CN112831484A

CN112831484A - T7-RNA polymerase mutant and application thereof

Info

Publication number: CN112831484A
Application number: CN202110044261.3A
Authority: CN
Inventors: 朱斌; 吴慧
Original assignee: Huazhong University of Science and Technology
Current assignee: Suzhou Inshore Protein Technology Co ltd
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2021-05-25
Anticipated expiration: 2041-01-13
Also published as: CN112831484B

Abstract

The invention discloses a T7-RNA polymerase mutant, which is obtained by replacing the serine at the 43 th position from the N end of the amino acid sequence of wild T7-RNA polymerase with A amino acid or B amino acid. The amino acid A is tyrosine, phenylalanine, leucine, lysine or aspartic acid, and the amino acid B is tryptophan, isoleucine, arginine, asparagine, glutamine, glutamic acid or proline. The T7-RNA polymerase mutant is suitable for synthesizing RNA containing a termination signal inside and RNA of which the tail end forms a hairpin structure, and can be widely applied to aspects of in vitro transcription, RNA synthesis, gene editing, RNA drug synthesis, in vivo protein expression or cell-free protein expression in vitro translation system, transcription terminator research, RNA-dependent RNA polymerase activity research, biological transcription regulatory element synthesis and the like.

Description

T7-RNA polymerase mutant and application thereof

Technical Field

The invention belongs to the field of nucleic acid tool enzymes and nucleic acid biology, and particularly relates to a phage T7-RNA polymerase mutant and application thereof.

Background

RNA (ribonucleic acid), a class of biological macromolecules for the transmission of genetic information, is widely found in eukaryotes, prokaryotes, partial viruses and viroids, and has many different species and functions. Except for the three major classes of RNA initially identified: in addition to mRNA, rRNA and tRNA, several novel types of RNAs discovered later, such as microrna (mirna) (Cheng et al, 2005), long non-coding RNA (lncRNA) (Dey et al, 2014), Circular RNAs (circRNA) (Memczak et al, 2013), etc., have become popular fields in RNA research in recent years. In addition, the intensive and gradual research on RNA related researches reveals that RNA has very important application value in the aspect of disease treatment. siRNA and mRNA synthesized in vitro can become important drugs for RNA targeted therapy (Sahin et al, 2014; Wittrup et al, 2015), and large pharmaceutical companies such as Merck, Shire and the like are all dedicated to developing RNA drugs for disease treatment. In addition, mRNA synthesized in vitro has been currently popularized and applied as a class of brand new vaccines due to its advantages such as transient expression of proteins in vivo (Pardi et al, 2018).

With the extensive development of RNA-related research and applications, RNA synthesis poses a high challenge. The in vitro synthesis of RNA comprises two methods of chemical synthesis and enzymatic synthesis. The chemical synthesis method is only suitable for synthesizing RNA of several tens of nucleotides in length, and the production cost is sharply increased due to the increase in length. When the number of nucleotides reaches more than a hundred, chemical synthesis is no longer suitable, and mRNA encoding proteins usually contains thousands of nucleotides, so enzymatic synthesis is currently the only method for preparing long-chain RNA. The single-subunit RNA polymerase from the short-tail phage code has the advantages of simple structure, high transcription efficiency and the like, is widely used for synthesizing RNA by in vitro transcription, is an important tool enzyme for RNA related research, and is most widely applied to the single-subunit RNA polymerase from the Escherichia coli phage T7. T7-RNA polymerase was identified in the last 70 th century and has been widely used in RNA synthesis in vitro, protein expression in vivo (bacterial overexpression system) and the like after first cloning (Davanloo et al, 1984), and in recent years T7-RNA polymerase transcription system has also shown an important role in synthetic biology (Wang et al, 2018). However, T7-RNA polymerase has some non-negligible disadvantages as an in vitro RNA synthesis tool, besides the advantages of high transcription efficiency and strong extension capability. It may produce a number of by-products during RNA synthesis, including oligonucleotides produced during transcription initiation, disrupted RNA products due to termination signals, 3' terminal extension products due to RdRp activity, etc. (Katalin et al, 2011). Two types of termination signals have been found to cause transcription termination by T7-RNA polymerase, one type of terminator being termination due to the formation of a distinct loop structure by the encoding RNA, and the other type of terminator being a specific type of sequence-specific termination due to the presence of a common sequence (HAUCUGUU) in the encoding RNA (Macdonald et al, 1994). Whereas the end extension products caused by the RdRp activity of T7-RNA polymerase occur with a pronounced secondary structure at the 3' end of the transcribed RNA product (Nacheva and Berzal-Herranz, 2010). Heterogeneity of in vitro synthesized RNA products can lead to activation of innate immunity after delivery of RNA drugs into vertebrates, which is also a key issue to be addressed by current RNA-targeted therapies. Although there are some purification methods such as High Performance Liquid Chromatography (HPLC) that can remove such immune activation due to heterogeneity of RNA products, a large increase in production cost is caused in large-scale production synthesis, and an increase in purification procedures is also not beneficial to stability of RNA drugs. Therefore, the current development of new RNA synthesis tool enzymes to maintain efficient transcription and simultaneously reduce RNA product heterogeneity has very important application value.

