T7 RNA polymerase mutant for synthesizing single-stranded DNA as well as screening method and application of T7 RNA polymerase mutant

Abstract

The invention relates to a T7RNA polymerase mutant for synthesizing single-stranded DNA as well as a screening method and application of the T7RNA polymerase mutant, and belongs to the technical field of enzyme engineering. The invention provides a method for directed evolution and screening of a T7RNA polymerase mutant, which comprises the following steps: knocking out exonuclease and mismatch repair protein from a chassis strain genome, knocking in single-stranded annealing protein, resistance gene containing deletion mutation and a single-stranded DNA synthesis template, and constructing to obtain a genetically engineered bacterium for screening; a T7RNA polymerase mutant library is constructed, recombinant plasmids with the mutant library are transferred into engineering bacteria, T7RNA polymerase mutants synthesize single-stranded DNA to repair deletion mutation of resistance genes, the transformed engineering bacteria can grow on a resistance plate, the T7RNA polymerase mutants obtained through multiple rounds of screening can efficiently synthesize the single-stranded DNA through in-vitro verification, and the T7RNA polymerase mutant can be used for preparing the single-stranded DNA. A brand new tool is provided for synthesis of single-stranded DNA, and the method has the potential of being applied to the fields of gene editing, evolutionary engineering and the like.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering technology, in particular to a T7 RNA polymerase mutant for synthesizing single-stranded DNA and a screening method and application thereof. Background Art

[0002] Single-stranded DNA (ssDNA) refers to a DNA molecule composed of only one deoxyribonucleotide chain. Under certain conditions, including DNA replication, unwinding during transcription, and local unwinding during DNA damage repair, double-stranded DNA will temporarily unwind to form single-stranded DNA. ssDNA has multiple functions. During DNA replication, ssDNA acts as a template chain to guide the synthesis of new chains; during transcription, the ssDNA template chain is recognized by RNA polymerase and synthesized into a complementary RNA chain; when the DNA double strand is damaged, such as a double-strand break, the cell will initiate a repair mechanism and use ssDNA as a template or intermediate product for repair synthesis. ssDNA can also participate in the formation of non-B-type structures such as R-loops and G-quadruplexes, which play an important role in regulating gene expression.

[0003] During the gene editing process, the single-stranded DNA template can be used as part of the repair mechanism to integrate into the genome, thereby achieving precise editing of the target gene. For example, PE (prime editing) technology can integrate a single-stranded DNA into the genome through the cell's self-repair mechanism, and the original sequence will be removed by the cell's repair mechanism. By studying and utilizing the structural and functional characteristics of ssDNA, the parameters and conditions of gene editing technology can be optimized to improve the efficiency and accuracy of gene editing. Understanding the dynamic changes of ssDNA during DNA replication and transcription will help to better control the timing and location of gene editing. Many gene editing technologies, such as CRISPR / Cas9, TALENs, and ZFNs, require the recognition and cutting of specific DNA sequences. As a potential target for these technologies, ssDNA provides more possibilities and flexibility for gene editing.

[0004] Traditional ssDNA synthesis methods mainly rely on reverse transcriptase. Although this strategy has achieved the in vivo synthesis of ssDNA to a certain extent, the length of its product is often limited to dozens of nucleotides, which is obviously inadequate for editing tasks that need to cover longer gene fragments or the entire genome. In addition, the use of reverse transcriptase is also limited by host specificity, which means that the same enzyme may not work effectively in different organisms, which greatly limits the versatility and application scope of ssDNA synthesis technology.

[0005] Therefore, a new method is urgently needed to achieve a more efficient, more versatile in vivo synthesis method that can generate longer ssDNA. T7 RNA polymerase has been widely used in the field of synthetic biology due to its high sequence specificity, ability to accurately identify and initiate transcription of DNA sequences downstream of the T7 promoter, and excellent transcription efficiency. In recent years, studies have shown that T7 RNA polymerase can synthesize ssDNA using deoxyribonucleotides in the reaction system under in vitro conditions. However, its synthesis efficiency and action scenarios have certain limitations, and it cannot be truly put into widespread application in the fields of gene editing and synthetic biology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for directed evolution and screening of T7 RNA polymerase mutants and mutants screened using the method. First, exonucleases and mismatch repair proteins are knocked out in the genome of a chassis strain, and single-stranded annealing proteins, resistance genes containing deletion mutations, and single-stranded DNA synthesis templates are knocked in to construct genetically engineered bacteria for screening. Then, a T7 RNA polymerase mutant library is constructed, and a recombinant plasmid carrying the mutant library is transferred into the engineered bacteria. The T7 RNA polymerase mutant synthesizes single-stranded DNA to repair the deletion mutation of the resistance gene, so that the transformed engineered bacteria can grow on a resistance plate. After multiple rounds of screening, a T7 RNA polymerase mutant with high recombination efficiency is obtained, and its ability to synthesize single-stranded DNA is verified in vitro.

