Modified linear arRNA or cyclized arRNA and application thereof in RNA editing

Abstract

The invention discloses a modified linear arRNA (Ribonucleic Acid) or cyclized arRNA and an application of the modified linear arRNA or cyclized arRNA in RNA (Ribonucleic Acid) editing. The invention provides an RNA (referred to as a modified arRNA), comprising n copies of arRNA truncated bodies; and n is a natural number greater than 2. The modified arRNA can be a linear RNA (Ribonucleic Acid) or a circular RNA (Ribonucleic Acid). To minimize the effect of bystanders without reducing the editing efficiency. The RNA sequence structure is modified by minimizing the specific sequence, the recruitment structural domain is combined, and the cyclization mechanism is optimized. Compared with the existing ADAR technology, the method and the device have the advantage that the editing efficiency is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to modified linear or circular arRNAs and their applications in RNA editing. Background Technology

[0002] RNA editing, as the name suggests, involves editing at the RNA level. Compared to DNA editing, the biggest advantage of RNA editing is that RNA has a very short lifespan, making RNA modifications reversible and safer. Especially when facing diseases caused by abnormal alternative splicing of transcripts, DNA editing is ineffective, and RNA editing is the only treatment option. Therefore, in many fields, RNA editing is a complementary method to DNA editing, and in specific areas, RNA editing has unparalleled advantages.

[0003] Human cells contain a protein called ADAR (Adenosine Deaminases Acting on RNA), which can deaminate adenosine nucleotides, converting them into inosine (A→I). During translation, I is treated as G (Guanosine). Therefore, ADAR possesses the ability to edit A→G.

[0004] LEAPER (Leveraging endogenous ADAR for programmable editing of RNA) refers to the use of specially designed arRNAs (ADAR-recruiting RNA) to recruit endogenous ADAR proteins to edit mRNA from A to G. However, the low editing efficiency and potential bystander editing have hindered the widespread application of circular RNA editors. Summary of the Invention

[0005] The purpose of this invention is to provide modified linear or circular arRNAs and their applications in RNA editing.

[0006] This invention provides an RNA (referred to as modified arRNA) comprising n copies of a truncated arRNA; n is a natural number greater than 2.

[0007] arRNA (ADAR-recruiting RNA): RNA that recruits ADAR proteins.

[0008] The arRNA is inversely complementary to the target sequence in the target RNA, thereby recruiting the ADAR protein to the vicinity of the target sequence.

[0009] The RNA containing the target nucleotides of RNA editing in LEAPER is called the target RNA.

[0010] The nucleotide corresponding to the target nucleotide in the arRNA is C, and the other parts are inversely complementary to the target sequence in the target RNA.

[0011] circ-arRNA (circular ARAR-recruiting RNA): A circular RNA that recruits ADAR.

[0012] The modified arRNA can be either linear RNA or circular RNA.

[0013] The modified arRNA can be a linear RNA formed by sequentially linking n copies of truncated arRNA.

[0014] The modified arRNA can be a circular RNA formed by joining n copies of truncated arRNA end to end.

[0015] In the existing technology, arRNA is 151 nt.

[0016] arRNA truncated form is the RNA obtained by truncating arRNA.

[0017] Specifically, the length of the truncated arRNA can be 15-150 nt. Specifically, the length of the truncated arRNA can be 15-120 nt. Specifically, the length of the truncated arRNA can be 20-100 nt. Specifically, the length of the truncated arRNA can be 20-80 nt. Specifically, the length of the truncated arRNA can be 21-71 nt. Specifically, the length of the truncated arRNA can be 51-71 nt. Specifically, the length of the truncated arRNA can be 61-71 nt. Specifically, the length of the truncated arRNA can be 21-51 nt. Specifically, the length of the truncated arRNA can be 21-41 nt. Specifically, the length of the truncated arRNA can be 21-31 nt, 31-41 nt, 41-51 nt, 51-61 nt, or 61-71 nt. Specifically, the length of the truncated arRNA can be 21nt, 31nt, 41nt, 51nt, 61nt, or 71nt.

[0018] Specifically, n is a natural number between 2 and 10 (i.e., n is 2, 3, 4, 5, 6, 7, 8, 9, or 10). Specifically, n is a natural number between 2 and 5 (i.e., n is 2, 3, 4, or 5). Specifically, n is a natural number between 3 and 5 (i.e., n is 3, 4, or 5).

[0019] In the truncated arRNA, the nucleotide corresponding to the target nucleotide is C, and the other parts are reverse complementary to the target sequence in the target RNA.

[0020] As a preferred embodiment, the nucleotide corresponding to the target nucleotide in the arRNA truncated form is located in the middle.

[0021] RNA molecules containing modified arRNA are also within the scope of protection of this invention. That is, in addition to the modified arRNA, the RNA molecule may also contain other nucleotides (e.g., nucleotides used for RNA cyclization). As an example, the RNA molecule is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 29, the modified arRNA, and the segment shown in SEQ ID NO: 30.