In the prior art, chinese granted patent CN102177236B provides a function-improved RNA polymerase mutant, in which at least 1 amino acid residue of glutamine at position 786, lysine at position 179 and valine at position 685 in the amino acid sequence constituting wild-type T7RNA polymerase is substituted with other amino acids, so as to improve the thermostability and/or specific activity of the T7RNA polymerase mutant. For example, chinese patent application CN107460177A provides an RNA polymerase mutant that can utilize chemically modified nucleotides, and a mutant in which the arginine at position 632 in the amino acid sequence constituting wild-type T7RNA polymerase is substituted with cysteine, whereby the transcription activity is improved and nucleoside triphosphates can be 2' modified. However, these prior art techniques also fail to maintain efficient transcription while reducing RNA product heterogeneity.

Disclosure of Invention

Aiming at the defects of the prior art, the mutant of the T7-RNA polymerase is provided, can be used for in vitro RNA synthesis, has obvious difference with the prior T7-RNA polymerase, provides an effective enzyme candidate tool for RNA research and application, and is specifically realized by the following technology.

T7-RNA polymerase mutant, wherein the serine at position 43 from the N-terminus of the amino acid sequence of the wild-type T7-RNA polymerase shown in SEQ ID NO.1 is replaced by tyrosine, phenylalanine, leucine, lysine or aspartic acid.

The applicants have screened the ability of T7-RNA polymerase to transcribe a class of terminators T7T Φ spanning T7 by a phage assisted directed evolution (PACE) system (esselt et al, 2010), and phages containing mutants with reduced termination capacity will express giii protein in e.coli to obtain normal infectivity and proliferation capacity, and will eventually survive the screening system. The mutant gene obtained after evolution is inserted into a prokaryotic expression vector pQE82L by sequencing and molecular cloning methods, and protein expression and purification are carried out in escherichia coli.

Then, the applicant detects the termination effect of the mutants obtained by evolution in vitro by an in vitro transcription method, and finds that the mutants with obviously reduced termination efficiency all contain the change of amino acid at the S43 site, so that the applicant deduces that the site is probably the key site for influencing termination, and then constructs 6 mutants S43A, S43L, S43K, S43D, S43Y and S43F in which single amino acid at the site is respectively replaced by alanine, leucine, lysine, aspartic acid, tyrosine and phenylalanine, and similarly performs protein expression and purification in vitro and detects the termination efficiency of transcription, and finds that the mutants S43L, S43K, S43D, S43Y and S43F can reduce the termination efficiency to different degrees except S43A. Among them, the mutant S43Y had the greatest effect on the termination efficiency, resulting in about 25% decrease in the termination efficiency of RNA polymerase on the terminator T7T Φ. Analysis of this result revealed that the molecular size of the site was positively correlated with the attenuation of termination. Then, the applicant found in further studies on the mutant S43Y that the mutant has a significantly reduced termination capability for all other type I terminators and type II terminators tested, indicating that S43Y can simultaneously reduce the termination efficiency of both types of termination signals.

In addition, the applicant also unexpectedly found that the mutant has significantly lower RdRp activity than the wild type, and can obtain RNA with higher purity and better yield when transcribing RNA with a 3' terminal higher structure, such as sgRNA. In the transcription of Cas9 mRNA, the S43Y mutant can obviously remove the interrupt RNA product generated in the wild type transcription, and can maintain the advantages of the wild type high-efficiency transcription.

