Tasr protein optimization mutants and their gene editing applications

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a TasR protein optimized mutant and gene editing application thereof. The TasR protein optimized mutant has one or more amino acid substitutions relative to the wild-type TasR protein shown in SEQ ID NO. 1. The amino acid substitutions are selected from E217W, E217F, E217Y, Q239F, Q239W, L256R, D276Q, D276H, S280K or S280R. The mutant of the application has significantly improved editing activity while maintaining PAM independence and a compact volume of about 340 amino acids: the single-point mutant is 2.53-3.53 times higher than the wild type. When p3.5 and tigRNA are packaged into a single AAV8 vector and delivered to the liver of a mouse, the editing efficiency of the mutant can reach PCSK9 Gene efficient editing. The application provides an efficient and compact TasR mutant and gene editing system for in vivo gene editing treatment of diseases such as hypercholesterolemia.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an optimized mutant of the TasR protein and its gene editing applications. Background Technology

[0002] RNA-guided nucleases (such as CRISPR-Cas9 and Cas12) have been widely used in genome engineering. However, their therapeutic applications are limited by two major factors: firstly, most nucleases rely on PAM / TAM recognition sequences, limiting the number of genomic sites they can target; secondly, Cas9, Cas12a, and their derivatives are too large, exceeding the packaging capacity of a single adeno-associated virus vector, leading to difficulties in in vivo delivery. While PAM-free Cas9 variants such as SpRY almost eliminate PAM limitations, they are still based on large Cas9 proteins, limiting single AAV delivery; and while mini nucleases such as Cas12f, Cas12j, IscB, TnpB, and Fanzor are compact, most still rely on PAM / TAM and require extensive engineering modifications to achieve usable editing activity in mammalian cells.

[0003] The TIGR-Tas system is a newly discovered class of compact RNA-guided nucleases. The TIGR array processes short tandem spacer guide RNAs (tigRNAs), which recognize DNA through a tandem spacer targeting mechanism. TasR (approximately 340 amino acids) contains a Nop domain (RNA-binding domain RBD, binding guide RNA), a RuvC catalytic domain, and a C-terminal coiled-coil dimerization domain. It generates staggered double-strand breaks through the two RuvC domains without requiring PAM. Although wild-type TasR possesses advantages such as PAM-free independence and compact size, its editing activity in mammalian cells is very low. Therefore, there is an urgent need for a TasR protein mutant with significantly enhanced editing activity while maintaining PAM-free independence and compact size to meet the application requirements of single AAV packaging and in vivo gene editing. Summary of the Invention

[0004] To address the issue of low editing activity of wild-type ParTasR in mammalian cells, this invention performs a deep mutation scan of the entire ParTasR protein, analyzes its sequence-function map, and identifies key gain mutation sites located at the protein-nucleic acid interface. This provides a TasR protein mutant with significantly enhanced editing activity while maintaining PAM-free and compact size, thus meeting the application requirements of single AAV packaging and in vivo gene editing.

[0005] The technical solution adopted in this invention is: This invention provides an optimized mutant of the TasR protein, wherein the optimized mutant has one or more amino acid substitutions relative to the wild-type TasR protein shown in SEQ ID NO. 1; The amino acid substitutions are selected from E217W, E217F, E217Y, Q239F, Q239W, L256R, D276Q, D276H, S280K, or S280R.

[0006] A second aspect of the present invention provides a nucleic acid molecule that encodes an optimized mutant of the TasR protein.

[0007] Preferably, the nucleic acid molecule is DNA or RNA.

[0008] A third aspect of the present invention provides an expression cassette containing the aforementioned nucleic acid molecule; The expression box also contains a promoter.

[0009] Preferably, the promoter includes at least one of the U6 promoter and the EF1α promoter.

[0010] A fourth aspect of the present invention provides a gene editing system comprising the aforementioned expression cassette; The gene editing system also includes guide RNA.

[0011] A fifth aspect of the present invention provides a recombinant expression vector comprising the gene editing system described above.

[0012] Preferably, the starting vector of the recombinant expression vector is the pAAV8 vector.

