A base editing system targeting mitochondrial RNA and application thereof

Abstract

The application provides a base editing system targeting mitochondrial RNA and application. The base editing system comprises a fusion protein, wherein the fusion protein comprises a mitochondrial localization signal peptide, a PUF RNA binding domain and a RNA deaminase domain; and the amino acid sequence of the mitochondrial localization signal peptide is shown as SEQ ID NO:1. The application uses the PUF RNA binding domain to replace the Cas13-gRNA system, directly recruits the deaminase to the vicinity of the target RNA of the mitochondria, and realizes the targeted editing through the direct recognition of the protein-RNA, so as to overcome the technical bottleneck that the gRNA cannot enter the mitochondria.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a base editing system for targeting mitochondrial RNA and its applications. Background Technology

[0002] Mitochondria are semi-autonomous organelles in eukaryotic cells, possessing their own circular genome. The mitochondrial genome encodes multiple core proteins in the respiratory chain complex, and mutations in these proteins are closely associated with various human genetic diseases and plant cytoplasmic male sterility. Mitochondrial gene expression is regulated at multiple levels, with post-transcriptional modifications and base editing at the RNA level playing crucial roles in gene function regulation.

[0003] In recent years, RNA base editing technology has developed rapidly. The REPAIR system, based on ADAR2 deaminase, and the RESCUE system, based on TadA deaminase, can achieve A-to-I (equivalent to A-to-G) and C-to-U (equivalent to C-to-T) base conversions at the RNA level. These systems typically utilize the Cas13 protein to recognize target RNA under the guidance of guide RNA (gRNA) and recruit deaminases for site-specific editing. Meanwhile, RNA editing technology based on programmable RNA-binding proteins has also made significant progress. The REWIRE system fuses the RNA-binding domain of PUF (Pumilio / FBF family protein) with human ADAR deaminase or APOBEC3A deaminase to construct a single-protein RNA A-to-I and C-to-U base editor that does not require guide RNA. Subsequent research further optimized the CU-REWIRE system. It should be noted that the aforementioned REWIRE and its derivative systems mainly target mRNA in the cytoplasm or nucleus of animal cells; there are no experimental reports on combining them with organelle-localizing peptides for mitochondrial or chloroplast RNA editing.

[0004] Furthermore, significant progress has been made in organelle RNA editing technology based on PPR (pentatricopeptide repeat) proteins. PPR proteins are a family of naturally occurring RNA-binding proteins in plants, and their RNA recognition specificity is determined by the 5th and 35th amino acids in each repeat unit (i.e., the "PPR code"). Researchers have engineered PPR proteins and fused them with the DYW domain, which has cytosine deaminase activity, to construct a variety of guide RNA-independent organelle RNA C-to-U editing tools.

[0005] However, existing organelle RNA editing tools still have the following shortcomings: First, Cas13-type systems rely on exogenous guide RNA, which is difficult to enter mitochondria, making the system unsuitable for direct mitochondrial RNA editing; Second, while PPR-DYW-type systems have solved the guide RNA delivery problem and achieved C-to-U editing in mitochondria and chloroplasts, their editing type is limited to C-to-U and cannot achieve A-to-I editing, which is also of great value in mitochondrial RNA research; Third, although REWIRE and its derivative systems have achieved A-to-I and C-to-U editing using PUF, their application is limited to mRNA in the cytoplasm and nucleus, and there are no specific implementation plans or experimental verifications to extend them to organelle RNA editing; Fourth, mitochondrial DNA editing tools such as DdCBE and TALED act on DNA rather than RNA and cannot be used to study post-transcriptional regulation at the RNA level.

[0006] Therefore, there is an urgent need for an RNA base editing system that does not rely on guide RNA, can achieve both A-to-I and C-to-U editing, and can function in mitochondria. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a base editing system for targeting mitochondrial RNA and its applications. This invention utilizes the PUF (Pumilio / FBF family protein) RNA-binding domain to replace the Cas13-gRNA system, directly recruiting deaminases to the vicinity of the target mitochondrial RNA. Targeted editing is achieved through direct protein-RNA recognition, thus overcoming the technical bottleneck of gRNA's inability to enter mitochondria.

