Photosensitive RNA adenosine base editor and use thereof

Abstract

The present invention relates to a photosensitive RNA adenosine base editor and the use thereof. The photosensitive RNA adenosine base editor can cause the deamination of adenosine (A) in a target double-stranded RNA into inosine (I), thereby achieving the editing of A into G. The photosensitive RNA adenosine base editor comprises: (1) a dCasl3-ADAR2ddC-nMag fusion protein; and (2) a pMag-ADAR2ddN fusion protein.

Description

A photosensitive RNA adenosine base editor and its applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a photosensitive RNA adenosine base editor and its applications. Background Technology

[0002] Base editing is an emerging gene modification technology that can precisely convert specific bases directly into DNA or RNA without causing breaks in the nucleic acid strand. 1 Compared to DNA base editing, RNA base editing does not alter the genome sequence and has excellent reversibility. This characteristic makes RNA base editing tools a safer and more flexible treatment method for gene therapy, and to some extent addresses the ethical issues caused by genome modification. Currently, existing RNA base editors can achieve base conversions of A (adenosine) > I (inosine) and C (cytidine) > U (uridine) on RNA. 2 Among them, the A>I RNA base editor has highly efficient RNA-targeted editing capabilities and has broad application prospects in disease treatment, protein and RNA function research, and other fields.

[0003] RNA base conversion from adenosine to inosine is primarily achieved through naturally occurring or engineered adenosine deaminases (ADARs). The working principle is that ADAR catalyzes the deamination of adenosine (A) in double-stranded RNA to inosine (I). Because inosine is structurally similar to guanosine (G), it can pair with cytidine (C) during protein translation, thus achieving A>G editing. Based on the source of the ADAR in the editor, these RNA base editing tools can be divided into two main categories: the first category recruits endogenous ADAR to the vicinity of the target RNA by delivering antisense RNA complementary to the target RNA into the cell, achieving A>I editing, such as the LEAPER system. 3 The second type involves fusing exogenous ADAR or its catalytic domain ADARdd (deaminase domain of adenosine deaminase acting on RNA) with a specific functional protein. This allows the exogenous ADAR to be recruited to the target site via the interaction between the functional protein and a specific RNA sequence, achieving A>I base conversion. For example, the REPAIR system fuses catalytically inactivated dCas13b with ADAR2dd protein for A-to-I editing. 4 These RNA base editors can achieve efficient editing in cells and animals, but persistent expression of ADAR limits their application. Overexpression of ADAR has potential oncogenicity. 5And it will cause relatively serious off-target editing. 4 Furthermore, these RNA base editors lack spatiotemporal specificity, making them difficult to use for regulating biological processes based on temporal and spatial gene expression patterns, especially in animals.

[0004] Currently, methods for achieving time- and space-specific RNA base editing mainly utilize small chemical molecule regulation and photoregulation. Small chemical molecule regulation strategies achieve time-specific A-to-I base editing within cells by adjusting the timing of inducing drug addition, such as the abscisic acid-induced CIRTS system. 6 and gibberellin-induced SNAP-ADAR system 7 However, the high diffusion and long metabolic cycle of chemical drugs make it difficult for this method to precisely target single cells or localized regions of specific tissues. Therefore, these chemically induced RNA base editors lack high spatial specificity and cannot achieve more precise and reversible gene manipulation in localized regions of specific tissues or single cells. Recently, the Won Do Heo team in South Korea constructed a blue light-activated A-to-I RNA base editor, or padCas13 editor, by fusing the Magnets system-driven recombinant segmented dCas13b protein with the full-length ADAR2dd. 8 The padCas13 editor can restore the activity of exogenous luciferase in mouse liver, achieving spatiotemporally specific base editing in vivo. However, this system still has many limitations. Therefore, there is an urgent need to develop an RNA base editing tool that is more precise, efficient, small in size, and highly spatiotemporally specific.

[0005] Based on this, the present invention is proposed.

[0006] [References]

[0007] Porto, EM, Komor, AC, Slaymaker, IM & Yeo, GWBase editing: advances and therapeutic opportunities. Nat. Rev. Drug Discov. 19, 839–859 (2020).

