Chain displacement-responsive adar rna editing system and applications thereof

Abstract

The application discloses a kind of chain displacement response type ADAR RNA editing system (SPRING), belong to gene editing technical field.The system is made of effector fusion protein and hairpin guide RNA, and guide RNA forms hairpin structure by blocking sequence, and only when target RNA exists, editing is activated by chain displacement reaction, and invalid complex is greatly reduced, and efficiency and specificity are improved.The system can realize A-to-I and C-to-U double type editing, and the efficiency of exogenous editing reaches 67%, the editing of endogenous gene is improved by more than 40%, and off-target effect is significantly reduced.The application has the advantages of high efficiency, precision, safety, universality, etc., and is suitable for gene function research, genetic disease treatment, RNA regulation and biotechnology development, and has important scientific research and clinical value.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biotechnology, specifically relating to an ADAR-based strand substitution-responsive RNA editing system and its application in efficient and highly specific RNA single-base editing. Background Technology

[0002] RNA editing is a post-transcriptional regulatory technique that modifies bases on RNA molecules at specific sites without altering the genomic DNA sequence. Due to its reversibility, high safety, and lack of permanent genomic alteration, it has become an important research direction for the treatment of genetic diseases and the regulation of gene function. Among numerous RNA editing tools, A-to-I editing mediated by human ADAR (adenosine deaminase acting on RNA) protein has the greatest potential for clinical translation. ADAR is derived from the human body, has extremely low immunogenicity, and can deaminate adenine (A) at the target site into hypoxanthine (I), which is recognized as guanine (G) during translation and splicing, thus achieving site-specific A→G correction. Currently, ADAR-based RNA editing systems typically consist of two parts: The expression combines an ADAR deaminase effector protein with a guide RNA (gRNA) that can target and bind to RNA.

[0003] Current mainstream ADAR-mediated RNA editing systems, including MS2-MCP-ADAR, BoxB-λN-ADAR, and Cas13-ADAR, generally suffer from two major drawbacks: low editing efficiency and severe off-target effects. The main reasons are: free guide RNA (gRNA) readily forms ineffective binary complexes with ADAR fusion proteins or target mRNAs, crowding out effective editing resources and triggering non-specific binding; simultaneously, traditional gRNAs have a high tolerance for target sequence mismatches, further exacerbating off-target effects.

[0004] Current strategies for improving ADAR editing performance mainly focus on modifying ADAR protein mutants, such as ADAR2E488Q. However, screening for highly efficient protein variants with low off-target effects is difficult, time-consuming, and fails to fundamentally inhibit ineffective binary complexes and mismatch binding. Therefore, developing a guide RNA modification strategy that does not rely on ADAR protein modification and can simultaneously improve editing efficiency and specificity has significant scientific research and clinical translational value. Summary of the Invention

[0005] RNA editing, used to correct harmful mutations, offers advantages such as reversibility and tunability, and does not affect the genome sequence, making it a promising therapeutic approach. However, current RNA editing tools still suffer from relatively low editing efficiency and off-target effects, limiting their application as therapeutic tools. To address these shortcomings, we introduce a novel strategy based on existing RNA editing tools: a strand displacement-responsive ADAR RNA editing system. This system involves adding a "closing sequence" to form a guide RNA with a hairpin structure. The ADAR system is activated for RNA editing through a strand displacement reaction upon binding to the substrate. Results show that this modification significantly improves the efficiency of site-specific RNA editing at various target sites. Furthermore, the system possesses a stable stem-loop structure, which needs to be opened upon binding to the target RNA. This necessitates a more precise match between the guide RNA and the target sequence, allowing for minimal mismatches and thus enhancing the specificity of RNA editing. In principle, this method can be applied to various ADAR-based editing systems, providing a novel RNA editing platform with broad potential for research, therapy, and biotechnology applications.