[0022] DNA molecules expressing modified arRNA or DNA molecules expressing RNA molecules with modified arRNA are also within the scope of protection of this invention. As an example, the DNA molecule is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 2, the segment for expressing the modified arRNA, and the segment shown in SEQ ID NO: 3. As an example, the DNA molecule is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment for expressing the modified arRNA, and the segment shown in SEQ ID NO: 15.

[0023] Recombinant vectors containing any of the DNA molecules described above are also within the scope of protection of this invention. As an example, the recombinant vector may be a recombinant plasmid obtained by inserting any of the DNA molecules described above into the BsmBⅠ restriction site of the pcg 2.0-BFP-circ-arRNA backbone plasmid.

[0024] Recombinant microorganisms containing any of the DNA molecules described above are also within the scope of protection of this invention.

[0025] This invention also protects the use of any of the modified arRNAs described above as a LEAPER tool. This invention also protects the use of any of the modified arRNAs described above as an RNA editing tool. This invention also protects the use of any of the modified arRNAs described above in RNA editing. This invention also protects the use of any of the modified arRNAs described above in the preparation of LEAPER kits. This invention also protects the use of any of the modified arRNAs described above in the preparation of RNA editing kits.

[0026] This invention also protects the use of RNA molecules with modified arRNA as LEAPER tools. This invention also protects the use of RNA molecules with modified arRNA as RNA editing tools. This invention also protects the use of RNA molecules with modified arRNA in RNA editing. This invention also protects the use of RNA molecules with modified arRNA in the preparation of LEAPER kits. This invention also protects the use of RNA molecules with modified arRNA in the preparation of RNA editing kits.

[0027] This invention also protects the use of any of the above-described DNA molecules, recombinant vectors, or recombinant microorganisms as LEAPER tools. This invention also protects the use of any of the above-described DNA molecules, recombinant vectors, or recombinant microorganisms as RNA editing tools. This invention also protects the use of any of the above-described DNA molecules, recombinant vectors, or recombinant microorganisms in RNA editing. This invention also protects the use of any of the above-described DNA molecules, recombinant vectors, or recombinant microorganisms in the preparation of LEAPER kits. This invention also protects the use of any of the above-described DNA molecules, recombinant vectors, or recombinant microorganisms in the preparation of RNA editing kits.

[0028] Any of the above-described RNA editing methods involves RNA editing resulting from a mutation from A to G.

[0029] Linear 3*arRNA 61 Editor and circular 3*arRNA 61 See the structural diagram of the editor. Figure 1 .

[0030] Previous versions of arRNAs (LEAPER) primarily utilize 151nt arRNAs to recruit endogenous ADAR for A→G editing. However, ADAR1 / 2 enzymes tend to indiscriminately amino-ammonitropic adenosines in the RNA double strand, meaning all adenosines present in the target transcript within the arRNA coverage area are susceptible to editing to varying degrees. Increased arRNA length increases the likelihood of bystander effects. Conversely, shortened arRNA length may lead to reduced editing efficiency. To address these issues, the inventors proposed a novel strategy: shortening arRNA length while increasing arRNA copy number. This new strategy aims to minimize bystander effects without reducing editing efficiency. This invention aims to improve A→I (A→G) editing efficiency by minimizing specific sequence modifications to the RNA sequence structure, incorporating recruitment domains, and optimizing the circularization mechanism. Compared to existing ADAR technologies, this invention significantly improves editing efficiency. Attached Figure Description

[0031] Figure 1 Linear 3*arRNA 61 Editor and circular 3*arRNA 61 A schematic diagram of the editor's structure.

[0032] Figure 2 The result diagram is from Example 1.

[0033] Figure 3 This is a result diagram of Example 2.

[0034] Figure 4 This is a result diagram of Example 3.

[0035] Figure 5 This is a result diagram of Example 4.

[0036] Figure 6 This is a result diagram of Example 5. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. Complete culture medium: DMEM medium containing 10% FBS and 1% penicillin-streptomycin. PEI (full name Hieff) PEI transfection reagent: Yisheng Biotechnology Co., Ltd., product catalog number 40820ES04. Lipofectamine TM3000 transfection reagent: Thermo Fisher Scientific, catalog number L3000015. In the examples, RNA was quantified by measuring A260 readings. HEK-293T cells, also known as HEK293T cells. pcg 2.0-BFP-circ-arRNA backbone plasmid: AddGene Scientific, catalog number 213965. All recombinant plasmids in the examples have been sequenced and verified. The RNA is the DNA-corresponding RNA, i.e., RNA in which the T in the DNA is replaced with U. psPAX2 plasmid and pMD2.G plasmid are described in the following literature: Naxin Sun, Dongdong Zhao, Siwei Li, Ziteng Zhang, Changhao Bi, Xueli Zhang, Reconstructed glycosylase baseeditors GBE2.0 with enhanced C-to-G base editing efficiency and purity, Molecular Therapy, Volume 30, Issue 7, 2022. The mcherry-gfp plasmid is a circular plasmid, and its full sequence is shown in SEQ ID NO: 1. The mcherry-gfp plasmid contains a reporter gene structure (the reporter gene structure is located at positions 1265-2770 in SEQ ID NO: 1; in SEQ ID NO: 1, nucleotides 1265-1972 encode the mcherry protein, positions 2039-2041 are the stop codon TAG, and positions 2054-2767 encode the gfp protein), which includes the upstream mCherry gene and the downstream eGFP gene. Between the two genes is a 3×GGGGS coding sequence and a TAG.

