CN112226453B - Schizochytrium CRISPR/Cas9 gene editing system and application thereof - Google Patents
Schizochytrium CRISPR/Cas9 gene editing system and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/70—Vector systems having a special element relevant for transcription from fungi
Abstract
The invention relates to a schizochytrium CRISPR/Cas9 gene editing system and application thereof. The gene editing system is obtained by introducing a regulatory element comprising schizochytrium P into pBS-Zeo plasmid A8047 Cas9 protein regulated by promoter, schizochytrium endogenous nuclear localization signal and terminator T A8047 And from schizochytrium P U6‑2 A promoter-regulated sgRNA transcription cassette. The gene editing system of the invention can realize efficient gene editing in schizochytrium.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to schizochytrium limacinumSchizochytriumsp.) CRISPR/Cas9 gene editing system and application thereof.
Background
Schizochytrium is marine fungus with high DHA yield, and unsaturated fatty acid in grease produced by fermentation has simple components and high DHA content, so that the schizochytrium is a research hot spot. The schizochytrium is mainly synthesized into saturated fatty acid and unsaturated fatty acid by two ways of PKS and FAS, the content of unsaturated fatty acid in the schizochytrium is improved by strain breeding, culture medium optimization and fermentation regulation at present to be in a bottleneck, key enzymes and regulating factors of the intracellular fatty acid synthesis way of the schizochytrium need to be studied deeply, and a schizochytrium genetic transformation system is an important means for researching metabolic mechanism. Therefore, there is a need for a tool that is efficient and allows multiple edits to be made to achieve multiple gene edits within schizochytrium. The CRISPR/Cas9 system is an emerging gene editing tool, has the advantages of low off-target rate, simple and convenient operation, capability of realizing multiple editing and the like, and is widely applied.
Currently, aiming at gene modification of schizochytrium, students at home and abroad mainly adopt a homologous recombination method to carry out over-expression or knockout of genes, for example, a patent 201710327763. X is based on the homologous recombination method, and acyl transferase functional domains in the schizochytrium PKS enzyme are replaced by acyl transferase functional domains in the Shewanella PKS enzyme, so that EPA content in strains is improved; the patent 201710747980.5 and the patent 201710078025.7 also respectively over-express malonate monoacyltransferase genes and knock-out ketosynthase in schizochytrium by adopting a homologous recombination technology, and finally regulate and control the grease accumulation and fatty acid synthesis of strains, but the homologous recombination method has low efficiency. In recent years, CRISPR/Cas technology has remarkable achievement in gene editing and is widely applied to the field of gene editing. CRISPR/Cas systems are the defense systems present in bacteria and archaea to immunize phage and foreign DNA invasion. The CRISPR/Cas system consists of repeated spacer sequences (clustered regularly interspaced short palindromic repeats) and CRISPR-associated genes (Cas). According to different strains, the repeated interval sequence consists of a plurality of repeated sequences of 21-48 bp palindromic sequences and non-repeated interval sequences between 26-72 bp repeated sequences. When the exogenous DNA first invades the bacteria, the Cas nuclease can bind to the exogenous sequence and cleave the DNA duplex under the guidance of the sgRNA, creating a new spacer sequence and inserting into the existing spacer sequence. If the same DNA invades again, the CASCADE complex recognizes, binds and shears, degrading the foreign DNA specifically. However, at present, the gene editing method of Crispr/Cas9 is not widely applied to schizochytrium, so that the CRISPR/Cas9 gene editing system of the schizochytrium is constructed for improving the gene editing efficiency of the schizochytrium, and enzymes of the schizochytrium uracil synthetic pathway are successfully knocked out, so that a uracil-deficient strain is obtained, and technical guidance is provided for the application of the following schizochytrium in synthetic biology.
Disclosure of Invention
The invention aims to provide a CRISPR/Cas 9-based schizochytrium limacinum strainSchizochytriumsp.), the problem of difficult genetic modification of the important oleaginous microorganism is effectively solved.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
CRISPR/Cas9 gene editing system applied to schizochytrium, wherein the CRISPR/Cas9 gene editing system is obtained by introducing a regulatory element into a pBS-Zeo plasmid, and the regulatory element comprises schizochytrium P A8047 Cas9 protein regulated by promoter, schizochytrium endogenous nuclear localization signal and terminator T A8047 And from schizochytrium P U6-2 A promoter-regulated sgRNA transcription cassette.
The P is A8047 The nucleotide sequence of the promoter is shown in SEQ ID NO:1 is shown in the specification; said T A8047 The nucleotide sequence of the terminator is shown in SEQ ID NO: 2. P (P) A8047 Promoters and T A8047 The terminator is a schizochytrium endogenous promoter obtained through screening, belongs to a medium-strength promoter, and can well express a Cas9 sequence.
The nucleotide sequence of the Cas9 protein is shown as SEQ ID NO:3, the nucleotide series of the schizochytrium endogenous nuclear localization signal is shown as SEQ ID NO: 4.
The sgRNA transcription cassette comprises P U6-2 A promoter, a target sequence recognized by sgRNA, and a gRNA scaffold (gRNA scaffold);
further, the P is U6-2 The nucleotide sequence of the promoter is shown in SEQ ID NO:5 is shown in the figure; the nucleotide sequence of the sgRNA scaffold is shown as SEQ ID NO: shown at 6.