[0006] The technical solution described in this invention is as follows: Construction of a strand displacement-responsive ADAR RNA editing system: The MS2-MCP-ADAR system was selected as the base editing system for modification. The MS2-MCP-ADAR system includes an ADAR-MCP fusion protein module as the effector and an MS2-gRNA portion as the guide RNA. The MS2 domain is derived from the thermophilic Escherichia coli MS2 phage and can interact tightly with the MCP protein. The MS2-gRNA consists of an MS2 aptamer and a guide sequence, which can specifically bind to the fusion protein ADAR-MCP (… Figure 1This configuration enables the ADAR deaminase to be recruited to the target site for editing by forming a double strand with the target mRNA through base pairing. The MCP protein is fused with the deaminase domain of the human ADAR2 mutant (E488Q). This specific mutation (E488Q) in ADAR2 is known to enhance enzyme activity and affinity. The ADAR protein hydrolyzes and deaminates the target adenosine (A) at the CA mismatch site, converting it to inosine (I), which can be interpreted as guanosine (G) through RNA splicing and translation. A strand substitution-responsive ADAR RNA editing system is constructed by integrating a closed sequence (red) into the 5' end of the MS2-gRNA. This sequence is complementary to the MS2 sequence of the recruiting effector, forming a hairpin structure. Therefore, the BSM-gRNA remains closed when the mRNA is absent because MS2 is enclosed within the hairpin structure. When substrate mRNA is present, the antisense sequence in BSM-gRNA can pair complementaryally with the substrate to form an RNA double strand, subsequently releasing MS2 to promote the recruitment of the editing site ADAR-MCP. Figure 2 ).

[0007] The structural features of the strand substitution-responsive ADAR RNA editing system of the present invention are as follows: (1) Closure sequence length: 14–19 nt, preferably 19 nt; (2) Antisense target sequence length: 21–25 nt, preferably 25 nt; (3) Editing site: located in the middle of the complementary sequence; (4) The closure sequence and the aptamer sequence are completely complementary to form a stable hairpin structure. Attached Figure Description

[0008] Figure 1 It is a traditional MS2-MCP-ADAR system.

[0009] Figure 2 It is a strand displacement-responsive ADAR RNA editing system.

[0010] Figure 3 This is the plasmid map used in the examples.

[0011] Figure 4 This is an editing efficiency analysis chart from the embodiment. Detailed Implementation

[0012] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an artificial long non-coding RNA provided by the present invention and its application in targeted protein degradation, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0013] Example: Construction and validation of four artificial long non-coding RNAs targeting the degradation of RBFOX1 protein To assess their editing efficiency, we subsequently created vectors containing these modified guide RNAs and GFP (W58X) and MCP-DD (E488Q) effectors, respectively. All of these constructs were confirmed by DNA sequencing before transfection into human embryonic kidney cells (293T).

[0014] plasmid construction The *E. coli* strain used in the experiment was Trelief™ 5α chemocompetent cells, purchased from Qingke Biotechnology (Beijing, China) Co., Ltd. Figure 3 As shown, the guide RNA gene used in this study was cloned between the Eco-RV and Not-I restriction endonucleases of the plasmid vector pcDNA3.1 zeo(+)-U6. The DNA sequence of the target gene was first amplified using primers, and the resulting fragment was purified using a PCR purification kit. Subsequently, the purified DNA fragment and the pcDNA3.1 zeo(+)-U6 vector were digested overnight at 37°C using the restriction endonucleases Eco-RV (NEB, USA) and Not-I (NEB, USA). The digested vector and fragment were recovered using a gel extraction kit. Finally, the two were ligated using T4 ligase (Vazyme) to form a ligation product, which was then transformed into *E. coli* Trelief™ 5α chemocompetent cells. Finally, DNA sequencing analysis was performed to confirm the presence of the inserted sequence in the plasmid. The 4λN-DD (E488Q), 4λN-ADARRESCUE effector genes, and dead-GFP reporter genes were cloned between the Hind-III and Eco-RI restriction endonuclease sites in the modified pcDNA3.1 zeo (+)-U6 vector. The target gene fragments were amplified using Fidelity DNA Polymerase (Vazyme) and purified using a PCR purification kit. The purified samples were then digested overnight at 37°C, with the purified DNA fragments and pcDNA3.1 (+) vector digested using Eco-RI (NEB, USA) and Hind-III (NEB, USA). Subsequently, the purified inserts and vector DNA were ligated together using T4 ligase (Vazyme) according to the reagent instructions. All constructed vectors were sequenced and validated by Zhejiang Youkang Biotechnology Co., Ltd.