[0008] This invention provides a base editing system targeting mitochondrial RNA, comprising a fusion protein, wherein the fusion protein includes a mitochondrial localization signal peptide (MTS), a PUF RNA-binding domain, and an RNA deaminase domain; The amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO:1.

[0009] Mitochondrial localization signal peptides do not merely play a single role in introducing fusion proteins into mitochondria; rather, they directly influence the localization efficiency of the fusion protein within the cell, its enrichment level in mitochondria, and the function of the PUF RNA-binding domain and RNA deaminase domain. This application uses the mitochondrial localization signal peptide shown in SEQ ID NO:1. After constructing a fusion protein by combining it with the PUF RNA-binding domain and RNA deaminase domain, it can effectively mediate the entry of the fusion protein into the mitochondria. Furthermore, without disrupting the specific recognition of target RNA by PUF and the catalytic activity of RNA deaminase, it enables PUF and RNA deaminase to co-localize near the target RNA in the mitochondria, thereby achieving site-specific deamination editing of the target RNA.

[0010] In this invention, the PUF RNA-binding domain is used to specifically recognize the target sequence of mitochondrial target RNA, and the RNA deaminase domain is used to deaminate and edit the target bases in the mitochondrial target RNA. The mitochondrial localization signal peptide of this application has a functional matching relationship with both the PUF RNA-binding domain and the RNA deaminase domain.

[0011] In this invention, the RNA recognition specificity of the PUF domain is determined by amino acids at specific positions within each repeat unit, particularly amino acids at positions 12 and 16. By altering the amino acid combinations at these positions, the recognition specificity of the PUF domain for RNA sequences can be reprogrammed to recognize arbitrarily defined RNA target sequences.

[0012] In some embodiments, the PUF RNA-binding domain includes two or more PUF repeat units, each containing 36 to 43 amino acids.

[0013] Preferably, the PUF RNA-binding domain comprises eight or more PUF repeat units. PUF variants obtained by adjusting the amino acid composition of the repeat units according to different target sequences and PUF recognition rules are all within the scope of protection of this invention.

[0014] In some embodiments, the amino acid sequence of the PUF RNA-binding domain is shown in SEQ ID NO:2.

[0015] In some embodiments, the RNA deaminase domain is adenosine deaminase or cytidine deaminase.

[0016] In some embodiments, the RNA deaminase domain is TadA8e adenosine deaminase or a variant thereof that retains RNA adenosine deaminase activity.

[0017] In some embodiments, the amino acid sequence of the RNA deaminase domain is as shown in SEQ ID NO:3 or SEQ ID NO:4.

[0018] In some embodiments, the mitochondrial localization signal peptide, the PUF RNA-binding domain, and the RNA deaminase domain are linked by a linker peptide. The linker peptide is a flexible linker peptide, a rigid linker peptide, or a combination of a flexible linker peptide and a rigid linker peptide, and the linker peptide comprises 2 to 50 amino acids.

[0019] Preferably, the linker peptide is 2 to 7 amino acids in length; more preferably, the sequence of the linker peptide is VDTGNGS.

[0020] In some embodiments, the fusion protein is arranged as follows: Mitochondrial localization signal peptide-PUF RNA-binding domain-RNA deaminase domain; or, Mitochondrial localization signal peptide-RNA deaminase domain-PUF RNA binding domain.

[0021] In this invention, the mitochondrial localization signal peptide is preferably located at the N-terminus to facilitate mitochondrial introduction.