The T7-RNA polymerase mutant is applied to in vitro transcription.

The use of the T7-RNA polymerase mutant in the synthesis of non-coding RNA.

Preferably, the non-coding RNA is a sgRNA, tRNA, mRNA, siRNA, snoRNA, or oligonucleotide.

The T7-RNA polymerase mutant is applied to gene editing.

The T7-RNA polymerase mutant is applied to RNA drug synthesis.

The T7-RNA polymerase mutant is applied to an in vivo protein expression or cell-free protein expression in vitro translation system.

The T7-RNA polymerase mutant is applied to the research of transcription terminators or the research of RNA-dependent RNA polymerase activity.

The T7-RNA polymerase mutant is applied to the synthesis of biological transcription regulatory elements.

Compared with the prior art, the invention has the advantages that: the key amino acid sites which influence the termination of two types of T7-RNA polymerase and the key sites which influence the activity of T7-RNA polymerase RdRp are discovered and identified, so that a tool enzyme (namely the T7-RNA polymerase mutant of the invention) which is more suitable for synthesizing RNA with a 3' terminal higher-order structure such as sgRNA in yield and purity is developed, and the enzyme is also more suitable for synthesizing RNA containing a termination signal.

Drawings

FIG. 1 is a graph showing the effect of 6 mutants of T7-RNA polymerase on termination of class I terminator T7T Φ, prepared in example 1;

FIG. 2 is a comparison of the termination efficiency of T7-RNA polymerase mutant S43Y with that of the wild type against class I terminators T3T Φ, thr attenuator, rrnBT1 terminator and rrnC terminator;

FIG. 3 is a comparison of the termination efficiency of T7-RNA polymerase mutant S43Y with wild-type class II termination signals for PTH, VSV, Adeno5 and CJ sites;

FIG. 4 is a comparison of the transcription effect of the sgRNA of T7-RNA polymerase mutant S43Y for transcribing different genes with that of the wild type;

fig. 5 is a comparison of the effect of T7RNA polymerase mutant S43Y and wild type in transcribing Cas9 mRNA, respectively.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: in vitro termination efficiency assay Using T7-RNA polymerase mutant

(1) Expression and purification of T7-RNA polymerase mutant

T7-RNA polymerase mutant S43A, S43L, S43K, S43D, S43Y and S43F are respectively constructed by a molecular cloning method, then, prokaryotic expression vector pQE82L containing the mutants is transformed into E.coli BL21 expression strain, the strain is selected for amplification culture, the strain is placed in LB culture medium containing 100 mug/ml ampicillin and is subjected to shake culture at 37 ℃ until OD600 value is close to 1.2, then isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.5mM is added to be induced and expressed for 4h at 30 ℃ by a shaking table, then the thalli sediment is collected by centrifugation at 4 ℃ and 5000rpm for 20min, the thalli is fully suspended in a lysis solution containing 300mM NaCl, 20mM Tris-HCl (pH value 7.5), 0.5mg/ml lysozyme and 0.5mM DTT, freezing at-80 deg.C for half an hour, taking out, thawing on ice for 1h, and repeatedly freezing and thawing at-80 deg.C for two times.

And (3) placing the protein lysate subjected to freeze thawing for three times in a high-speed refrigerated centrifuge for centrifugation for 1h at the temperature of 4 ℃ and the rpm of 18000, then separating and filtering the supernatant to remove impurities, and temporarily storing the filtered supernatant on ice to be subjected to subsequent nickel column purification.

Before passing through the nickel column, the nickel column is equilibrated by 10 times of elution buffer (20mM Tris-HCl (pH7.5), 300mM NaCl and 0.5mM DTT), then the filtered protein liquid is added into the nickel column, after all the protein liquid passes through the nickel column packing, imidazole solution (20mM-50mM-100mM) with different gradients is used for elution, and effluent is collected after a plurality of test tubes are used for numbering according to the flowing sequence. All the above operations are carried out on ice or at 4 ℃.