Further, the system further comprises an SV40 nuclear localization signal (PKKKRKV), wherein the SV40 nuclear localization signal and the schizochytrium endogenous nuclear localization signal are respectively connected with two ends of the Cas9 protein, so that the efficiency can be improved.
The invention also provides a construction method of the CRISPR/Cas9 gene editing system, which comprises the following steps:
(1) Will P A8047 Promoter, schizochytrium endogenous nuclear localization signal, cas9 protein gene and terminator T A8047 Fusion and original vector pBS-Zeo are carried out to carry out recombination connection to construct vector pBS-Cas9.
(2) Single enzyme cutting the recombinant vector pBS-Cas9 constructed in the step (1) and schizochytrium P U6-2 The sgRNA transcription cassette regulated by the promoter is recombined and connected to construct a CRISPR/Cas9 recombinant vector;
(3) Transferring the CRISPR/Cas9 recombinant vector constructed in the step (2) toE.coliDH5 alpha competent cells, extracting transformant plasmid DNA and verifying; picking up the transformant which is verified to be correct for liquid enrichment, and extracting a large amount of recombinant vector plasmid DNA after culturing, thereby obtaining the CRISPR/Cas9 gene editing system.
In the step (3), the constructed CRISPR/Cas9 recombinant vector is transformed into escherichia coli DH5 alpha competent cells by a heat shock method.
The culture mode is as follows:
single colony is picked in 50mL LB liquid medium, shake cultured at 200rpm and 37 ℃ for 12 hours overnight, and extracted by AXYGENE plasmid small extraction kit to obtain recombinant vector plasmid DNA.
The invention also provides application of the CRISPR/Cas9 gene editing system in schizochytrium gene editing, constructed plasmids are transformed into schizochytrium competent cells, an incubation liquid is coated on a resistance screening flat plate for inversion culture, transformants are selected and enriched in a schizochytrium seed culture medium by single colony, genomic DNA is extracted from the enriched positive clone strain, and PCR verification is carried out to obtain the recombinant strain with knocked-out genes.
After the primary construction of the gene editing system is completed, if the knocked-out target gene needs to be changed, the gene editing system can be realized only by replacing the target sequence identified by the sgRNA in the system.
The invention has the following beneficial effects:
(1) All regulatory elements involved in the knockout vector (Cas 9 expression promoter P A8047 Cas9 gene, terminator T A8047 Promoter P of sgRNA transcription cassette U6-2 The sgRNA recognition target sequence N20 and the transcription box of the gRNA scaffold) are constructed on the same vector, so that the operation is convenient and quick; the constructed CRISPR/Cas9 gene editing system has simple design and low cost.
(2) The expression of Cas9 protein and sgRNA is controlled by adopting an endogenous promoter of schizochytrium, so that the expression effect can be improved;
(3) The Cas9 protein is designed to be more efficiently introduced into the nucleus for cleavage by introducing an endogenous nuclear localization signal into the vector. In addition, the general SV40 nuclear positioning signal can be added to assist positioning, so that the efficiency is improved.
(4) Screened Cas9 expression promoter P A8047 Terminator T A8047 Cas9 can be well expressed, belongs to a medium-strength promoter, and does not generate cytotoxicity due to over-expression of Cas9 caused by over-expression.
(5) The schizochytrium strain with uracil deficiency is obtained based on the Crispr/Cas9 system, and an excellent experimental material is provided for subsequent genetic modification of the schizochytrium strain.
Drawings
FIG. 1 is a schematic diagram of the schizochytrium Crispr gene knockout plasmid constructed in this patent.
FIG. 2 shows growth of primordial and knock-out bacteria on 5-FOA and uracil-deficient plates.
FIG. 3 is a morphological diagram of the original bacteria and the knocked-out bacteria.
Detailed Description
In order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. The specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the invention.
The following examples are directed to gene knockout systems and gene knockout recombinant bacteria using the gene pyrF encoding schizochytrium OPRT enzyme as an example.
The sources of the biological materials in the invention are as follows:
schizochytrium limacinum @Schizochytriumsp.) HX-308, which has been disclosed in patent CN104974944B of the previous application of the laboratory, with a preservation number of CCTCC No. m 209059;
plasmid pBS-Zeo, disclosed in the present laboratory prior application, patent CN 104974944B;
the plasmid pUC-Cas9 is constructed by connecting a codon optimized Cas9 sequence to a pUC57 plasmid, and specific construction modes can be referred to the molecular cloning experiment guidelines; wherein the Cas9 sequence is a codon-optimized Cas9 sequence synthesized by a consignment gene company, and the nucleotide sequence of the Cas9 sequence is shown as SEQ ID NO:3 is shown in the figure;
plasmid pUC-fFuCas9-HTB NLS Hph has been disclosed in the prior paper ACS synth. Biol 2019, 8, 445-454 by the present laboratory.