[0013] The sixth aspect of this invention provides an application of the TasR protein optimized mutant, the nucleic acid molecule, the expression cassette, the gene editing system, and / or the recombinant expression vector, wherein the application refers to any one of the following: 1) Prepare gene-edited products; 2) Prepare gene therapy drugs.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention identifies key gain mutation sites at the protein-nucleic acid interface by performing a deep mutation scan of the entire ParTasR protein. The obtained TasR protein mutants, while maintaining PAM-independent editing and a compact size of approximately 340 amino acids, exhibit significantly enhanced editing activity. Single-point mutations can increase editing activity by 2.53–3.53 times compared to the wild type, while combined mutations can increase it by approximately 10 times. Among these, the preferred combined mutant p3.5 exhibits the highest activity at all tested doses.CXCR4 The site-editing activity was approximately 5.81 times that of the wild type. B2M The site size is approximately 9.07 times larger. This mutant retains the PAM-independent nature of TasR, with a compact size of approximately 340 amino acids. Its dimer is also smaller than most RNA-guided nucleases, allowing it to be co-packaged with tigRNA into a single AAV vector, meeting in vivo delivery requirements. In in vivo applications, PCSK9 gene editing is highly efficient via single AAV8 delivery. After single AAV8 delivery, liver tissue indels decreased by approximately 25.36%, circulating PCSK9 decreased by approximately 82%, and LDL-C decreased by approximately 47%. Therefore, this invention provides a highly efficient and compact TasR mutant and gene editing system for in vivo gene editing therapy of diseases such as hypercholesterolemia. Attached Figure Description

[0015] Figure 1 A schematic diagram illustrating the identification of activity-enhancing mutations in the TasR protein using a deep mutation scan. A is a schematic diagram of the ccdB reverse screening experiment with cut-dependency; B represents the reproducibility of the enrichment spectra obtained from three independent screenings (3D scatter plot). C is the average Log2FC heatmap of all single amino acid substitutions (labeled with RBD, RuvC, CC and C-terminal loop domain partitions). D represents the Log2FC distribution of single amino acid substitutions in each domain; E represents the enrichment score mapped to the structure of the TasR–tigRNA–DNA ternary complex predicted by AlphaFold3.

[0016] Figure 2 The figure shows the verification and testing results of the candidate protein mutants; A represents the editing activity scatter points (including domain color bands) of single-point mutants; B represents the fold-change distribution of single amino acid variants in each domain; C is a partial view of the protein structure at five preferred mutation sites; D is a heatmap of the fold-change matrix of preferred mutation sites in pairs; E represents a combined mutant in CXCR4 and B2M Fold-change histogram of loci; F represents the dose-response curve of mRNA at the human CXCR4 site in HEK293T cells; G represents the dose-response curve of mRNA at the mouse Pcsk9 site.

[0017] Figure 3 Editing of mouse livers via PIVOT delivery via single AAV8 PCSK9Genetic results diagram; A is a schematic diagram of the timeline of single AAV8 tail vein delivery and blood / skin collection; B represents liver tissue. PCSK9 Editing efficiency (indel); C represents serum. PCSK9 The level changes over time; D represents the serum PCSK9 level on day 63; E represents PCSK9 immunofluorescence staining of liver tissue; F represents the change in serum LDL-C levels over time; G represents the serum LDL-C level on day 63. Detailed Implementation

[0018] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0019] The inventive concept of this invention is as follows: While wild-type ParTasR has advantages such as being PAM-free, small in size, and easy to deliver with a single AAV, its editing activity in mammalian cells is very low. This invention selects to perform a deep mutation scan on the entire ParTasR protein, systematically analyze its sequence-function map, and focus on identifying key gain mutation sites located at the protein-nucleic acid interface, thereby obtaining a TasR mutant that retains the original structural advantages and can significantly improve editing activity.

[0020] In terms of specific technical approach, this invention first identifies a batch of single-point gain mutations, including E217W, E217F, E217Y, Q239F, Q239W, L256R, D276Q, D276H, S280K, and S280R, through deep mutation scanning and cleavage-dependent ccdB reverse screening. These single-point mutations increase editing activity by approximately 2.53 to 3.53 times compared to the wild type. Subsequently, the E217W, Q239F, L256R, D276Q, and S280R sites are further optimized for pairwise and higher-order combinations to construct p1.1 to p5.1 combined mutants, resulting in combined mutation activity increases of up to 10 times or more. Among these, the preferred combined mutant p3.5 (E217W+L256R+S280R) exhibits... CXCR4 and B2MThe site activity reached approximately 5.81-fold and 9.07-fold that of the wild type, respectively, and exhibited the highest activity in dose-response experiments. Simultaneously, this mutant retained TasR's PAM-independent, compact size of approximately 340 amino acids, and its dimer was smaller than most RNA-guided nucleases, allowing it to be co-packaged with tigRNA into a single AAV vector, meeting in vivo delivery requirements. Based on this, this invention successfully delivered p3.5 and tigRNA as a single AAV8 vector to mouse liver. PCSK9 Gene editing resulted in approximately 25.36% indels in liver tissue and circulating in the liver. PCSK9 The cholesterol levels decreased by approximately 82%, and LDL-C decreased by approximately 47%, demonstrating that this regimen can be used for in vivo gene editing therapy for diseases such as hypercholesterolemia.

