CN111850011B - Improved Csy4 sequence, improvement method and application - Google Patents

Improved Csy4 sequence, improvement method and application Download PDF

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CN111850011B
CN111850011B CN202010591446.1A CN202010591446A CN111850011B CN 111850011 B CN111850011 B CN 111850011B CN 202010591446 A CN202010591446 A CN 202010591446A CN 111850011 B CN111850011 B CN 111850011B
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张运生
杨品红
刘良国
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Hunan University of Arts and Science
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Abstract

The invention discloses an improved Csy4 sequence, an improvement method and an application thereof in optimization and multi-gene knock-out of zebra fish so as to improve the expression efficiency of the Csy4 sequence in the zebra fish.

Description

Improved Csy4 sequence, improvement method and application
Technical Field
The invention relates to an improved Csy4 sequence, an improvement method and application thereof in optimization of zebra fish and multi-gene knock-out, belonging to the field of molecular biology.
Background
The ZFN, TALEN and CRISPR/Cas9 technologies are utilized to realize the directional knockout of a single gene in a plurality of species, but for the research on the aspects related to family genes, the interaction between genes and the regulation and control of multiple genes, two or even a plurality of genes need to be researched simultaneously in one individual, and the acquisition of a multigene mutant is particularly important. Multiple editing of a single gene or hybridization of a single gene mutant can also be carried out by a gene editing technology to obtain a multigene mutant, but the workload is heavy, the difficulty is increased, and some linked genes cannot be realized by a hybridization method. The CRISPR/Cas9 technology has great advantages in multi-gene multi-site targeting, and a plurality of multi-gene editing technologies based on the CRISPR/Cas9 technology are successively developed. The multiple gene editing technology mediated by the Csy4 is a fast-developing one with wide application.
Csy4, also known as Cas6f, is an rnase that functions during crRNA generation in the original CRISPR/Cas9 system, but is particularly characterized by recognition and cleavage of only one specific RNA sequence, and by cleavage at a specific site in this sequence (Haurwitz et al, 2012). This specificity of Csy4 is the basis on which it is used for multigene editing. The U6 promoter drives the Csy4 target sequence to be expressed in tandem with two or even multiple sgRNA intervals, and multiple sgRNAs separated by the Csy4 target sequence can be cut by Csy4 protein to form multiple active sgRNAs, so that the simultaneous editing of multiple genes can be realized (Tsai et al, 2014; Nissim et al, 2014; Wyvekens et al, 2015; Qin et al, 2015).
The zebra fish breeding cost is low, the breeding cycle is short, the egg laying amount is large, and the zebra fish breeding technology has the advantages of transparent embryos, in vitro fertilization, synchronous embryo development and the like. However, most of the existing Csy4 gene sequences are designed aiming at codon preference of mammals, the expression efficiency in zebra fish is low, further application of Csy4 in the multi-gene knockout of zebra fish is prevented, and in order to solve the problem, the Csy4 gene is optimized, so that the efficiency of Csy4 in the multi-gene knockout of zebra fish is improved.
Disclosure of Invention
In view of the defects in the prior art, the invention aims to provide an improved Csy4 sequence, an improved method and an application thereof in optimization and multi-gene knock-out of zebra fish so as to improve the expression efficiency of the Csy4 sequence in zebra fish.
In order to achieve the purpose, the invention adopts the technical scheme that: an improved Csy4 sequence, wherein the base sequence is shown in SEQ ID NO. 1.
Preferably, a Kozak sequence is added to the 5' end of the initiation codon of the modified Csy4 sequence, and the base sequence of the Kozak sequence is as follows: gccgccacc.
Preferably, a beta-globin 5 ' mRNA sequence is added to the 5 ' end of the Kozak sequence, and the base sequence of the beta-globin 5 ' mRNA is shown as SEQ ID NO. 2.
A method for improving a Csy4 sequence, comprising the steps of:
(1) optimizing and improving the Csy4 sequence according to codon preference, GC content, mRNA secondary structure and homeopathic elements;
(2) a Kozak sequence was added to the 5' end of the start codon of the modified Csy4 base sequence: gccgcccc, meanwhile, in order to increase the stability of mRNA, a beta-globin 5 'mRNA sequence is added at the 5' end of a Kozak sequence, the optimized Csy4 is called GKCsy4, and the base sequence of GKCsy4 is shown as SEQ ID NO. 1;
(3) the vector is convenient to construct, and ClaI enzyme cutting sites and AvaI enzyme cutting sites are respectively added at two ends of a GKCsy4 sequence.
The invention aims to provide an improved Csy4 sequence for optimization in zebrafish and application in multi-gene knock-out.