Example 1 CRISPR/Cas9 Gene editing System and method of constructing the same
Construction of Cas expression vectors according to the plasmid map of fig. 1 with schizochytrium endogenous promoter P4 (i.e., P A8047 ) Cas9 expression is guided, schizochytrium endogenous promoter U6-2 directs expression of the sgRNA sequence, and endogenous nuclear localization signal e-NLs is expressed, for example, as follows:
(1) Expression of Cas9 promoter P A8047 NLS, cas9 Gene, terminator T A8047 To pBS-Zeo;
the primers pbs-lb-uc pro F/pro-cas 9R, egfp-ter F/egfp-ter-RB-pbs R, cas9-nls F/e-nls R are used as template for amplifying the promoter P, respectively A8047 Terminator T A8047 And NLS, amplifying Cas9 gene by using primer 40aa EDNLS-dCS 9F/Cas 9-egfp R and taking plasmid pUC-Cas9 as template, purifying and recovering the four fragments, recombining the four fragments into pBS-Zeo vector recovered by double digestion (BgI II/EcoRI) by using one-step cloning enzyme, and connecting the products by a heat shock methodTransformed into E.coli DH 5. Alpha. Competent cells. After colony PCR and sequencing verification, the positive clone is picked, which shows that the construction of the Cas9 expression element is successful, and the recombinant vector pBS-Cas9 is obtained.
(2) sgRNA transcription cassette promoter P U6-2 The sgRNA recognizes the target sequence N20 and the gRNA scafold recombines into the pBS-Cas9 vector.
The schizochytrium genome DNA is used as a template, and a primer U6-2F/U6-2R is adopted to amplify a promoter P U6-2 In pUC-fFuCas9-HTB NLS The hph plasmid is used as a template, a primer gRNA-U62F/U6-2 gRNA R is adopted to amplify a gRNA scaffold fragment, an sgRNA recognition target sequence N20 is artificially synthesized, the PCR fragment is purified and recovered, then the PCR fragment is connected with a single-enzyme-digested (Not I) pBS-Cas9 carrier, and a connection product is converted into escherichia coli DH5 alpha competent cells by a heat shock method. After colony PCR and sequencing verification, the positive clone is selected to show that the construction of the sgRNA expression cassette is successful, and after further sequencing verification, the recombinant vector pBS-Cas 9-gRNA 1 (U6-2) is obtained (as shown in figure 1).
The connection system and the conditions of the step (1) and the step (2) are as follows: one Step Cloning Kit kit from Nanjinofizan Co., ltd, vector n bp after cleavage 0.02 ng, fragment n bp 0.02 ng,ExnaseMultiS 2uL, 5 XCE MultiS Buffer 4 uL, ddH2O up to 20uL,37℃for 30 min.
After the system is initially constructed, if the knocked-out target gene needs to be changed, the N20 sequence in the system is only needed to be replaced, and the specific operation method is as follows:
the N20 sequence is replaced by taking the designed gene sequence of the target site as a template, synthesizing 1 primer, directly amplifying by taking the pBS-Cas 9-gRNA 1 (U6-2) plasmid as the template by utilizing the synthesized primer and the primer U6-2 gRNA R, and directly recombining and connecting the amplified primer into the pBS-Cas9 plasmid to further guide the specific cutting of the Cas9 protein to a new target gene in the genome.
If the promoter for guiding the expression of the Cas9 protein needs to be changed, the forward sequence of the Cas9 is directly replaced and is re-integrated into the vector;
if the promoter for guiding the gRNA expression is required to be changed, directly replacing the sequence in front of the gRNA and re-integrating the sequence into the vector;
if different nuclear localization signals need to be compared, the nuclear localization signal sequences at the N end and the C end of the Cas9 sequence need to be replaced.
The primers used in the present invention are shown in the following table:
table 1 major primers and nucleotide sequences
| Primer name | Sequence information (5 '-3') | SEQ ID |
| pbs-lb-uc pro F | ttttggtcatgcatgagatcagatctgtttacaccacaatatatcctgcca | 7 |
| pro-cas9 R | gtacttcttgtccatcttggccttgtcttgtttcctgc | 8 |
| cas9-nls F | agacaaggccaagatggacaagaagtactcgattggc | 9 |
| e-nls R | caccttgtaaatgtacgaggagtag | 10 |
| 40aa EDNLS -dCas9 F | acaaggtgatggacaagaagtactcgattggc | 11 |
| cas9-egfp R | cccttggagaccatgactttacgttttttcttcggcgaacc | 12 |
| egfp-ter F | aaaaacgtaaagtcatggtctccaagggcgagg | 13 |
| egfp-ter-RB-pbs R | ataagcttgatatcgaattctgacaggatatattggcgggtaaaccggaaa | 14 |
| U6-1 F | agctccaccgcggtggcggccgctgacgtcgattttattggcagt | 15 |
| U6-1R | ctgcgtgggcgagagtgggtaactcttacttatatagtataagc | 16 |
| gRNA-U61 F | ttacccactctcgcccacgcagaccagcgagt | 17 |
| U6-1 gRNA R | tccactagttctagagcggccgcaaaaaaagcaccgactcggtgcc | 18 |
| U6-2 F | agctccaccgcggtggcggccgctgacgtcgattttattggcagt | 19 |
| U6-2 R | ctgcgtgggcgagagtgggtaactcttacttatatagtataagc | 20 |
| gRNA-U62F | ttacccactctcgcccacgcagaccagcgagt | 21 |
| U6-2 gRNA R | tccactagttctagagcggccgcaaaaaaagcaccgactcggtgcc | 22 |
| OPRT F | atcctctgcagcggagtcgaga | 23 |
| OPRT R | ttcggcttgtacgcggcggcca | 24 |
| P1-T-F | ttagtccgacttggccttggttacttgtagagctcgtcca | 25 |
| P1-T-R | ggtttgaattctcggtaccaccgcgtaatacgact | 26 |
| P2-P-F | gctttagatcttctcatcttggtcatcttgc | 27 |
| P2-P-R | tcctcgcccttggagaccatcttttcacgagctgttgtgg | 28 |
| P3-P-F | gctttagatctgctcgagaccgagctctcctcct | 29 |
| P3-P-R | gcccttggagaccatcttcggttcttctcgctgc | 30 |
| P4-P-F | ggtttagatctatgtccagtggaggcagagagcc | 31 |
| P4-P-R | cccttggagaccatcttggccttgtcttgtttcctgc | 32 |
Example 2 Effect of different Nuclear localization signals on knockout efficiency
Different endogenous nuclear localization signal sequences were replaced according to the method of example 1, and the effect of different combinations of nuclear localization signals on the knockout effect was examined, and the genome editing effect was obtained from the number of positive clones. The results are shown in Table 2. (endogenous localization signal was linked to the N-terminus of Cas9 protein and SV40 was linked to the C-terminus in the examples)
TABLE 2 genome editing effects of different Nuclear localization signals
| Nuclear localization signal | Genome editing effects |
| e-NLS | ++ |
| SV40 | + |
| e-NLS+SV40 | +++ |
Therefore, the editing efficiency of the schizochytrium endogenous nuclear localization signal sequence is higher than that of the traditional SV40 sequence, and the editing efficiency can be greatly increased by using the two nuclear localization signals together.