[0008] 2.Pfeiffer, LS&Stafforst, T. Precision RNA base editing with engineered and endogenous effectors. Nat. Biotechnol. 41, 1526–1542 (2023).

[0009] 3. Qu, L., et al. Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat. Biotechnol. 37, 1059–1069 (2019).

[0010] 4. Cox, D. B. T., et al. RNA editing with CRISPR-Cas13. Science(80-.). 358, 1019–1027 (2017).

[0011] 5. Anadón, C., et al. Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis. Oncogene 35, 4407–4413 (2016).

[0012] 6. Rauch, S., Jones, K. A. & Dickinson, B. C. Small Molecule-Inducible RNA-Targeting Systems for Temporal Control of RNA Regulation. ACS Cent. Sci. 6, 1987–1996 (2020).

[0013] 7. Stroppel, A. S., Lappalainen, R. & Stafforst, T. Controlling Site-Directed RNA Editing by Chemically Induced Dimerization. Chem.-A Eur. J. 27, 12300–12304 (2021).

[0014] 8. Yu, J., et al. Programmable RNA base editing with photoactivatable CRISPR-Cas13. Nat. Commun. 15, 1–14 (2024).

[0015] Summary of the Invention

[0016] This invention first relates to a photoactivatable RNA base editor (PA-rABE), which can cause adenosine (A) in the target double-stranded RNA to deaminate and become inosine (I), thereby achieving the editing of A into G;

[0017] The light-sensitive RNA adenosine base editor includes:

[0018] (1)dCas13-ADAR2dd C -nMag fusion protein, a C-terminal truncated fragment of the ADAR2dd catalytic domain of dCas13 protein and adenosine deaminase acting on RNA (ADAR). C The Magnets system, composed of nMagHigh monomers induced by blue light dimerization, is composed of nMagHigh monomers.

[0019] (2)pMag-ADAR2dd N The fusion protein consists of a pMag monomer of the Magnets system, dimerized by blue light, and an N-terminal truncated fragment of the adenosine deaminase acting on RNA (ADAR) catalytic domain ADAR2dd. N )constitute;

[0020] The nMagHigh and pMag can be induced by blue light to dimerize into the Magnets system (see PMID:25708714), thereby driving the C-terminus and N-terminus of the segmented ADAR2dd protein to recombine into an active adenine deaminase, which catalyzes the deamination of adenosine (A) of the target RNA into inosine (I).

[0021] The dCas13 protein is used to bind target RNA. The dCas13 protein includes, but is not limited to, mini dCas13X, dCas13b, dCas13X or dCas13d proteins, preferably the mini dCas13X protein with a smaller molecular weight.

[0022] Preferably, the sequence of ADAR2dd is as shown in SEQ ID NO.1; the ADAR2dd C The sequence is shown in SEQ ID NO.2; the ADAR2dd N The sequence is shown in SEQ ID NO.3;

[0023] The sequence of pMag is shown in SEQ ID NO.4; the sequence of nMagHigh is shown in SEQ ID NO.5;

[0024] The sequence of the mini dCas13X is shown in SEQ ID NO.6.

[0025] Furthermore, the various functional blocks of the fusion protein are linked together by a linker; the linker is:

[0026] L1, the sequence is shown in SEQ ID NO.7;

[0027] L2, the sequence is shown in SEQ ID NO.8;

[0028] L3, the sequence is shown in SEQ ID NO.9;

[0029] L4, the sequence is shown in SEQ ID NO.10;

[0030] L5, the sequence is shown in SEQ ID NO.11;

[0031] L6, the sequence is shown in SEQ ID NO.12;

[0032] L7, the sequence is shown in SEQ ID NO.13;

[0033] L8, the sequence is shown in SEQ ID NO.14;

[0034] L9, the sequence is shown in SEQ ID NO.15;

[0035] Preferably, the Linker is L2;

[0036] Furthermore, the blue light induction condition is: 0.25 mW / cm². 2 Turn on in 1 minute and turn off in 3 minutes. Blue light wavelength is 470nm.

[0037] Preferably, the length of the target double-stranded RNA is 10-100 nt, more preferably 50-60 nt, and most preferably 50 nt;

[0038] Preferably, the editing site is located at position 25 of the target double-stranded RNA.