[0039] Example 1: Reducing the bystander effect through tandem repeats of arRNA

[0040] I. Lentiviral Packaging

[0041] 1. HEK-293T cells were seeded into cell culture dishes with a diameter of 15 cm and cultured in complete culture medium until the cell density reached 70-80%.

[0042] 2. Take a 1.5 mL EP tube, add 15 μg mcherry-gfp plasmid, 10 μg psPAX2 plasmid, and 5 μg pMD2.G plasmid, then add 500 μL serum-free DMEM medium and incubate at room temperature for 5 min.

[0043] 3. Take a 1.5 mL EP tube, add 90 μg PEI, then add 500 μL serum-free DMEM medium, and incubate at room temperature for 5 min.

[0044] 4. Mix the liquid phase in the EP tube after completing step 2 with the liquid phase in the EP tube after completing step 3, and incubate at room temperature for 20 minutes to obtain the transfection solution (transfection solution for one culture dish).

[0045] 5. After completing step 1, discard the supernatant, add the transfection solution obtained in step 4 to the culture dish, and then add 10 mL of serum-free DMEM medium. Incubate for 4-6 hours.

[0046] 6. After completing step 5, discard the supernatant, add 25 mL of complete culture medium to the culture dish, and incubate for 24 h.

[0047] 7. After completing step 6, add 10 mL of complete culture medium to the culture dish and incubate for 24 h.

[0048] 8. After completing step 7, collect the liquid phase in the culture dish into a 50mL centrifuge tube, centrifuge at 6000g for 5min, and collect the supernatant.

[0049] 9. Transfer the supernatant obtained in step 8 to an ultracentrifuge tube, centrifuge at 120,000 rpm for 2 hours, and collect the precipitate.

[0050] 10. Resuspend the precipitate obtained in step 9 in 1 mL of serum-free DMEM medium and transfer it to a 1.5 mL EP tube. Incubate on ice for 30-60 min, then centrifuge at 6000 g for 5 min. Take the supernatant, which is the concentrated and purified lentivirus solution. Store at -80℃.

[0051] II. Slow Viral Infection

[0052] 1. HEK-293T cells were seeded into 6-well plates and cultured in complete culture medium until the cell density reached 30-40%.

[0053] 2. After completing step 1, discard the supernatant, add 1 mL of virus filtrate to each well, and incubate for 12 h.

[0054] Viral filtrate: The concentrated and purified lentivirus solution obtained in step one is filtered through a filter membrane with a pore size of 0.45 μm.

[0055] 3. After completing step 2, discard the supernatant, add 2 mL of complete culture medium to each well, and incubate for 72 h (mCherry fluorescence can be observed at this time).

[0056] 4. After completing step 3, collect and digest the cells, then inoculate them into a 10cm diameter cell culture dish, add 10mL of complete culture medium, and culture for 24h.

[0057] 5. After completing step 4, discard the supernatant, add new complete culture medium to the culture dish, and incubate for 24 hours.

[0058] 6. After completing step 5, collect and digest the cells, seed them into 6-well plates, and culture them in complete culture medium until the cell density reaches 40-50%.

[0059] III. Preparation of Editing Plasmids

[0060] Editing plasmid: A specific DNA molecule is inserted into the BsmBI restriction site of the pcg 2.0-BFP-circ-arRNA backbone plasmid to obtain the editing plasmid.

[0061] The specific DNA molecules are double-stranded DNA molecules, namely specific DNA molecule 1-1 (transcribed to arRNA151), specific DNA molecule 1-2 (transcribed to arRNA21*3), specific DNA molecule 1-3 (transcribed to arRNA31*3), specific DNA molecule 1-4 (transcribed to arRNA41*3), specific DNA molecule 1-5 (transcribed to arRNA51*3), specific DNA molecule 1-6 (transcribed to arRNA61*3), specific DNA molecule 1-7 (transcribed to arRNA71*3), or specific DNA molecule 1-8 (transcribed to arRNA71*2), specific DNA molecule 1-9 (transcribed to arRNA71*4), or specific DNA molecule 1-10 (transcribed to arRNA71*5).