[0022] This invention also provides a method for site-specific base editing of mitochondrial RNA, comprising the following steps: (1) Identify the mitochondrial target RNA and the base to be edited, and determine that the base to be edited is adenosine or cytidine; (2) Design a PUF RNA-binding domain based on the target sequence in the mitochondrial target RNA; (3) When the base to be edited is adenosine, adenosine deaminase is selected as the RNA deaminase domain; Alternatively, when the base to be edited is cytidine, cytidine deaminase is selected as the RNA deaminase domain; (4) The mitochondrial localization signal peptide, the PUF RNA binding domain and the RNA deaminase domain are linked together to form a fusion protein; (5) Introduce the nucleic acid molecule encoding the fusion protein into a host cell or organism and edit the target bases; The amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO:1.

[0023] In some embodiments, the amino acid sequence of the adenosine deaminase is shown in SEQ ID NO:3; The amino acid sequence of the cytidine deaminase is shown in SEQ ID NO:4.

[0024] The present invention also provides a nucleic acid molecule encoding the fusion protein described above.

[0025] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule, wherein the vector is a plant expression vector, a mammalian expression vector, or an insect cell expression vector.

[0026] The present invention also provides a host cell containing the expression vector described above, wherein the host cell is a plant cell, a mammalian cell, or an insect cell.

[0027] The present invention also provides the application of the base editing system described herein in mitochondrial RNA editing, mitochondrial gene function research, regulation of plant cytoplasmic male sterility, or crop trait improvement.

[0028] The present invention also provides a kit comprising the fusion protein or the nucleic acid molecule described herein.

[0029] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention uses the PUF RNA-binding domain as a universal targeting module. By modularly replacing the RNA deaminase, both A-to-I and C-to-U RNA base editing can be achieved under the same PUF targeting architecture. Specifically, using TadA8e as the RNA deaminase enables A-to-I editing, while using TadA8e-Cmax as the RNA deaminase enables C-to-U editing. Thus, without changing the PUF targeting module, different types of RNA base editing can be achieved simply by replacing the effector module. Compared to organelle RNA editing systems that can only achieve specific types of base editing, this invention has a stronger functional expansion capability.

[0030] (2) This invention employs a modular fusion system composed of a specific mitochondrial localization signal peptide, a PUF RNA-binding domain, a linker peptide, and an RNA deaminase domain. Each functional module undertakes mitochondrial localization, RNA targeting, and base editing functions, respectively. Different modules can be selected or replaced according to the target RNA and the desired editing type, thereby constructing different mitochondrial RNA base editing systems. Furthermore, compared to highly repetitive RNA-binding protein modules, the PUF domain has a more defined sequence design rule, and its repeating units can be customized according to the target RNA sequence, which is beneficial for constructing different target sequence editing systems. Simultaneously, the repetitiveness of the PUF repeating unit sequence is relatively low, which is beneficial for gene sequence design and synthesis.

[0031] (3) This invention utilizes the PUF RNA-binding domain to directly recognize target RNA without the need for additional gRNA introduction or co-expression, thus avoiding the delivery limitations faced by guide RNAs entering mitochondria. Simultaneously, the PUF RNA-binding domain has well-defined RNA recognition rules, allowing for the selection and combination of corresponding PUF repeat units based on the target RNA sequence, thereby achieving recognition capabilities for different mitochondrial RNA target sequences. Therefore, this invention integrates RNA targeting and base editing into the same fusion protein, exhibiting strong targeting programmability.

[0032] (4) This invention operates at the RNA level, achieving site-specific alterations of target transcripts by deamination editing of specific bases in mitochondrial RNA, without directly changing the mitochondrial DNA sequence. Therefore, this invention can be used for site-specific editing of plant mitochondrial RNA, mitochondrial gene function research, regulation of plant cytoplasmic male sterility, and crop trait improvement, providing new technical means for post-transcriptional regulation of mitochondria and related plant trait research. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The PUF in Arabidopsis thaliana in Embodiment 1 of the present invention ATP1 Gene targets.

[0035] Figure 2 This is a schematic diagram of the expression boxes for MTS-PUF-TadA8e and MTS-PUF-TadA8e-Cmax of the present invention.

