Enhanced gene knockdown tool and applications thereof

Abstract

The application provides an enhanced gene knockdown tool and application thereof, and belongs to the technical field of biotechnology. Based on the function research of RNAi effector protein, two plasmid tools, a basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) and an enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA), are constructed. The tool can overcome the dependence restriction of RNAi on the expression amount of endogenous Ago2 protein, synchronously deliver the effector protein and shRNA through single-plasmid co-expression, directly enhance the cleavage activity of RISC to the target gene to improve the gene knockdown efficiency, and can show the technical effect of significantly enhancing the gene knockdown efficiency on various target genes such as virus RNA, non-coding RNA and endogenous oncogene. The two tool plasmids of the application have the comprehensive advantages of simple cloning operation, high target gene universality, strong shRNA recognition specificity, high biological safety and high gene silencing efficiency.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and discloses an enhanced gene knockdown tool, particularly a method and its application that can enhance gene knockdown expression through an exogenous protein complementation mechanism, which is especially suitable for specific knockdown expression of specific proteins or viruses in vitro. Background Technology

[0002] RNA interference (RNAi) is a typical, representative, and extremely important gene knockdown method that has shown great potential in fields such as cancer, genetic diseases, and viral infections, and is considered a revolutionary breakthrough in biomedicine. RNAi relies on the guiding role of small RNAs, including siRNA and miRNA, to bind complementary to the target mRNA sequence, and then cleaves and degrades or inhibits translation through the RNA-induced silencing complex (RISC), thereby downregulating gene expression at the posttranscriptional level. Among them, the Ago2 protein, as the effector protein of RISC, cleaves the target mRNA that is completely complementary to the siRNA through its own endonuclease activity, or regulates the translation expression and stability of incompletely complementary mRNAs through non-cleavage mechanisms by loading miRNA.

[0003] As the final effector of RNAi knockdown expression, traditional functional studies of the Ago2 protein suggest that the catalytic tetrad (D597, E637, D669, H807) in the PIWI domain directly participates in the cleavage of target RNA. However, the highly Ago2-dependent mechanism of action in RNAi exposes multiple rate-limiting bottlenecks in antiviral applications and precision medicine. First, Ago2 is an endogenous cellular protein, performing both the silencing function of exogenous siRNA and physiological tasks such as regulating the homeostasis of endogenous miRNAs, resulting in limited endogenous expression levels. Second, during viral infection, viral suppressors of RNAsilencing (VSRs), such as the NS4B protein encoded by Zika virus (ZIKV) and non-coding RNA (ncRNA), can directly inhibit Ago2 activity or induce its degradation by binding to the PIWI or PAZ domains, thus disarming the effector Ago2. Third, mammalian somatic cells lack RNA-dependent RNA polymerase (RdRP), which is present in plants and invertebrates, making it impossible to achieve cascade amplification of siRNA signals. Furthermore, a single Ago2 / RISC complex can only cleave one target at a time. Finally, Ago2 knockdown efficiency is relatively weakened. For example, when facing highly replicative viruses (ZIKV can replicate more than 300-fold in 3 days in mouse embryonic brains), the rate-limiting bottleneck of RNAi is significantly slower than the viral replication rate. Therefore, the limited endogenous expression of the Ago2 effector, its inhibition by exogenous viruses, lack of signal amplification, and relatively slow knockdown efficiency—these technical bottlenecks stemming from the Ago2 protein constitute limitations of existing RNAi technology in antiviral and other specific applications. Therefore, this invention aims to address the rate-limiting bottleneck of Ago2 protein in RNAi based on the study of its structure and function, further improving the efficiency and application scope of RNAi.

[0004] In recent years, mosquito-borne viruses have received increasing attention. Zinc-Induced Kibble Virus (ZIKV) is a mosquito-borne flavivirus, a single-stranded positive-sense RNA virus with spherical particles typically around 40-50 nm in diameter. ZIKV may be associated with congenital microcephaly in newborns and autoimmune neurological disorders (Guillain-Barré syndrome). In recent years, sporadic outbreaks have continued in parts of Central America, Southeast Asia, and Africa, and climate change has expanded the suitable habitat for mosquitoes. It has been listed by the WHO as a pathogen with epidemic potential. Currently, there are no approved ZIKV-specific vaccines or antiviral drugs, and clinical treatment is mainly symptomatic and supportive. Therefore, this invention, based on functional optimization of the Ago2 protein in the RNAi pathway, provides a novel and efficient anti-ZIKV strategy, which will have significant clinical application value.

[0005] C-MYC is a transcription factor involved in regulating multiple core biological processes, including cell proliferation, differentiation, apoptosis, metabolism, and angiogenesis. In normal cells, its expression is strictly controlled. However, in many cancers, the C-MYC gene becomes dysregulated due to amplification, translocation, or persistent activation of upstream signaling pathways, leading to overexpression of its protein products. This overexpression is closely associated with many malignant characteristics of tumors, including promoting tumor development and progression, enhancing invasiveness and metastasis, inducing chemotherapy resistance, and causing poor patient prognosis. Although C-MYC is an ideal anti-cancer target, direct targeting of it was once considered "untreatable," primarily due to its protein structural characteristics and broad physiological functions. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical bottleneck of RNAi and provide an enhanced gene knockdown tool that can overcome the rate-limiting bottleneck in RNAi protein knockdown expression.

[0007] The present invention also aims to provide the application of enhanced gene knockdown tools in antiviral therapy.

[0008] To achieve the above objectives, the present invention also provides an enhanced gene knockdown expression recombinant plasmid and a method for its construction and use.

[0009] In a first aspect, the present invention provides an enhanced gene knockdown tool that enhances the gene knockdown effect on a target gene by co-expressing shRNA for recognizing target mRNA and an effector protein for performing cleavage and degradation on a single plasmid; wherein the shRNA sequence includes recognition sequences for the restriction enzymes EcoRI and NheI; the effector protein is Ago2 protein or its mutant S798A, the effector protein is a tagged fusion protein, and the fusion protein encoding a gene further includes restriction enzyme recognition sequences at both ends; the plasmid is either the basic gene knockdown plasmid V2-Flag-Ago2-shRNA or the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA.

[0010] According to a preferred embodiment of the present invention, the basal gene knockdown plasmid V2-Flag-Ago2-shRNA has the sequence shown in SEQ ID NO:7, and the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA has the sequence shown in SEQ ID NO:8.

[0011] According to a preferred embodiment of the present invention, the structure of the plasmid is as follows: the expression cassette of the fusion protein is driven by the EF-1α promoter, the tag of the fusion protein is a Flag tag, the expressed fusion protein is Flag-Ago2 or Flag-S798A, and the two ends of the fusion protein encoding gene are the recognition sequences of the endonucleases AgeI and BamHI, respectively; the expression cassette of the shRNA is driven by the U6 promoter, and the shRNA sequence includes the recognition sequences of the endonucleases EcoRI and NheI.

[0012] Secondly, the present invention provides a method for constructing an enhanced gene knockdown tool, which is based on the pLentiCRISPRV2 plasmid and modified to obtain the enhanced gene knockdown tool described in the first aspect. The steps include: 1) using primers AgeI-Flag-Ago2-F and BamHI-Flag-Ago2-R with the sequences shown in SEQ ID NO:1-2 to obtain the PCR amplification product Flag-Ago2 with the recognition sequences of the restriction enzymes AgeI and BamHI, double digesting the recovered PCR amplification product Flag-Ago2 and the pLentiCRISPR V2 plasmid with the restriction enzymes AgeI and BamHI respectively, and inserting the digested Flag-Ago2 fragment into the linearized pLentiCRISPR V2 plasmid using T4 ligase, and obtaining the V2-Flag-Ago2 plasmid through gene cloning screening and sequencing verification; 2) using the V2-Flag-Ago2 plasmid as a template, according to SEQ ID NO:1-2... 3) Using primers U6-EcoRI-F and U6-EcoRI-R with the sequences shown in NO:3-4, circular PCR was performed, and the basic gene knockdown plasmid V2-Flag-Ago2-shRNA plasmid was obtained through gene cloning screening and sequencing verification; 4) Using V2-Flag-Ago2-shRNA plasmid as a template, circular PCR was performed using primers S798A-F and S798A-R with the sequences shown in SEQ ID NO:5-6, and the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA was verified through gene cloning screening and sequencing.

[0013] According to a preferred embodiment of the present invention, in the method for constructing the enhanced gene knockdown tool, in step 1), the Cas9 sequence on the original pLentiCRISPR V2 plasmid is completely replaced with the coding sequence of Flag-Ago2 on the modified V2-Flag-Ago2 plasmid; in step 2), all gRNA scaffold sequences after the U6 promoter on the original pLentiCRISPR V2 plasmid are deleted on the modified V2-Flag-Ago2-shRNA plasmid and replaced with EcoRI and NheI recognition sites for inserting shRNA.

[0014] Preferably, the deletion after the U6 promoter also includes all irrelevant sequences after the transcription start site and between the gRNA scaffold sequence.

[0015] Further optimization involves ensuring that the 3' end of the U6 promoter is directly fused and connected to the EcoRI and NheI recognition sites after deletion and replacement.

[0016] Preferably, in step 2), the fusion protein of the basal gene knockdown plasmid V2-Flag-Ago2-shRNA plasmid is Flag-Ago2.

[0017] Preferably, in step 3), the fusion protein of the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA is Flag-S798A.

[0018] Thirdly, the present invention provides a recombinant plasmid for enhanced gene knockdown expression, which is constructed based on the enhanced gene knockdown tool described in the first aspect. The structure of the shRNA framework sequence is: sense strand - stem-loop sequence - antisense strand, wherein the sense strand is identical to the target sequence of the target gene, and the antisense strand is complementary to the target sequence of the target gene. The sense strand and antisense strand sequences are complementary and each has a length of 20±3nt. The stem-loop sequence is a short sequence with a length of 9nt that can be recognized by the intracellular Dicer enzyme and can flexibly connect the sense strand and antisense strand to form a stable hairpin structure.

[0019] According to a preferred embodiment of the present invention, the stem-loop sequence of the recombinant plasmid for enhanced gene knockdown expression is TTCAAGAGA or GGCCACACC.

[0020] Preferably, the recombinant plasmids for enhanced gene knockdown expression include, but are not limited to, V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, V2-Flag-Ago2-shZIKV3, V2-Flag-S798A-shZIKV1, V2-Flag-S798A-shZIKV3, V2-Flag-Ago2-shC-MYC, and V2-Flag-S798A-shC-MYC.

[0021] Fourthly, the present invention provides a method for constructing an enhanced gene knockdown expression recombinant plasmid. The steps for constructing the enhanced gene knockdown expression recombinant plasmid described in the third aspect include: 1) designing a target sequence and shRNA framework sequence for the target gene; 2) designing shRNA primers for annealing to obtain target sequence-related shRNA double strands; 3) purifying the shRNA double strands, and simultaneously purifying the enhanced gene knockdown tool described in any one of claims 1-3 by digestion with EcoRI and NheI enzymes; 4) ligating and transforming the purified shRNA double strands with the digested and purified enhanced gene knockdown tool described in any one of claims 1-3, and inserting the shRNA into the basic gene knockdown plasmid V2-Flag-Ago2-shRNA or the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA; 5) obtaining the enhanced gene knockdown expression recombinant plasmid after screening and verification of the transformed positive clones.