[0021] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0022] Example 1 An optimized mutant of the TasR protein and its gene editing application are detailed below: 1. Deep mutation scanning of TasR protein.

[0023] A DMS library covering all single amino acid substitutions of ParTasR was constructed: the coding sequence was divided into seven fragments of approximately 150 bp, and a pool of saturated mutant oligonucleotides was synthesized by Twist Bioscience. The library was then assembled using Gibson. The library was transformed into E. coli TOP10 containing an arabinose-induced ccdB virus plasmid, and active variants were enriched using cleavage-dependent ccdB reverse screening. The fitness of each variant was quantified by Illumina NovaSeq 6000 sequencing with log2FC relative to the wild type. The Spearman correlation coefficients between the three independent repeats were 0.90–0.95.

[0024] A total of 125 candidate gain mutations (mean log2FC>1) were identified, distributed as follows: 83 in the RBD domain, 24 in the RuvC domain, and 18 in the CC domain. Figure 1 ).

[0025] 2. Construction and validation of single mutants.

[0026] Targeting plasmids were constructed from 125 candidate mutants and used in HEK293T cells. CXCR4Sites were targeted, and the indel percentage was calculated using NGS sequencing. This percentage was then normalized to the wild-type ParTasR protein indel percentage to identify the top ten single-point mutants with the greatest activity enhancement. These mutants exhibited editing activity 2.53–3.53 times higher than the wild-type. Figure 2 (A and B). The first ten gain abrupt changes are E217W, E217F, E217Y, Q239F, Q239W, L256R, D276Q, D276H, S280K and S280R (Table 1).

[0027] Table 1. Editing efficiency of the top ten single mutants with the largest ParTasR improvements. WT 0.87 0.92 1.21 1.00 D276H 2.36 2.47 2.77 2.53 D276Q 2.42 2.74 2.47 2.54 L256R 2.57 2.67 2.99 2.74 S280R 2.95 2.73 3.22 2.97 Q239W 3.23 2.62 3.09 2.98 S280K 2.92 2.99 3.19 3.03 E217Y 3.00 3.27 3.24 3.17 Q239F 3.17 3.20 3.17 3.18 E217F 2.73 3.21 3.81 3.25 E217W 3.52 3.82 3.25 3.53 3. Construction and validation of combined mutants.

[0028] The above mutants were combined in pairs, and the five mutation sites E217W, Q239F, L256R, D276Q, and S280R that showed the greatest improvement when combined with other variants were selected. Figure 2 (D); These five preferred sites were combined in a higher order and named p1.1~p5.1 (see Table 5 for specific sequence information), and then applied to HEK293T cells. CXCR4 , B2M Site testing showed that editing efficiency was significantly improved compared to wild-type. Figure 2 (E and Table 2). Among them, p3.5 (E217W+L256R+S280R) is in CXCR4 The site-editing activity was approximately 5.81 times that of the wild type. B2M The number of sites is approximately 9.07 times.

[0029] Table 2 Editing efficiency of ParTasR's preferred high-order combination mutations of 5 mutation types 4. Dose-response determination.

[0030] For the top five mutant combinations with the highest improvement in editing efficiency (p2.4, p3.3, p3.5, p3.6, and p4.2), mRNA was prepared via in vitro transcription (IVT) and then chemically synthesized targeting human cells. CXCR4 Genes and mice PCSK9 The tigRNA of the gene was used to determine the editing efficiency after gradient transfection and dose-response curve fitting. Figure 2 (F and G). The results showed that p3.5 in humans CXCR4 The peak indel for target fitting was 58.97% in mice. PCSK9 The target concentration was 62.19%, and the activity was the highest at all tested doses.

[0031] The combined mutant mRNA was prepared via in vitro transcription (IVT): using a linearized plasmid containing the ParTasR coding sequence as a template, transcription was performed using a T7 RNA polymerase in vitro transcription system (Synthgene, catalog number 1030M-1). The transcription product was purified by column chromatography and then used for transfection. Targeted human... CXCR4 Genes and mice PCSK9 The tigRNA of the gene was chemically synthesized by GenScript.