Example 3 Effect of different promoters on knockout efficiency by controlling Cas9 protein and sgRNA expression
Four promoters P1-P4 (SEQ ID NOS: 33-36) were obtained with the primer sequences in Table 1 according to the method of example 1 to initiate the expression of Cas9 protein, examine the effect thereof on the knockdown effect, the expression level was obtained by RNA sequencing or RT-PCR method, and the genome editing effect was obtained according to the number of positive clones. The results are shown in Table 3.
TABLE 3 editing effects of different promoter genome
| Different promoters | Expression level | Genome editing effects |
| P1 | 277.11 | ++ |
| P2 | 132.56 | + |
| P3 | 37302.13 | + |
| P4 | 914.51 | +++ |
| U6-1 | - | + |
| U6-2 | - | +++ |
It can be seen that the use of the medium strength promoter P4 (i.e.P A8047 ) Starting the expression of the Cas9 protein, wherein the editing efficiency is highest; the U6-2 promoter facilitates the expression of sgRNA.
Example 4 CRISPR/Cas 9-based schizochytrium pyrF gene knockout
The pyrF gene knockout vector successfully constructed in example 1 was transformed into schizochytrium HX-308 competent cells (sucking the competent cells and vector DNA mixture into a 2min pre-cooled electric rotating cup, standing in ice bath for 10min, performing electric shock at a voltage of 1.65kv, rapidly adding into 1mL of schizochytrium seed culture medium, mixing well, incubating, and resuscitating at 30 ℃ and 170rpm for 4 h); and (3) coating a proper amount of incubation liquid on a 5-FOA resistance screening plate with the concentration of 1.0g/L, inversely culturing at 28 ℃ for 48 hours, picking up transformants, enriching in a single colony and schizochytrium limacinum seed culture medium (50 mL), extracting genome DNA from the enriched positive clone strain, and carrying out PCR verification by using OPTR-F/OPRT R primers to obtain pyrF gene knocked-out recombinant strains MT10-1 and MT 9-2. The strain shows uracil deficiency character, and proves that uracil genes in recombinant bacteria are destroyed (shown in figures 2 and 3).
The above examples only represent one embodiment of the invention, and several variations, combinations and modifications of the above-described embodiments, both more specific and detailed, are possible, all falling within the scope of protection of the present patent. Therefore, the protection scope of the patent is subject to the claims.
Sequence listing
<110> university of Nanjing Industrial science
<120> schizochytrium CRISPR/Cas9 gene editing system and application thereof
<130> xb20102901
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 800
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tttgtttctt acatcaattc attcgttcgc tcacgcactc cttctttatt actattagaa 60
ggcgcgcaca cccgcacacc cacactctct ccccacaccc gccccagcgc gcgcgcaaag 120
agctagagtg aagaagggag agggggggca gagagagaga aagaaaccaa agaaaaagaa 180
ggaaagaaag aaagaaagaa agaaagaaag aaagaaagaa aacaaaacaa aggatggtaa 240
aagaagtccc ctccactcta ctccactcca ctcctctcca ctccactcca ctccactcct 300
ctccactcct cgccactcag ctgctctcca gtccgctcga cgctgcgctc tctccagtct 360
cgggtctcgc cgtgtccgca ggcgcacgcg gcgcagcacg cggctctgtg cgcggccccg 420
tcttctctgg cgccgcccgc agggaggctc cctctgctcc gcaggaaggg aggaaggtgc 480
tcgcgcggcc cgcgctctct cctctctcct ttcctctctg tgtctgctgc tcgcttgggt 540
gaactttttt ttaaactttg ttacctttgc acccatttcc cgctcgcctc ggctgtttct 600
cgcctccggg gccagagaga gaggcggcct ctgcacgcag caaacaccat tcgagggcca 660
gcaggcagtc ccgtgcagaa cgagaccggc cgtgctttcg ccttgctttg cgttgccagg 720
aacagcagca gcagcagcaa gagcaagagc aagagcaacg gcagcaggtt taccttcgga 780
ggaccagaac cagagaaacc 800
<210> 2
<211> 1189
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggtctcca agggcgagga gctctttacc ggcgtcgtcc ctattctcgt cgagctcgac 60
ggcgacgtca acggccacaa gttttccgtc tccggcgagg gcgagggcga cgccacctac 120
ggcaagctca ccctcaagtt tatttgcacc accggcaagc tccccgtccc ttggcctacc 180