[0022] Fifthly, the present invention provides a method for using an enhanced gene knockdown expression recombinant plasmid, wherein the enhanced gene knockdown expression recombinant plasmid described in the third aspect is transfected into target cells and cultured for 24-72 hours.

[0023] According to a preferred embodiment of the present invention, the target cell is a mammalian cell.

[0024] According to a preferred embodiment of the present invention, after culturing for 24 hours, sampling is performed to verify the knockdown effect of the recombinant plasmid expressing the enhanced gene knockdown on the endogenous target gene; or after culturing for 24 hours, the exogenous target gene is expressed and resampling is performed to verify the knockdown effect of the recombinant plasmid expressing the enhanced gene knockdown on the exogenous target gene.

[0025] Preferably, after culturing for 24 hours, the individual is infected with a foreign virus, and after culturing for another 24 hours post-infection, samples are taken to verify the antiviral effect of the recombinant plasmid with enhanced gene knockdown expression.

[0026] Preferably, the verification method includes Western blot detection, Nortern blot detection, and RT-qPCR detection.

[0027] In a sixth aspect, the present invention provides the application of the enhanced gene knockdown tool described in the first aspect, or the method for constructing the enhanced gene knockdown tool described in the second aspect, or the recombinant plasmid for enhanced gene knockdown expression described in the third aspect, or the method for constructing the recombinant plasmid for enhanced gene knockdown expression described in the fourth aspect, or the method for using the recombinant plasmid for enhanced gene knockdown expression described in the fifth aspect, in the knockdown expression of a target gene.

[0028] According to a preferred embodiment of the invention, the application includes an antiviral application.

[0029] The beneficial effects of this invention are: This invention provides an enhanced gene knockdown tool and its construction method, a recombinant plasmid for enhanced gene knockdown expression and its construction and usage method, and its application in target gene knockdown expression. Based on functional studies of RNAi effector proteins, this invention constructs two plasmid tools: a basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) and an enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA). This tool can overcome the dependence of RNAi on the expression level of endogenous Ago2 protein. By co-expressing and simultaneously delivering effector proteins and shRNA with a single plasmid, it directly enhances the cleavage activity of RISC on target genes to improve gene knockdown efficiency. It can significantly enhance gene knockdown efficiency on various target genes, including viral RNA, non-coding RNA, and endogenous oncogenes. The fusion proteins Flag-Ago2 and Flag-S798A expressed in these two plasmids serve as effector proteins that enhance gene knockout, overcoming the rate-limiting bottleneck in traditional RNAi knockdown expression. Moreover, they possess comprehensive advantages in construction and application, including simple operation, high target gene universality, strong shRNA recognition specificity, high biosafety, and high gene silencing efficiency. They have broad application prospects in multiple technical fields such as gene function research, gene expression regulation, and antiviral therapy.

[0030] The enhanced gene knockdown tools constructed in this invention (V2-Flag-Ago2-shRNA plasmid and V2-Flag-S798A-shRNA plasmid) possess high efficiency and specificity. By transfecting cells with a single plasmid tool, the expression level of Ago2 protein is increased, thereby directly improving the cleavage activity of Ago2. Furthermore, it has successfully achieved efficient knockdown on a variety of different types of targets, including exogenous reporter genes (such as EGFP), viral genes (such as ZIKV), and proto-oncogenes (such as C-MYC). Moreover, this enhanced specific knockdown is more accurate in identifying target genes and has no significant impact on non-target genes, demonstrating "broad applicability" and high specificity in target gene recognition.

[0031] The enhanced gene knockdown tool constructed in this invention has been experimentally verified to be multifunctional. Two specific plasmids (V2-Flag-Ago2-shRNA plasmid and V2-Flag-S798A-shRNA plasmid) can not only efficiently knock down reporter genes (such as EGFP), but also coding genes (such as C-MYC) and viral genes (such as shZIKV1, shZIKV2, and shZIKV3), demonstrating significant knockdown effects on both coding and non-coding sequences, and efficiently knocking down RNA molecules with specific sequences. Furthermore, the enhanced gene knockdown tool has been proven effective on both EGFP (exogenous reporter gene) and C-MYC (endogenous key oncogene), not only efficiently silencing exogenously introduced reporter genes, but also inhibiting highly structured or actively translated endogenous oncogenes, directly verifying that the tool is active against target genes in both endogenous and exogenous pathways, and is not limited to specific types of target genes. Regarding C-MYC, unlike traditional inhibitors (which only "lock" a specific functional domain), the method of this invention, which targets and reduces C-MYC expression, achieves superior anti-cancer effects and overcomes the common drug resistance problem of inhibitors by knocking down the C-MYC protein. Therefore, the enhanced gene knockdown tool of this invention can specifically knock down transcripts of multiple types of target genes, and has broader application prospects, which is conducive to its application in multiple fields such as gene function research, gene expression regulation, viral diseases, and anti-cancer treatment.

[0032] The enhanced gene knockdown tool of this invention exhibits high stability. Two specific plasmids (V2-Flag-Ago2-shRNA plasmid and V2-Flag-S798A-shRNA plasmid) have been experimentally verified to be transfected in different cell lines such as HEK293T, BHK21, and HeLa, successfully achieving specific knockdown expression. Therefore, the application of the two core plasmid tools constructed in this invention is not limited to specific cell types, demonstrating its high reliability and cross-cell line applicability. The tool's applicability, stability, and reliability are all relatively high.

[0033] The enhanced gene knockdown tool of this invention exhibits strong antiviral capabilities. Specific experiments demonstrate that the viral knockdown expression of shZIKV (shZIKV1, shZIKV2, shZIKV3) designed targeting different independent regions of the Zika virus genome showed statistically significant differences. Compared to the negative control VEC empty vector, the two plasmids constructed by inserting the three shZIKV vectors (V2-Flag-Ago2-shZIKV plasmid and V2-Flag-S798A-shZIKV plasmid) significantly inhibited ZIKV RNA expression levels (P < 0.01). In particular, the V2-Flag-S798A-shZIKV1 plasmid showed a more significant difference in specific knockdown inhibition of ZIKV RNA: the difference was extremely significant compared to VEC (P < 0.0001); and the difference was also very significant compared to the V2-Flag-Ago2-shZIKV1 plasmid (P < 0.01). Therefore, this invention demonstrates that the enhanced gene knockdown tool, through two single plasmids, can significantly enhance the knockdown effect on the RNA-level expression of viral target genes by achieving exogenous overexpression of Ago2 protein (or its mutant S798A) and shRNA. Compared to traditional RNAi, this invention can significantly improve the cleavage activity against direct viral gene expression. This enhanced knockdown avoids high dependence on shRNA design, and the knockdown effect is stable and highly reproducible. Furthermore, in addition to directly enhancing antiviral ability, the mutant S798A exhibits higher cleavage activity than the wild-type Ago2 protein, laying a solid functional foundation for further applications in antiviral research against highly replicative viruses and virus-encoded RNAi repressors.

[0034] Regarding antiviral efficacy, this invention reveals that shRNA knockdown effects differ depending on the target sequence. For example, the enhanced gene knockdown tool of this invention exhibits varying knockdown efficiencies for the three targets shZIKV1, shZIKV2, and shZIKV3: shZIKV1 > shZIKV3 > shZIKV2. This difference primarily stems from: variations in shRNA sequences leading to different binding affinities to the target mRNA and varying loading efficiency by the RISC complex; differences in the secondary structure and accessibility of the mRNA corresponding to different target sequences; the potential existence of multiple splicing isoforms in the target gene; and differences in the stability of the target protein itself. Despite these sequence-dependent differences, the enhanced gene knockdown tool of this invention still demonstrates significant advantages. This invention's enhanced gene knockdown tool directly increases the expression levels of key effector proteins (Ago2 and S798A) in target cells using two specific plasmids (V2-Flag-Ago2-shRNA plasmid and V2-Flag-S798A-shRNA plasmid). This directly enhances the catalytic activity of the effector proteins, thereby improving the turnover efficiency of the RISC complex, increasing the specificity of gene silencing, and avoiding dependence on high concentrations of siRNA. More importantly, the enhanced effect on the effector proteins is independent of the binding process between siRNA and target mRNA, and does not depend on the affinity between the two. Therefore, even for targets with poor binding efficiency, this invention's plasmid tool can still achieve enhanced knockdown efficiency, overcoming the rate-limiting effect of traditional RNAi that directly depends on the expression level of endogenous effector proteins.

[0035] This invention provides an enhanced gene knockdown tool, designed based on research into the function of the PIWI domain of the Ago2 (Argonaute 2) protein, particularly the discovery that mutations at the S798A site enhance the direct cleavage activity of Ago2. In a comparison of two plasmids (V2-Flag-Ago2-shRNA plasmid and V2-Flag-S798A-shRNA plasmid), experiments demonstrated that the V2-Flag-S798A-shRNA plasmid constructed based on the S798A mutant exhibited significantly better knockdown performance on the target gene than the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) based on wild-type Ago2. Previous experiments using the EGFP reporter gene knockdown system and pre-miR-451 non-coding RNA cleavage have also verified that the cleavage activity of the S798A mutant protein is significantly enhanced compared to the wild-type Ago2 protein. Therefore, the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) constructed in this invention can significantly improve the efficacy in applications such as gene silencing and antiviral therapy mediated by Ago2 protein and shRNA.

[0036] The enhanced gene knockdown tool of this invention utilizes two plasmids that represent a direct application of a single plasmid expression system. By co-expressing key effector proteins (Ago2 protein and mutant S798A) with shRNA on the same vector, it achieves simultaneous delivery and stable co-localization of key effector molecules and target sequences. First, the co-expression design significantly improves RISC assembly efficiency and intracellular concentration, thereby enhancing overall gene knockdown efficiency. In particular, it reduces dependence on shRNA framework design, enabling precise identification of specific target genes. For example, this tool can be widely used to target tumor genes, significantly reducing damage to non-target normal tissues. Simultaneously, stable exogenous expression facilitates the acquisition of a long-acting RISC complex, thus reducing the frequency of tumor treatment and alleviating the burden on patients. Second, the co-expression design ensures 100% synergistic action between wild-type AGO2 protein and mutant S798A with shRNA in target cells, avoiding the imbalanced expression ratios and the problem of some cells receiving only a single, ineffective element due to separate delivery. Third, compared with antiviral therapy using shRNA alone, the co-expression design approach can reduce dependence on high doses of shRNA, further reducing off-target effects and cytotoxicity. Fourth, both plasmids employ a modular design of effector protein (Ago2 and S798A) expression cassettes and shRNA expression cassettes. The two expression cassettes are relatively independent, facilitating rapid replacement with target gene-specific shRNA sequences to adapt to different target genes or antiviral needs.