And finally, detecting all eluted protein effluent through SDS-PAGE electrophoresis and Coomassie brilliant blue staining, comprehensively selecting proteins with higher concentration and better purity, adding the proteins into a dialysis bag, sealing, dialyzing in 1L of dialysate, and replacing fresh and clean dialysate after 6-8 h. After three rounds of dialysis, the protein was collected and stored at-20 ℃. The dialysate contained 100mM NaCl, 50mM Tris-HCl pH7.5, 1mM DTT, 0.1mM EDTA, 50% glycerol, and 0.1% Triton X-100.

(2) Transcription reaction template acquisition and termination efficiency detection

Designing a universal primer, wherein the sequence of the primer is as follows:

pETsnrs-F:5’-ATCAGGCGCCATTCGCCATTCAGG-3’

pETsnrs-1R:5’-TCGGTGAGTTTTCTCCTTCATTAC-3’

the universal primer is used for amplifying a DNA fragment containing a T7 promoter and a classical I terminator T7T phi of T7 in an existing plasmid pETsnrs-1 (shown as a sequence table SEQ ID NO.2) in a laboratory, and a purification kit DNA Clean is used&Concentrator^TM-5(ZYMO RESEARCH) the PCR product was purified.

The in vitro transcription reaction components contained 40mM Tris-HCl (pH8.0), 16mM MgCl₂2mM spermidine, 5mM DTT, 4mM ATP, GTP, CTP, UTP, 0.3. mu.L RNase inhibitor, 0.2. mu.L pyrophosphatase, 50nM RNA polymerase and 14nM PCR template, supplemented with DEPC water to 10. mu.L. The reaction was incubated at 37 ℃ for 1h, and the template was removed by DNase I and the RNA product was purified by RNA purification kit (ZYMO RESEARCH). After the concentration measurement of RNA products, 400ng of RNA was taken from each reaction, mixed with 3 XRNA loading buffer, heated at 80 ℃ for 2min, placed on ice, and then electrophoresed at 100V using 1.5% agarose gel 45min, and EB staining was performed.

The termination efficiency of each lane reaction was calculated using ImageJ software to count the band gray values and based on the RNA product size. The experiment was repeated 3 times independently and then statistically analyzed using Prism software, and the results are shown in FIG. 1. In FIG. 1, template represents transcription template DNA, run off represents full-length transcribed RNA product, terminated represents disrupted RNA product, M represents GeneRuler DNA Ladder (Thermo Scientific), and WT represents T7RNA polymerase wild type. FIG. 1 (right) is a bar graph of termination efficiency measured in three independent replicates. As can be seen from fig. 1, mutant S43Y had the greatest effect on termination efficiency, reducing termination efficiency by about 25%. Mutants S43L, S43K, S43D and S43F reduced the termination efficiency by about 10%, 12%, 5% and 17%, respectively, and mutant S43A had no significant effect on the termination efficiency.

Example 2: comparison of T7-RNA polymerase mutant S43Y and wild type for termination efficiency of class I from different sources

(1) Obtaining a template for a transcription reaction

The sequences of several I type terminators T3T phi, thr attenuator, rrnBT1 terminator and rrnC terminator which are reported to exist in T3 bacteriophage and Escherichia coli by T7 (respectively shown in sequence tables SEQ ID NO.3-6) are replaced by a molecular cloning method for the neck ring structure sequence of the I type terminator T7T phi in the carrier pETsnrs-1 (shown in sequence table SEQ ID NO. 7). After successful vector construction, PCR amplification was performed using the universal primers of example 1, followed by Clean using DNA purification kit&Concentrator^TM-5, purifying and measuring the concentration of the PCR product.

(2) In vitro transcription termination efficiency assay

In vitro transcription reactions (10. mu.L) were performed in the presence of 40mM Tris-HCl (pH8.0), 16mM MgCl₂2mM spermidine, 5mM DTT, 4mM ATP, GTP, CTP, UTP, 0.3. mu.L RNase inhibitor, 0.2. mu.L pyrophosphatase, 50nM RNA polymerase, 14nM PCR template and incubation at 37 ℃ for 1h, then digesting the template with DNase I and purifying the RNA product with RNA purification kit, determining the RNA concentration, then mixing 400ng RNA per reaction with 3 XRNA loading buffer, heating at 80 ℃ for 2min and placing on ice, then usingA1.5% agarose gel was electrophoresed at 100V for 45min and EB staining was performed. The termination efficiency of each lane reaction was calculated according to the described method, and the detection results and termination efficiency are shown in FIG. 2. In FIG. 2, run off represents the RNA product of full-length transcription, terminated represents the RNA product of interruption, and M represents Low Range ssRNA Ladder (New England Biolabs). Mutant S43Y was able to significantly reduce the termination efficiency of all class I terminators tested, as compared to wild-type T7 RNAP.

