccag cacattccgg tattttaatt gatgcagcta attccgtttt tattttggat 1140 gaattacatg cttacgatgc acggaggttg ggttatattt tagcttccgc cagattgtgg 1200 gaaagattgg gtggtaggat tactgttttg tccgcaactt taccaagggc cttagccgat 1260 ttgtttgaat ctactttaac agcacctatt acgtttttgg atactcctga tttgggttta 1320 ccagccaggc atttgttaca tactaggggt catcatttga ctgatcctgc aactttggaa 1380 gaaattaggt tgagattgtc cagggatgaa tccgttttag ttattgctaa caacgtttct 1440 caagctattg ccttgtatga acaattagca cctgatgttt gtgaaaggtt tggtcaagat 1500 gcagccttgt tattacattc tcgctttaga cggatggatc gcagcaggat tgaacaaaaa 1560 attgccgata gatttgccac agttgctcct gatgctcaaa atagtaggaa accaggttta 1620 gttgttgcta cacaagttgt tgaagtttcc ttggatgttg attttgatgt tttgtttaca 1680 ggtgctgccc cattggaagc cttattgcaa agatttggta ggactaatag agttggtgcc 1740 agacctccag ccgatgttat tgttcatcat ccagcatgga ctacacgtcg tagacaacct 1800 ggtgaatatg ccgatggtat ctatccaagg gaacctgttg aatccgcttg gcatatttta 1860 actagaaatc atggtagagt tattgatgaa gccgatgcca ctgcatggtt ggatgaagtt 1920 tatgcaactg attggggtag gcaatggcat agggaagttt tggaaagacg tgaaagattt 1980 gatagagcct ttttgcagtt tagataccca tttgaagata ggactgatct cgcagatact 2040 tttgatgaat tatttgatgg ttccgaagca attttagcag aagatcaaga tgcctatagt 2100 gccgccttag ctgccccaga tggagaccat ccaggtgccg gtaggttatt agccgaagaa 2160 tatttgattc cagttcctca ttgggcttca ccattatcta ggtatgaaaa acaattaaaa 2220 gttagagtta ttaatggcga ctatcatcca gatcatggtt taatggccgt taggggttta 2280 cctcaacctg catatagggc cggtgaagtt ttacccaaga agaagaggaa ggtgtga 2337 <210> 3 <211> 908 <212> DNA <213> Artificial Sequence () <400> 3 atcgtttgcg tcgggtgttc tcctttgccg ttatgtatat gggtcaatca ccatatagtg 60 cccctggtac atattcctta ttacaatata cagaattaac agaaggtatt tggtatcctc 120 gtggtggttt ttggcaagtt cctaatacat tgttgcaaat tgttaaacgt aacaatccat 180 ccgccaagtt taattttaat gcaccagttt cacaagtttt attatcacct gccaaagatc 240 gtgccacagg tgttcgttta gaatccggtg aagaacatca tgccgatgtt gttattgtta 300 atgccgattt ggtttatgcc tccgaacatc tcattccagc agctccagaa ggtaaagatg 360 ctattgttat tttagttcca tgtggtcata ttgatacttc taatccacaa gattataaca 420 agttagttgc tcgtgctcgt aaatttgtta ttcaaacatt aagtgctaaa ttaggtttac 480 ctgattttga aaaaatgatt gttgccgaaa aagttcatga tgcaccatca tgggaaaaag 540 agtttaattt gaaagatggt agtattttgg gtttagcaca taattttatg caagttttgg 600 gttttcgtcc tagtactcgt catcctaaat atgataaatt gtttttcgtt ggtgcttcta 660 ctcatccagg tacaggtgtt ccaattgttt tggccggtgc caaattaact gccaatcaag 720 ttttggaatc ctttaatcgt tctccagcac ctgatcctaa tatgtccttg tccgttccat 780 acggtaaacc attgaaatct aatggtacag gtattgattc tcaagttcaa ttgaagttta 840 tggatttgga aaaatgggtt tatcttttag ttttgttaat tggtgccgtt attgctcgtt 900 cagttggt 908 <210> 4 <211> 889 <212> DNA <213> Artificial Sequence () <400> 4 gccttaccta aaactcggtc ttctgcacgt tcattagcct taaaagcatt aattccatta 60 ccaattatct acctgtttac agttcatcca tcaccatcac cagatccatt agttactgat 120 cattattttt atatgcgtgc cttgtcattg ttaattacac cacctacaat gttattagcc 180 gccttaagtg gtgaatatgc ctttgattgg aaatcaggtc gtgccaaatc tactattgca 240 gctattatga ttccaacagt ttatctgatt tgggttgatt atgttgccgt tggtcaagat 300 tcatggagta ttaatgatga aaaaattgtt ggttggcgtt tgggtggtgt tttaccaatt 360 gaagaagcaa tgttcttctt gttgactaat ttgatgattg ttttgggttt gagtgcctgt 420 gatcatacac aagccttgta tcttttgcat ggtccaagtc gtccaactgg tatgtatcca 480 ttgccaccac ctccatcttt atcaccagcg gaattagttc gttttttgac agaacgtgtt 540 ccagttcaat atcattttgc ctttcgttta ttagctaaat tgcaaggttt aattcctcgt 600 tatccattag atgaattgtt acgtggttat acaacagacc tgacatttcc attaagtaca 660 gaagccgttc aagcccgtaa aactcctatt gaaacaactg ccgatttgtt agattatggt 720 ttatgtgttg ctggttcagt tgccgaatta ttagtttatg tttcatgggc ctcagcccca 780 tcacaagttc cagctactat tgaagaacgt gaagccgttt tagttgctag tcgtgaaatg 840 ggtacagcct tacaattagt taatattgct cgtgatatta aaggcgatg 889 <210> 5 <211> 552 <212> DNA <213> Artificial Sequence () <400> 5 ggttatgttt gaatttgaat gctggtctca ctgtgatttt tgtgtttggt ctatgcctac 60 atggtttgtt tttttttttg tttttttttt tttgtttgtt tttttgagac tgtcgcccag 120 gctggaatgc agtggcgcca tctcggctca ctgcaagctc tgcctcccag gttcatgtca 180 ttctcctgcc tcagcctccc gagtagctgg gactacaggc gcccaccacc acgcccggct 240 aagtttttgt atttttagta gagacaggat ttcactgtgt tagccaggat ggtctccacc 300 tcctgccctc gtgatccacc tgcctcgacc tcctggaaac tttaacacag ttcaacatgc 360 ttttaatttt gtttgtaggc taagtgtatt ggaaactaca aaattagtgt tatggtaatt 420 gcagaaatat tttgtagcat tggcagtttg ggaaacatgt ttatgtctgt tttattcttt 480 ctgtttttta gagcttatat tctctgtgga agatgtgaca tatccaggcg gaacatcatg 540 atgcagggaa ac 552