ctcgtcacca ccctcaccta cggcgtccag tgcttttccc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gagggctacg tccaggagcg caccattttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtca agtttgaggg cgacaccctc 360
gtcaaccgca ttgagctcaa gggcatcgac tttaaggagg acggcaacat tctcggccac 420
aagctcgagt acaactacaa ctcccacaac gtctacatca tggccgacaa gcagaagaac 480
ggcatcaagg tcaacttcaa gatccgccac aacattgagg acggctccgt ccagctcgcc 540
gaccactacc agcagaacac ccctattggc gacggccctg tcctcctccc cgacaaccac 600
tacctctcca cccagtccgc cctctccaag gaccctaacg agaagcgcga ccacatggtc 660
ctcctcgagt ttgtcaccgc cgccggcatt accctcggca tggacgagct ctacaagtaa 720
atggtaaacc cggtttttgt acttttctga ctttttcact cctctctctt gctccttttg 780
tctttttgct gattgtttga gcctgaggcg agttattggt gactggacct tgtgtgcctt 840
gttttgcccc cttcttgttt tcaagggtga tcaagccgaa gcagagctga tttttgcttg 900
ggtccacaac caagatcgag gatggaggat ggcgcgccct cggaggacgt cttgcgcgag 960
agcggcggca gcgcaggggg cctcgtgacc gtggtgctgg atatgaacga agctctttgg 1020
gcgcaagtgt cggccaaggg cggcgaggac aacgtgacgc tcgcgagcgc cctcagcagc 1080
gtggtggtct ttctcaacgc cgtgcagctc atggaccgtg aaagcgaggt ctgtgtcgtg 1140
gcagagtgcc ccgacggcac tgcgcgcgta cttgctcgtg gtgtttccg 1189
<210> 3
<211> 4131
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atggacaaga agtactcgat tggcctcgat atcggcacca acagcgtcgg ttgggcggtg 60
atcacggacg agtacaaggt ccccagcaag aagttcaagg tcctcggcaa tacggatcgc 120
cattcgatta agaaaaacct tatcggcgcc ctcctctttg atagcggcga gacggccgag 180
gcgacccgcc tcaaacgcac cgcccgccgt cgctacaccc gtcgtaagaa ccgcatctgc 240
tacctccaag agatcttttc gaatgagatg gccaaggtgg acgactcgtt tttccaccgc 300
cttgaggagt cgttcctcgt cgaggaggat aagaagcacg agcgccatcc gattttcggt 360
aatatcgtcg acgaggtggc gtaccacgag aaatatccca ccatctacca cctccgcaag 420
aaactcgtgg actcgacgga caaggcggac ctccgcctca tctacctcgc gctcgcccac 480
atgattaaat tccgcggcca tttcctcatc gagggcgatc ttaaccccga caacagcgac 540
gtggacaagc tcttcatcca gctcgtccag acgtacaacc aactctttga ggagaacccc 600
attaatgcct cgggcgtgga tgcgaaggcc atcctttcgg cgcgtctcag caagagccgc 660
cgcctcgaaa atcttatcgc ccagctcccc ggtgagaaaa agaacggtct cttcggcaac 720
cttattgcgc tttcgcttgg cctcaccccc aacttcaaat cgaatttcga cctcgcggag 780
gacgccaagc tccagctctc gaaggacacg tacgatgatg acctcgacaa ccttctcgcc 840
cagatcggtg accagtacgc cgacctcttc ctcgcggcca aaaaccttag cgacgccatc 900
ctcctttcgg acattctccg cgtcaacacc gaaattacga aggccccgct ctcggccagc 960
atgatcaaac gctacgacga gcatcaccaa gatcttacgc ttctcaaagc cctcgtccgt 1020
caacagctcc cggagaagta caaggagatc ttcttcgacc aatcgaagaa cggttacgcc 1080
ggctatatcg atggcggcgc ctcgcaagaa gagttctaca agttcatcaa gcccatcctc 1140
gaaaagatgg acggtacgga ggagctcctc gtcaagctca accgcgaaga ccttctccgt 1200
aagcaacgca cgtttgataa cggcagcatc ccgcaccaga tccaccttgg cgagcttcat 1260
gccattcttc gtcgtcaaga agacttttat ccctttctca aggacaaccg cgagaagatt 1320
gagaagattc tcaccttccg catcccctac tacgtgggtc cccttgcccg cggcaattcg 1380
cgcttcgcgt ggatgacccg caagagcgag gagaccatca cgccttggaa ctttgaggaa 1440
gtcgtggaca agggcgcgtc ggcccagagc ttcattgagc gtatgacgaa cttcgacaag 1500
aacctcccca atgagaaggt cctccccaag cattcgctcc tctacgagta tttcacggtc 1560
tacaatgaac tcaccaaggt caaatacgtc accgagggca tgcgtaagcc cgcctttctt 1620
tcgggcgaac agaaaaaggc catcgtcgac ctccttttca agacgaaccg taaggtcacg 1680
gtcaagcaac tcaaagagga ttatttcaag aagatcgagt gcttcgactc ggtcgagatt 1740
tcgggcgtgg aagaccgttt taacgcctcg cttggtacct atcacgacct tctcaagatc 1800
attaaggata aggacttcct cgacaacgag gagaatgagg acatccttga ggacatcgtc 1860
cttacgctta cccttttcga agaccgtgag atgattgagg agcgcctcaa aacctacgcc 1920
cacctcttcg acgataaggt catgaaacaa ttaaaacgtc gccgttatac gggttggggc 1980
cgcctctcgc gcaagctcat caacggcatc cgtgacaagc aaagcggcaa gaccattctc 2040
gacttcctca agtcggacgg ctttgccaat cgtaacttca tgcagctcat ccacgacgat 2100
tcgctcacgt tcaaggagga tattcagaaa gcccaagtct cgggccaagg tgatagcctt 2160
cacgagcata ttgccaacct cgcgggttcg cccgccatta agaagggtat ccttcagacg 2220
gtcaaggtgg tggacgagct cgtcaaggtc atgggtcgtc acaagcccga gaacatcgtc 2280
atcgaaatgg cgcgtgagaa tcagaccacc cagaagggcc aaaaaaattc gcgtgagcgt 2340
atgaaacgca ttgaggaggg tatcaaggag ctcggctcgc agatcctcaa ggaacacccg 2400
gtggagaaca cgcaactcca gaatgagaag ctctaccttt actacctcca gaatggccgt 2460