[0062] Specific DNA molecule 1-1 is composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 4, and the segment shown in SEQ ID NO: 3. Specific DNA molecule 1-2 is composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 5, and the segment shown in SEQ ID NO: 3. Specific DNA molecule 1-3 is composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 6, and the segment shown in SEQ ID NO: 3. Specific DNA molecule 1-4 is composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 7, and the segment shown in SEQ ID NO: 3. Specific DNA molecule 1-5 is composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 8, and the segment shown in SEQ ID NO: 3. Specific DNA molecules 1-6 are composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 9, and the segment shown in SEQ ID NO: 3. Specific DNA molecules 1-7 are composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 10, and the segment shown in SEQ ID NO: 3. Specific DNA molecules 1-8 are composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 11, and the segment shown in SEQ ID NO: 3. Specific DNA molecules 1-9 are composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 12, and the segment shown in SEQ ID NO: 3. Specific DNA molecules 1-10 are composed, from upstream to downstream, of the following segments: the segment shown in SEQ ID NO: 2, the segment shown in SEQ ID NO: 13, and the segment shown in SEQ ID NO: 3.

[0063] Ten editing plasmids were obtained. Following the principle of corresponding to the names of specific DNA molecules, the editing plasmids were named sequentially from Editing Plasmid 1-1 to Editing Plasmid 1-10.

[0064] IV. The impact of arRNA length on editing efficiency

[0065] In this step, the inventors reduced the length of the arRNAs from 151nt to different lengths (21, 31, 41, 51, 61 or 71nt), while retaining three copies of each arRNA, and locating the target site in the middle of each repeating sequence.

[0066] The seven editing plasmids (i.e., editing plasmids 1-1 to 1-7) prepared in step three were processed separately.

[0067] 1. Take a 1.5 mL EP tube, add 4 μg of editing plasmid, then add 500 μL of serum-free DMEM medium, and incubate at room temperature for 5 min.

[0068] 2. Take a 1.5 mL EP tube, add 12 μg PEI, then add 500 μL serum-free DMEM medium, and incubate at room temperature for 5 min.

[0069] 3. Mix the liquid phase in the EP tube after completing step 1 with the liquid phase in the EP tube after completing step 2, and incubate at room temperature for 20 minutes to obtain the transfection solution (transfection solution for one well).

[0070] 4. Take the 6-well plate from step 2, discard the supernatant, add the transfection solution prepared in step 3, and add 500 μL of serum-free DMEM medium to each well. Incubate for 4-6 hours.

[0071] 5. After completing step 4, discard the supernatant, add 2 mL of complete culture medium to each well, and incubate for 24 h.

[0072] 6. After completing step 5, discard the supernatant, add 2 mL of complete culture medium containing 4 mg / mL PURO to each well, and incubate for 24 h.

[0073] 7. After completing step 6, discard the supernatant, resuspend the cells in 10×PBS, and then centrifuge at 4000g for 5 min to collect the cells. At this point, it can be observed that arRNA151, arRNA21*3, arRNA31*3, arRNA41*3, arRNA51*3, arRNA61*3, and arRNA71*3 all light up eGFP, indicating that the truncated tandem linear arRNA editors can all edit the target sites.

[0074] 8. Take the cells collected in step 7, extract total RNA, and reverse transcribe it to obtain cDNA. Use the cDNA as a template for PCR amplification, recover the specific bands and sequence them, and calculate the RNA editing efficiency based on the sequencing results.

[0075] The primers used for PCR amplification are as follows:

[0076] Upstream primer: 5'-CACTCCACCGGCGGCATGGACGAG-3';

[0077] Downstream primer: 5'-CACGCTGAACTTGTGGCCGTTTACGTCG-3'.

[0078] RNA editing efficiency = number of target sequences with the target mutation ÷ total number of target sequences × 100%.

[0079] The target mutation refers to the mutation of the stop codon TAG (i.e., positions 2039-2041 in SEQ ID NO: 1) in the target sequence into TGG.

[0080] The results for RNA editing efficiency are shown in [link to data]. Figure 2 ArRNAs A, arRNA151, arRNA21*3, arRNA31*3, arRNA41*3, arRNA51*3, arRNA61*3, and arRNA71*3 all possess RNA editing capabilities. Compared to arRNA151, arRNA71*3 exhibits significantly improved RNA editing efficiency.

[0081] V. The effect of arRNA copy number on editing efficiency

[0082] In this step, the inventors set the copy number of arRNAs to 2, 3, 4 or 5, respectively, and located the target site in the middle of each repeating sequence.

[0083] The five editing plasmids prepared in step three (i.e., editing plasmid 1-1, editing plasmid 1-7, editing plasmid 1-8, editing plasmid 1-9 and editing plasmid 1-10) were processed separately.

[0084] The method is the same as step four.