[0032] Dose-response assays were performed in 48-well plates: human HEK293T cells (human...) were used to... CXCR4 (target) or mouse N2a cells (mouse) PCSK9 Target points) at 4.0 × 10 per well 4 After seeding cells at a density of 1,000 cells and culturing for 24 h, mRNA and tigRNA were co-transfected into cells in Opti-MEM medium using Lipofectamine MessengerMAX transfection reagent (Thermo Fisher, catalog number LMRNA003). The mRNA dose was set at 250 ng per well, with 7 dose points (250, 83.33, 27.78, 9.26, 3.09, 1.03, 0.343 ng / well) using a 3-fold serial dilution series, with 3 replicates per dose point. Cells were collected and genomic DNA was extracted 48 h after transfection. Nested PCR was used to amplify the flanking regions of the editing target sites using primers carrying sample-specific barcodes. Sequencing was performed on an Illumina NovaSeq 6000 platform, and the indel frequency for each sample was calculated using CRISPResso2 software. The dose-response data for each mutant combination were fitted using a four-parameter Hill (variable slope) dose-response equation to obtain the fitted peak value and EC50 parameters. The obtained fitting peak values ​​and parameters such as EC50 are shown in Tables 3 and 4.

[0033] Table 3. Parameters for fitting dose-response curves of the top 5 ParTasR-efficient mutant combinations (human) CXCR4 (target) p2.4 (E217W / S280R) 57.26 8.81 p3.3 (E217W / Q239F / S280R) 42.02 9.46 p3.5 (E217W / L256R / S280R) 58.97 8.22 p3.6 (E217W / D276Q / S280R) 55.31 12.35 p4.2 (E217W / Q239F / L256R / S280R) 49.54 11.31 Table 4. Parameters for fitting dose-response curves of the top 5 ParTasR-efficient mutant combinations (mice) PCSK9 (target) p2.4 (E217W / S280R) 58.51 25.36 p3.3 (E217W / Q239F / S280R) 49.25 39.63 p3.5 (E217W / L256R / S280R) 62.19 21.71 p3.6 (E217W / D276Q / S280R) 60.34 23.29 p4.2 (E217W / Q239F / L256R / S280R) 49.56 30.25 5. Single AAV delivery.

[0034] In this experiment, tigRNA and p3.5 were packaged into a single AAV8 vector and delivered to the liver of adult mice to achieve... PCSK9 Efficient gene editing significantly reduced circulating PCSK9 and LDL-C levels. Specifically:

[0035] A pAAV8 vector containing a tigRNA expression cassette driven by the U6 promoter and a p3.5 coding sequence driven by the EF1α promoter was constructed and packaged as AAV8 virus (AAV8-PIVOT, or PIVOT for short) by PackGene. The titer was quantified by qPCR (ITR primers). Six-week-old male C57BL / 6 mice were injected intravenously via tail vein with AAV8-PIVOT at a dose of 5 × 10⁻⁶. 13 Blood samples were collected at a dose of vg / kg before injection (day -1) and on days 7, 14, 21, 28, 35, 49, and 63 after injection. Liver tissue was collected in batches on days 35, 49, and 63.

[0036] Targeted amplicon sequencing of liver tissue genomic DNA showed that the liver tissue of PIVOT group mice... PCSK9 The average indel was approximately 25.36%; by day 63, the serum PCSK9 level in the PIVOT group was approximately 82% lower than that in the AAV8-EGFP control group (using the Sangon Biotech mouse PCSK9 ELISA kit, catalog number D721080-0096), and the serum LDL-C level was approximately 47% lower (using the Sangon Biotech low-density lipoprotein cholesterol micro-assay kit, catalog number D799859-0096); immunofluorescence staining of liver tissue showed that the liver of the PIVOT group... PCSK9 The signal almost disappeared (see Figure 3 These results demonstrate that the present invention has successfully achieved the effect of [the following] in mice. PCSK9 Gene editing.

[0037] The amino acid sequences of the wild-type and mutant ParTasR protein described in this invention are shown in Table 5.