[0007] The first object of the present invention is to provide a T7 RNA polymerase mutant, wherein the T7 RNA polymerase mutant has the aspartic acid at position 10 mutated to glutamic acid, the isoleucine at position 581 mutated to phenylalanine, the alanine at position 586 mutated to valine and the alanine at position 615 mutated to threonine in the starting sequence as shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the T7 RNA polymerase mutant is shown in SEQ ID NO.2.

[0009] The second object of the present invention is to provide a gene encoding the above-mentioned T7 RNA polymerase mutant.

[0010] The third object of the present invention is to provide an expression vector comprising the above gene.

[0011] The fourth object of the present invention is to provide a method for synthesizing single-stranded DNA in a host cell by using the mutant, the gene or the expression vector.

[0012] The fifth object of the present invention is to provide a method for repairing a deletion mutation in the host cell genome comprising the mutant, the gene or the expression vector.

[0013] The sixth object of the present invention is to provide a host cell comprising the mutant, the gene or the expression vector.

[0014] The seventh object of the present invention is to provide the use of the mutant, the gene, the expression vector or the host cell in in vitro synthesis of single-stranded DNA.

[0015] The eighth object of the present invention is to provide a method for screening T7 RNA polymerase mutants, comprising the following steps:

[0016] Step S1, constructing a genetically engineered bacterium for screening, wherein the genetically engineered bacterium knocks out a nuclease and a mismatch repair protein in the genome of the chassis strain, knocks in a single-stranded annealing protein, a first resistance gene containing a deletion mutation, and a single-stranded DNA synthesis template, wherein the template includes a T7 promoter and a T7 terminator;

[0017] Step S2, constructing a recombinant plasmid, wherein the plasmid includes an inducible promoter and a second resistance gene;

[0018] Step S3, using the recombinant plasmid as a template, constructing a T7 RNA polymerase mutant library;

[0019] Step S4, transforming the plasmid containing the T7 RNA polymerase mutant library into genetically engineered bacteria, coating the first resistance plate, and allowing the T7 RNA polymerase mutant to synthesize single-stranded DNA to repair the deletion mutation of the first resistance gene, so that the genetically engineered bacteria grow in the first resistance plate;

[0020] Step S5, repeating step S4 to carry out directed evolution of the T7 RNA polymerase mutant in the engineered bacteria, the number of repetitions is 2-4 times, extracting the enriched plasmid, and sequencing to obtain the T7 RNA polymerase mutant.

[0021] In one embodiment of the present invention, the first resistance gene is a chloramphenicol resistance gene.

[0022] In one embodiment of the present invention, the chloramphenicol resistance gene lacks the 75th amino acid.

[0023] In one embodiment of the present invention, the inducible promoter is an arabinose inducible promoter.

[0024] In one embodiment of the present invention, the second resistance gene is a spectinomycin resistance gene.

[0025] In one embodiment of the present invention, the single-stranded DNA synthesis template is shown as SEQ ID NO.3.

[0026] In one embodiment of the present invention, step S4 is repeated twice.

[0027] Furthermore, the chassis strain is Escherichia coli TOP10.

[0028] Furthermore, the genes encoding exonucleases are recJ and sbcB.

[0029] Beneficial effects of the present invention:

[0030] The T7 RNA polymerase mutant provided by the present invention has more optimized catalytic activity and substrate recognition ability, thereby showing higher efficiency and accuracy in specific biosynthesis or gene repair applications. By encoding the mutant gene and its expression vector, a powerful tool is provided for synthesizing single-stranded DNA in host cells, thereby meeting the needs of various biotechnology and synthetic biology applications, and showing a wide range of application prospects in in vitro synthesis of single-stranded DNA and in vivo repair of host cell genome deletion mutations. These applications are not limited to academic research, but can also be extended to the fields of biotechnology industry, gene therapy, etc. The present invention also provides an efficient T7 RNA polymerase mutant screening method. By constructing a genetically engineered bacterium, a recombinant plasmid and a T7 RNA polymerase mutant library, and utilizing the principles of single-stranded DNA synthesis and resistance gene repair, a T7 RNA polymerase mutant with excellent performance can be quickly screened out, providing strong support for subsequent research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0032] Figure 1 This is a schematic diagram of the principle of T7 RNA polymerase mutant synthesizing single-stranded DNA to repair deletion mutations in the present invention;

[0033] Figure 2 is a map of the recombinant plasmid pTM in Example 2 of the present invention;