[0037] The plasmid design for co-expression of the enhanced gene knockdown tool in this invention, which uses shRNA (short hairpinRNA) instead of traditional chemically synthesized siRNA, has several potential benefits. First, shRNA is continuously transcribed within cells from a plasmid vector expression cassette, achieving long-lasting and stable gene silencing. A single cell transfection can maintain expression for several days or even longer, avoiding the limitations of chemically synthesized siRNA which requires repeated administration or multiple transfections. Second, shRNA can be packaged and delivered to difficult-to-transfect cells (such as primary neurons, stem cells, or in vivo tissues) via viral vectors (e.g., lentivirus, adeno-associated virus AAV) to achieve long-term gene silencing in vivo, whereas traditional siRNA is difficult to apply in vivo and is easily degraded by nucleases. Third, shRNA expression cassettes are typically driven by RNA polymerase type III promoters (e.g., U6), resulting in high transcription efficiency and controllable expression abundance. In the future, it is hoped that polycistronic design can be used to simultaneously express multiple shRNAs on the same vector, achieving multi-target silencing. Fourth, shRNA is transcribed in a short hairpin structure and then processed into mature siRNA by Dicer. This processing endows shRNA with an additional screening mechanism specific to the sequence, reducing non-specific silencing or off-target effects. Fifth, as part of the plasmid system, shRNA is co-expressed with the effector protein (Ago2 or S798A) on the same vector, ensuring 100% co-delivery and constant expression ratio of key molecules in target cells, avoiding the expression imbalance problem caused by separate delivery of siRNA and protein expression vectors in dual plasmid systems. Sixth, shRNA sequence design and cloning are relatively simple. This invention uses shRNA primer design, annealing to generate shRNA double strands, and then directly inserting them into the plasmid vector via EcoRI and NheI sticky ends, facilitating rapid cloning, screening, and replacement of target sequences, greatly improving experimental flexibility and throughput. Finally, from the perspective of gene knockdown tool cost, once the shRNA expression plasmid is constructed, its amplification and extraction cost is far lower than that of repeated chemical synthesis of siRNA, making it particularly suitable for large-scale screening or long-term applications. Therefore, compared to the advantages of traditional siRNA in transient transduction experiments, which are rapid onset of action and do not require cloning, the design concept of the enhanced gene knockdown tool of this invention, which co-expresses effector proteins and shRNA, has more advantages in terms of long-term effectiveness, delivery capability, in vivo applicability, synergistic expression, and economy. It is especially suitable for future scenarios that require continuous gene silencing or in vivo therapy.

[0038] This invention's enhanced gene knockdown tool exhibits superior safety. Compared to chemical enhancers such as Enoxacin, the key effector proteins (Ago2 and S798A) are directly enhanced through exogenous overexpression via protein engineering, resulting in a purer and more specific mechanism of action. It does not interfere with the global miRNA regulatory network and avoids the rate-limiting bottleneck of endogenous expression. Compared to dual-plasmid delivery systems, this invention is a typical single-plasmid expression system, specifically a single-plasmid expression system based on the pLentiCRISPR V2 vector. It is more compatible with delivery vectors such as adeno-associated virus (AAV), which improves the efficiency of viral crossing of the blood-brain barrier and infection of neural progenitor cells, providing a novel technical platform for developing broad-spectrum, efficient, and safe RNAi antiviral therapies. Attached Figure Description

[0039] Figure 1 This is a graph showing the RT-qPCR detection results of the knockdown effect of the Ago2 S798A mutant on EGFP mRNA in this invention; Figure 2 The figure shows the Northern Blot results of the knockdown effect of the Ago2 S798A mutant on pre-miR451 in this invention; in the figure, pre-miR451 is the precursor of miR451, miR451 is the mature cleavage product, and U6 is the internal control. Figure 3 The diagram shows the key structures of two plasmids constructed using the enhanced gene knockdown tool of this invention. A is the key structure of the basic gene knockdown plasmid, and B is the key structure of the enhanced gene knockdown plasmid. Figure 4 This is a Western blot validation result of the expression of Flag-Ago2 fusion protein by tool plasmids constructed based on three different shZIKVs using the basic gene knockdown plasmid of this invention; VEC is the negative control with no vector inserted, Ago2 is the control without shZIKV insertion, GAPDH is the internal control, and "ZIKV" at the top horizontal indicates that ZIKV infection culture is required after plasmid transfection; Figure 5 The figure shows the RT-qPCR detection results of three different shZIKV strains inserted into the basic gene knockdown plasmid of this invention to combat ZIKV infection. Figure 6 The figure shows the Western blot results of expressing the Flag-Ago2 fusion protein using two tool plasmids constructed by inserting the basic gene knockdown plasmid and the enhanced gene knockdown plasmid into shZIKV1, respectively; VEC is the negative control for the empty vector, and Tubulin is the internal control; Figure 7The figure shows the qRT-PCR detection results of two tool plasmids constructed by inserting the basic gene knockdown plasmid and the enhanced gene knockdown plasmid into shZIKV1 to resist ZIKV infection. Figure 8 The figure shows the Western blot results of expressing the Flag-Ago2 fusion protein using two tool plasmids constructed by inserting the basic gene knockdown plasmid and the enhanced gene knockdown plasmid into shZIKV3, respectively; VEC is the negative control for the empty vector, and Tubulin is the internal control; Figure 9 The figure shows the qRT-PCR detection results of two tool plasmids constructed by inserting the basic gene knockdown plasmid and the enhanced gene knockdown plasmid into shZIKV3 to resist ZIKV infection. Figure 10 This is a Western blot validation result of the enhanced gene knockdown tool of the present invention on the knockdown expression level of C-MYC protein; VEC is the empty vector control, GAPDH is the internal control, and the primary antibodies include Flag and C-MYC respectively; In the figure, the sample group transfected with the V2-Flag-Ago2-shRNA plasmid is abbreviated as "Ago2-shRNA", and the sample group transfected with the V2-Flag-S798A-shRNA plasmid is abbreviated as "S798-shRNA"; the sample groups transfected with plasmids without added or inserted target gene-specific shRNA are abbreviated as "Ago2" and "S798", respectively; Figure 1 , Figure 5 , Figure 7 , Figure 9 In the bar chart, VEC, representing the negative control of the empty plasmid vector, was set as 1 (baseline). The vertical axis represents the fold change in the RT-qPCR quantitative detection value of the target gene relative to the RNA of VEC (mean ± standard deviation, n = 2-3 replicates). The t-test was used to analyze the significance of the differences, where "ns" indicates no significant difference, "*" represents P < 0.05 (significant difference), "**" represents P < 0.01 (extremely significant difference), "***" represents P < 0.001, and "****" represents P < 0.0001. Detailed Implementation

[0040] The exemplary embodiments of the present invention are described in detail below with reference to practical applications. For ease of understanding, many specific details are set forth to provide a comprehensive understanding of the present invention. In this context, "same" or "similar" refers to the same concepts, such as Ago2 mutant protein = Ago2 S798A = S798A, short hairpin RNA = shRNA, and stem-loop sequence = LOOP sequence.

[0041] The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods, and the materials and reagents used are commercially available. For example, primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (hereinafter referred to as Sangon Biotech); siEGFP and pre-miR451 RNA were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.; miR451 probe and internal control U6 probe were purchased from Wuhan Youming Biotechnology Co., Ltd.; cell culture plates (6-well, 12-well, and 24-well) and 10cm culture dishes were purchased from Wuhan Diyue Innovation Biotechnology Co., Ltd.; transfection reagents, DMEM medium (Gibco), and fetal bovine serum FBS (Gibco) were all purchased from Invitrogen Trading Co., Ltd.; HEK293T cells (including HEK293T cells with the Ago2 protein coding gene knocked out through gene editing technology, referred to as 293T-Ago2 KO cells), BHK21 cells, and HeLa cells were directly purchased from Wuhan Diyue Innovation Biotechnology Co., Ltd., with the original source being ATCC (American Type Culture Collection); pLentiCRISPR V2 plasmid (catalog number P0114) was purchased from Wuhan Miaoling Biotechnology Co., Ltd. (Miaoling Plasmid Platform). However, it is obvious that one or more embodiments can be implemented without these specific details, and conditions not specifically specified in the embodiments should be performed under standard conditions or conditions recommended by the manufacturer. Molecular biology experimental methods not specifically described in the following embodiments are performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0042] The objects of detection in this invention are genes and proteins expressed through in vitro experiments (such as cell culture and viral infection), or genetically engineered bacteria and commercially available cell lines (such as HEK293T, BHK21, and HeLa). The detection objects are cell samples or engineered bacteria cultured in vitro. The cell samples are ex vivo biological samples that have been separated from living human or animal bodies and are intended for commercial use. The detection process is entirely conducted in an in vitro experimental environment and does not involve direct manipulation or in vivo experiments from any living human or animal body. The direct purpose of detection in this invention includes: verifying the correctness of the construction of plasmid vectors obtained through molecular cloning (e.g., expressing Flag-Ago2 fusion protein and shRNA), and quantitatively or qualitatively analyzing the expression level of target genes in in vitro expression systems (such as Northern Blot, RT-qPCR, and Western blot) to evaluate the effect of the constructed tool plasmids on the specific knockdown expression of target genes. Therefore, the detection or determination described in this invention does not constitute a disease diagnosis method and meets the basic requirements for patent protection subject matter under the Patent Law.

[0043] While studying the function of the Ago2 protein, the final effector of RNAi knockdown expression, the inventors unexpectedly discovered that an Ago2 protein mutant (Ago2 S798A) exhibits a stronger cleavage effect on target RNA. Based on this discovery, we developed an enhanced gene knockdown tool. By introducing exogenous expression of the Ago2 protein (wild-type and mutant S798A), it can not only be used to specifically cleave target RNAs in both endogenous and exogenous pathways, but also, through optimized design of specific shRNAs, can be extended to specifically knock down the expression levels of coding sequences (such as C-MYC mRNA, genomic RNA of viral proteins) and non-coding sequences (such as pre-miR451, exogenously transformed EGFP). This enhanced gene knockdown tool contains two different gene knockdown plasmid forms, and the construction, optimization, and validation processes are detailed below.

[0044] Example 1: Functional study of Ago2 protein Regarding the effect of Ago2 protein in knocking down EGFP expression: By reintroducing different forms of Ago2 protein into HEK293T cells with Ago2 gene knockout, the results showed that the Ago2 S798A mutant exhibited significantly different knockdown effects in mediating siRNA silencing of EGFP mRNA. The specific experimental procedures and results are as follows.

[0045] 1) Cell model construction: Using CRISPR / Cas9 gene editing technology, a stable HEK293T cell line (293T-Ago2 KO) with the Ago2 protein gene knocked out was constructed as a cell background with Ago2 function loss to eliminate interference from the limited expression of endogenous Ago2 protein.

[0046] 2) Experimental grouping and plasmid preparation: Prepare 6-well cell culture plates pre-filled with 5 × 10⁶ cells per well. 5293T-Ago2 KO cells were seeded at the appropriate cell density and cultured in DMEM complete medium (containing 10% FBS) at 37°C and 5% CO2 until the cells reached 70-80% confluence. Three co-transfection experiments were set up, each containing: ① Expression plasmid (0.1 μg pEGFP-C1 plasmid, expressing GFP), used to provide target mRNA that can be silenced; ② siRNA: 40 pmol siEGFP RNA, specifically guiding the RISC complex to recognize EGFP mRNA to trigger EGFP knockdown expression, used to specifically trigger interference with the target gene; ③ Complementary protein: pRK-FLAG-C plasmid of the empty vector (VEC) group as a negative control; pRK-FLAG-Ago2 plasmid of the wild-type Ago2 (WT Ago2) group; pRK-FLAG-S798A plasmid of the Ago2 S798A mutant group (Ago2 protein with serine mutated to alanine at position 798). The three different plasmids corresponded to three groups, and 1 μg of each complementary protein plasmid was taken from each well for co-transfection, with each group containing at least two replicates.