[0036] Figure 3 This is an example of the editing of MTS-ATP1PUF-TadA8e in Arabidopsis protoplasts in Example 4 of the present invention.

[0037] Figure 4 This describes the editing of MTS-ATP1PUF-TadA8e in transgenic Arabidopsis plants in Example 4 of this invention.

[0038] Figure 5 This is an example of the editing of MTS-ATP1PUF-TadA8e-Cmax in Arabidopsis protoplasts in Example 4 of the present invention. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0040] This invention provides a base editing system targeting mitochondrial RNA. This system achieves site-specific base editing of mitochondrial RNA using a single fusion protein, without the need for guide RNA co-expression. The fusion protein consists of three core functional modules: a mitochondrial localization signal peptide (MTS), a PUF RNA-binding domain, and an RNA deaminase domain. These three modules work synergistically: the MTS is responsible for transporting the fusion protein from the cytoplasm to the mitochondria; the PUF domain is responsible for recognizing specific sequences in the target mitochondrial RNA; and the deaminase is responsible for deaminating and editing target bases near the PUF recognition site.

[0041] This invention's system achieves two editing modes by replacing the deaminase module: when the deaminase module is TadA8e, the system catalyzes the deamination of adenosine (A) into inosine (I), achieving A-to-I editing (equivalent to A-to-G conversion at the DNA level); this invention combines it with PUF and MTS for A-to-I editing of mitochondrial RNA. The TadA8e can be wild-type TadA8e, its truncated form, its point mutant, or an engineered variant that retains RNA adenosine deamination activity.

[0042] When the deaminase module is TadA8e-Cmax, the system catalyzes the deamination of cytidine (C) into uridine (U), achieving C-to-U editing (equivalent to C-to-T conversion at the DNA level); the TadA8e-Cmax can be wild-type TadA8e-Cmax, its truncated form, its point mutant, or an engineered variant that retains the RNA cytidine deamination activity.

[0043] This invention is the first to combine a PUF RNA-binding domain with a deaminase domain and a mitochondrial localization signal peptide to form a fusion protein, constructing a novel system for site-specific RNA base editing in mitochondria without the need for guide RNA. This combination design and its application in organelle RNA editing are both innovative and practical.

[0044] The editing mechanism of this invention is as follows: After the fusion protein is expressed in the cytoplasm, MTS guides it into the mitochondrial matrix; after the MTS is cleaved, the mature fusion protein recognizes the target sequence in the mitochondrial target RNA through the PUF domain; after PUF binding, the deaminase is localized to the vicinity of the target base and catalyzes its deamination. When TadA8e is used, the target adenosine is converted to inosine (A-to-I); when TadA8e-Cmax is used, the target cytidine is converted to uridine (C-to-U). The editing window is located within a certain range of bases near the PUF recognition sequence.

[0045] Unless otherwise specified, all experimental materials used in the following examples are commercially available products; and all methods used are conventional molecular biology or biochemistry experimental methods in the field. Primers used in the examples were synthesized by Anshengda Biotechnology Co., Ltd., and gene sequencing was performed by Kangwei Century Biotechnology Co., Ltd. Phanta high-fidelity DNA polymerase was purchased from Nanjing Novizan Biotechnology Co., Ltd.; RNA extraction kits and reverse transcription kits were purchased from Takara Bio Engineering (Dalian) Co., Ltd. (TaKaRa). All procedures involving the use of reagent kits were strictly performed according to the product instructions provided by the respective manufacturers. The specific correspondence between each repeat unit (module) of the PUF protein and the target bases was determined according to Table 1; the arrangement of each repeat unit in the PUF domain can be freely combined, and the full-length PUF-encoding nucleic acid sequence determined after combination was artificially synthesized by Anshengda Biotechnology Co., Ltd.

[0046] Example 1: Construction of A-to-I Editing and Recombination Vector This embodiment provides an exemplary method for constructing a recombinant vector for A-to-I editing of mitochondrial target genes.