gatatgtacg tggatcaaga gctcgatatc aaccgcctca gcgactacga tgtggaccac 2520
atcgtcccgc agagctttct taaggatgat agcatcgaca acaaggtcct tacccgtagc 2580
gacaagaacc gtggtaagtc ggacaacgtc ccgtcggagg aggttgtgaa gaaaatgaag 2640
aactactggc gccagctcct taacgcgaaa cttatcaccc agcgcaagtt cgataacctt 2700
accaaggcgg agcgcggcgg tctctcggaa cttgacaagg cgggcttcat caaacgccag 2760
ctcgtcgaaa cgcgccagat cacgaagcac gtggcgcaaa tcctcgacag ccgcatgaac 2820
accaaatatg atgaaaacga caaacttatt cgcgaagtga aggtgatcac gctcaaatcg 2880
aagctcgtgt cggacttccg caaggacttc cagttctaca aggtgcgcga gatcaataac 2940
taccaccatg cgcacgacgc ctacctcaac gcggtcgtcg gtacggccct tattaagaag 3000
tacccgaaac tcgaatcgga gtttgtgtac ggtgactaca aggtgtacga tgtgcgcaag 3060
atgatcgcca agagcgagca agaaatcggc aaggcgacgg cgaagtactt cttttactcg 3120
aacatcatga acttcttcaa gacggaaatt acgcttgcga acggcgagat ccgcaaacgt 3180
ccccttattg agacgaacgg tgagacgggt gaaatcgtgt gggacaaagg tcgcgacttt 3240
gccaccgtgc gcaaggtcct ctcgatgccg caagttaaca tcgtgaaaaa aaccgaggtc 3300
caaaccggtg gtttcagcaa agagagcatc cttcccaagc gtaactcgga caaacttatc 3360
gcccgcaaga aggattggga tcccaagaag tacggcggct ttgatagccc gaccgtggcc 3420
tattcggtcc tcgtcgtggc caaggtcgaa aagggcaagt cgaagaaact taaaagcgtc 3480
aaggaactcc tcggcattac gatcatggag cgtagcagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaagggtta caaagaagtg aaaaaggatc tcattattaa gctcccgaag 3600
tacagcctct ttgagctcga gaacggccgt aagcgtatgc tcgccagcgc cggcgaactt 3660
cagaagggca acgagcttgc cctcccgtcg aaatacgtca acttcctcta cctcgccagc 3720
cactacgaaa agctcaaagg ttcgcccgag gataacgagc agaaacagct ctttgtcgag 3780
cagcataaac actacctcga tgagattatt gagcaaattt cggagttttc gaagcgcgtg 3840
atcctcgcgg acgcgaacct cgacaaagtc ctctcggcct acaataagca ccgcgataaa 3900
ccgattcgcg aacaagccga aaacatcatc catctcttca cgctcacgaa cctcggcgcc 3960
ccggccgcgt ttaaatactt tgataccacc attgaccgca agcgctatac gagcaccaag 4020
gaggtcctcg atgccacgct tatccaccag agcattacgg gcctttacga gacgcgcatc 4080
gatcttagcc aactcggtgg cgacggttcg ccgaagaaaa aacgtaaagt c 4131
<210> 4
<211> 120
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atggccggcg gatccaaggg cgtgagcaag aaggcagcca aggccaccaa ggcctcgggc 60
gacaagtcga agaagcgctc gaagcgcacg gagacctact cctcgtacat ttacaaggtg 120
<210> 5
<211> 300
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tgacgtcgat tttattggca gtcacaagac gatcacgctc agcacggggc cttacacgcc 60
ctttacgcac tggcaccaat gccgcatgct cctccccgag cccattgccg tgaacaaggg 120
tcagacagtg gagggaacga tccacatggt tgccaacgag aagttttcct acacgatcaa 180
tctcgaggtt gcactcaagg gcaccgacgt gaagtcgtcc aacgtcattc ttttgcagga 240
gcaattctac cactatcttc agggctcgcc gtctttctct gccgagtgaa cgcagctgga 300
<210> 6
<211> 80
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt 80
<210> 7
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ttttggtcat gcatgagatc agatctgttt acaccacaat atatcctgcc a 51
<210> 8
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gtacttcttg tccatcttgg ccttgtcttg tttcctgc 38
<210> 9
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
agacaaggcc aagatggaca agaagtactc gattggc 37
<210> 10
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
caccttgtaa atgtacgagg agtag 25
<210> 11
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
acaaggtgat ggacaagaag tactcgattg gc 32
<210> 12
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
cccttggaga ccatgacttt acgttttttc ttcggcgaac c 41
<210> 13
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
aaaaacgtaa agtcatggtc tccaagggcg agg 33
<210> 14
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ataagcttga tatcgaattc tgacaggata tattggcggg taaaccggaa a 51
<210> 15
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
agctccaccg cggtggcggc cgctgacgtc gattttattg gcagt 45
<210> 16
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ctgcgtgggc gagagtgggt aactcttact tatatagtat aagc 44
<210> 17
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ttacccactc tcgcccacgc agaccagcga gt 32
<210> 18
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
tccactagtt ctagagcggc cgcaaaaaaa gcaccgactc ggtgcc 46