[0085] The results for RNA editing efficiency are shown in [link to data]. Figure 2 B. arRNA151, arRNA71*2, arRNA71*3, arRNA71*4, and arRNA71*5 all possess RNA editing capabilities. Compared to arRNA151, the RNA editing efficiency of arRNA71*3, arRNA71*4, and arRNA71*5 is significantly improved.

[0086] Example 2: Reducing bystanders and further improving efficiency through tandem repeats of circ-arRNA

[0087] Circ-arRNA editing efficiency is generally higher than that of linear arRNA editing. In this embodiment, the inventors verified the editing efficiency of Circ-arRNA.

[0088] I. Lentiviral Packaging

[0089] Same as step one in Example 1.

[0090] II. Slow Viral Infection

[0091] Same as step two in Example 1

[0092] III. Preparation of Editing Plasmids

[0093] Editing plasmid: A specific DNA molecule is inserted into the BsmBI restriction site of the pcg 2.0-BFP-circ-arRNA backbone plasmid to obtain the editing plasmid.

[0094] The specific DNA molecules are double-stranded DNA molecules, namely specific DNA molecule 2-1 (transcribed to Circ-arRNA151), specific DNA molecule 2-2 (transcribed to Circ-arRNA21*3), specific DNA molecule 2-3 (transcribed to Circ-arRNA31*3), specific DNA molecule 2-4 (transcribed to Circ-arRNA41*3), specific DNA molecule 2-5 (transcribed to Circ-arRNA51*3), specific DNA molecule 2-6 (transcribed to Circ-arRNA61*3), specific DNA molecule 2-7 (transcribed to Circ-arRNA71*3), specific DNA molecule 2-8 (transcribed to Circ-arRNA61*2), specific DNA molecule 2-9 (transcribed to Circ-arRNA61*4), or specific DNA molecule 2-10 (transcribed to Circ-arRNA61*5).

[0095] Specific DNA molecule 2-1, from upstream to downstream, consists of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 4, and the segment shown in SEQ ID NO: 15. Specific DNA molecule 2-2, from upstream to downstream, consists of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 5, and the segment shown in SEQ ID NO: 15. Specific DNA molecule 2-3, from upstream to downstream, consists of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 6, and the segment shown in SEQ ID NO: 15. Specific DNA molecule 2-4, from upstream to downstream, consists of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 7, and the segment shown in SEQ ID NO: 15. Specific DNA molecule 2-5, from upstream to downstream, consists of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 8, and the segment shown in SEQ ID NO: 15. Specific DNA molecules 2-6, from upstream to downstream, are composed of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 9, and the segment shown in SEQ ID NO: 15. Specific DNA molecules 2-7, from upstream to downstream, are composed of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 10, and the segment shown in SEQ ID NO: 15. Specific DNA molecules 2-8, from upstream to downstream, are composed of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 16, and the segment shown in SEQ ID NO: 15. Specific DNA molecules 2-9, from upstream to downstream, are composed of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 17, and the segment shown in SEQ ID NO: 15. Specific DNA molecules 2-10, from upstream to downstream, are composed of the following segments in sequence: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 18, and the segment shown in SEQ ID NO: 15.

[0096] Ten editing plasmids were obtained. Following the principle of corresponding to the names of specific DNA molecules, the editing plasmids were named sequentially from Editing Plasmid 2-1 to Editing Plasmid 2-10.

[0097] IV. The impact of arRNA length on editing efficiency

[0098] The seven editing plasmids (i.e., editing plasmids 2-1 to 2-7) prepared in step three were processed separately.

[0099] Same as step four in Example 1.

[0100] See GFP fluorescence image Figure 3 A. It can be observed that, compared with Circ-arRNA151, Circ-arRNA51*3, Circ-arRNA61*3 and Circ-arRNA71*3 have significantly improved efficiency in illuminating eGFP.

[0101] The results for RNA editing efficiency are shown in [link to data]. Figure 3 B. Circ-arRNA151, Circ-arRNA21*3, Circ-arRNA31*3, Circ-arRNA41*3, Circ-arRNA51*3, Circ-arRNA61*3, and Circ-arRNA71*3 all possess RNA editing capabilities. Compared to Circ-arRNA151, Circ-arRNA51*3, Circ-arRNA61*3, and Circ-arRNA71*3 exhibit significantly improved RNA editing efficiency. Circ-3*arRNA61 showed the highest editing efficiency (average 48.03%), a 2.14-fold improvement over Circ-arRNA151, with the potential bystander region shrinking from 150 nt to 60 nt.

[0102] V. The effect of arRNA copy number on editing efficiency

[0103] The five editing plasmids prepared in step three (i.e., editing plasmid 2-1, editing plasmid 2-6, editing plasmid 2-8, editing plasmid 2-9, and editing plasmid 2-10) were processed separately.

[0104] The same as step four in Method Example 1.

[0105] The results for RNA editing efficiency are shown in [link to data]. Figure 3 C. Circ-arRNA151, Circ-arRNA61*2, Circ-arRNA61*3, Circ-arRNA61*4, and Circ-arRNA61*5 all possess RNA editing capabilities.