[0047] 3) Co-transfection of cells: The above three types of molecules (expression plasmid, siRNA, and complement protein) were co-introduced into the corresponding cell wells of the cell culture plate using liposome transfection.

[0048] 4) Cell culture and cell sample collection and detection: After co-transfecting 293T-Ago2 KO cells were incubated at 37℃, 5% CO2 and DMEM+10% FBS medium for 24 h, cells from each well of the cell culture plate were collected, total RNA was extracted, and EGFP mRNA level was quantitatively detected by RT-qPCR to evaluate the differences in EGFP RNA level caused by different Ago2 protein forms.

[0049] 5) Experimental results: RT-qPCR results are as follows Figure 1 As shown in the bar chart, the fold change (mean ± standard deviation, n = 2 replicates) relative to the VEC negative control group (=1) shows that: among all the replacement groups, the EGFP mRNA level in the plasmid cells co-transfected with the Ago2S798A mutant was the lowest, indicating that the Ago2S798A mutant had the strongest specific knockdown effect on EGFP expression, which was significantly better than the WT Ago2 group (P < 0.0001, extremely significant difference). The WT Ago2 group was further significantly better than the VEC group (P < 0.001, extremely significant difference).

[0050] The above results suggest that position 798 of Ago2 may be a negatively regulated phosphorylation site. Existing studies have only demonstrated that the PIWI domain (containing S798) of the Ago2 protein is the core region of its cleavage activity. However, this invention demonstrates that the Ago2 S798A mutant exhibits stronger RNA-specific cleavage activity by mutating serine (S) to non-phosphorylated alanine (A). This indicates that the S798 site may be regulated by specific kinases / phosphatases. S798 phosphorylation inhibits Ago2 activity by connecting intracellular and extracellular signals with the RNAi pathway. If Ago2 mutants with antiphosphorylation or dephosphorylation of the S798 site, such as the Ago2 S798A mutant, can be designed, the RNAi ability of cells can be enhanced, and it may also have more significant potential value in antiviral responses or the promotion and application of siRNA.

[0051] Regarding the effect of Ago2 protein in knocking down pre-miR451 expression: miR451 matures in erythrocytes after the cleavage of the precursor pre-miR451 and plays a key regulatory role in oxidative stress response and tumor suppression. The process from precursor pre-miR451 to mature miR451 follows the classic Dicer-independent processing model. Ago2 protein can directly bind to pre-miR451 and mediate its degradation and maturation. Referring to the above experimental procedure for knocking down EGFP expression, in the constructed 293T-Ago2 KO cell model, different forms of Ago2 protein were reintroduced before transfection with endogenous non-coding RNA precursor molecule pre-miR451 RNA. Northern blotting was used to verify whether there was a difference in the cleavage effect of Ago2 S798A mutant on pre-miR451 RNA. The specific experimental procedure and results are as follows.

[0052] 1) Cell preparation: Prepare 6-well cell culture plates and pre-culture 293T-Ago2 KO cells until the cell confluence reaches 70-80% for use.

[0053] 2) Plasmid transfection for protein replenishment: Three groups were set up, each containing at least two replicates. Each well was transfected with three plasmids (1 μg each): ① Negative control group (VEC): pRK-FLAG-C plasmid; ② Wild-type Ago2 protein group (WT): pRK-FLAG-Ago2 plasmid; ③ Ago2 S798A mutant protein group (S798A): pRK-FLAG-S798A plasmid; The above three plasmids were mixed with PEI transfection reagent at a ratio of plasmid:PEI = 1:3 w / w and incubated at room temperature for 15 minutes before being added to the corresponding cell culture wells and incubated at 37℃ and 5% CO2 for 12 hours.

[0054] 3) RNA transfection: Using the RNA transfection reagent (Lipofectamine™ 2000), transfect 40 pmol pre-miR451 RNA into each well according to the instructions, and continue to incubate at 37℃ and 5% CO2 for 24 h.

[0055] 4) Sample collection and RNA extraction: Collect cells, extract total RNA using the TRIZOL method, quantify the RNA, and take an equal amount of RNA (e.g., 10 μg) for Northern Blot analysis.

[0056] 5) Northern Blot Detection: RNA was separated using 18% urea-PAGE electrophoresis. After transfer to a membrane, hybridization was performed using digoxigenin-labeled miR451 probe and U6 snRNA probe (as internal controls), respectively. The Northern Blot analysis diagram is shown below. Figure 2 As shown, under normal internal control conditions, the VEC group showed almost no bands for pre-miR451 or mature miR451, the WT group showed a moderately strong mature miR451 band, and the S798A group showed a significantly enhanced mature miR451 band. This result demonstrates that the VEC group's lack of Ago2 supplementation led to miR451 processing blockage, while the WT group, having been supplemented with wild-type Ago2 protein, was able to function in the miR451 maturation pathway, thus restoring cleavage function. The S798A group, having been supplemented with the Ago2 mutant protein (Ago2 S798A) with enhanced cleavage activity, showed the strongest mature miR451 band signal. Therefore, this further verifies that Ago2 S798A can enhance cleavage activity against non-coding RNA and can be used as a preferred tool for constructing enhanced RNAi. Example 2: Construction of an Enhanced Gene Knockdown Tool The above verification demonstrated that, compared to the wild-type Ago2 protein, the Ago2 mutant protein (Ago2 S798A) significantly enhances the cleavage activity against EGFP mRNA and pre-miR451 RNA. To overcome the rate-limiting bottleneck caused by the endogenous expression level of the Ago2 effector, this invention constructs a universal gene knockdown tool capable of exogenous Ago2 protein complementation. Furthermore, to further verify the cleavage capabilities of different Ago2 protein forms, two sets of parallel plasmid tools were simultaneously set up: a basic gene knockdown plasmid and an enhanced gene knockdown plasmid, based on both wild-type and Ago2 mutant proteins (Ago2 S798A) for exogenous Ago2 protein complementation. First, the construction of the basic gene knockdown plasmid mainly involves two steps: introducing the Flag-Ago2 sequence and deleting irrelevant sequences and gRNA scaffold sequences (to facilitate shRNA transcription). The plasmid was modified based on the pLentiCRISPR V2 plasmid, and the specific construction process and results are as follows.

[0057] The first step was to introduce the Flag-Ago2 sequence. ① Primer design and target fragment amplification: Primer pairs AgeI-Flag-Ago2-F and BamHI-Flag-Ago2-R were designed. The forward primer (AgeI-Flag-Ago2-F) introduced an AgeI restriction site at its 5' end, and the reverse primer (BamHI-Flag-Ago2-R) introduced a BamHI restriction site at its 5' end. Using a template plasmid containing the Flag-Ago2 sequence as the amplification template, the Flag-Ago2 target fragment with AgeI and BamHI restriction sites at both ends was amplified by PCR. After identifying the band size by agarose gel electrophoresis, the Flag-Ago2 target fragment was purified and recovered using a gel extraction kit. ② Enzyme digestion and ligation of vector and target fragment: The original pLentiCRISPR V2 vector plasmid and the purified and recovered Flag-Ago2 target fragment were digested with AgeI and BamHI restriction endonucleases, respectively. The double digestion removed the original Cas9 coding sequence in the pLentiCRISPR V2 vector plasmid. After separation of the digestion products by agarose gel electrophoresis, the backbone fragment of the linearized vector plasmid was recovered and separated by agarose gel electrophoresis. The backbone fragment of the recovered linearized vector plasmid and the digested Flag-Ago2 target fragment were mixed at a ratio of vector:target fragment = 1:3, and T4 ligase and corresponding buffer were added. The mixture was ligated overnight at 4℃. The ligation product was transformed into competent Escherichia coli (DH5α) cells and plated on LB solid medium containing ampicillin. The cells were incubated upside down at 37℃ for 12-16 hours. ③ Screening and Validation of Positive Clones: Single colonies were picked and inoculated into LB liquid medium containing ampicillin. After overnight incubation at 37°C, plasmids were extracted. The size of the inserted fragment was identified by double digestion with AgeI and BamHI to preliminarily screen positive clones. Finally, candidate clones were sent to a sequencing company for Sanger sequencing to confirm the insertion position and sequence accuracy of the Flag-Ago2 sequence, thus obtaining the successfully modified V2-Flag-Ago2 plasmid.

[0058] The first step in modifying the pLentiCRISPR V2 vector plasmid into the V2-Flag-Ago2 plasmid is based on the following design: the pLentiCRISPR V2 plasmid's core function is Cas9 protein-mediated CRISPR gene editing, while the goal of this invention is to construct a gene knockdown tool based on RNA interference (RNAi). Therefore, the Cas9 sequence needs to be replaced with endogenously expressed Ago2 protein to overcome the rate-limiting bottleneck of RNAi. Ago2 protein is a core component of the RNA-induced silencing complex (RISC), responsible for siRNA / shRNA-mediated cleavage of target mRNA; thus, Ago2 protein is a key protein for improving gene knockdown efficiency. The Flag tag carried when introducing the Flag-Ago2 target fragment facilitates subsequent experiments such as Western blotting and immunofluorescence to detect the expression and localization of the Flag-Ago2 target protein using the Ago2 tag. The AgeI and BamHI double digestion system was chosen because the restriction sites of these two restriction enzymes are naturally present at both ends of the Cas9 coding region of the original pLentiCRISPR V2 plasmid. This allows for directional and seamless sequence replacement through enzyme digestion and ligation, while avoiding damage to other functional elements of the plasmid (such as lentiviral packaging signals, resistance genes, etc.).

[0059] The second step involves deleting irrelevant sequences following the U6 promoter and all gRNA scaffold sequences. ① Primer design and circular PCR: Design inverse complementary primer pairs U6-EcoRI-F and U6-EcoRI-R. Use the V2-Flag-Ago2 plasmid (verified by sequencing above) as a template for circular PCR amplification. Obtain a circular PCR amplification product with the 3' end of the U6 promoter directly linked to the EcoRI and NheI recognition sites, while ensuring the deletion of irrelevant sequences following the U6 promoter transcription start site and all gRNA scaffold sequences. The 5' end of the forward primer covers part of the 3' end region of the U6 promoter and part of the downstream region of NheI. The 3' end of the reverse primer covers part of the downstream region of NheI and part of the 3' end region of the U6 promoter. The middle region of the primers contains the EcoRI and NheI recognition sequences. ② Purification and ligation of circularized PCR amplification products: After identification by agarose gel electrophoresis, the amplified target fragment was recovered. The PCR product was treated with DpnI enzyme (10 U / μg DNA) (incubated at 37℃ for 1 hour) to remove methylated template plasmids. The DpnI-treated product was purified and self-circulated overnight at 4℃ using T4 ligase. The ligation product was transformed into competent Escherichia coli (DH5α) cells and plated on LB solid medium containing ampicillin. The medium was incubated upside down at 37℃ for 12-16 hours. ③ Positive clone screening and verification: Single colonies were picked and inoculated into LB liquid medium containing ampicillin. After overnight incubation at 37°C, plasmids were extracted and digested with EcoRI. The plasmid size was then determined by agarose gel electrophoresis to check if it met expectations (the size of the plasmid will decrease after digestion due to the deletion of irrelevant sequences and the complete gRNA scaffold sequence). This preliminary screening was used to identify whether the plasmid modification was successful. Finally, candidate clones were sent to a sequencing company for Sanger sequencing to confirm whether the original irrelevant sequences and gRNA scaffold sequences had been deleted and whether the EcoRI and NheI recognition sites were correct. Positive clones confirmed by sequencing are the successfully modified V2-Flag-Ago2-shRNA plasmids of this invention. Here, "shRNA" refers to the sequence between the recognition sequences of the two restriction sites used to conveniently introduce the target gene shRNA into the 2 restriction sites through double digestion with EcoRI and NheI.