A method for optimizing an improved Csy4 sequence in zebrafish and applying the improved Csy4 sequence in multi-gene knock-out, which is characterized by comprising the following steps:
(1) synthesis of GKCsy4 sequence;
(2) construction of pCS2-GKCsy4 vector: respectively double-digesting pCS2 plasmid and GKCsy4 sequence with ClaI and AvaI restriction sites on two sides by ClaI and AvaI DNA endonuclease (TAKARA), reacting at 37 ℃ for 2 hours, after tapping and recycling, connecting digested pCS2 skeleton and GKCsy4 sequence by T4 DNA ligase to obtain a pCS2-GKCsy4 expression vector, namely CMV-GKCsy4-polyA expression vector;
(3) designing a zebra fish dnd gene target sequence and synthesizing a DgRNA sequence: designing a knockout site aiming at a zebra fish dnd gene, respectively designing a target site on a first exon and a second exon, wherein the distance between the two target sites is 199bp, the target site sequence on the first exon is Ccagcagcaggagcttcagc and is called as site 1, the target site sequence on the second exon is Ctctgcaggaatggatgcag and is called as site 2, the gRNA containing the site 1 and the site 2 is called as DgRNA, the DgRNA sequence is shown as SEQ ID NO.12, and the DgRNA sequence is transcribed in vitro;
(4) in vitro transcription to obtain Cas9 mRNA: using the Cas9 plasmid, Cas9 mRNA was synthesized;
(5) cas9 mRNA and DgRNA and pCS2-GKCsy4 plasmid vector are injected together in a micro-injection mode, then GFP-nos 3' UTR mRNA is injected for the second time to the embryo which is injected together in a micro-injection mode, the zebra fish embryo is cultured for 48 hours, and the quantity change of PGCs is observed.
Compared with the prior art, the invention has the following beneficial effects: the invention optimizes and improves the original Csy4 sequence to obtain an improved Csy4 sequence (namely GKCsy4 sequence), improves the expression efficiency of the Csy4 sequence in the zebra fish, and provides a basis and guarantee for further application of the Csy4 in the polygene knock-out of the zebra fish.
Drawings
FIG. 1 shows a comparison of the nucleotide sequences of Csy4 before and after optimization;
FIG. 2 is a histogram of expression levels of Csy4 before and after sequence modification at different embryonic developmental stages, where Csy4 is before modification and GKCsy4 is after modification;
FIG. 3 is the two site mutation rate mediated before and after modification of the Csy4 sequence, where Csy4 is before modification and GKCsy4 is after modification;
FIG. 4 is a fiber diagram showing the result of the reduction of primordial germ cells by the mediated two-site co-knockout of dnd gene before and after the sequence modification of Csy4, wherein WT is an individual injected with GFP-nos 3' UTR mRNA alone, Csy4 is before modification, and GKCsy4 is after modification.
Detailed Description
The technical solutions and effects of the present invention will be described with reference to specific embodiments, but the invention is not limited by the scope of the present invention.
The base sequence of the improved Csy4 sequence, namely GKCsy4 sequence, is shown as SEQ ID NO. 1:
CGCatcgatcttgttctttttgcagccgccaccATGGGGGACCATTACCTGGATATCCGACTGAGACCAGACCCCGAGTTCCCACCTGCACAGCTGATGAGCGTGCTGTTTGGCAAGCTGCACCAGGCACTGGTGGCACAGGGAGGGGATAGAATCGGGGTCTCCTTCCCCGACCTGGATGAATCCAGGTCTCGCCTGGGAGAGCGACTGCGGATTCACGCCTCTGCTGACGATCTGAGGGCTCTGCTGGCACGCCCTTGGCTGGAAGGACTGAGGGACCACCTCCAGTTTGGCGAGCCAGCTGTGGTCCCTCATCCAACCCCCTACAGGCAGGTGTCACGCGTCCAGGCAAAGAGCAATCCTGAGCGCCTGAGAAGGCGCCTGATGCGACGGCACGATCTGTCTGAGGAAGAGGCCAGAAAAAGAATCCCCGACACAGTGGCACGGGCCCTGGATCTGCCCTTCGTCACACTGAGAAGTCAGTCAACTGGACAGCACTTCCGACTGTTTATTCGGCACGGCCCCCTCCAGGTCACTGCTGAAGAGGGAGGCTTTACCTGCTATGGCCTGAGCAAGGGGGGATTCGTCCCTTGGTTTctcgagCATG
the sequence of SEQ ID NO.1, wherein 1-9: a ClaI restriction enzyme site; 10-24: a beta-globin 5' mRNA sequence; 25-33: a KozaK sequence; 34-597: GKCsy4 sequence; 598-607: AvaI cleavage site.
The specific improvement method of the Csy4 sequence comprises the following steps:
(1) the original Csy4 gene coding sequence information was from an article (Tsai SQ, Wyvekens N, Khayter C, Foden JA, thapal V, Reyon D, Goodwin MJ, Aree MJ, Joung JK. 2014, Dimeric CRISPR RNA-formulated FokI genes for high-level genetic testing, Nat Biotechnol, 32, 569-76.); optimizing and improving the Csy4 sequence according to the preference of codons, GC content, mRNA secondary structure and homeopathic action elements, wherein the sequence comparison results before and after optimization are shown in figure 1, and the corresponding amino acid sequences before and after optimization are unchanged;
(2) in order to improve the translation efficiency, a Kozak sequence was added to the 5' end of the start codon of the modified Csy4 base sequence: gccgcccc, meanwhile, in order to increase the stability of mRNA, a beta-globin 5 'mRNA sequence (SEQ ID NO. 2: cttgttctttttgca) is added at the 5' end of a Kozak sequence, and the optimized Csy4 is called GKCsy 4;
(3) the vector is convenient to construct, and ClaI enzyme cutting sites and AvaI enzyme cutting sites are respectively added at two ends of a GKCsy4 sequence.