[0034] Figure 3 is the size of 10 libraries in the directed evolution process in Example 2 of the present invention;

[0035] Figure 4 It is the mutant enrichment of 10 libraries and the control group (Y639F) during the directed evolution process in Example 3 of the present invention;

[0036] Figure 5 is the probability of strain recombination after culturing the 12 enriched mutants and the control group (Y639F) in Example 4 of the present invention;

[0037] Figure 6 is the concentration of single-stranded DNA synthesized by the wild type (WT), control group (Y639F) and experimental group (TM10) in Example 4 of the present invention;

[0038] Figure 7 This is the gel electrophoresis of the single-stranded DNA synthesized by the wild type (WT), control group (Y639F) and experimental group (TM10) in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] The culture medium involved in the following examples is as follows:

[0041] Culture medium (g / L): Tryptone 10, yeast powder 5, NaCl 10.

[0042] The inducing agent concentrations involved in the following examples are as follows:

[0043] L-arabinose: 0.01 M, isopropylthiogalactoside (IPTG) 0.5 μM.

[0044] The culture methods involved in the following embodiments are as follows:

[0045] The seeds cultured at 37°C and 220 rpm for 12 h were transferred into the fermentation medium at a 1% inoculum amount and induced for 12 h at 37°C and 220 rpm.

[0046] Example 1: Construction of engineered bacteria E. coli TOP13

[0047] The CRISPR / Cas9 gene editing tool was used to knock out the exonucleases (recJ, Sequence ID: CP160468.1 and sbcB, Sequence ID: CP080620.1) and mismatch repair protein (MutS, Sequence ID: CP048330.1) in the Escherichia coli TOP10 genome. The chloramphenicol resistance CmR (Sequence ID: OR992664.1) with a missing amino acid at position 75, the single-stranded annealing protein Csp (GenBank: OP072625.1) and a single-stranded DNA synthesis template containing a T7 promoter and a T7 terminator (the nucleotide sequence is shown in SEQ ID NO.3) were inserted into the knocked-out gene MutS. Only by restoring chloramphenicol resistance through the synthesis of single-stranded DNA can the cell grow under the screening conditions ( Figure 1 ).

[0048] Table 1 Primer list

[0049]

[0050]

[0051] Example 2: Construction of recombinant plasmid and mutant library

[0052] The recombinant expression vector pTM was constructed using the Gibson assembly method to encode T7 RNA polymerase and its mutants induced by arabinose. pTM carries spectinomycin resistance, allowing the host to grow on plates containing spectinomycin resistance. The plasmid map is shown in Figure 2 shown.

[0053] Based on the recombinant expression vector pTM, a T7 RNA polymerase (SequenceID: AM946981.2) mutant library was constructed. The mutations included saturation mutations and random mutations. The mutant library and its characteristics are shown in Table 2. Degenerate base NNK was used to introduce saturation mutations, mismatch PCR kit (Biyuntian) was used to introduce random mutations, and a one-step cloning kit was used to construct plasmids. The constructed recombinant expression vector with mutant library was transformed into the engineering bacteria E. coli TOP13, and the expression of T7 RNA polymerase mutants was induced in a medium containing an inducer (arabinose) for 6 hours. After dilution in a certain gradient, it was spread on a spectinomycin resistance plate, and the library size was calculated by colony number × dilution multiple ( Figure 3 ).

[0054] Table 2 Mutation library and its characteristics

[0055]

[0056]

[0057] Example 3: Directed evolution of T7 RNA polymerase mutants

[0058] The pTM-T7 RNAP recombinant plasmid was used to induce the expression of T7 RNA polymerase mutant Y639F as a control. After sequencing verification, the recombinant wild-type T7 RNA polymerase expression vector plasmid was successfully constructed and then transformed into E. coli TOP13. Colony PCR was used to verify the successful transformation of the plasmid.

[0059] The recombinant plasmid containing the mutant library constructed in Example 2 was transformed into the engineered bacteria E. coli TOP13, and after 6 hours of induction culture, a chloramphenicol resistance plate was coated to obtain a recombinant strain that can grow on the resistance plate as the first round of screening; the plate colony was washed and the plasmid was extracted, and it was re-transformed into the engineered bacteria E. coli TOP13 and then coated with a chloramphenicol resistance plate for the second round of screening; the third round of screening was continued in the same way. During the screening process, the editing efficiency was obtained by dividing the number of colonies that obtained chloramphenicol resistance by the sum of the strains that obtained and did not obtain chloramphenicol resistance. It was found that variants carrying T7 RNA polymerase mutations that can efficiently synthesize ssDNA in cells were enriched during the cycle, which was manifested as a continuous increase in the probability of recombination, such as Figure 4 Subsequently, about 200 mutants were selected on the third round of chloramphenicol resistance plates generated by 10 libraries for Sanger sequencing, and 12 T7 RNA polymerase mutants with large enrichment were selected for further characterization.