[0060] The second step involves modifying the V2-Flag-Ago2 plasmid into a basic gene knockdown plasmid (V2-Flag-Ago2-shRNA). The design concept is as follows: The gRNA scaffold sequence contained in the original pLentiCRISPR V2 plasmid is a key element for Cas9 protein to bind to and locate target DNA. However, the core function of the universal gene knockdown tool constructed in this invention is to specifically knock down specific target genes through shRNA-mediated knockdown. The presence of the original gRNA scaffold sequence will cause non-specific binding with the Ago2 protein, interfere with the normal assembly of shRNA and Ago2, and thus affect the knockdown efficiency of the target gene. Therefore, this invention requires the deletion of all irrelevant sequences from downstream of the U6 promoter to downstream of the original gRNA scaffold sequence, as well as the gRNA scaffold sequence, retaining only the original EcoRI and NheI recognition sequences downstream of the U6 promoter and gRNA scaffold sequence. This allows the target gene's shRNA sequence to be directionally inserted downstream of the U6 promoter via double digestion with EcoRI and NheI. This leverages the efficient transcription of shRNA by the U6 promoter to achieve efficient knockdown of the target gene. This not only simplifies the subsequent cloning process for introducing shRNA sequences corresponding to different target genes but also utilizes the efficient control of the U6 promoter, further expanding the versatility and knockdown efficiency of the gene knockdown tool (V2-Flag-Ago2-shRNA).

[0061] Construction of Enhanced Gene Knockdown Plasmid. Using the successfully validated basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) as a template, the gene sequence at position 798 of Ago2 was targeted. Circular PCR amplification was performed using designed primers to replace the TCC coding sequence at this amino acid position with GCA, thus constructing the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA plasmid). The specific construction process and results are as follows: ① Primer Design and Circular PCR: Reverse complementary primer pairs S798A-F and S798A-R were designed. Using the correctly sequenced V2-Flag-Ago2-shRNA plasmid as a template, circular PCR amplification was performed. Replacing the TCC sequence with GCA allowed for the circular PCR amplification product. Both the forward primer (S798A-F) and the reverse primer (S798A-R) included complementary GCA sequences. ② Purification and ligation of circularized PCR amplification products: Following the second step of constructing the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA), the PCR product with introduced mutation obtained by circularized PCR amplification was recovered, digested with DpnI, self-circulated, and ligated into competent cells (DH5α); ③ Screening and verification of positive clones: Positive single colonies were selected through antibiotic selection and inoculated into LB liquid medium containing ampicillin. After overnight culture at 37°C on a shaker, the plasmid was extracted; candidate clones were sent to a sequencing company for Sanger sequencing to confirm whether the original TCC sequence had been successfully replaced with the GCA sequence. The positive clones confirmed by sequencing are the enhanced gene knockdown plasmids (V2-Flag-S798A-shRNA) successfully modified in this invention.

[0062] Figure 3 The key structures of two different tool plasmids (basic gene knockdown plasmid V2-Flag-Ago2-shRNA and enhanced gene knockdown plasmid V2-Flag-S798A-shRNA) of the enhanced gene knockdown tool of the present invention are shown. It can be seen that both plasmids can express two different forms of Ago2 fusion protein (wild-type Flag-Ago2 and mutant Flag-S798A) under the control of the EF-1α core promoter, thereby overcoming the limitation of endogenous Ago2 protein expression. In addition, the plasmid provides EcoRI and NheI insertion sites for shRNA of different target genes to improve the universality and convenience of the plasmid. At the same time, the expression of the inserted specific shRNA can be efficiently transcribed and controlled by the U6 promoter, overcoming the dependence of traditional RNAi on transfection of high doses of chemically synthesized siRNA, and further improving the specific silencing efficiency against the target gene.

[0063] The primers for constructing the enhanced gene knockdown tool, the sequences of two tool plasmids, and the relevant sequences used for subsequent validation are summarized in Table 1 below.

[0064] Table 1 Sequence List

[0065] Example 3: Composition of the enhanced gene knockdown tool and the operation steps for plasmid construction. The enhanced gene knockdown tool constructed in this invention is a single plasmid tool capable of co-expressing Flag-Ago2 fusion protein and shRNA. Specifically, based on the structural form of Ago2 in the Flag-Ago2 fusion protein (wild-type Ago2 protein and Ago2 protein mutant S798A), it is divided into a basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) and an enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA). These two core plasmid tool vectors can be used as a universal gene knockdown tool for direct transfection into cells. Among them, the shRNAs (short hairpin RNAs) carried by the two core plasmids follow the classic hairpin structure design. The standardized structure is bounded by EcoRI and NheI restriction sites at both ends (5' end and 3' end). The core functional region in the middle consists of three parts, in the following order from 5' end to 3' end: 1) Homologous sequence of the target gene (sense strand): 20±3nt in length, completely complementary to the target gene mRNA sequence, and is the core recognition region mediating gene silencing; 2) Stem-loop sequence (LOOP): as an unpaired flexible linker, it enables the sense and antisense strands to form a stable hairpin structure, and can be recognized by the intracellular Dicer enzyme, improving the processing efficiency of shRNA. The classic LOOP sequence (TTCAAGAGA, 9nt in length) is preferred; 3) Complementary sequence of the target gene (antisense strand): the length is completely consistent with the sense strand, and it is complementary to the sense strand bases, together forming the "stem" of the hairpin structure, ensuring that the shRNA folds into a functional secondary structure. Therefore, the complete shRNA sequence framework is: EcoRI-sense strand-LOOP sequence-antisense strand-NheI.

[0066] The process for obtaining shRNA and constructing recombinant plasmids is as follows: ① Target sequence and shRNA framework sequence design: Target sequences are obtained by screening based on target genes, and shRNA framework sequences (EcoRI-sense strand-LOOP sequence-antisense strand-NheI) are designed based on these sequences; ② shRNA primer design and shRNA double-strand preparation: shRNA amplification primer pairs are designed based on the designed shRNA framework sequences. The 5' ends of the forward and reverse primers are EcoRI and NheI sticky end sequences, respectively, to ensure the amplified shRNA... The double-stranded DNA fragments of shRNA are directly amplified by annealing with primer pairs to form shRNA double-stranded DNA fragments. ③ shRNA purification and enzyme digestion: After verification by agarose gel electrophoresis, the amplified products are purified using a gel extraction kit to remove impurities such as primer dimers to obtain purified shRNA double strands. The two core plasmids are first digested with EcoRI and NheI enzymes and then purified. ④ Ligation and transformation: The purified shRNA double strands with EcoRI and NheI sticky ends are ligated with the two enzyme-digested and purified plasmids using T4 DNA ligase overnight at 4°C. The purpose is to insert the shRNA double-stranded fragments into the EcoRI and NheI sites at both ends of the shRNA in the enzyme-digested plasmids. The ligation products are transformed into competent E. coli cells (DH5α). ⑤ Recombinant plasmid screening and verification: Positive clones after successful ligation and transformation are obtained through antibiotic selection. Colony PCR and sequencing verification confirm that the shRNA sequence insertion is correct and there are no mutations. These are recombinant plasmids that can be directly used for target gene knockdown expression.

[0067] The two core plasmid vectors constructed in this invention simultaneously contain shRNA that mediates gene knockdown and Ago2 protein that performs cleavage. The difference lies in the slightly different functional localizations of the two core plasmids: the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) carries the wild-type Ago2 protein coding sequence. Protein expression can be tracked through the Flag tag, overcoming the limitation of endogenous Ago2 protein and avoiding the bottleneck of a single Ago2 / RISC complex cleaving only one target at a time, thus improving gene knockdown efficiency. The enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) carries the mutant Ago2 protein (Ago2 S798A, or S798A for short). Compared to the wild-type Ago2 protein, S798A has enhanced binding ability to the RNA-induced silencing complex (RISC), thereby significantly improving the cleavage efficiency of target mRNA and achieving a stronger gene knockdown effect than the basic gene knockdown plasmid.

[0068] The specific steps for using enhanced gene knockdown tools include: 1) shRNA design: For specific target genes, potential target sequences that can be used to form shRNA are screened from the genome. The length is 20±3nt and high recognition specificity is required. The target sequence is used to specifically recognize the target gene and design to form the shRNA framework sequence. The shRNA framework sequence is designed based on the target sequence: EcoRI-sense strand-LOOP sequence-antisense strand-NheI.

[0069] 2) shRNA primer design and shRNA double-strand preparation: The shRNA amplification primers are equipped with EcoRI and NheI recognition sequences at the 5' end, respectively; the designed shRNA primers are annealed to obtain shRNA double-stranded DNA with sticky ends.

[0070] 3) shRNA purification and enzyme digestion: The annealed shRNA double-stranded DNA was purified and recovered. Two plasmids (V2-Flag-Ago2-shRNA and V2-Flag-S798A-shRNA) were digested with EcoRI and NheI enzymes respectively. Incompletely digested fragments were removed by gel purification to prevent self-ligation or false positive clones.

[0071] 4) Ligation and transformation: The shRNA double strand and the two enzyme-digested plasmids were ligated overnight at 4°C using T4 DNA ligase. The molar ratio of shRNA fragment to plasmid was controlled at 3:1 to 5:1 to improve ligation efficiency. Highly competent cells were used for transformation and amplification. DH5α cells that could be screened with ampicillin were preferred as competent cells. An empty vector digestion-ligation-transformation was set up as a negative control to eliminate background contamination.

[0072] 5) Clone screening and amplification: After the positive clones obtained from the resistance screening are verified to be correct by colony PCR and sequencing, the positive clones are used as recombinant plasmids for knocking down the target gene. After the recombinant plasmids are expanded and cultured, the plasmids are extracted and stored at -20℃ for later use.

[0073] 6) Plasmid transfection of cells: The extracted recombinant plasmid was transfected into the target cells to verify the expression of the recombinant plasmid and the knockdown effect of the target gene.

[0074] Example 4: Application and Effect of Enhanced Gene Knockdown Tools in Antiviral Treatment 4.1 Construction of the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) 1) Target sequence and shZIKV framework sequence design: The target gene is the genomic sequence of Zika virus (ZIKV). The genomic sequence of Zika virus (ZIKV) was obtained from databases such as GenBank. Online tools (siRNA DesignTools, Block-iT™ RNAi Designer) were used to screen potential ZIKV target sequences. The GC content of candidate target sequences was required to be between 40% and 60%, and the length was about 15-25 nt. Sequences such as SEQ ID NO:9-SEQ ID were obtained. NO:11 shows three target sequences ZIKV1, ZIKV2, and ZIKV3. Based on these three target sequences and the LOOP sequence, the shZIKV framework sequence is designed as follows: the shZIKV1 framework sequence is EcoRI-positive chain-LOOP sequence-antisense chain-AGTTTT-NheI; the shZIKV2 and shZIKV3 framework sequences are both EcoRI-positive chain-LOOP sequence-antisense chain-TTTTTT-NheI; among them, the preferred LOOP sequence is TTCAAGAGA.