Example one
The invention discloses a method for optimizing a modified Csy4 sequence, namely a GKCsy4 sequence in zebra fish and applying the sequence in multi-gene knock-out, which comprises the following steps:
(1) the synthesis of the sequence GKCsy4, and the comparison with the original Csy4 sequence in transcription efficiency, a set of comparative experiments are set, namely ClaI and AvaI enzyme cutting sites are respectively added at two ends of the original Csy4 sequence, the Csy4 sequence and the GKCsy4 sequence are directly synthesized by Shanghai Yingjun biotechnology limited, and the original Csy4 sequence is specifically shown as SEQ ID NO. 3:
CGCatcgatATGGGTGATCATTATCTGGATATTCGGCTGAGGCCTGATCCAGAGTTCCCACCTGCGCAGCTGATGTCTGTCCTTTTTGGCAAACTTCATCAGGCCCTGGTTGCCCAGGGCGGAGATCGGATAGGGGTAAGCTTTCCAGACCTCGACGAAAGCCGGAGCCGCCTGGGAGAACGCCTGCGGATCCACGCTTCTGCCGACGATCTGAGAGCCTTGCTGGCAAGGCCATGGCTTGAGGGGCTCCGGGATCACCTGCAGTTTGGCGAACCCGCCGTTGTTCCCCACCCAACCCCTTATCGGCAGGTGTCTAGAGTGCAGGCCAAATCTAATCCAGAACGGCTGCGACGGCGACTCATGCGGCGACATGATCTTAGCGAGGAAGAGGCCCGAAAAAGAATCCCTGATACCGTGGCCCGCGCCCTTGACTTGCCTTTTGTCACACTGCGGTCCCAGAGTACGGGGCAGCATTTCAGACTTTTCATTCGACACGGGCCACTGCAAGTTACCGCCGAAGAAGGAGGCTTTACTTGTTATGGACTCTCCAAGGGAGGTTTCGTGCCCTGGTTTctcgagCATG
the sequence of the Csy4 is specifically shown in SEQ ID NO.3, wherein: 1-9: a ClaI restriction enzyme site; 10-572: a Csy4 sequence; 573-583: an AvaI cleavage site;
(2) construction of pCS2-Csy4 vector and pCS2-GKCsy4 vector:
a. construction of pCS2-Csy4 vector: respectively double-digesting the pCS2 plasmid and an original Csy4 sequence with ClaI and AvaI restriction sites on both sides by ClaI and AvaI endonucleases (TAKARA), reacting at 37 ℃ for 2 hours, after tapping and recycling, connecting the digested pCS2 skeleton and the original Csy4 sequence by using T4 DNA ligase to obtain a pCS2-Csy4(CMV-Csy4-polyA) expression vector, wherein the sequence of the expression vector is shown as SEQ ID NO. 4:
tcgaccatagccaattcaatatggcgtatatggactcatgccaattcaatatggtggatctggacctgtgccaattcaatatggcgtatatggactcgtgccaattcaatatggtggatctggaccccagccaattcaatatggcggacttggcaccatgccaattcaatatggcggacttggcactgtgccaactggggaggggtctacttggcacggtgccaagtttgaggaggggtcttggccctgtgccaagtccgccatattgaattggcatggtgccaataatggcggccatattggctatatgccaggatcaatatataggcaatatccaatatggccctatgccaatatggctattggccaggttcaatactatgtattggccctatgccatatagtattccatatatgggttttcctattgacgtagatagcccctcccaatgggcggtcccatataccatatatggggcttcctaataccgcccatagccactcccccattgacgtcaatggtctctatatatggtctttcctattgacgtcatatgggcggtcctattgacgtatatggcgcctcccccattgacgtcaattacggtaaatggcccgcctggctcaatgcccattgacgtcaataggaccacccaccattgacgtcaatgggatggctcattgcccattcatatccgttctcacgccccctattgacgtcaatgacggtaaatggcccacttggcagtacatcaatatctattaatagtaacttggcaagtacattactattggaaggacgccagggtacattggcagtactcccattgacgtcaatggcggtaaatggcccgcgatggctgccaagtacatccccattgacgtcaatggggaggggcaatgacgcaaatgggcgttccattgacgtaaatgggcggtaggcgtgcctaatgggaggtctatataagcaatgctcgtttagggaaccgccattctgcctggggacgtcggagcaagcttgatttaggtgacactatagaatacaagctacttgttctttttgcaggatcccatcgatATGGGTGATCATTATCTGGATATTCGGCTGAGGCCTGATCCAGAGTTCCCACCTGCGCAGCTGATGTCTGTCCTTTTTGGCAAACTTCATCAGGCCCTGGTTGCCCAGGGCGGAGATCGGATAGGGGTAAGCTTTCCAGACCTCGACGAAAGCCGGAGCCGCCTGGGAGAACGCCTGCGGATCCACGCTTCTGCCGACGATCTGAGAGCCTTGCTGGCAAGGCCATGGCTTGAGGGGCTCCGGGATCACCTGCAGTTTGGCGAACCCGCCGTTGTTCCCCACCCAACCCCTTATCGGCAGGTGTCTAGAGTGCAGGCCAAATCTAATCCAGAACGGCTGCGACGGCGACTCATGCGGCGACATGATCTTAGCGAGGAAGAGGCCCGAAAAAGAATCCCTGATACCGTGGCCCGCGCCCTTGACTTGCCTTTTGTCACACTGCGGTCCCAGAGTACGGGGCAGCATTTCAGACTTTTCATTCGACACGGGCCACTGCAAGTTACCGCCGAAGAAGGAGGCTTTACTTGTTATGGACTCTCCAAGGGAGGTTTCGTGCCCTGGTTTtcgagcctctagaccctatagtgagtcgtattacgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggtttttt
the sequence (SEQ ID NO. 4) of the pCS2-Csy4(CMV-Csy4-polyA) expression vector, wherein 1-987: CMV promoter; 988-1078: a spacer sequence; 1079-1642: a Csy4 sequence; 1643-1679: 1680-1875: poly A;
b. construction of pCS2-GKCsy4 vector: wherein ClaI and AvaI DNA endonuclease (TAKARA) are respectively used for double enzyme digestion of pCS2 plasmid and GKCsy4 sequence with ClaI and AvaI enzyme digestion sites on two sides, reaction conditions are carried out at 37 ℃ for 2 hours, after gel tapping recovery, T4 DNA ligase is used for connecting pCS2 skeleton after enzyme digestion and GKCsy4 sequence, thus obtaining pCS2-GKCsy4 expression vector, namely CMV-GKCsy4-polyA expression vector, the sequence of which is shown as SEQ ID NO. 5:
tcgaccatagccaattcaatatggcgtatatggactcatgccaattcaatatggtggatctggacctgtgccaattcaatatggcgtatatggactcgtgccaattcaatatggtggatctggaccccagccaattcaatatggcggacttggcaccatgccaattcaatatggcggacttggcactgtgccaactggggaggggtctacttggcacggtgccaagtttgaggaggggtcttggccctgtgccaagtccgccatattgaattggcatggtgccaataatggcggccatattggctatatgccaggatcaatatataggcaatatccaatatggccctatgccaatatggctattggccaggttcaatactatgtattggccctatgccatatagtattccatatatgggttttcctattgacgtagatagcccctcccaatgggcggtcccatataccatatatggggcttcctaataccgcccatagccactcccccattgacgtcaatggtctctatatatggtctttcctattgacgtcatatgggcggtcctattgacgtatatggcgcctcccccattgacgtcaattacggtaaatggcccgcctggctcaatgcccattgacgtcaataggaccacccaccattgacgtcaatgggatggctcattgcccattcatatccgttctcacgccccctattgacgtcaatgacggtaaatggcccacttggcagtacatcaatatctattaatagtaacttggcaagtacattactattggaaggacgccagggtacattggcagtactcccattgacgtcaatggcggtaaatggcccgcgatggctgccaagtacatccccattgacgtcaatggggaggggcaatgacgcaaatgggcgttccattgacgtaaatgggcggtaggcgtgcctaatgggaggtctatataagcaatgctcgtttagggaaccgccattctgcctggggacgtcggagcaagcttgatttaggtgacactatagaatacaagctacttgttctttttgcaggatcccatcgatcttgttctttttgcagccgccaccATGGGGGACCATTACCTGGATATCCGACTGAGACCAGACCCCGAGTTCCCACCTGCACAGCTGATGAGCGTGCTGTTTGGCAAGCTGCACCAGGCACTGGTGGCACAGGGAGGGGATAGAATCGGGGTCTCCTTCCCCGACCTGGATGAATCCAGGTCTCGCCTGGGAGAGCGACTGCGGATTCACGCCTCTGCTGACGATCTGAGGGCTCTGCTGGCACGCCCTTGGCTGGAAGGACTGAGGGACCACCTCCAGTTTGGCGAGCCAGCTGTGGTCCCTCATCCAACCCCCTACAGGCAGGTGTCACGCGTCCAGGCAAAGAGCAATCCTGAGCGCCTGAGAAGGCGCCTGATGCGACGGCACGATCTGTCTGAGGAAGAGGCCAGAAAAAGAATCCCCGACACAGTGGCACGGGCCCTGGATCTGCCCTTCGTCACACTGAGAAGTCAGTCAACTGGACAGCACTTCCGACTGTTTATTCGGCACGGCCCCCTCCAGGTCACTGCTGAAGAGGGAGGCTTTACCTGCTATGGCCTGAGCAAGGGGGGATTCGTCCCTTGGTTTtcgagcctctagaccctatagtgagtcgtattacgtagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggtttttt
the sequence is SEQ ID NO.5, wherein 1-987 is CMV promoter; 988-1078: a spacer sequence; 1079-1093: a beta-globin 5' mRNA sequence; 1094-1102: a KozaK sequence; 1103-1666: GKCsy4 sequence; 1667-1703: interval sequence 1704-1899: poly A;
(3) csy4 and GKCsy4 transcription efficiency and stability test: injecting 50 ng/ul pCS2-Csy4 vector and 50 ng/ul pCS2-GKCsy4 vector into zebra fish fertilized eggs of one cell period respectively, wherein the injection dose of each embryo is about 0.002 ul, collecting zebra fish embryos at 12h, 18h, 24h, 30h, 36h and 48h after injection, and extracting total RNA of embryos of different periods by using Trizol reagent (Invitrogen) specifically: homogenizing 15 collected embryos in 1ml of Trizol, centrifuging at 4 ℃ and 11000rpm for 10min, transferring supernatant into another Ep tube of RNAase-free, standing at room temperature for 5min, adding 200 mu l of trichloromethane, violently shaking for 10s, standing at room temperature for 5min, then centrifuging at 4 ℃ and 11000rpm for 12min, absorbing supernatant liquid into another Ep tube of RNAse-free, adding 500 mu l of isopropanol, gently reversing and mixing evenly, standing at