[0106] Example 3: Verifying the editing efficiency of Circ-3*arRNA61 on endogenous gene RNA.

[0107] The five endogenous genes are: GUSB gene (Gene ID: 2990 in NCBI), KRAS gene (Gene ID: 3845 in NCBI), PPIB gene (Gene ID: 5479 in NCBI), DAXX gene (Gene ID: 1616 in NCBI), and ALDOA gene (Gene ID: 226 in NCBI).

[0108] I. Preparation of Editing Plasmids

[0109] Editing plasmid: A specific DNA molecule is inserted into the BsmBI restriction site of the pcg 2.0-BFP-circ-arRNA backbone plasmid to obtain the editing plasmid.

[0110] The specific DNA molecules are double-stranded DNA molecules, namely: GUSB-Circ-arRNA151 (transcribed to obtain Circ-arRNA151 targeting the GUSB gene), GUSB-Circ-arRNA61*3 (transcribed to obtain Circ-arRNA61*3 targeting the GUSB gene), KRAS-Circ-arRNA151 (transcribed to obtain Circ-arRNA151 targeting the KRAS gene), KRAS-Circ-arRNA61*3 (transcribed to obtain Circ-arRNA61*3 targeting the KRAS gene), and PPIB-Circ-arRNA151 (transcribed to obtain Circ-arRNA61*3 targeting the PPIB gene). NA151), specific DNA molecule PPIB-Circ-arRNA61*3 (transcribed to obtain Circ-arRNA61*3 targeting the PPIB gene), specific DNA molecule DAXX-Circ-arRNA151 (transcribed to obtain Circ-arRNA151 targeting the DAXX gene), specific DNA molecule DAXX-Circ-arRNA61*3 (transcribed to obtain Circ-arRNA61*3 targeting the DAXX gene), specific DNA molecule ALDOA-Circ-arRNA151 (transcribed to obtain Circ-arRNA151 targeting the ALDOA gene), specific DNA molecule ALDOA-Circ-arRNA61*3 (transcribed to obtain Circ-arRNA61*3 targeting the ALDOA gene).

[0111] The specific DNA molecule GUSB-Circ-arRNA151 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 19, and the segment shown in SEQ ID NO: 15.

[0112] The specific DNA molecule GUSB-Circ-arRNA61*3 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 20, and the segment shown in SEQ ID NO: 15.

[0113] The specific DNA molecule KRAS-Circ-arRNA151 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 21, and the segment shown in SEQ ID NO: 15.

[0114] The specific DNA molecule KRAS-Circ-arRNA61*3 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 22, and the segment shown in SEQ ID NO: 15.

[0115] The specific DNA molecule PPIB-Circ-arRNA151 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 23, and the segment shown in SEQ ID NO: 15.

[0116] The specific DNA molecule PPIB-Circ-arRNA61*3 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 24, and the segment shown in SEQ ID NO: 15.

[0117] The specific DNA molecule DAXX-Circ-arRNA151 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 25, and the segment shown in SEQ ID NO: 15.

[0118] The specific DNA molecule DAXX-Circ-arRNA61*3 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 26, and the segment shown in SEQ ID NO: 15.

[0119] The specific DNA molecule ALDOA-Circ-arRNA151 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 27, and the segment shown in SEQ ID NO: 15.

[0120] The specific DNA molecule ALDOA-Circ-arRNA61*3 is composed of the following segments from upstream to downstream: the segment shown in SEQ ID NO: 14, the segment shown in SEQ ID NO: 28, and the segment shown in SEQ ID NO: 15.

[0121] A total of 10 editing plasmids were obtained.

[0122] II. Plasmid Transfection

[0123] The 10 editing plasmids prepared in step one were then processed separately.

[0124] 1. Take a 1.5 mL EP tube, add 4 μg of editing plasmid, then add 500 μL of serum-free DMEM medium, and incubate at room temperature for 5 min.

[0125] 2. Take a 1.5 mL EP tube, add 12 μg PEI, then add 500 μL serum-free DMEM medium, and incubate at room temperature for 5 min.

[0126] 3. Mix the liquid phase in the EP tube after completing step 1 with the liquid phase in the EP tube after completing step 2, and incubate at room temperature for 20 minutes to obtain the transfection solution (transfection solution for one well).

[0127] 4. Seed HEK-293T cells into 6-well plates and culture them in complete medium until the cell density reaches 40-50%. Then discard the supernatant, add the transfection solution prepared in step 3, and add 500 μL of serum-free DMEM medium to each well. Culture for 4-6 hours.

[0128] 5. After completing step 4, discard the supernatant, add 2 mL of complete culture medium to each well, and incubate for 24 h.

[0129] 6. After completing step 5, discard the supernatant, add 2 mL of complete culture medium containing 4 mg / mL PURO to each well, and incubate for 24 h.