[0075] 2) shZIKV primer design and shZIKV double-stranded DNA preparation: The shZIKV amplification primers have EcoRI and NheI recognition sequences at the 5' end, respectively. The shRNA primer sequences corresponding to the above three target sequences are shown in SEQ ID NO:12-SEQ ID NO:17. Equal volumes of forward and reverse primer stock solutions were mixed in pairs and placed in a PCR instrument for annealing. The program was as follows: incubation at 95℃ for 5 min, and then slow cooling to 25℃ at a rate of 1℃ per minute to obtain three shZIKV double-stranded DNA segments with EcoRI and NheI sticky ends.

[0076] 3) shZIKV purification and enzyme digestion: The annealed shZIKV double-stranded DNA was purified using an agarose gel extraction kit. The purified shZIKV double-stranded DNA contained EcoRI and NheI sticky ends. The basal gene knockdown plasmid (V2-Flag-Ago2-shRNA) was digested in a 50 μL enzyme digestion system (1 μg plasmid, 5 μL 10× digestion buffer, 2 μL EcoRI restriction endonuclease, 2 μL NheI restriction endonuclease, ddH2O) at 37°C for 2 hours. 50 μL of the digestion product was subjected to 0.8% agarose gel electrophoresis to determine the size of the linearized vector (the band should migrate to the expected 1.4K). The digestion product was purified using a gel extraction kit, with an elution volume of 30 μL, and the concentration was determined (NanoDrop).

[0077] 4) Ligation and transformation of shZIKV with plasmid: Take approximately 5 μL of shZIKV double-stranded DNA, approximately 2 μL of linearized basal gene knockdown plasmid (V2-Flag-Ago2-shRNA), 1 μL of 10×T4 DNA ligase buffer, approximately 1 μL of T4 DNA ligase, and 1 μL of PEG4000 and incubate overnight at 4℃ (12–16 hours). The molar ratio of plasmid to shZIKV is 1:3 to improve ligation efficiency. Add 10 μL of the ligation product to 100 μL of DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and then quickly incubate on ice for 2 min. Add 900 μL of antibiotic-free LB medium, and culture at 37℃ with shaking at 200 rpm for 1 h. Spread 200 μL of the bacterial culture onto LB agar plates containing 100 µg / mL ampicillin and incubate upside down at 37℃ overnight.

[0078] 5) Positive clone amplification and screening: Single colonies from the plate were picked and inoculated into 5 mL of LB medium containing ampicillin, and cultured at 37°C with shaking for 6 h. 1 µL of the bacterial culture was used as a template for PCR amplification using primers U6-F. The PCR program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 60 s, for a total of 25 cycles; final extension at 72°C for 10 min. The PCR products were preliminarily verified by 0.8% agarose gel electrophoresis, and positive clones with the correct band size were screened. The bacterial culture corresponding to the correct positive clone was sent to a sequencing company for sequencing using sequencing primers U6-F (sequence shown in SEQ ID NO:21). After further amplification and culture of the positive clones verified by sequencing, plasmids were extracted using a plasmid extraction kit, yielding three corresponding plasmids: V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, and V2-Flag-Ago2-shZIKV3. The extracted plasmids were then measured for concentration (NanoDrop) and stored at -20°C for later use.

[0079] 6) Transfection of BHK21 cells and Western blot detection: BHK21 cells were transfected at a concentration of 1×10⁻⁶ cells / mL. 6Cells were seeded at a density of 100 cells / well in 6-well plates, and 2 mL of DMEM medium containing 10% fetal bovine serum was added. The plates were incubated at 37°C with 5% CO2 for 24 h. Transfection was performed when the cell confluence reached 70%-80%. 2 µg of each of the three plasmids was added to each well according to the PEI transfection reagent instructions. An empty VEC plasmid was used as a negative control. 24 h after transfection, the culture medium was discarded, and the cells were washed twice with PBS. 55 µL of RIPA lysis buffer (containing 1% PMSF) was added, and the cells were sonicated at 60 W for 30 s. The cells were centrifuged at 12000 rpm for 10 min at 4°C, and 50 µL of the supernatant was collected as total protein. Western blot analysis: After protein quantification, 10 µL of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h, then incubated overnight at 4°C with rabbit anti-Flag monoclonal antibody (1:10000 dilution). The membrane was washed three times with TBST for 10 min each time, then incubated for 1 h at room temperature with HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution). The membrane was washed three times with TBST for 10 min each time, developed using an ECL chemiluminescence kit, and images were acquired using a chemiluminescence imaging system (Bio-Rad). Western blot results are shown below. Figure 4 As shown, with the internal control GAPDH function correctly, compared to the negative control VEC empty vector, the V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, and V2-Flag-Ago2-shZIKV3 transfection groups all showed obvious Flag-Ago2 protein bands. This indicates that the three constructed plasmids (V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, and V2-Flag-Ago2-shZIKV3) can correctly express Flag-Ago2 protein in BHK21 cells. In other words, the exogenous expression plasmids were successfully constructed and can successfully express Ago2 protein, avoiding the limitation imposed by the limited expression level of endogenous Ago2 protein. In particular, V2-Flag-Ago2-shZIKV1 showed the highest amount of Ago2 protein expression.

[0080] 4.2 Application of basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) transfection The three successfully constructed basal gene knockdown plasmids (V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, and V2-Flag-Ago2-shZIKV3) capable of expressing exogenous Ago2 protein were transfected into BHK21 cells. After 24 hours, the cells were infected with ZIKV. Cell samples were collected 24 hours after ZIKV infection, and total RNA was extracted. The extracted RNA was reverse transcribed into cDNA. Using cDNA as a template, RT-qPCR was performed using primers specific to the ZIKV genome to verify the knockdown level (antiviral efficiency) of the three different basal gene knockdown plasmids (V2-Flag-Ago2-shRNA) on the ZIKV genome. A bar chart was plotted based on the RT-qPCR quantitative data of ZIKV RNA knocked down by the three plasmids. Figure 5 As shown.

[0081] Compared with the negative control VEC empty vector, the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) group, which did not design and insert ZIKV-specific shRNA framework sequences (shZIKV1, shZIKV2, shZIKV3), did not show any identifiable specific knockdown expression of RAN levels after ZIKV infection. This indicates that, due to the absence of shRNA mediation, even if the interference of Ago2 protein limit is avoided through exogenous expression, the specific gene knockdown program cannot be initiated. Therefore, inserting and constructing specific shRNA is the basis for the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) to achieve specific gene knockdown effect, and it excludes the interference of non-specific knockdown effect of this plasmid.

[0082] Conversely, based on the successful expression of the specific shZIKV framework (shZIKV1, shZIKV2, shZIKV3) sequences under the control of the highly efficient promoter U6 while simultaneously considering Ago2 protein expression, the three basic gene knockdown plasmids all exhibited significantly different knockdown effects (different degrees of antiviral effect). This result indicates that the enhanced gene knockdown of specific genes (such as ZIKV) by the three successfully constructed basic gene knockdown plasmids is feasible in principle, design, and technical effect. They can all interfere with the normal replication process of ZIKV in cells through exogenously expressed Ago2 protein and specific shZIKV, thereby achieving a very significant (minimum P < 0.01) reduction in viral genome level (ZIKV RNA). The difference lies in the antiviral efficiency of the three different plasmids: V2-Flag-Ago2-shZIKV1 had the highest antiviral efficiency (P < 0.001), with an extremely significant difference, while V2-Flag-Ago2-shZIKV3 had the second highest antiviral efficiency (P < 0.01), with a very significant difference, which is highly statistically significant. The results indicate that the basic gene knockdown plasmid constructed in this invention has high reliability in enhancing gene knockdown effects. The possible reasons for this beneficial technical effect are: firstly, the complementary matching degree between different shRNAs (shZIKV1, shZIKV2, shZIKV3) and the target sequences on the ZIKV genome leads to different binding stability of the formed Ago2 / RISC complex; secondly, the difference in Ago2 protein expression among the three plasmids (V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, V2-Flag-Ago2-shZIKV3), with V2-Flag-Ago2-shZIKV1 expressing the highest amount of Ago2 protein, thus resulting in a correspondingly higher amount of Ago2 / RISC complex formed; these combined factors lead to differences in the technical effects of viral genome cleavage and degradation.

[0083] 4.3 Construction of the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) Following the construction steps of the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA), shZIKV1, which showed the best technical performance among the three shRNAs (shZIKV1, shZIKV2, and shZIKV3) mentioned above, was inserted into the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA). The enhanced gene knockdown plasmid V2-Flag-S798A-shZIKV1 was successfully constructed through molecular cloning. PCR identification and sequencing results confirmed that shZIKV1 was correctly inserted into the correct positive clone, which was the enhanced gene knockdown plasmid V2-Flag-S798A-shZIKV1. Both V2-Flag-S798A-shZIKV1 and the previously constructed V2-Flag-Ago2-shZIKV1 were transfected into BHK21 cells in parallel. Western blot analysis was performed 24 hours later, and the results are shown below. Figure 6 As shown, under the condition that the internal control Tubulin is expressed correctly and with the same shRNA framework structure, the expression of Flag-Ago2 fusion protein can be successfully detected in cell samples transfected by both the successfully constructed enhanced gene knockdown plasmid V2-Flag-S798A-shZIKV1 and the basic gene knockdown plasmid V2-Flag-Ago2-shZIKV1. However, no Flag-Ago2 fusion protein band was observed in the negative control VEC with empty vector. This indicates that the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNAZIKV1) can correctly express the Flag-Ago2 fusion protein in BHK21 cells. It can be seen that the concentration of the Flag-Ago2 fusion protein band expressed by the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV1) and the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV1) is consistent.

[0084] 4.4 Transfection Application of Enhanced Gene Knockdown Plasmid (V2-Flag-S798A-shRNA) Referring to the transfection application of the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV) described above, the concentration and purity of the successfully constructed enhanced gene knockdown plasmid (V2-Flag-S798A-shRNAZIKV1) and the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV1) were determined using NanoDrop. Under the same conditions, the two plasmids (V2-Flag-Ago2-shZIKV1 and V2-Flag-S798A-shRNA) were transfected in parallel into BHK21 cells in good growth condition. After culturing in a cell culture incubator at 37°C and 5% CO2 for 24 h, ZIKV virus solution was added at an appropriate multiple of infection (MOI=0.1), and the cells were then incubated at 37°C and 5% CO2. After culturing cells in a CO2-filled cell incubator for 24 hours, cell samples were collected, total RNA was extracted and reverse transcribed into cDNA, and bar charts were plotted based on the RT-qPCR quantitative data of ZIKV RNA. Figure 7 As shown.