room temperature for 15min, centrifuging at 4 ℃ and 11000rpm for 10min, discarding supernatant, adding 1ml of 75% (volume ratio) ethanol, shaking and mixing evenly, centrifuging at 4 ℃ and 7000rpm for 15min, then discarding ethanol, drying in the open air for 15min, and adding 30 mu l of RNAase-free water to obtain the total RNA. Mu.g of total RNA was digested with RNAase-free DNase (TOYOBO) for 30min and then inverted to cDNA (TOYOBO). Adding 1 mul of cDNA diluted 4 times in each qPCR reaction as a template, taking beta-actin gene as an internal reference, and carrying out upstream primer: SEQ ID NO.6, β -actin-F (5'-ATGGCTTCTGCTCTGTATGGC-3'), downstream primer: SEQ ID NO.7, β -actin-R (5'-GAGGAGGGCAAAGTGGTAAAC-3'); the Csy4 upstream primer is: SEQ ID No.,8, Csy4-F (5'-CTGGATATTCGGCTGAGGCCT-3'), downstream primers: SEQ ID NO.9, Csy4-R (5'-AAACTGCAGGTGATCCCGGAG-3'); the upstream primer of GKCsy4 is: SEQ ID NO.10, GKCsy4-F (5'-TGCACAGCTGATGAGCGTGCT-3'), downstream primer: SEQ ID NO.11, GKCsy4-R (5'-AACTGGAGGTGGTCCCTCAGT-3'). Prepare 20 μ l system with 2xSYBR green real time PCR mix (TOYOBO), react on real time quantitative PCR instrument (bIO-RAD) under the following conditions: one cycle at 95 ℃ for 2min, 15s at 94 ℃, 15s at 57 ℃ and 40 cycles at 72 ℃ for 40 s. The results are shown in FIG. 2: the GKCsy4 mRNA level is significantly higher than that of Csy4 mRNA (p < 0.01) from 12 hours to 48 hours after fertilization, and the GKCsy4 mRNA still has higher level at 48 hours after fertilization, but the Csy4 mRNA is hardly detected at the moment, and the result shows that the transcription efficiency and stability of the GKCsy4 gene in zebra fish are higher than those of the original Csy 4.
(4) Designing a zebra fish dnd gene target sequence and synthesizing a DgRNA sequence: the design of a knockout site is carried out aiming at zebra fish dnd gene, wherein the dnd base sequence is shown as SEQ ID NO. 15:
tttaatgaccttttcttgacttttccaccaatttacaggtgtgtctatcatcatcatcacagatggtcggagacatggatgcccagcagcaggagcttcagcaggtaagcgagtttatttacacgtttataaacaacacgcctgttttgcaaacagtttaacttttcggtcgaatgtttattagtatgtgtgtgtcggtttgacacttgaaaggccgtcaattcctcaatacaccacgccaaaaacattaacatcccctctctaatttgatggcagattctgaacccgcagaaactcaagtctctgcaggaatggatgcagaggaactccatcactttaacccaagtcaatgggcagaggaaatatggtggtcctcctccaggtaagtgcccctccatcgacctgagagcag；
then, respectively designing a target site on a first exon and a second exon, wherein the distance between the two target sites is 199bp, the target site sequence on the first exon is Ccagcagcaggagcttcagc, named as site 1, the target site sequence on the second exon is Ctctgcaggaatggatgcag, named as site 2, and the gRNA containing the site 1 and the site 2 is named as DgRNA, wherein the base sequence of the DgRNA is shown as SEQ ID NO. 12:
taatacgactcactataggGTTCACTGCCGTATAGGCAGCcagcagcaggagcttcagcGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTTCACTGCCGTATAGGCAGCtctgcaggaatggatgcagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTTCACTGCCGTATAGGCAGGTTCACTGCCGTATAGGCAG
the sequence is shown in SEQ ID NO.12, wherein 1-19: the T7 promoter; 20-39: csy4 recognition sequence; 40-59: a target site 1; 60-135: a gRNA sequence; 136-155: csy4 recognition sequence; 156-175: a target site 2; 176-251: a gRNA sequence; 252-291: csy4 identifies a sequence. The DgRNA sequence was synthesized directly by Shanghai Yingjun Biotechnology Ltd. In vitro transcription kit (Invitrogen) was then used TM : MEGAscript T7 Transcription Kit) transcribes the synthesized dnd-gRNA in vitro, and a 20 ul reaction system is as follows:
10x Reaction Buffer 2ｕl；
DNA 4 ul (about 1 ug);
T7 Enzyme 1ｕl；
10mmol/L NTP 1ｕl；
DEPC water 12ｕl；