Example 3: comparison of T7-RNA polymerase mutant S43Y and wild type against class II termination efficiency from different sources

(1) Obtaining and purifying transcription reaction template

The termination signal sequences of PTH, VSV, Adeno5 and CJ sites class II (respectively shown in the sequence table SEQ ID NO.8-11) reported in different species in nature are replaced by the termination sequence in the vector pETsnrs-1 through a molecular cloning method by T7. The constructed vector was also subjected to PCR amplification using the universal primers described in example 1, and then the PCR product was purified and concentration determined using a DNA purification kit.

(2) In vitro transcription termination efficiency assay

The in vitro transcription reaction components contained 40mM Tris-HCl (pH8.0), 16mM MgCl₂2mM spermidine, 5mM DTT, 4mM ATP, GTP, CTP, UTP, 0.3. mu.L RNase inhibitor, 0.2. mu.L pyrophosphatase, 50nM RNA polymerase, 14nM PCR template, and 10. mu.L DEPC water supplemented. The transcription reaction was incubated at 37 ℃ for 1h, followed by DNase I to remove the template and RNA purification using an RNA purification kit. And (3) measuring the concentration of the RNA product, then adding 400ng of RNA into 3x RNA loading buffer solution, mixing, heating at 80 ℃ for 2min, placing on ice, performing electrophoresis at 100V for 45min by using 1.5% agarose gel, and performing EB detection and termination efficiency calculation. The results are shown in FIG. 3, where run off in FIG. 3 represents the full-length transcribed RNA product, terminated represents the disrupted RNA product, and M represents the Low Range ssRNA Ladder. Mutant S43Y was able to significantly reduce the termination efficiency of all class II terminators tested by comparison with wild-type T7 RNAP.

Example 4: comparison of the transcription effects of T7-RNA polymerase S43Y and the wild type on different sgRNAs

(1) Obtaining and purifying transcription reaction template

By inserting sgRNA sequences of different genes such as a T7 promoter and eGFP into a vector pUC19 by molecular cloning, the first 20nt of the sgRNA sequences of the different genes differ in sequence depending on the type of the gene, and the latter sequence remains unchanged as the gRNA backbone. The sequence table SEQ ID NO.12-19 shows the sequence information of different targeted genes (PLK1, BMPR1B, TBCE, eGFP, Nod, Lsd, mCherry and MosDT 1). The constructed plasmid was amplified using PCR, and the purified PCR product was also used as a template for subsequent transcription reactions.

(2) In vitro transcription termination efficiency assay

The in vitro transcription reaction components contained 40mM Tris-HCl (pH8.0), 16mM MgCl₂2mM spermidine, 5mM DTT, 4mM ATP, GTP, CTP, UTP, 0.3. mu.L RNase inhibitor, 0.2. mu.L pyrophosphatase, 150nM RNA polymerase and 70nM PCR template, supplemented with DEPC water to 10. mu.L. The reaction system is placed at 37 ℃ for incubation for 1h, 1 mu L of the transcribed product is directly taken to be mixed with RNA loading buffer solution, heated at 80 ℃ for 2min and then placed on ice, and then electrophoresis is carried out for 1h at 100V by utilizing 12% TBE PAGE for EB detection. As shown in FIG. 4, M represents Low Range ssRNA Ladder, and compared with T7 RNAP wild type, the sgRNA transcribed by mutant S43Y is significantly better than the wild type in yield and purity.