[0060] Example 4: Characterization of T7 RNA polymerase mutants

[0061] The pTM-T7 RNAP recombinant plasmid was used to induce the expression of T7 RNA polymerase mutant Y639F and wild-type T7 RNA polymerase WT as controls. After sequencing verification, the recombinant wild-type T7 RNA polymerase expression vector plasmid was successfully constructed and then transformed into E. coli TOP13. Colony PCR was used to verify the successful transformation of the plasmid.

[0062] The seeds cultured at 37°C and 220 rpm for 12 h were transferred to the fermentation medium at an inoculum of 0.1% and cultured at 37°C and 220 rpm for 12 h. They were spread on plates containing chloramphenicol and plates without chloramphenicol, and the editing efficiency was obtained by dividing the number of colonies with chloramphenicol resistance by the sum of the number of strains with and without chloramphenicol resistance. The recombination probability was calculated as Figure 5 As shown, it can be seen that the mutant Y639F has a lower editing probability than TM1-12 screened by the present invention, wherein mutant T1-7 is derived from library 6 and TM8-12 is derived from library 7.

[0063] T7 RNA polymerase and its mutants were expressed using strain E. coli BL21 and pET-28A vector (Sequence ID: MK847899.1). After overnight induction culture, the cells were broken, the supernatant was centrifuged and filtered through a 0.45 μm filter membrane, and then T7 RNA polymerase and its mutants were purified using a gravity column (Sangon Biological Order NO. C600801). After SDS-PAGE verification of the correct size, single-stranded DNA synthesis was performed according to the T7 RNA polymerase in vitro transcription kit operating procedure (NEB), wherein the template used for in vitro transcription was replaced with the single-stranded DNA synthesis template containing the T7 promoter and terminator on the genome of the engineering bacteria TOP13 described in Example 1, and the single-stranded DNA concentration was determined using Qubit4 ( Figure 6 ), gel electrophoresis confirmed the synthesis product ( Figure 7 ). It can be seen from the results that the T7 RNA polymerase mutant screened by the present invention has better single-stranded DNA synthesis ability than the wild type and mutant Y639F.

[0064] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A T7 RNA polymerase mutant, characterized in that: The T7 RNA polymerase mutant has a starting sequence as shown in SEQ ID NO.1 in which the 10th aspartic acid is mutated to glutamic acid, the 581st isoleucine is mutated to phenylalanine, the 586th alanine is mutated to valine, and the 615th alanine is mutated to threonine.

2. A gene encoding the T7 RNA polymerase mutant according to claim 1.

3. An expression vector comprising the gene according to claim 2.

4. Use of the mutant according to claim 1, the gene according to claim 2 or the expression vector according to claim 3 in synthesizing single-stranded DNA in a host cell.

5. Use of the mutant according to claim 1, the gene according to claim 2 or the expression vector according to claim 3 in repairing deletion mutations in the host cell genome.

6. A host cell comprising the mutant according to claim 1, the gene according to claim 2 or the expression vector according to claim 3.

7. Use of the mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3 or the host cell according to claim 6 in in vitro synthesis of single-stranded DNA.

8. A method for directed evolution and screening of T7 RNA polymerase mutants, characterized in that: The following steps are involved: Step S1, constructing a genetically engineered bacterium for screening, wherein the genetically engineered bacterium knocks out a nuclease and a mismatch repair protein in the genome of the chassis strain, knocks in a single-stranded annealing protein, a first resistance gene containing a deletion mutation, and a single-stranded DNA synthesis template, wherein the template includes a T7 promoter and a T7 terminator; Step S2, constructing a recombinant plasmid, wherein the plasmid includes an inducible promoter and a second resistance gene; Step S3, using the recombinant plasmid as a template, constructing a T7 RNA polymerase mutant library; Step S4, transforming the plasmid containing the T7 RNA polymerase mutant library into genetically engineered bacteria, coating the first resistance plate, and allowing the T7 RNA polymerase mutant to synthesize single-stranded DNA to repair the deletion mutation of the first resistance gene, so that the genetically engineered bacteria grow in the first resistance plate; Step S5, repeating step S4 to carry out directed evolution of the T7 RNA polymerase mutant in the engineered bacteria, the number of repetitions is 2-4 times, extracting the enriched plasmid, and sequencing to obtain the T7 RNA polymerase mutant.

9. The method according to claim 8, characterized in that: The chassis strain is Escherichia coli TOP10.

10. The method according to claim 8, characterized in that: The genes encoding exonucleases are recJ and sbcB.

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