[0039] This invention also relates to the following applications of the aforementioned photosensitive RNA adenosine base editor:

[0040] (1) Targeted editing of eukaryotic or prokaryotic cells;

[0041] (2) To prepare drugs for treating diseases caused by gene mutations;

[0042] (3) Used for crop genetic breeding;

[0043] (4) Used to prepare animal models.

[0044] The beneficial effects of this invention are as follows:

[0045] (1) A photoactivated A-to-I RNA base editor (PA-rABE) was constructed using a photo-induced segmented ADAR2dd protein recombination strategy. This system fuses the inactive segmented ADAR2dd with a blue light-induced dimerizing Magnets system, using a small mini dCas13X protein as the RNA binding platform. Only after blue light stimulation can the dimerization of pMag and nMagHigh restore the deaminase activity of the recombined segmented ADAR2dd fragments, thus achieving A-to-I editing at the RNA level.

[0046] (2) To obtain an RNA base editor with high editing efficiency and no leakage, the Cas protein, ADAR2dd segmentation sites, relative positions of various gene elements, linker sequences, number of DRs, and Magnet mutants in the PA-rABE system were systematically optimized and modified. The results showed that PA-rABE can respond sensitively to blue light, not only achieving spatiotemporally specific RNA base editing within cells, but also sensitively regulating the intensity of editing activity by adjusting the time and intensity of light.

[0047] (3) When targeting transcripts of exogenous reporter or endogenous genes, the average editing efficiency of PA-rABE is higher than that of the mxABE and REPAIRv2 systems. Unlike the padCas13 editor, PA-rABE directly regulates ADAR2dd activity, enabling not only spatiotemporally specific RNA base editing but also strict regulation of ADAR2dd activity with almost no off-target effects across the transcriptome, demonstrating high fidelity.

[0048] In summary, the PA-rABE system is a novel RNA base editing tool that integrates high-efficiency editing, low off-target effects, and precise control, laying a technological foundation for the precise manipulation of important physiological and pathological processes from an RNA perspective. Attached Figure Description

[0049] Figure 1. Construction of the PA-rABE system. (a) Working principle of PA-rABE. (b) Schematic diagram of evaluating RNA base editing efficiency using luciferase (Gluc W160X) as a reporter. (c) dCas13-ADARdd CFour candidate vectors for -nMag and pMag-ADARdd N Schematic diagram of the plasmid. (d) Test of dCas13-ADARdd C Luciferase repair efficiency of the -nMag candidate vector. (e) Screening for segmented ADAR2dd for RNA base editing. Data are expressed as mean ± sd, n = 3.

[0050] Figure 2. Optimization of the PA-rABE system. (a) Evaluation of the relative positions of each gene element. (bc) Optimization of the linker sequence between the ADAR2dd fragment and nMag or pMag. (d) Testing the effect of the number of DRs on editing efficiency. (e) Evaluation of the effectiveness of the Magnets mutant for RNA base editing. (f) Comparison of the editing efficiency of PA-rABE with the mxABE, REPAIRv2, and REPAIRx systems. Data are expressed as mean ± sd, n = 3.

[0051] Figure 3. Characterization of PA-rABE. (a) Effect of crRNA length on editing efficiency. (b) Effect of mismatched base positions on editing efficiency. (c) Comparison of PA-rABE editing efficiency using single and tandem dual targets. (d) Light intensity-dependent PA-rABE editing activity. (e) Light time-dependent PA-rABE editing activity. (f) PA-rABE reversibility test. (g) PA-rABE-induced space-specific RNA base editing. Data are expressed as mean ± SD, n = 3.

[0052] Figure 4. Editing of endogenous transcripts using PA-rABE. (a) Editing efficiency of PA-rABE, mxABE, and REPAIRv2 on various endogenous transcripts. (b) Average editing efficiency of each RNA base editor on 10 endogenous transcripts. (c) Quantitative analysis of the number of off-target sites in the PA-rABE and mxABE systems under targeted Kras and non-targeted (NT) conditions. EGFP was used as a negative control. (d) Off-target editing analysis of PA-rABE across the transcriptome. All experiments were performed in HEK293T cells, and editing efficiency was quantified by NGS. Data are expressed as mean ± SD, n = 3. Detailed Implementation