Example 5: comparison of transcription of Cas9 mRNA by T7-RNA polymerase S43Y and wild type

Amplifying an existing vector in a laboratory by a PCR method, wherein the existing vector contains a T7 promoter and a Cas9 mRNA coding sequence (shown in a sequence table SEQ ID NO.20), taking a purified PCR product as a transcription template, and performing in vitro transcription reaction in a reaction medium containing 40mM Tris-HCL (pH8.0) and 16mM MgCl₂2mM spermidine, 5mM DTT, 4mM ATP, GTP, CTP, UTP, 0.3. mu.L RNase inhibitor, 0.2. mu.L pyrophosphatase, 150nM RNA polymerase, and 20 ng/. mu.L PCR template in a 10. mu.L system, incubating at 37 ℃ for 1h, directly taking 1. mu.L of the transcribed product, mixing with RNA loading buffer, heating at 80 ℃ for 2min, placing on ice, and performing EB detection by electrophoresis on 1.5% agarose gel at 100V for 45 min. The results are shown in FIG. 5, where run off in FIG. 5 represents full length transcriptionRNA products, terminated represents disrupted RNA products; when transcribed with the T7 RNAP wild type, a prematurely aborted RNA product appeared, whereas when transcribed with mutant S43Y, this aborted RNA product was almost negligible in the gel plot. This result also reflects that mutant S43Y was able to transcribe with high efficiency as the wild type.

Sequence listing

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agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa 1500

tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag 1560

tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa 1620

caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc 1680

gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag 1740

gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat 1800

caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc 1860

atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca 1920

gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt 1980

tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt 2040

gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta 2100

atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac 2160

tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg 2220

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 2280

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 2340

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 2400

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2460

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2520

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 2580

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 2640

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 2700

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 2760

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 2820

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 2880

ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 2940

gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 3000

gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 3060

acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca atctgctctg 3120

atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 3180

gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 3240

cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 3300

atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc 3360

acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt 3420

ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc 3480

ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat 3540

gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa 3600

acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg 3660

cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat 3720

ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa 3780

accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca 3840

cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag 3900

ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat 3960

aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag 4020

ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa 4080

gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat 4140

aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct 4200

gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta 4260

acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 4320

gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg 4380

tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct 4440

gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga 4500

tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg 4560

agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca 4620

tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg 4680

tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt 4740

gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta 4800

atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca 4860

gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat 4920

caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat 4980

ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg 5040

ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt 5100

gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg 5160

aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg 5220

gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt 5280

ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga 5340

catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct 5400

atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct 5460

cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc 5520

gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg 5580

cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct 5640

tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg 5700

ccggccacga tgcgtccggc gtagaggatc ga 5732

<210> 3

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cctagcataa accccttggg ttccctcttt aggagtctga ggggtttttt gctgaaaga 59

<210> 4

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cacagaaaaa agcccgcacc tgacagtgcg ggcttttttt ttcgactc 48

<210> 5

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 60

gctgc 65

<210> 6

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tctccgcccc tgccagaaat catccttagc gaaagctaag gatttttttt atctgaaat 59

<210> 7

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttg 47

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgtgtcccta tctgttacag tctcct 26

<210> 9

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgcttgcca tctgttttct tgcaag 26

<210> 10

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atccatgata tctgttagtt tttttc 26

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tagttttgta tctgttttgc agcagc 26

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gccaagcaca atttgccgt 19

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggagacagaa atatatcaga 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggaacccagg cactggtatt 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gggcacgggc agcttgccgg 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggaaagtgtg gaactccaag 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggtggtataa cccttgacgg 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggagccgtac atgaactgag 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gtcaagcact tgatcgacgg 20

<210> 20

<211> 4258

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gggagagccg ccaccaugga uaaaaaguau ucuauugguu uagacaucgg cacuaauucc 60