<210> 19
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
agctccaccg cggtggcggc cgctgacgtc gattttattg gcagt 45
<210> 20
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
ctgcgtgggc gagagtgggt aactcttact tatatagtat aagc 44
<210> 21
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
ttacccactc tcgcccacgc agaccagcga gt 32
<210> 22
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
tccactagtt ctagagcggc cgcaaaaaaa gcaccgactc ggtgcc 46
<210> 23
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
atcctctgca gcggagtcga ga 22
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ttcggcttgt acgcggcggc ca 22
<210> 25
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ttagtccgac ttggccttgg ttacttgtag agctcgtcca 40
<210> 26
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggtttgaatt ctcggtacca ccgcgtaata cgact 35
<210> 27
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gctttagatc ttctcatctt ggtcatcttg c 31
<210> 28
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tcctcgccct tggagaccat cttttcacga gctgttgtgg 40
<210> 29
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gctttagatc tgctcgagac cgagctctcc tcct 34
<210> 30
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gcccttggag accatcttcg gttcttctcg ctgc 34
<210> 31
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ggtttagatc tatgtccagt ggaggcagag agcc 34
<210> 32
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
cccttggaga ccatcttggc cttgtcttgt ttcctgc 37
<210> 33
<211> 800
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tttgtttctt acatcaattc attcgttcgc tcacgcactc cttctttatt actattagaa 60
ggcgcgcaca cccgcacacc cacactctct ccccacaccc gccccagcgc gcgcgcaaag 120
agctagagtg aagaagggag agggggggca gagagagaga aagaaaccaa agaaaaagaa 180
ggaaagaaag aaagaaagaa agaaagaaag aaagaaagaa aacaaaacaa aggatggtaa 240
aagaagtccc ctccactcta ctccactcca ctcctctcca ctccactcca ctccactcct 300
ctccactcct cgccactcag ctgctctcca gtccgctcga cgctgcgctc tctccagtct 360
cgggtctcgc cgtgtccgca ggcgcacgcg gcgcagcacg cggctctgtg cgcggccccg 420
tcttctctgg cgccgcccgc agggaggctc cctctgctcc gcaggaaggg aggaaggtgc 480
tcgcgcggcc cgcgctctct cctctctcct ttcctctctg tgtctgctgc tcgcttgggt 540
gaactttttt ttaaactttg ttacctttgc acccatttcc cgctcgcctc ggctgtttct 600
cgcctccggg gccagagaga gaggcggcct ctgcacgcag caaacaccat tcgagggcca 660
gcaggcagtc ccgtgcagaa cgagaccggc cgtgctttcg ccttgctttg cgttgccagg 720
aacagcagca gcagcagcaa gagcaagagc aagagcaacg gcagcaggtt taccttcgga 780
ggaccagaac cagagaaacc 800
<210> 34
<211> 800
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
tctcatcttg gtcatcttgc gcgtctcgtc tcatctcatc tcgtcccagc ccgtcgtggc 60
cgtcctgctc gccgcgtctc gctcgacgct ggccggcggg gcgcgagaaa cgcccggctg 120
cgcgtccgcc cgtgcgtccg tcggccgcgg tccgagccgg ggcggggtcg cacgcactcg 180
aggtgacgga gcagcggaga agagaggcag tcccggccaa gattccgcag cttgcaagcg 240
gctgcggagt cgaacgacgg caagcgggag gaagttcaag ttggacgggg aagaaaaagc 300
tcctaagctg acctgcgggc tgccaagacg gccgagcttg aagccttgat ggtctgggga 360
cggcgagcga gatccaggct gggccggcca aggacgaact cgacgccgtc ctggccttgt 420
tgctgctgct gatgatggat gaatgacgga tgatggatga tggatgatgc tgggcttgag 480
cgtcgcgttg aagaaggacg acttcaggaa tggtgctttt cgaggtcttg gccgcgagcg 540
tggcgggggg cacaagaaac gactcctggc gggacgagat gatctgcttt gcgtcctggc 600
aggctcacgg gacacggagt cggcgtatcg gatagtagac tgactggcag acaggtcgag 660
gaaggatcac aaagaaagat tccacacgca gatctggaga gcagaaccgc aacgctgagc 720
tgaaagatcg cgaccctacg aggcggaagc aggaccagca caagaggcga agaagcttta 780
ccacaacagc tcgtgaaaag 800
<210> 35
<211> 812
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
tcggtacccg ttagaacgcg taatacgact cactataggg agagtcgact gagcacaact 60
ctgctgcgag cgggcctcga gagcgtttgc ttcgagccgc ggagcaaggg ggatggatcg 120
ctcatgcggt cgtgcggccc tcggtcaccc ggtgggtcct gcactgacgc atctgttctg 180
atcagacaca cgaacgaaca aaccgaggag ccgcagcgcc tggtgcaccc gccgggcgtt 240
gttgtgtgct cttcttgcct ccgagagaga gagcggagcg gatgcatagg aaatcgggcc 300
acgcgggagg gccatgcgtt cgccccacac gccactttcc acgcccgctc tctctccggc 360
cggcaggcag cgcataactc tccgacgctg gcaggctggt agcaactggc agggacaact 420