[0047] (1) Determination of target sequence: Obtain the target mitochondrial gene (Arabidopsis thaliana mitochondrial gene) ATP1 The RNA transcript sequence information of the gene. Based on this transcript sequence, the target recognition sequence that the PUF protein recognizes and binds to is screened and determined (its location is shown in the diagram). Figure 1 (As shown).

[0048] (2) Design of PUF structural domains: Based on the base type of each site in the determined target sequence, and referring to the PUF-base correspondence table (Table 1), the amino acid combinations at positions 12 and 16 of each repeat unit in the PUF protein are determined. Repeat units of equal length to the target sequence are tandemly linked to obtain the amino acid sequence of the PUF domain that specifically recognizes the target sequence; the order of each repeat unit can be arbitrarily combined and adjusted according to the sequence of bases corresponding to the 5' to 3' directions of the target sequence. The specific amino acid sequence of the PUF domain described in this embodiment is shown in SEQ ID NO: 2.

[0049] Table 1

[0050] (3) Construction of recombinant expression vectors: The amino acid sequences encoding the mitochondrial localization signal peptide (MTS), the PUF domain described in step (2), the flexible linker peptide (Linker), and TadA8e are provided respectively. The above amino acid sequences are codon optimized according to the codon preference of the host cell (such as Arabidopsis thaliana), and the optimized fragments are fused and spliced ​​in the order of MTS-PUF-Linker-TadA8e to obtain the fusion coding sequence, which is then artificially synthesized into the fusion gene fragment.

[0051] Using the artificially synthesized fragment as a template, PCR amplification was performed using Phanta DNA polymerase. The reaction system was prepared according to the product instructions (50 μL system). The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 5 min. The amplified product was purified by agarose gel electrophoresis to obtain the MTS-PUF-TadA8e fusion expression cassette fragment.

[0052] Simultaneously, the plant expression vector pCambia backbone plasmid was double-digested with restriction endonucleases (such as Kpn I and Sac I), and the linearized vector fragment was recovered. The obtained fusion expression cassette fragment was seamlessly ligated to the linearized pCambia vector backbone using a homologous recombinase (such as ClonExpress II) at 37°C for 30 min. The ligation product was transformed into *E. coli* DH5α competent cells, and positive clone plasmids were extracted after kanamycin resistance screening and colony PCR identification to obtain the recombinant vector pCambia-pAtUBQ10-MTS-PUF-TadA8e-tNOS. The amino acid sequences of each functional module of the fusion protein in this vector are shown in SEQ ID NO: 1 (MTS), SEQ ID NO: 2 (PUF), and SEQ ID NO: 3 (TadA8e), respectively. A schematic diagram of its expression cassette structure is shown below. Figure 2 As shown, the specific sequence is as follows: SEQ ID NO: 1 MFKQASRLLSRSVAAASSKSVTTRAFSTELPSTLDS SEQ ID NO: 2 GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGNRFIQLKLERATPAERQLVFNEILQHTEQLVQDQYGSYVIEHVLEHGRPEDKSKIVAEIRG AAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGCRVIQKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSHVVEKCIECVQP QSLQFIIDAFKGQVFALSTHPYGSRVIERILEHCLPDQTLPILEELHQHTEQLVQDQYGNYVIQHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFAS NVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG SEQ ID NO: 3 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN SEQ ID NO: 4 SEVEFSHEYWMRHALTLAKRARDEGEAPVGAVLVLNNRVIGEGWPRGIGLHDPTAHAEIMALRQGGLVMQNPRLYDATLYTTFEPCVMCAGAMINSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCRFYRMPRRVFNAQKKAQSSIN.

[0053] Example 2 Construction of C-to-U Editing and Recombination Vector This embodiment provides a method for constructing a recombinant vector for C-to-U editing of mitochondrial target genes.

[0054] Using the pCambia-pAtUBQ10-MTS-PUF-TadA8e-tNOS recombinant vector constructed in Example 1 as a template, conventional reverse PCR site-directed mutagenesis technology was used to mutate specific bases in the nucleic acid sequence encoding the TadA8e domain to obtain the mutant TadA8e-Cmax, which has deaminase activity converted to C-to-U editing activity.