guuggauggg cugucauaac cgaugaauac aaaguaccuu caaagaaauu uaagguguug 120

gggaacacag accgucauuc gauuaaaaag aaucuuaucg gugcccuccu auucgauagu 180

ggcgaaacgg cagaggcgac ucgccugaaa cgaaccgcuc ggagaaggua uacacgucgc 240

aagaaccgaa uauguuacuu acaagaaauu uuuagcaaug agauggccaa aguugacgau 300

ucuuucuuuc accguuugga agaguccuuc cuugucgaag aggacaagaa acaugaacgg 360

caccccaucu uuggaaacau aguagaugag guggcauauc augaaaagua cccaacgauu 420

uaucaccuca gaaaaaagcu aguugacuca acugauaaag cggaccugag guuaaucuac 480

uuggcucuug cccauaugau aaaguuccgu gggcacuuuc ucauugaggg ugaucuaaau 540

ccggacaacu cggaugucga caaacuguuc auccaguuag uacaaaccua uaaucaguug 600

uuugaagaga acccuauaaa ugcaaguggc guggaugcga aggcuauucu uagcgcccgc 660

cucucuaaau cccgacggcu agaaaaccug aucgcacaau uacccggaga gaagaaaaau 720

ggguuguucg guaaccuuau agcgcucuca cuaggccuga caccaaauuu uaagucgaac 780

uucgacuuag cugaagaugc caaauugcag cuuaguaagg acacguacga ugacgaucuc 840

gacaaucuac uggcacaaau uggagaucag uaugcggacu uauuuuuggc ugccaaaaac 900

cuuagcgaug caauccuccu aucugacaua cugagaguua auacugagau uaccaaggcg 960

ccguuauccg cuucaaugau caaaagguac gaugaacauc accaagacuu gacacuucuc 1020

aaggcccuag uccgucagca acugccugag aaauauaagg aaauauucuu ugaucagucg 1080

aaaaacgggu acgcagguua uauugacggc ggagcgaguc aagaggaauu cuacaaguuu 1140

aucaaaccca uauuagagaa gauggauggg acggaagagu ugcuuguaaa acucaaucgc 1200

gaagaucuac ugcgaaagca gcggacuuuc gacaacggua gcauuccaca ucaaauccac 1260

uuaggcgaau ugcaugcuau acuuagaagg caggaggauu uuuauccguu ccucaaagac 1320

aaucgugaaa agauugagaa aauccuaacc uuucgcauac cuuacuaugu gggaccccug 1380

gcccgaggga acucucgguu cgcauggaug acaagaaagu ccgaagaaac gauuacuccc 1440

uggaauuuug aggaaguugu cgauaaaggu gcgucagcuc aaucguucau cgagaggaug 1500

accgccuuug acaagaauuu accgaacgaa aaaguauugc cuaagcacag uuuacuuuac 1560

gaguauuuca caguguacaa ugaacucacg aaaguuaagu augucacuga gggcaugcgu 1620

aaacccgccu uucuaagcgg agaacagaag aaagcaauag uagaucuguu auucaagacc 1680

aaccgcaaag ugacaguuaa gcaauugaaa gaggacuacu uuaagaaaau ugaaugcuuc 1740

gauucugucg agaucuccgg gguagaagau cgauuuaaug cgucacuugg uacguaucau 1800

gaccuccuaa agauaauuaa agauaaggac uuccuggaua acgaagagaa ugaagauauc 1860

uuagaagaua uaguguugac ucuuacccuc uuugaagauc gggaaaugau ugaggaaaga 1920

cuaaaaacau acgcucaccu guucgacgau aagguuauga aacaguuaaa gaggcgucgc 1980

uauacgggcu ggggagccuu gucgcggaaa cuuaucaacg ggauaagaga caagcaaagu 2040

gguaaaacua uucucgauuu ucuaaagagc gacggcuucg ccaauaggaa cuuuauggcc 2100

cugauccaug augacucuuu aaccuucaaa gaggauauac aaaaggcaca gguuuccgga 2160

caaggggacu cauugcacga acauauugcg aaucuugcug guucgccagc caucaaaaag 2220

ggcauacucc agacagucaa aguaguggau gagcuaguua aggucauggg acgucacaaa 2280

ccggaaaaca uuguaaucga gauggcacgc gaaaaucaaa cgacucagaa ggggcaaaaa 2340

aacagucgag agcggaugaa gagaauagaa gaggguauua aagaacuggg cagccagauc 2400

uuaaaggagc auccugugga aaauacccaa uugcagaacg agaaacuuua ccucuauuac 2460

cuacaaaaug gaagggacau guauguugau caggaacugg acauaaaccg uuuaucugau 2520

uacgacgucg aucacauugu accccaaucc uuuuugaagg acgauucaau cgacaauaaa 2580