cgcgcgcggg tcccggtcgt tcgatgtgcc aacccgagag aatccagcca gcagggcggt 480
tggcctcatc gcccacctgc tatggtgcag cgaaccaact cccgaagcgg ccggttctgc 540
gattccctct tctgaattct gaattctgaa ctgattccgg aggagaaccc tctggaagcg 600
cgggttgcct ctccagttct gccgaactag acaggggagt gagcagagag tgaccctgac 660
gcggagcgag ctggttgctg gaaaagtcgc gaacgctggg ctgtgtcacg cgtccacttc 720
gggcagaccc caaacgacaa gcagaacaag caacaccagc agcagcaagc gacctaagca 780
acactagcca acatgactga ggataagacg aa 812
<210> 36
<211> 500
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gaagaggggg taagcgagtc tgaacaagga gaagcggaag ttgacattga ggtcggcgag 60
aaccagccta aacaagatgg ctcgaagggt gatcagccgc aggggactgt ccttgcatcg 120
acttcctccg gtactcaagc ttcttcatac aagggtgacg atctcggcat tgacagttcg 180
gtttgcgagg aagacgccgt atccacttca caaacttcgc acctctcgca aaagcgccga 240
gcatcgagtg gcagacttcg cccgatgcgc aactccatcg catcttcgta atttacacgg 300
ggcttgtgtg accctatttc ggacaaaaga ttgcttcaca cgcttccatc tctttttttc 360
cattcacatt tatactatga cccaataagt tactcgtttt gttttgttgg ctcgtctttc 420
tttatgcggc tgtttcgctt cggcagcaca attgcctttt gataccgccg caatcgtgtc 480
acatcagtct aatttctgat 500
Claims (6)
1. A first partThe schizochytrium CRISPR/Cas9 gene editing system is characterized in that the system is obtained by introducing a regulatory element into pBS-Zeo plasmid, wherein the regulatory element comprises schizochytrium P A8047 Cas9 protein regulated by promoter, schizochytrium endogenous nuclear localization signal and terminator T A8047 And from schizochytrium P U6-2 A promoter-regulated sgRNA transcription cassette;
the P is A8047 The nucleotide sequence of the promoter is shown in SEQ ID NO:1 is shown in the specification; said T A8047 The nucleotide sequence of the terminator is shown in SEQ ID NO:2 is shown in the figure; the nucleotide sequence of the Cas9 protein is shown as SEQ ID NO:3, the nucleotide series of the schizochytrium endogenous nuclear localization signal is shown as SEQ ID NO:4 is shown in the figure;
the sgRNA transcription cassette comprises P U6-2 A promoter, a target sequence recognized by sgRNA, and a gRNA scaffold; the P is U6-2 The nucleotide sequence of the promoter is shown in SEQ ID NO:5 is shown in the figure; the nucleotide sequence of the sgRNA scaffold is shown as SEQ ID NO: shown at 6.
2. The schizochytrium CRISPR/Cas9 gene editing system of claim 1, further comprising an SV40 nuclear localization signal, the SV40 nuclear localization signal and the schizochytrium endogenous nuclear localization signal being linked to both ends of the Cas9 protein, respectively.
3. The method for constructing a CRISPR/Cas9 gene editing system according to any one of claims 1 to 2, characterized by comprising the steps of:
(1) Will P A8047 Promoter, schizochytrium endogenous nuclear localization signal, cas9 protein gene and terminator T A8047 Recombinant ligation to pBS-Zeo vector construction recombinant vector pBS-Cas9;
(2) Single enzyme cutting the recombinant vector pBS-Cas9 constructed in the step (1) and schizochytrium P U6-2 The sgRNA transcription cassette regulated by the promoter is recombined and connected to construct a CRISPR/Cas9 recombinant vector;
(3) And (3) converting the CRISPR/Cas9 recombinant vector constructed in the step (2) into a competent cell of escherichia coli DH5 alpha, and extracting recombinant plasmid DNA after culturing to obtain the CRISPR/Cas9 gene editing system.
4. The method of claim 3, wherein in step (3), the constructed CRISPR/Cas9 recombinant vector is transformed into escherichia coli DH5 a competent cells by a heat shock method.
5. The method of claim 3, wherein in step (3), the culturing is performed as follows:
single colony is picked in 50mL LB liquid medium, shake cultured at 200rpm and 37 ℃ for 12 hours overnight, and extracted by AXYGENE plasmid small extraction kit to obtain recombinant vector plasmid DNA.
6. Use of the CRISPR/Cas9 gene editing system of any one of claims 1-2 in schizochytrium gene editing.
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