[0055] Specifically, a pair of complementary primers containing the target mutation site were designed, and whole-plasmid PCR amplification was performed using Phanta high-fidelity DNA polymerase. The PCR product was digested with Dpn I restriction endonuclease at 37°C for 1 h to remove methylated template plasmid, followed by transformation into *E. coli* DH5α. Positive clones were screened and sequenced for verification. The verified mutant vector was named pCambia-pMTS-PUF-TadA8e-Cmax-tNOS, where the amino acid sequence of the TadA8e-Cmax mutant is shown in SEQ ID NO: 4, and the remaining MTS and PUF sequences are consistent with those in Example 1. Its expression cassette structure is shown in the diagram below. Figure 2 As shown.

[0056] Example 3: Obtaining transgenic positive Arabidopsis plants This embodiment provides a method for obtaining stably genetically inherited Arabidopsis transgenic plants using the above-described recombinant vector.

[0057] The recombinant vectors (pCambia-pAtUBQ10-MTS-PUF-TadA8e-tNOS or pCambia-pAtUBQ10-MTS-PUF-TadA8e-Cmax-tNOS) successfully constructed in Example 1 or Example 2 were transformed into Agrobacterium using the freeze-thaw method. Agrobacterium tumefaciens GV3101 competent cells. Positive Agrobacterium clones were selected and inoculated into LB liquid medium containing the corresponding antibiotics (kanamycin and rifampin). The cells were cultured at 28°C with shaking until the logarithmic growth phase. After centrifugation, the cells were collected and resuspended in infection buffer containing 5% sucrose and 0.02% Silwet L-77. The OD600 of the bacterial culture was adjusted to approximately 0.8.

[0058] Wild-type Arabidopsis thaliana (Col-0 ecotype) was transformed using the inflorescence dip method: Arabidopsis plants were inverted so that the above-ground inflorescences were completely immersed in the Agrobacterium-mediated transformation solution. The plants were gently shaken for 1 minute, then removed and cultured in the dark and humidified environment for 24 hours. After normal culture and seed harvest, the harvested T1 generation seeds were surface-sterilized with 75% ethanol and sodium hypochlorite solution, and then sown on MS selection medium containing hygromycin (final concentration 50 mg / L). The seeds were cultured at 22℃ under long-day conditions (16 h light / 8 h dark). Once the resistant seedlings developed true leaves, they were transplanted into nutrient soil for further culture to obtain T1 generation transgenic positive plants. Genomic DNA was extracted from the transgenic plants, and PCR amplification was used to verify the integration of the exogenous gene.

[0059] Example 4: A-to-I editing efficiency detection based on pMTS-PUF-TadA8e vector This embodiment tests the A-to-I editing efficiency of the editing tool constructed in Example 1 in Arabidopsis protoplasts and transgenic plants.

[0060] (1) Detection of transient protoplast transformation: Arabidopsis mesophyll protoplasts were prepared using PEG-Ca 2+ The mediated transformation method involved transforming the pCambia-pAtUBQ10-MTS-PUF-TadA8e-tNOS recombinant vector obtained in Example 1 into protoplasts. The transformed protoplasts were placed in protoplast culture medium and cultured in the dark at 25°C for 24–48 h. (See the edit results for further information.) Figure 3 .

[0061] (2) Detection of stable transgenic plants: T1 generation positive transgenic Arabidopsis plants obtained in Example 3 were cultured under normal growth conditions for 30 days. Rosette leaf tissue was then sampled. (See the edited results for details.) Figure 4 .

[0062] (3) RNA extraction, reverse transcription and target amplification: Protoplasts or rosette leaf samples from 30-day-old seedlings were collected after the above culture, and total RNA was extracted using an RNA extraction kit. The extracted RNA was treated with DNase I (RNase-free) at 37°C for 30 min to remove genomic DNA contamination, and then the purified RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit (reverse transcription reaction conditions: incubation at 42°C for 15 min, inactivation at 85°C for 5 sec).