[0015] Cell culture HEK293T cells were grown in DMEM medium (Gibco) containing specific components including 200 units / mL penicillin, 200 units / mL streptomycin sulfate, and 2 units / mL L-glutamine, supplemented with 10% (v / v) fetal bovine serum (provided by Invitrogen). Culture conditions were set at a constant temperature of 37°C in an incubator filled with a humidified 5% CO2 atmosphere. The day before transfection, cells were trypsinized and seeded into 24-well plates for subsequent experimental procedures.

[0016] Cell transfection When cells in the culture dish reach approximately 70% to 80% confluence, wash with PBS buffer, add trypsin (0.25%), incubate at 37°C for 120 seconds, and then seed into 24-well plates. After culturing for 24 hours, the cells in the 24-well plates should be at least 80% confluence. Transfect 1 μg of 4λN-DD (E488Q) and guide RNA into each well. All transfections were performed using Hieff Trans™ Liposomal Transfection Reagent (Yeasen) according to the manufacturer's instructions.

[0017] Editing efficiency analysis Transfected HEK 293T cells were used to quantify editing efficiency in mammalian cells. In in vivo RNA editing experiments involving gRNA+4λN-DD (E488Q), a reporter plasmid was used, with 250 ng of the reporter plasmid and 250 g of the gRNA+4λN-DD (E488Q) plasmid employed. All in vivo RNA editing experiments targeting endogenous transcripts or disease mimic reporter genes were performed in 24-well plates using 500 ng of the gRNA / enzyme plasmid. Transfection was performed when cells reached 80% confluence, followed by medium replacement after 24 hours and incubation for another 48 hours (medium replacement every 24 hours) until further treatment. After cell isolation from the samples, total RNA was extracted using the Cell Total RNA Isolation Kit V2 (Vazyme). Following RNA purification, the obtained RNA was reverse transcribed, and cDNA (Vazyme) was synthesized using oligo-dT and random primers according to the manufacturer's instructions. The obtained cDNA product was amplified by PCR using target-specific primers, and the PCR product was sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. The first-generation sequencing results were further quantified using Software Scanner 1.0 (Applied Biosystems) combined with EditR software (baseditr.com). The peak heights of adenosine and guanosine were measured, and the editing ratio of each site was calculated according to the formula A / [A + G]. The editing efficiency is as follows: Figure 4 As shown.

Claims

1. A strand substitution-responsive ADAR RNA editing system, characterized in that, It consists of an effector fusion protein and a hairpin guide RNA; the effector fusion protein is selected from λN-ADAR2 deaminase, MCP-ADAR2 deaminase, and λN-ADARRESCUE; the hairpin guide RNA includes, from the 5′ end to the 3′ end, a blocking sequence, an RNA aptamer sequence, and an antisense targeting sequence; the blocking sequence is completely complementary to the RNA aptamer sequence to form a hairpin structure; the antisense targeting sequence is complementary to the target RNA.

2. The system according to claim 1, characterized in that, The length of the closed sequence is 12–19 nt, preferably 14 nt or 19 nt, and most preferably 19 nt.

3. The system according to claim 1, characterized in that, The antisense targeting sequence is 15–31 nt in length, preferably 21–25 nt, and most preferably 25 nt.

4. The system according to claim 1, characterized in that, The RNA aptamer is BoxB or MS2.

5. The system according to claim 1, characterized in that the ADAR2 contains the highly active E488Q mutation.

6. The system according to claim 1, characterized in that the ADARRESCUE is capable of simultaneously catalyzing A-to-I and C-to-U editing.

7. The system according to claim 1, characterized in that the guide RNA is driven by a U6, CMV or U6-tRNA promoter, with the U6-tRNA promoter being the most preferred.

8. The system according to claim 1, characterized in that, in the absence of target RNA, the hairpin structure is in a closed state; and in the presence of target RNA, editing is activated by a strand displacement reaction.

9. The application of the system according to any one of claims 1–8 in precise RNA editing, wherein the editing includes A-to-I editing and C-to-U editing.

10. Use of the system according to any one of claims 1–8 in the preparation of a medicament for treating monogenic genetic diseases.

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