[0038] Table 5. Amino acid sequences of wild-type and mutant ParTasR protein ParTasR_wild-type MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKGIGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 1 ParTasR_E217F MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEFVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 2 ParTasR_E217Y MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEYVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 3 ParTasR_Q239W MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKWRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 4 ParTasR_D276H MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSHTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 5 ParTasR_S280K MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLKLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 6 ParTasR_E217W(p1.1) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 7 ParTasR_Q239F(p1.2) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 8 ParTasR_L256R(p1.3) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 9 ParTasR_D276Q(p1.4) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 10 ParTasR_S280R(p1.5) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 11 ParTasR_E217W+Q239F (p2.1) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 12 ParTasR_E217W+L256R (p2.2) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 13 ParTasR_E217W+D276Q (p2.3) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 14 ParTasR_E217W+S280R (p2.4) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 15 ParTasR_Q239F+L256R (p2.5) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 16 ParTasR_Q239F+D276Q (p2.6) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 17 ParTasR_Q239F+S280R (p2.7) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 18 ParTasR_L256R+D276Q (p2.8) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 19 ParTasR_L256R+S280R (p2.9) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKYKIFVQHVWLAWRTCEGLETREEPPLQYCHPEGHEGS 20 ParTasR_D276Q+S280R (p2.10) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKMMKIFVQHVWLAWRTCEGLEKYPYPYCHGHEGFIG 21 ParTasR_E217W+Q239F+L256R(p3.1) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYDQEKARQREIEYPEGELASKFLGYKKSDTHSLNHAHRRAIRKMMKYKIFVQHVWLAWRTCEGLEKYPYPYGHEGFIG 22 ParTasR_E217W+Q239F+D276Q(p3.2) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKA RQLEIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 23 ParTasR_E217W+Q239F+S280R(p3.3) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYDQEKARQLEIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKMMKIFVQHVWLAWRTCEGLEKYKYGPHYGFIG 24 ParTasR_E217W+L256R+D276Q(p3.4) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKA RQREIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 25 ParTasR_E217W+L256R+S280R(p3.5) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKMMKIFVQHVWLAWRTCEGLEKYPYPYGPHYGFIGHE 26 ParTasR_E217W+D276Q+S280R(p3.6) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIYDQEKA RQLEIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 27 ParTasR_Q239F+L256R+D276Q(p3.7) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKMMKIFVQHVWLAWRTCEGLEKYPYPYGFIGHE 28 ParTasR_Q239F+L256R+S280R(p3.8) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDLKIVKKKVKKVKWKWKWKWKWKWKWKWKWKW IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPVLQEPLQYHGFIGCHE 29 ParTasR_Q239F+D276Q+S280R(p3.9) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 30 ParTasR_L256R+D276Q+S280R(p3.10) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKQRSPVYRDIIDQEKARQREIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 31 ParTasR_E217W+Q239F+L256R+D276Q(p4.1) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSQTHLSLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 32 ParTasR_E217W+Q239F+L256R+S280R(p4.2) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSDTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 33 ParTasR_E217W+Q239F+D276Q+S280R(p4.3) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQLEIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 34 ParTasR_E217W+L256R+D276Q+S280R(p4.4) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKQRSPVYRDIIDQEKARQREIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEHFIEPPVKIQPEGS 35 ParTasR_Q239F+L256R+D276Q+S280R(p4.5) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEEVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 36 ParTasR_E217W+Q239F+L256R+D276Q+S280R (p5.1) MRQVAIDWAFAEEKLAVYDGKKVLKAVPKLKAGDEIFAENIPMKHAAQWLKDGIIIRRCRPNDTAALRKEIGQEKTDALDAKLIWQLAEAHPEKFREWKGDPQLTTLYRAFKEVQRCRVGQSNRVWAKGEETATAVLDDLEKTERKIVKAIEKELKSYKVWDWLSQIKG IGVATGGGLVGLIAKYGIENIRQVSSLWHLFGLHVVEGKAPRRTKGEWVSYVPEAKTLVLGVIADCFIKFRSPVYRDIYDQEKARQREIEYPEGELASKFLGYKKSQTHLRLNHAHRRAIRKMMKIFVQHVWLAWRTCEGLETRPLYCHEYLGHEFIEPPVKIQPEGS 37 The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0039] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An optimized mutant of the TasR protein, characterized in that, The optimized mutant of the TasR protein has one or more amino acid substitutions relative to the wild-type TasR protein shown in SEQ ID NO. 1; The amino acid substitutions are selected from E217W, E217F, E217Y, Q239F, Q239W, L256R, D276Q, D276H, S280K, or S280R.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the TasR protein optimized mutant as described in claim 1.

3. An expression box, characterized in that, The expression cassette contains the nucleic acid molecule as described in claim 2; The expression box also contains a promoter.

4. The expression box according to claim 3, characterized in that, The promoter includes at least one of the U6 promoter and the EF1α promoter.

5. A gene editing system, characterized in that, The gene editing system includes the expression cassette as described in claim 3; The gene editing system also contains guide RNA.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene editing system of claim 5.

7. The recombinant expression vector according to claim 6, characterized in that, The starting vector for the recombinant expression vector is the pAAV8 vector.

8. The application of the TasR protein optimized mutant of claim 1, the nucleic acid molecule of claim 2, the expression cassette of claim 3, the gene editing system of claim 5, and / or the recombinant expression vector of claim 6, characterized in that, The application refers to any one of the following: 1) Prepare gene-edited products; 2) Prepare gene therapy drugs.

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