reacting the above system at 37 ℃ for one hour, and purifying for later use;
(5) in vitro transcription to obtain Cas9 mRNA: cas9 plasmid from Addgene TM (#63154), using MAXiScript T7/T3 Transcription Kit (Invitrogen) TM ) Synthesizing Cas9 mRNA, and carrying out the same system and reaction conditions as the step 4;
(5) cas9 mRNA and DgRNA and pCS2-GKCsy4 plasmid vectors were microinjected: firstly, a microinjection system is prepared as follows:
Cas9 mRNA 300 ng/ｕl；
DgRNA 50ng/ｕl；
pCS2-GKCsy4/pCS2-Csy4 50ng/ｕl
Phenol-red 0.2ｕl；
DEPC Water up to 2ｕl；
and then mixing the solutions, injecting the mixed solution into zebra fish fertilized eggs at a single cell stage in a microinjection manner, and detecting the mutation efficiency of the dnd gene after culturing for 24 hours. The method comprises the following steps: randomly selecting 8 embryos to extract genome DNA, taking F-dnd/R-dnd as a primer, F-dnd: SEQ ID NO.13, 5'-aggtgtgtctatcatcatca-3', R-dnd, SEQ ID NO.14, 5'-TGCCATGTGCTCGTCTTTAT-3', using KOD PLUS high fidelity enzyme (TAKARA) to perform PCR amplification, after the PCR product is connected with pMD18-T vector (TAKARA), transforming competent Escherichia coli, respectively selecting 20 Escherichia coli to perform monoclonal amplification culture, extracting plasmid DNA, then obtaining mutation efficiency after plasmid DNA sequencing comparison, and the result is shown in figure 3: cas9 mRNA and DgRNA and plasmid pCS2-GKCsy4 were co-injected, the mutation rate of target site 1 and target site 2 was higher than that of plasmid pCS2-Csy4, and about 10% of the embryos co-injected with plasmid pCS2-GKCsy4 were mutated into target site 1 and target site 2 at the same time, indicating that Csy4 has higher activity than GKCsy4 of the present invention.
Dnd gene plays an important role in the survival of the primordial germ cells of zebra fish, therefore, the change of the primordial germ cells in the embryo after injection is detected by the following method: in vitro transcription (Ambion) is carried out by using an SP6 in vitro transcription kit by taking 355 vector (SP 6-GFP-nos 3 'UTR) with GFP gene as a template, and GFP-nos 3' UTR mRNA is obtained after transcription, wherein the system is as follows:
plasmid vector 0.5 ug;
2x NTP/CAP 10ul；
10x Buffer 2 ul；
SP6 transcriptase MIX 2 ul;
RNase-free Water make-up to 20 ul;
reaction conditions are as follows: water bath is carried out for 2h at 37 ℃;
then, the embryos after microinjection are subjected to secondary injection of GFP-nos 3' UTR mRNA, and a secondary microinjection system is configured as follows:
GFP-nanos3’UTR mRNA 50ng/ｕl
Phenol-red 0.2ｕl；
DEPC Water up to 2ｕl；
after the injection, the zebra fish embryos are cultured for 48h, and the change of the quantity of PGCs is observed. Photographing with a Digital camera (Nikon, Digital sight DS-SMC) mounted on a fluoroscope (olympus MVX 10) under the condition of completely consistent aperture size, exposure time and magnification, the result is shown in fig. 4, compared with wild control (WT), the number of primordial germ cells of the embryo after microinjection of Cas9 mRNA and DgRNA with pCS2-Csy4 plasmid vector and the number of primordial germ cells of the embryo after microinjection of Cas9 mRNA and DgRNA with pCS 2-gksy 4 plasmid vector are both significantly reduced, but the number of primordial germ cells of the embryo after microinjection of Cas9 mRNA and DgRNA with pCS 2-gksy 4 plasmid vector is much reduced than that of the embryo after microinjection of Cas9 mRNA and DgRNA with pCS2-Csy4 vector, which indicates that the activity of the gksy 4 designed by us in zebra Csy4 is much greater than that of the zebra fish.