[0085] Figure 7As can be seen, firstly, compared with the negative control of empty vector VEC, the basal gene knockdown plasmid (V2-Flag-Ago2-shZIKV1) has been able to interfere with the normal replication process of ZIKV in cells through exogenously expressed wild-type Ago2 protein and specific shZIKV1, reducing the ZIKV genomic RNA level to 46% (0.46), thereby achieving a technical effect of extremely significant difference (P<0.0001 vs VEC) in reducing the viral genomic level (ZIKV RNA), with a knockdown efficiency of 54%. That is, the basal gene knockdown plasmid (V2-Flag-Ago2-shRNA) has achieved the technical effect of significantly enhancing the specific knockdown of ZIKV RNA expression in anti-ZIKV. Furthermore, the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) constructed in this invention, at the level of superimposed beneficial effects of the preferred shRNA framework sequence (shZIKV1), further reduces the ZIKV genomic RNA level to 36% (0.36) compared to the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV1), and can further demonstrate a very significant (P<0.01 vs V2-Flag-Ago2-shRNA) technical effect of further reducing the viral genome level (ZIKV RNA), with a knockdown efficiency of 64%. It is evident that compared to the gene knockdown plasmid (V2-Flag-Ago2-shRNA), the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) significantly improved antiviral efficiency (from 54% to 64%), and the difference was highly statistically significant. This demonstrates that the Ago2 mutant protein (Ago2 S798A, or S798A for short) is a key functional site for further enhancing the antiviral effect of the Ago2 fusion protein, providing a more efficient and preferred tool for subsequent in vivo antiviral applications.

[0086] 4.5 Universality of the Different Expression of Ago2 Protein Using Two Gene Knockdown Plasmid Tools with Different shRNAs First, the reason why the expression of Flag-Ago2 fusion protein differs among the three different shRNA basal gene knockdown plasmids (V2-Flag-Ago2-shZIKV1, V2-Flag-Ago2-shZIKV2, V2-Flag-Ago2-shZIKV3) successfully constructed above is mainly due to the different selection of target gene recognition sequences, namely, ZIKV1>ZIKV3>ZIKV2.

[0087] Secondly, the above demonstrates that the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV1) can significantly enhance the cleavage activity of the Ago2 protein while maintaining the same expression level of the Flag-Ago2 fusion protein, exhibiting the functional gain effect of S798A alone on specific gene knockdown. Therefore, is the enhanced cleavage activity of the Ago2 mutant protein related to shRNA?

[0088] To verify whether there is a difference in the expression level of Flag-Ago2 fusion protein after transfection of cells with the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) and the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA), the preferred shZIKV1 was replaced with shZIKV3. Under the same shRNA conditions, the difference in exogenous overexpression of Ago2 protein by the two enhanced gene knockdown tool plasmids (V2-Flag-S798A-shRNA and V2-Flag-Ago2-shRNA) was verified, referring to the operation steps of plasmid construction and application above. The validation experiment mainly included the following steps: 1) Transfecting HEK293T cells with the successfully constructed basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV3), enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV3), and empty vector VEC control, respectively; 2) Collecting cell lysates 24-48 h after transfection; 3) Western blot detection, using anti-Flag antibody to detect the expression of Flag-Ago2 fusion protein, with Tubulin as an internal control; 4) Comparing the Western blot results between the two groups. Figure 8 The grayscale values ​​of the Flag-Ago2 bands are used to determine whether there are differences in expression levels.

[0089] like Figure 8As shown, similar to the comparison results with the shZIKV1 framework above, when the internal control Tubulin expression was correct, no Flag-Ago2 fusion protein band was observed in the negative control using the empty VEC vector when the same framework structure was used. However, both the successfully constructed plasmid V2-Flag-S798A-shZIKV3 and the basal gene knockdown plasmid V2-Flag-Ago2-shZIKV3 successfully detected the Flag-Ago2 fusion protein. The expression results showed that both gene knockdown plasmids (V2-Flag-Ago2-shZIKV3 and V2-Flag-S798A-shZIKV3) could correctly express the Flag-Ago2 fusion protein in HEK293T cells, and the band concentration of the Flag-Ago2 fusion protein expressed by the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV3) was consistent with that of the basal gene knockdown plasmid (V2-Flag-Ago2-shZIKV3). It can be inferred that in different cells (HEK293T & BHK21), the expression trends of the Flag-Ago2 fusion protein in BHK21 cells using shZIKV3 (a completely independent shRNA targeting different regions of the ZIKV genome) and shZIKV1 plasmids are basically consistent. This indicates that the construction system of the two plasmids of this enhanced gene knockdown plasmid tool constructed in this invention has relatively stable consistency and difference in Ago2 protein expression under different shRNAs (shZIKV1 and shZIKV3) and different cells (HEK293T and BHK21), that is, it does not depend on the framework sequence of the shRNA, nor on different cell lines.

[0090] 4.6 Universality of the differences in antiviral efficacy between two gene knockdown plasmid tools for different shRNAs Given that different shRNAs target different gene genes and have different target sequences, is there a difference in antiviral efficiency? To this end, using the two tool plasmids successfully constructed above—the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV3) and the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV3)—we further verified the difference in knockdown efficiency of the two plasmids for ZIKVRNA. The main steps of this validation experiment included: 1) Seeding HEK293T cells into cell culture plates and culturing them to a suitable density, followed by transfection with the basal gene knockdown plasmid (V2-Flag-Ago2-shZIKV3), the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV3), and an empty vector VEC negative control; 2) 24 h after transfection, infecting the cells with ZIKV virus solution at a suitable multiplicity of infection (MOI=0.3) and culturing for another 24 h; 3) Collecting cells or supernatant, extracting RNA, and reverse transcribing it into cDNA; 4) Performing RT-qPCR detection, calculating the relative value of viral load (ZIKV RNA), and plotting a bar chart as shown below. Figure 9 As shown.

[0091] Figure 9 It can be seen that, compared with the negative control of empty vector VEC, the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV3) reduced the ZIKV genomic RNA level to 52% (0.52), thus achieving a technical effect of extremely significant reduction in viral genome level (ZIKV RNA) (P < 0.001 vs VEC), with a knockdown efficiency of 48%. Compared with the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV3), the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV3) further reduced the ZIKV genomic RNA level to 41% (0.41), and could further demonstrate a very significant technical effect of further reducing viral genome level (ZIKV RNA) (P < 0.05 vs V2-Flag-Ago2-shZIKV3), with a knockdown efficiency of 59%. It is evident that, regardless of whether it is shZIKV1 or shZIKV3, in BHK21 cells or HEK293T cells, the enhanced gene knockdown plasmid (V2-Flag-S798A-shZIKV3) exhibits a relatively stronger knockdown effect on Zika virus (ZIKV) RNA compared to the basic gene knockdown plasmid (V2-Flag-Ago2-shZIKV3). This suggests that the S798A mutant enhances RISC function or target cleavage efficiency, demonstrating a relatively better antiviral effect (antiviral efficiency increased from 48% to 59%).

[0092] As further verified above, the two plasmids of the enhanced gene knockdown tool constructed in this invention showed exogenous overexpression of Ago2 protein and significantly improved antiviral efficiency against Zika virus (ZIKV) in different shRNAs and different cells. It can be seen that the enhanced gene knockdown tool of this invention has universality across target sequences and cell types. The above comparison results also ruled out the randomness of "antiviral enhancement results being effective only for specific sequences". It can be seen that the beneficial effect of the enhanced gene knockdown tool is independent of shRNA sequence, proving that the core of its enhancement mechanism lies in improving the catalytic activity or stability of Ago2 protein, rather than optimizing the matching of specific sequences. Western blot analysis showed that the Ago2 protein expression levels in the enhanced gene knockdown plasmids (V2-Flag-S798A-shZIKV1, V2-Flag-S798A-shZIKV3) were consistent with those in the basal gene knockdown plasmids (V2-Flag-WT-shZIKV1, V2-Flag-WT-shZIKV3), but the antiviral efficiency was significantly improved (from 54% to 64%, from 48% to 59%). This suggests that the functional gain brought about by the S798A mutation is mainly a qualitative improvement (enhanced Ago2 cleavage activity) rather than a quantitative improvement.

[0093] Furthermore, traditional RNAi mainly relies on screening multiple shRNAs / siRNAs to obtain a highly efficient target sequence in protein knockdown expression. However, the enhanced gene knockdown tool constructed in this invention has higher basal cleavage activity due to overexpression of Ago2 protein. Therefore, it may enable shRNAs with originally medium to low efficiency (such as shZIKV3 and shZIKV2) to reach significantly different knockdown expression thresholds. Figure 9 52% of shZIKV3, Figure 5 The present invention achieves 68% of the shZIKV2 target, thereby reducing the difficulty and cost of shRNA screening. Furthermore, this invention has been verified to show consistent and significant advantages for different independent target sequences (shZIKV1, shZIKV3, shZIKV2). Even if the target gene identified by a certain target sequence is suppressed to some extent in vivo (e.g., by VSR interference), the knockdown advantage will still be significant (P < 0.01), resulting in a relatively lower risk of failure in future in vivo clinical trials (e.g., the advantage difference in animal models may be reduced from P < 0.01 to P < 0.05).

[0094] The enhanced gene knockdown tool constructed in this invention includes two specific tool plasmids: the basic gene knockdown plasmid (V2-Flag-Ago2-shRNA) and the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA). In particular, the preferred enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) can further enhance the functional synergistic effect of exogenous overexpression of Ago2 protein mutants in directly cleaving target RNA. The modification site S798A of the Ago2 protein mutant is located in the PIWI domain of the Ago2 protein. It is speculated that the mutation at this site may affect: ① the interaction interface between Ago2 and viral VSR protein; ② the conformational stability of Ago2; ③ the metal ion binding capacity of the catalytic center of the Ago2 / RISC complex. Therefore, this invention creatively discovers an optimized modification in which the proserine (S) at position 798 of the Ago2 protein is replaced with alanine (A), providing a new research approach for further understanding the cleavage mechanism of the Ago2 protein through structural biology or mutation scanning experiments.

[0095] Example 5: Universality of Enhanced Gene Knockdown Tools in Target Population The enhanced gene knockdown tools constructed in this invention (two plasmid tools, V2-Flag-Ago2-shRNA and V2-Flag-S798A-shRNA) not only verified the co-expression of two different forms of Ago2 protein and shRNA recognizing the target gene by a single plasmid through experiments, as mentioned above, and significantly enhanced the antiviral effect (anti-ZIKV RNA level) of cells, this invention further verified the specific knockdown effect of the enhanced gene knockdown tools on the proto-oncogene C-MYC target gene encoding the protein. The experimental steps and results are as follows.

[0096] 5.1 The knockdown effect of enhanced gene knockdown tools on proto-oncogene C-MYC 1) Target sequence and shC-MYC framework sequence design: The target gene is derived from the coding region of the human C-MYC gene. The sense strand sequence suitable for designing a target sequence for specific shRNA is shown in SEQ ID NO:18. The complete shC-MYC framework sequence designed based on this target sequence is: EcoRI-sense strand-LOOP sequence-antisense strand-TTTTT-NheI, where the LOOP sequence is preferably GGCCACACC.

[0097] 2) shC-MYC primer design and shC-MYC double-strand preparation: Primer pairs (shC-MYC-F and shC-MYC-R) for amplifying double-stranded shC-MYC were designed based on the shC-MYC framework sequence. The primer sequences are shown in SEQ ID NO:19-SEQ ID NO:20. Equal volumes of forward and reverse primer (shC-MYC-F and shC-MYC-R) stock solutions were mixed and placed in a PCR instrument for annealing. The program was: heating at 95℃ for 5 min, and then slowly cooling to 25℃ at a rate of 0.5℃ per minute to form shC-MYC double-stranded DNA fragments with EcoRI and NheI sticky ends.