[0063] Using the obtained cDNA as a template, PCR amplification was performed using specific detection primer pairs (forward primer: agaatgagtcccgcctcgaag; reverse primer: ccatcaataggtactcccatcgc). The reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min. The amplified product was purified and then sequenced and analyzed using nanopores to obtain the RNA mutation rate.

[0064] In the protoplast transfection results of pCambia-pAtUBQ10-MTS-PUF-TadA8e-tNOS, ATP1 Upstream of the gene target site, some A bases were detected to have been edited to I, with a maximum editing rate of approximately 15%. Figure 3 In transgenic plants of pCambia-pAtUBQ10-MTS-PUF-TadA8e-tNOS, ATP1 Upstream of the gene target site, some A bases were detected to have been edited to I, with a maximum editing rate of approximately 35%. Figure 4 In the protoplast transfection results of pCambia-pAtUBQ10-MTS-PUF-TadA8e-Cmax-tNOS, ATP1 Upstream of the gene target site, some C bases were detected to have been edited to U, with a maximum editing rate of approximately 11%. Figure 5 This indicates that the tool can function in transient expression systems or transgenic plants in Arabidopsis thaliana.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A base editing system targeting mitochondrial RNA, characterized in that, The fusion protein includes a mitochondrial localization signal peptide, a PUF RNA-binding domain, and an RNA deaminase domain. The amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO:

1.

2. The base editing system according to claim 1, characterized in that, The PUF RNA-binding domain includes two or more PUF repeat units, each containing 36 to 43 amino acids.

3. The base editing system according to claim 1, characterized in that, The amino acid sequence of the PUF RNA-binding domain is shown in SEQ ID NO:

2.

4. The base editing system according to claim 1, characterized in that, The RNA deaminase domain is either adenosine deaminase or cytidine deaminase.

5. The base editing system according to claim 1, characterized in that, The RNA deaminase domain is TadA8e adenosine deaminase or a variant thereof that retains RNA adenosine deaminase activity.

6. The base editing system according to claim 1, characterized in that, The amino acid sequence of the RNA deaminase domain is shown in SEQ ID NO:3 or SEQ ID NO:

4.

7. The base editing system according to claim 1, characterized in that, The mitochondrial localization signal peptide, PUF RNA binding domain and RNA deaminase domain are connected by a linker peptide. The linker peptide is a flexible linker peptide, a rigid linker peptide, or a combination of a flexible linker peptide and a rigid linker peptide, and the linker peptide comprises 2 to 50 amino acids.

8. The base editing system according to claim 1, characterized in that, The structural arrangement of the fusion protein is as follows: Mitochondrial localization signal peptide-PUF RNA-binding domain-RNA deaminase domain; or, Mitochondrial localization signal peptide-RNA deaminase domain-PUF RNA binding domain.

9. A method for site-specific base editing of mitochondrial RNA, characterized in that, Includes the following steps: (1) Identify the mitochondrial target RNA and the base to be edited, and determine that the base to be edited is adenosine or cytidine; (2) Design a PUF RNA-binding domain based on the target sequence in the mitochondrial target RNA; (3) When the base to be edited is adenosine, adenosine deaminase is selected as the RNA deaminase domain; Alternatively, when the base to be edited is cytidine, cytidine deaminase is selected as the RNA deaminase domain; (4) The mitochondrial localization signal peptide, the PUF RNA binding domain and the RNA deaminase domain are linked together to form a fusion protein; (5) Introduce the nucleic acid molecule encoding the fusion protein into a host cell or organism and edit the target bases; The amino acid sequence of the mitochondrial localization signal peptide is shown in SEQ ID NO:

1.

10. The application of the base editing system according to any one of claims 1 to 8 in mitochondrial RNA editing, mitochondrial gene function research, regulation of plant cytoplasmic male sterility, or crop trait improvement.

Images