Sequence listing
<110> Hunan institute of culture and literature
<120> improved Csy4 sequence, improved method and application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 607
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgcatcgatc ttgttctttt tgcagccgcc accatggggg accattacct ggatatccga 60
ctgagaccag accccgagtt cccacctgca cagctgatga gcgtgctgtt tggcaagctg 120
caccaggcac tggtggcaca gggaggggat agaatcgggg tctccttccc cgacctggat 180
gaatccaggt ctcgcctggg agagcgactg cggattcacg cctctgctga cgatctgagg 240
gctctgctgg cacgcccttg gctggaagga ctgagggacc acctccagtt tggcgagcca 300
gctgtggtcc ctcatccaac cccctacagg caggtgtcac gcgtccaggc aaagagcaat 360
cctgagcgcc tgagaaggcg cctgatgcga cggcacgatc tgtctgagga agaggccaga 420
aaaagaatcc ccgacacagt ggcacgggcc ctggatctgc ccttcgtcac actgagaagt 480
cagtcaactg gacagcactt ccgactgttt attcggcacg gccccctcca ggtcactgct 540
gaagagggag gctttacctg ctatggcctg agcaaggggg gattcgtccc ttggtttctc 600
gagcatg 607
<210> 2
<211> 15
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cttgttcttt ttgca 15
<210> 3
<211> 583
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cgcatcgata tgggtgatca ttatctggat attcggctga ggcctgatcc agagttccca 60
cctgcgcagc tgatgtctgt cctttttggc aaacttcatc aggccctggt tgcccagggc 120
ggagatcgga taggggtaag ctttccagac ctcgacgaaa gccggagccg cctgggagaa 180
cgcctgcgga tccacgcttc tgccgacgat ctgagagcct tgctggcaag gccatggctt 240
gaggggctcc gggatcacct gcagtttggc gaacccgccg ttgttcccca cccaacccct 300
tatcggcagg tgtctagagt gcaggccaaa tctaatccag aacggctgcg acggcgactc 360
atgcggcgac atgatcttag cgaggaagag gcccgaaaaa gaatccctga taccgtggcc 420
cgcgcccttg acttgccttt tgtcacactg cggtcccaga gtacggggca gcatttcaga 480
cttttcattc gacacgggcc actgcaagtt accgccgaag aaggaggctt tacttgttat 540
ggactctcca agggaggttt cgtgccctgg tttctcgagc atg 583
<210> 4
<211> 1875
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcgaccatag ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc 60
tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc 120
tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggact 180
tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc 240
ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat 300
tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc 360
tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt 420
tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct 480
tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt 540
cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca 600
attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat 660
tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt 720
caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa 780
gtacattact attggaagga cgccagggta cattggcagt actcccattg acgtcaatgg 840
cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca 900
atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc 960
tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt 1020
gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgatat 1080
gggtgatcat tatctggata ttcggctgag gcctgatcca gagttcccac ctgcgcagct 1140
gatgtctgtc ctttttggca aacttcatca ggccctggtt gcccagggcg gagatcggat 1200
aggggtaagc tttccagacc tcgacgaaag ccggagccgc ctgggagaac gcctgcggat 1260
ccacgcttct gccgacgatc tgagagcctt gctggcaagg ccatggcttg aggggctccg 1320
ggatcacctg cagtttggcg aacccgccgt tgttccccac ccaacccctt atcggcaggt 1380
gtctagagtg caggccaaat ctaatccaga acggctgcga cggcgactca tgcggcgaca 1440
tgatcttagc gaggaagagg cccgaaaaag aatccctgat accgtggccc gcgcccttga 1500
cttgcctttt gtcacactgc ggtcccagag tacggggcag catttcagac ttttcattcg 1560
acacgggcca ctgcaagtta ccgccgaaga aggaggcttt acttgttatg gactctccaa 1620
gggaggtttc gtgccctggt tttcgagcct ctagacccta tagtgagtcg tattacgtag 1680
atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 1740
aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 1800
gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 1860
tgtgggaggt ttttt 1875
<210> 5
<211> 1899
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcgaccatag ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc 60
tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc 120
tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggact 180
tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc 240
ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat 300
tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc 360
tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt 420
tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct 480
tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt 540
cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca 600
attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat 660
tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt 720
caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa 780
gtacattact attggaagga cgccagggta cattggcagt actcccattg acgtcaatgg 840
cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca 900
atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc 960
tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt 1020
gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc ccatcgatct 1080
tgttcttttt gcagccgcca ccatggggga ccattacctg gatatccgac tgagaccaga 1140
ccccgagttc ccacctgcac agctgatgag cgtgctgttt ggcaagctgc accaggcact 1200
ggtggcacag ggaggggata gaatcggggt ctccttcccc gacctggatg aatccaggtc 1260