[0130] 7. After completing step 6, discard the supernatant, resuspend the cells by pipetting with 10×PBS, then centrifuge at 4000g for 5min to collect the cells.

[0131] III. Testing Editing Efficiency

[0132] Collect the cells from step 2, step 7, extract total RNA, and reverse transcribe it to obtain cDNA. Use the cDNA as a template for PCR amplification, recover the specific bands, and sequence them. Calculate the RNA editing efficiency based on the sequencing results.

[0133] The primers used for PCR amplification of the GUSB gene are as follows:

[0134] Upstream primer: 5'-CCGCTATGGGATTGTGGTCA-3';

[0135] Downstream primer: 5'-CCCCTTGTCTGCTGCATAGT-3'.

[0136] The primers used for PCR amplification of the KRAS gene are as follows:

[0137] Upstream primer: 5'-AGGTGCGGGAGAGAGGC-3';

[0138] Downstream primer: 5'-CCCTCCCCAGTCCTCATGTA-3'.

[0139] The primers used for PCR amplification of the PPIB gene are as follows:

[0140] Upstream primer: 5'-GGAGATGGCACAGGAGGAAA-3';

[0141] Downstream primer: 5'-TTCTCCACCTCGATCTTGCC-3'.

[0142] The primers used for PCR amplification of the DAXX gene are as follows:

[0143] Upstream primer: 5'-TGATGGGAGAGCTGACCGGCCGTGTCATAGAGCAGC-3';

[0144] Downstream primer: 5'-GTGAGGTGGCAGCCAAAGTTGTAGATGA-3'.

[0145] The primers used for PCR amplification of the ALDOA gene are as follows:

[0146] Upstream primer: 5'-GACAGCTGACGACCGCGTGAACC-3';

[0147] Downstream primer: 5'-CCCCAATCTTCAGCACACAACGCCA-3'.

[0148] RNA editing efficiency = number of target sequences with the target mutation ÷ total number of target sequences × 100%.

[0149] Target mutation refers to a mutation in the target nucleotide from A to G.

[0150] RNA editing efficiency results are shown in Figure 4Compared to Circ-arRNA151, Circ-arRNA61*3 showed significantly improved RNA editing efficiency. The RNA editing efficiency was increased by 1.67-fold, 11.00-fold, 2.33-fold, 2.74-fold, and 1.27-fold, respectively.

[0151] Example 4: In vitro synthesis of linear arRNA and RNA editing

[0152] I. Lentiviral Packaging

[0153] Same as step one in Example 1.

[0154] II. Slow Viral Infection

[0155] Same as step two in Example 1

[0156] III. In vitro synthesis and preparation of linear arcRNA

[0157] Seven single-stranded RNA molecules were prepared by in vitro transcription synthesis (IVT), namely IVT-arRNA151 (the RNA corresponding to the DNA shown in SEQ ID NO: 4), IVT-arRNA21*3 (the RNA corresponding to the DNA shown in SEQ ID NO: 5), IVT-arRNA31*3 (the RNA corresponding to the DNA shown in SEQ ID NO: 6), IVT-arRNA41*3 (the RNA corresponding to the DNA shown in SEQ ID NO: 7), IVT-arRNA51*3 (the RNA corresponding to the DNA shown in SEQ ID NO: 8), IVT-arRNA61*3 (the RNA corresponding to the DNA shown in SEQ ID NO: 9), and IVT-arRNA71*3 (the RNA corresponding to the DNA shown in SEQ ID NO: 10).

[0158] IV. RNA Transfection

[0159] The seven RNA molecules prepared in step three were then processed separately.

[0160] 1. Seed HEK-293T cells into 6-well plates and culture them in complete culture medium until the cell density reaches 50-60%, then discard the supernatant.

[0161] 2. Take the 6-well plate from step 1 and use Lipofectamine... TM Use 3000 transfection reagents and follow the instructions to transfect the RNA molecules into the cells that have completed step 1, and culture for 24 hours.

[0162] 3. After completing step 2, discard the supernatant, add complete culture medium, and incubate for 48 hours.

[0163] 4. After completing step 3, discard the supernatant, resuspend the cells by pipetting with 10×PBS, then centrifuge at 4000g for 5min to collect the cells.

[0164] V. Testing Editing Efficiency

[0165] Step 8 of the same as in Example 1.

[0166] RNA editing efficiency results are shown in Figure 5 . Figure 5 In the diagram, the bars from left to right correspond to IVT-arRNA151, IVT-arRNA21*3, IVT-arRNA31*3, IVT-arRNA41*3, IVT-arRNA51*3, IVT-arRNA61*3, and IVT-arRNA71*3, respectively. All IVT-arRNA151, IVT-arRNA21*3, IVT-arRNA31*3, IVT-arRNA41*3, IVT-arRNA51*3, IVT-arRNA61*3, and IVT-arRNA71*3 possess RNA editing capabilities. Compared to IVT-arRNA151, the RNA editing efficiency of IVT-arRNA41*3, IVT-arRNA51*3, IVT-arRNA61*3, and IVT-arRNA71*3 is significantly improved.