Claims

1. A method for gene editing in Saccharomyces cerevisiae cells based on the gene cas7-3 in the type I CRISPR-Cas system, characterized in that: Gene editing of Saccharomyces cerevisiae cells was performed using protein expression plasmids and gene editing vectors constructed using cas7 and cas3 genes in the genome of Streptomyces virginica IBL14; Including the construction of protein expression plasmids, the construction of gene editing vectors, and the acquisition and testing of recombinants. The specific steps are as follows: (1) Construction of protein expression plasmid According to the gene cas7 and cas3 sequences and vector sequences in Streptomyces virginica IBL14, the codon-optimized genes cas7 and cas3 and related primers were synthesized, and the optimized genes cas7 and cas3 were connected to the vector to obtain the protein expression plasmid vector-cas7-3; (2) Construction of gene editing vector Primers were designed based on the DNA sequence information of the targeted gene of Saccharomyces cerevisiae. The genome of Saccharomyces cerevisiae was used as a template. The upstream and downstream homology arm fragments of the targeted gene with restriction endonuclease cutting sites and overlap PCR complementary sequences at the ends were amplified by PCR reaction, and the upstream and downstream homology arms were connected to the gene editing template t-DNA by overlap PCR. At the same time, the targeted gene fragment g-DNA containing a galactose promoter and an RNA terminator at the head and tail were designed and directly synthesized based on the sequence information of the targeted gene of Saccharomyces cerevisiae. The gene editing template t-DNA and the targeted gene fragment g-DNA were connected to the vector according to the sticky ends on the restriction enzyme cutting sites to obtain the gene editing vector vector-t / g-DNA; (3) Acquisition and testing of recombinants Prepare Saccharomyces cerevisiae competent cells, transform or transfect the protein expression plasmid vector-cas7-3 obtained in step (1) and the gene editing vector vector-t / g-DNA obtained in step (2) into target competent cells respectively to obtain candidate gene editing recombinants; perform PCR and gene sequencing or functional analysis on the recombinant chromosomes to obtain correct gene editing recombinants.

2. A method for gene editing in Saccharomyces cerevisiae cells based on gene cas7-3 in type I CRISPR-Cas system according to claim 1, characterized in that: The gene editing refers to the insertion, knockout, and traceless point mutation of the chromosome genes of the Saccharomyces cerevisiae cells.

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