gugcuuacac gcucggauaa gaaccgaggg aaaagugaca auguuccaag cgaggaaguc 2640

guaaagaaaa ugaagaacua uuggcggcag cuccuaaaug cgaaacugau aacgcaaaga 2700

aaguucgaua acuuaacuaa agcugagagg gguggcuugu cugaacuuga caaggccgga 2760

uuuauuaaac gucagcucgu ggaaacccgc gccaucacaa agcauguugc gcagauacua 2820

gauucccgaa ugaauacgaa auacgacgag aacgauaagc ugauucggga agucaaagua 2880

aucacuuuaa agucaaaauu ggugucggac uucagaaagg auuuucaauu cuauaaaguu 2940

agggagauaa auaacuacca ccaugcgcac gacgcuuauc uuaaugccgu cguagggacc 3000

gcacucauua agaaauaccc gaagcuagaa agugaguuug uguaugguga uuacaaaguu 3060

uaugacgucc guaagaugau cgcgaaaagc gaacaggaga uaggcaaggc uacagccaaa 3120

uacuucuuuu auucuaacau uaugaauuuc uuuaagacgg aaaucacucu ggcaaacgga 3180

gagauacgca aacgaccuuu aauugaaacc aauggggaga caggugaaau cguaugggau 3240

aagggccggg acuucgcgac ggugagaaaa guuuugucca ugccccaagu caacauagua 3300

aagaaaacug aggugcagac cggaggguuu ucaaaggaau cgauucuucc aaaaaggaau 3360

agugauaagc ucaucgcucg uaaaaaggac ugggacccga aaaaguacgg uggcuucgau 3420

agcccuacag uugccuauuc uguccuagua guggcaaaag uugagaaggg aaaauccaag 3480

aaacugaagu cagucaaaga auuauugggg auaacgauua uggagcgcuc gucuuuugaa 3540

aagaacccca ucgacuuccu ugaggcgaaa gguuacaagg aaguaaaaaa ggaucucaua 3600

auuaaacuac caaaguauag ucuguuugag uuagaaaaug gccgaaaacg gauguuggcu 3660

agcgccggag agcuucaaaa ggggaacgaa cucgcacuac cgucuaaaua cgugaauuuc 3720

cuguauuuag cgucccauua cgagaaguug aaagguucac cugaagauaa cgaacagaag 3780

caacuuuuug uugagcagca caaacauuau cucgacgaaa ucauagagca aauuucggaa 3840

uucaguaaga gagucauccu agcugaugcc aaucuggaca aaguauuaag cgcauacaac 3900

aagcacaggg auaaacccau acgugagcag gcggaaaaua uuauccauuu guuuacucuu 3960

accaaccucg gcgcuccagc cgcauucaag uauuuugaca caacgauaga ucgcaaacga 4020

uacacuucua ccaaggaggu gcuagacgcg acacugauuc accaauccau cacgggauua 4080

uaugaaacuc ggauagauuu gucacagcuu gggggugacg gaucccccaa gaagaagagg 4140

aaagucucga gcgacuacaa agaccaugac ggugauuaua aagaucauga caucgauuac 4200

aaggaugacg augacaaggc ugcaggauga ccggucauca ucaccaucac cauugagu 4258

Claims

T7-RNA polymerase mutant wherein the serine at position 43 from the N-terminus of the amino acid sequence of wild-type T7-RNA polymerase shown in SEQ ID No.1 is replaced by tyrosine, phenylalanine, leucine, lysine or aspartic acid.
2. Use of the T7-RNA polymerase mutant of claim 1 for in vitro transcription.
3. Use of the T7-RNA polymerase mutant of claim 1 for non-coding RNA synthesis.
4. The use of claim 3, wherein the non-coding RNA is a sgRNA, tRNA, mRNA, siRNA, snorRNA or oligonucleotide.
5. Use of the T7-RNA polymerase mutant of claim 1 for gene editing.
6. Use of the T7-RNA polymerase mutant of claim 1 in RNA drug synthesis.
7. Use of the T7-RNA polymerase mutant of claim 1 in an in vivo protein expression or in vitro translation system for cell-free protein expression.
8. Use of the mutant T7-RNA polymerase of claim 1 in the study of transcription terminators or in the study of RNA-dependent RNA polymerase activity.
9. Use of the T7-RNA polymerase mutant of claim 1 for the synthesis of a biological transcription regulatory element.