[0167] Example 5: In vitro synthesis of Circ-arRNA and RNA editing

[0168] I. Lentiviral Packaging

[0169] Same as step one in Example 1.

[0170] II. Slow Viral Infection

[0171] Same as step two in Example 1

[0172] III. Preparation of Circ-arRNA

[0173] Seven single-stranded RNA molecules (named RNA molecule 1, RNA molecule 2, RNA molecule 3, RNA molecule 4, RNA molecule 5, RNA molecule 6, and RNA molecule 7) were prepared by in vitro transcription synthesis (IVT). RNA molecule 1, from upstream to downstream, consists of the following segments: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 4, and the segment shown in SEQ ID NO: 30. RNA molecule 2, from upstream to downstream, consists of the following segments: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 5, and the segment shown in SEQ ID NO: 30. RNA molecule 3, from upstream to downstream, consists of the following segments: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 6, and the segment shown in SEQ ID NO: 30. RNA molecule 4, from upstream to downstream, consists of the following segments: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 7, and the segment shown in SEQ ID NO: 30. RNA molecule 5, from upstream to downstream, consists of the following segments sequentially: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 8, and the segment shown in SEQ ID NO: 30. RNA molecule 6, from upstream to downstream, consists of the following segments sequentially: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 9, and the segment shown in SEQ ID NO: 30. RNA molecule 7, from upstream to downstream, consists of the following segments sequentially: the segment shown in SEQ ID NO: 29, the RNA segment corresponding to the DNA shown in SEQ ID NO: 10, and the segment shown in SEQ ID NO: 30.

[0174] Seven single-stranded RNA molecules spontaneously circularized based on ribozymes in their intron sequences, yielding seven corresponding circular RNA molecules. Following the principle of corresponding to RNA molecules 1 through 7, the seven circularized RNA molecules were named IVT-Circ-arRNA151, IVT-Circ-arRNA21*3, IVT-Circ-arRNA31*3, IVT-Circ-arRNA41*3, IVT-Circ-arRNA51*3, IVT-Circ-arRNA61*3, and IVT-Circ-arRNA71*3, respectively.

[0175] IV. RNA Transfection

[0176] Same as step four in Example 4.

[0177] V. Testing Editing Efficiency

[0178] Step 8 of the same as in Example 1.

[0179] RNA editing efficiency results are shown in Figure 6 . Figure 6 In the diagram, the bars from left to right correspond to IVT-Circ-arRNA151, IVT-Circ-arRNA21*3, IVT-Circ-arRNA31*3, IVT-Circ-arRNA41*3, IVT-Circ-arRNA51*3, IVT-Circ-arRNA61*3, and IVT-Circ-arRNA71*3, respectively. All four IVT-Circ-arRNAs—151, 21*3, 31*3, 41*3, 51*3, 61*3, and 71*3—possess RNA editing capabilities. Compared to IVT-Circ-arRNA151, the RNA editing efficiency of IVT-Circ-arRNA51*3, 61*3, and 71*3 is significantly improved.

[0180] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An RNA comprising n copies of a truncated arRNA; where n is a natural number greater than 2.

2. The RNA as described in claim 1, characterized in that: The length of the truncated arRNA is 15-150 nt.

3. The RNA as described in claim 1 or 2, characterized in that: n is a natural number between 2 and 10.

4. The RNA as described in claim 1, 2, or 3, characterized in that: The RNA is either linear RNA or circular RNA.

5. An RNA molecule having any one of the RNAs described in claims 1 to 4.

6. A DNA molecule expressing the RNA of any one of claims 1 to 4 or a DNA molecule expressing the RNA molecule of claim 5.

7. A recombinant vector or recombinant microorganism having the DNA molecule of claim 6.

8. The use of any of the RNAs described in claims 1 to 4 or the RNA molecule described in claim 5, as follows (a1) or (a2) or (a3): (a1) As an application of the LEAPER tool; (a2) Application as an RNA editing tool; (a3) Application in RNA editing.

9. The use of the RNA molecule according to any one of claims 1 to 4 or the RNA molecule according to claim 5, as follows (b1) or (b2): (b1) Application in the preparation of the LEAPER kit; (b2) Application in the preparation of RNA editing kits.

10. The application of the DNA molecule of claim 6, the recombinant vector of claim 7, or the recombinant microorganism of claim 7, is as follows (b1) or (b2) or (b3) or (b4) or (b5): (b1) As an application of the LEAPER tool; (b2) Application as an RNA editing tool; (b3) Applications in RNA editing; (b4) Application in the preparation of the LEAPER kit; (b5) Application in the preparation of RNA editing kits.

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