tcgcctggga gagcgactgc ggattcacgc ctctgctgac gatctgaggg ctctgctggc 1320
acgcccttgg ctggaaggac tgagggacca cctccagttt ggcgagccag ctgtggtccc 1380
tcatccaacc ccctacaggc aggtgtcacg cgtccaggca aagagcaatc ctgagcgcct 1440
gagaaggcgc ctgatgcgac ggcacgatct gtctgaggaa gaggccagaa aaagaatccc 1500
cgacacagtg gcacgggccc tggatctgcc cttcgtcaca ctgagaagtc agtcaactgg 1560
acagcacttc cgactgttta ttcggcacgg ccccctccag gtcactgctg aagagggagg 1620
ctttacctgc tatggcctga gcaagggggg attcgtccct tggttttcga gcctctagac 1680
cctatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac 1740
aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg 1800
ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt 1860
ttatgtttca ggttcagggg gaggtgtggg aggtttttt 1899
<210> 6
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggcttctg ctctgtatgg c 21
<210> 7
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gaggagggca aagtggtaaa c 21
<210> 8
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctggatattc ggctgaggcc t 21
<210> 9
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aaactgcagg tgatcccgga g 21
<210> 10
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgcacagctg atgagcgtgc t 21
<210> 11
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aactggaggt ggtccctcag t 21
<210> 12
<211> 291
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
taatacgact cactataggg ttcactgccg tataggcagc cagcagcagg agcttcagcg 60
ttttagagct agaaatagca agttaaaata tggctagtcc gttatcaact tgaaaaagtg 120
gcaccgagtc ggtgcgttca ctgccgtata ggcagctctg caggaatgga tgcaggtttt 180
agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac 240
cgagtcggtg cgttcactgc cgtataggca ggttcactgc cgtataggca g 291
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aggtgtgtct atcatcatca 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgccatgtgc tcgtctttat 20
<210> 15
<211> 412
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tttaatgacc ttttcttgac ttttccacca atttacaggt gtgtctatca tcatcatcac 60
agatggtcgg agacatggat gcccagcagc aggagcttca gcaggtaagc gagtttattt 120
acacgtttat aaacaacacg cctgttttgc aaacagttta acttttcggt cgaatgttta 180
ttagtatgtg tgtgtcggtt tgacacttga aaggccgtca attcctcaat acaccacgcc 240
aaaaacatta acatcccctc tctaatttga tggcagattc tgaacccgca gaaactcaag 300
tctctgcagg aatggatgca gaggaactcc atcactttaa cccaagtcaa tgggcagagg 360
aaatatggtg gtcctcctcc aggtaagtgc ccctccatcg acctgagagc ag 412

Claims (4)

1. An improved Csy4 sequence, which is characterized in that a Kozak sequence is added at the 5' end of the initiation codon of the Csy4 sequence, and the base sequence of the Kozak is as follows: gccgccacc; a beta-globin 5 ' mRNA sequence is added at the 5 ' end of the Kozak sequence, and the base sequence of the beta-globin 5 ' mRNA is shown as SEQ ID NO. 2; the base sequence of the modified Csy4 sequence is shown in SEQ ID NO. 1.
2. An improved method for Csy4 sequence, characterized by comprising the following steps:
(1) optimizing and improving the Csy4 sequence according to codon preference, GC content, mRNA secondary structure and homeopathic elements;
(2) a Kozak sequence was added to the 5' end of the start codon of the modified Csy4 base sequence: gccgcccc, meanwhile, in order to increase the stability of mRNA, a beta-globin 5 'mRNA sequence is added at the 5' end of a Kozak sequence, the optimized Csy4 is called GKCsy4, and the base sequence of GKCsy4 is shown as SEQ ID NO. 1;
(3) the vector is convenient to construct, and ClaI and AvaI enzyme cutting sites are respectively added at two ends of a GKCsy4 sequence.
3. The use of the modified Csy4 sequence of claim 1, wherein the modified Csy4 sequence is GKCsy4 for the multigene knock-out of zebrafish.
4. The use of the improved Csy4 sequence of claim 3, in a polygene knock-out of zebrafish, comprising the steps of:
(1) synthesis of GKCsy4 sequence;
(2) construction of pCS2-GKCsy4 vector: respectively double-digesting the pCS2 plasmid and the GKCsy4 sequence with ClaI and AvaI restriction sites on two sides by ClaI and AvaI DNA endonucleases under the reaction condition of 37 ℃ for 2 hours, and after tapping and recycling, connecting the digested pCS2 skeleton and the sequence GKCsy4 by using T4 DNA ligase to obtain a pCS2-GKCsy4 expression vector, namely a CMV-GKCsy4-polyA expression vector;
(3) design aiming at a zebra fish dnd gene target sequence and DgRNA sequence synthesis: designing a knockout site aiming at a zebra fish dnd gene, respectively designing a target site on a first exon and a second exon, wherein the distance between the two target sites is 199bp, the target site sequence on the first exon is Ccagcagcaggagcttcagc and is called as site 1, the target site sequence on the second exon is Ctctgcaggaatggatgcag and is called as site 2, the gRNA containing the site 1 and the site 2 is called as DgRNA, the DgRNA sequence is shown as SEQ ID NO.12, and the DgRNA sequence is transcribed in vitro;
(4) in vitro transcription to obtain Cas9 mRNA: using the Cas9 plasmid, Cas9 mRNA was synthesized;
(5) cas9 mRNA and DgRNA and pCS2-GKCsy4 plasmid vector are injected together in a micro-injection mode, then GFP-nos 3' UTR mRNA is injected for the second time to the embryo which is injected together in a micro-injection mode, the zebra fish embryo is cultured for 48 hours, and the quantity change of PGCs is observed.
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