[0098] 3) shC-MYC purification and enzyme digestion: The annealed shC-MYC double-stranded DNA was purified using an agarose gel extraction kit. The purified shC-MYC double-stranded DNA contained EcoRI and NheI sticky ends. The basal gene knockdown plasmid (V2-Flag-Ago2-shRNA) and the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) were placed in a 50µL enzyme digestion system (1µg plasmid, 5µL 10× digestion buffer, 2µL EcoRI restriction endonuclease, 2µL...). The enzyme was digested in NheI restriction endonuclease (with an appropriate amount of ddH2O) in a 37°C water bath for 2 hours. 50 µL of the digestion product was subjected to 0.8% agarose gel electrophoresis to preliminarily determine the size of the linearized vector (the band should migrate to the expected 1.4K). The remaining digestion product was purified using a gel extraction kit with an elution volume of 30 µL, and the concentration was determined (NanoDrop).

[0099] 4) Ligation and transformation of shC-MYC with plasmids: Two purified and recovered linearized gene knockdown plasmids (V2-Flag-Ago2-shRNA and V2-Flag-S798A-shRNA) and shC-MYC double-stranded DNA were mixed at a plasmid:shC-MYC molar ratio of 1:3. Ligation was performed overnight at 4°C using T4 DNA ligase and its accompanying buffer. 10 µL of the ligation product was added to 100 µL of DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then immediately incubated on ice for 2 min. 900 µL of antibiotic-free LB medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 1 h. 200 µL of the bacterial culture was then spread onto LB agar plates containing 100 µg / mL ampicillin and incubated upside down at 37°C overnight.

[0100] 5) Positive clone amplification and screening: Single colonies from the plate were picked and inoculated into 5 mL of LB medium containing ampicillin, and cultured at 37°C with shaking for 6 h. 1 µL of the bacterial culture was used as a template for PCR amplification using U6-F primers (sequence shown in SEQ ID NO:21). The PCR program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 60 s, for a total of 25 cycles; final extension at 72°C for 10 min. The PCR products were preliminarily identified by 0.8% agarose gel electrophoresis. Positive clones with the correct band size were sent to a sequencing company for sequencing. The positive clones verified by sequencing were further amplified and cultured, and plasmids were extracted using a plasmid extraction kit to obtain two plasmid tools: V2-Flag-Ago2-shC-MYC and V2-Flag-S798A-shC-MYC. The extracted plasmids were measured for concentration (NanoDrop) and stored at -20°C for later use.

[0101] 6) Cell transfection and Western blot detection: HeLa cells were pre-seeded in 6-well cell culture plates and cultured until the cell confluence reached 70%-80% before transfection. Two plasmids (V2-Flag-Ago2-shC-MYC and V2-Flag-S798A-shC-MYC) were added at 2 µg / well according to the PEI transfection reagent instructions. An empty VEC vector was used as a negative control. Samples were collected 36 h after transfection: the culture medium was discarded, cells were washed twice with PBS, and 55 µL of RIPA lysis buffer (containing 1% PMSF) was added. Cells were sonicated at 60W for 30 seconds. The cells were centrifuged at 12000 rpm for 10 min at 4℃, and 50 µL of the supernatant was collected as the total protein sample. Western blot assay: After protein quantification, 10 µL of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h. Primary antibodies included Flag and C-MYC antibodies, specifically rabbit anti-Flag monoclonal antibody (1:10000 dilution) and rabbit anti-C-MYC monoclonal antibody (1:1000 dilution), respectively, and incubated overnight at 4°C. The membrane was washed three times with TBST for 10 min each time, and HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution) was added, followed by incubation at room temperature for 1 h. The membrane was washed three times with TBST for 10 min each time, developed using an ECL chemiluminescence kit, and images were acquired using a chemiluminescence imaging system (Bio-Rad).

[0102] Assuming the internal reference GAPDH is correct, the Western blot results are as follows: Figure 10As shown in the figure, firstly, when the primary antibody is a Flag antibody, compared with the negative control VEC empty vector (without a Flag-Ago2 fusion protein band), the protein samples expressed by cells transfected with the two plasmids V2-Flag-Ago2-shC-MYC and V2-Flag-S798A-shC-MYC both showed obvious Flag-Ago2 fusion protein bands. This indicates that the two successfully constructed plasmid tools (V2-Flag-Ago2-shC-MYC and V2-Flag-S798A-shC-MYC) can also express Flag-Ago2 protein in HeLa cells, and the expression level is not significantly different. Therefore, this tool plasmid can avoid the limitation of the limited expression level of endogenous Ago2 protein when performing gene knockdown function. Secondly, when the primary antibody is C-MYC, since C-MYC is an endogenous key oncogene, a distinct C-MYC protein band is visible in the protein sample treated with the negative control VEC empty vector cells. In contrast, the two plasmid tools successfully constructed in this invention (V2-Flag-Ago2-shC-MYC and V2-Flag-S798A-shC-MYC) exhibit significant knockdown effects, resulting in a significant reduction in the amount of C-MYC protein expressed compared to the negative control VEC, particularly... In the corresponding samples of cells transfected with V2-Flag-S798A-shC-MYC constructed by the enhanced gene knockdown plasmid, the C-MYC protein band was very weak. This indicates that while both plasmid tools (V2-Flag-Ago2-shC-MYC and V2-Flag-S798A-shC-MYC) significantly enhanced C-MYC gene expression through gene knockdown, the enhanced gene knockdown plasmid (V2-Flag-S798A-shRNA) has a relatively better gene knockdown efficiency.

[0103] Specific embodiments of the present invention have been described in detail, making them readily understandable to those skilled in the art. However, based on all the disclosed descriptions, various modifications or substitutions may be made to those details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An enhanced gene knockdown tool, characterized in that: Gene knockdown of target genes can be enhanced by co-expressing shRNA, which recognizes target mRNA, and effector proteins that perform cleavage and degradation on a single plasmid; among them, The shRNA sequence includes recognition sequences for the endonucleases EcoRI and NheI. The effector protein is Ago2 protein or its mutant S798A. The effector protein is a tagged fusion protein, and the fusion protein encoding gene also includes endonuclease recognition sequences at both ends. The plasmid is either the basic gene knockdown plasmid V2-Flag-Ago2-shRNA or the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA.

2. The enhanced gene knockdown tool according to claim 1, characterized in that: The basic gene knockdown plasmid V2-Flag-Ago2-shRNA has the sequence shown in SEQ ID NO:7, and the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA has the sequence shown in SEQ ID NO:

8.

3. The enhanced gene knockdown tool according to claim 1, characterized in that: The plasmid has the following structure: the expression cassette of the fusion protein is driven by the EF-1α promoter, the fusion protein is tagged with a Flag tag, the expressed fusion protein is Flag-Ago2 or Flag-S798A, and the two ends of the gene encoding the fusion protein are recognition sequences for the restriction enzymes AgeI and BamHI, respectively; the expression cassette of the shRNA is driven by the U6 promoter, and the shRNA sequence includes recognition sequences for the restriction enzymes EcoRI and NheI.

4. A method for constructing an enhanced gene knockdown tool, characterized in that: Based on the pLentiCRISPR V2 plasmid, modifications are made to obtain the enhanced gene knockdown tool as described in any one of claims 1-3, the steps of which include: 1) Using primers AgeI-Flag-Ago2-F and BamHI-Flag-Ago2-R with the sequences shown in SEQ ID NO:1-2, the PCR amplification product Flag-Ago2 with the recognition sequences of the restriction enzymes AgeI and BamHI was obtained by PCR amplification. The recovered PCR amplification product Flag-Ago2 and pLentiCRISPR V2 plasmid were double-digested with restriction enzymes AgeI and BamHI, respectively. The digested Flag-Ago2 fragment was inserted into the linearized pLentiCRISPR V2 plasmid by T4 ligase localization. The V2-Flag-Ago2 plasmid was obtained by gene cloning screening and sequencing verification. 2) Using V2-Flag-Ago2 plasmid as a template, circular PCR was performed using primers U6-EcoRI-F and U6-EcoRI-R with the sequences shown in SEQ ID NO:3-4. The basic gene knockdown plasmid V2-Flag-Ago2-shRNA plasmid was obtained through gene cloning screening and sequencing verification. 3) Using the V2-Flag-Ago2-shRNA plasmid as a template, circular PCR was performed using primers S798A-F and S798A-R with the sequences shown in SEQ ID NO:5-6. The enhanced gene knockdown plasmid V2-Flag-S798A-shRNA was verified by gene cloning screening and sequencing.

5. The method for constructing the enhanced gene knockdown tool according to claim 4, characterized in that: In step 1), the Cas9 sequence on the original pLentiCRISPR V2 plasmid is completely replaced with the coding sequence of Flag-Ago2 on the modified V2-Flag-Ago2 plasmid; in step 2), all gRNA scaffold sequences after the U6 promoter on the modified V2-Flag-Ago2-shRNA plasmid are deleted and replaced with EcoRI and NheI recognition sites for inserting shRNA on the modified V2-Flag-Ago2-shRNA plasmid.

6. A recombinant plasmid for enhanced gene knockdown expression, characterized in that: The recombinant plasmid constructed based on the enhanced gene knockdown tool described in any one of claims 1-3 has the following structure for the shRNA framework sequence: a sense strand-stem-loop sequence-antisense strand, wherein the sense strand is identical to the target sequence of the target gene, the antisense strand is complementary to the target sequence of the target gene, the sense strand and antisense strand are complementary, and both are 20±3nt in length; the stem-loop sequence is a short sequence that can be recognized by the intracellular Dicer enzyme and can flexibly connect the sense strand and antisense strand to form a stable hairpin structure, and is 9nt in length.

7. The recombinant plasmid for enhanced gene knockdown expression according to claim 6, characterized in that: The stem-loop sequence is either TTCAAGAGA or GGCCACACC.

8. A method for constructing an enhanced gene knockdown expression recombinant plasmid, characterized in that: The steps for constructing the recombinant plasmid for enhanced gene knockdown expression as described in any one of claims 6-7 include: 1) designing a target sequence and shRNA framework sequence for the target gene; 2) designing shRNA primers for annealing to obtain target sequence-related shRNA double strands; 3) purifying the shRNA double strands, and simultaneously purifying the enhanced gene knockdown tool as described in any one of claims 1-3 by digestion with EcoRI and NheI enzymes; 4) ligating and transforming the purified shRNA double strands with the digested and purified enhanced gene knockdown tool as described in any one of claims 1-3, and inserting the shRNA into the basic gene knockdown plasmid V2-Flag-Ago2-shRNA or the enhanced gene knockdown plasmid V2-Flag-S798A-shRNA; 5) obtaining the recombinant plasmid for enhanced gene knockdown expression after screening and verification of the transformed positive clones.

9. A method for using an enhanced gene knockdown expression recombinant plasmid, characterized in that: After transfecting the target cells with the recombinant plasmid of enhanced gene knockdown expression as described in any one of claims 6-7, culture for 24-72 hours.

10. The application of the enhanced gene knockdown tool as described in any one of claims 1-3, or the method for constructing the enhanced gene knockdown tool as described in claims 4-5, or the recombinant plasmid for enhanced gene knockdown expression as described in claims 6-7, or the method for constructing the recombinant plasmid for enhanced gene knockdown expression as described in claim 8, or the method for using the recombinant plasmid for enhanced gene knockdown expression as described in claim 9, in the knockdown expression of a target gene.

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