[0053] The sequence structure of each structural unit of the PA-rABE construct is as follows:

[0054] ADAR2dd, sequence as shown in SEQ ID NO.1; SEQ ID NO.1:

[0055] ADAR2dd316-466, sequence as shown in SEQ ID NO.2; SEQ ID NO.2:

[0056] ADAR2dd467-700, sequence as shown in SEQ ID NO.3; SEQ ID NO.3:

[0057] pMag, sequence as shown in SEQ ID NO.4; SEQ ID NO.4:

[0058] nMagHigh, sequence as shown in SEQ ID NO.5; SEQ ID NO.5:

[0059] mini dCas13X, sequence as shown in SEQ ID NO.6; SEQ ID NO.6:

[0060] The nine linker sequences are as follows:

[0061] L1, the sequence is shown in SEQ ID NO.7; SEQ ID NO.7: GS

[0062] L2, sequence as shown in SEQ ID NO.8; SEQ ID NO.8; GSGGGGS

[0063] L3, sequence as shown in SEQ ID NO.9; SEQ ID NO.9; GSGGGGSGGGGR

[0064] L4, the sequence is shown in SEQ ID NO.10; SEQ ID NO.10: GSGGGGSGGGGSGGGGR

[0065] L5, the sequence is shown in SEQ ID NO.11; SEQ ID NO.11: EAAAKEAAAKEAAA

[0066] L6, sequence as shown in SEQ ID NO.12; SEQ ID NO.12: LEASPSNPGASNGSGT

[0067] L7, sequence as shown in SEQ ID NO.13; SEQ ID NO.13: SGSETPGTSESATPES

[0068] L8, the sequence is shown in SEQ ID NO.14; SEQ ID NO.14: SGGSSGGSSGSETPGTSESATPESSGGSSGGS

[0069] L9, sequence as shown in SEQ ID NO.15; SEQ ID NO.15: APTLADLAVDLAALRPLEHPNPPLQRAAEALL

[0070] Material:

[0071] 1. CMV, U6, DR, dCas13b, dCas13X or dCas13d are all conventional components in the art and are well known to those skilled in the art.

[0072] 2. Gluc W160X system:

[0073] To facilitate rapid testing of PA-rABE editing efficiency, we mutated tryptophan (W, TGG) at position 160 of Gaussian luciferase to a stop codon (X, TAG), constructing a Gaussian luciferase reporter system carrying a nonsense mutation, abbreviated as Gluc W160X. Targeted editing of the UAG of the Gluc W160X transcript can convert the stop codon UAG to UIG, thereby functionally restoring the activity of Gaussian luciferase. The editing efficiency of PA-rABE can be determined by detecting the intensity of luciferase. The PA-rABE candidate plasmid and the Gluc W160X reporter plasmid were co-transfected into HEK-293T cells. Twelve hours after transfection, one group of cells was irradiated with blue light (0.25 mW / cm²). 2 One group was placed in the light-exposed group (on for 1 minute, off for 3 minutes), while the other group was placed in the dark for protection from light. Forty-eight hours after transfection, the cell supernatant was collected to detect luciferase activity. Theoretically, the higher the fluorescence value, the stronger the PA-rABE activity. The ratio of the fluorescence value of the light-exposed group to that of the dark-exposed group was used as the PA-rABE induction fold; a higher value indicated a stronger light dependence of PA-rABE.

[0074] Example 1: Construction of a blue light-activated A>I RNA base editing system

[0075] To construct a photoactivatable A-to-I RNA base editor (PA-rABE), we will use segmented inactivated ADAR2dd fragments (ADAR2dd... N and ADAR2dd C ), which are fused with pMag and nMag respectively.

[0076] pMag-ADAR2dd under dark conditions N It exists as an independent component in free form; ADAR2dd C -nMag is fused with catalytically inactivated dCas13 protein for expression (dCas13-ADAR2dd). C After -nMag), it is localized to the target RNA under the guidance of crRNA.

[0077] Upon blue light stimulation, pMag and nMag dimerize, thereby reducing pMag-ADAR2dd N Recruited to the vicinity of the target site. After recombination of the segmented ADAR2dd fragments, deaminase activity is restored, thereby achieving A-to-I editing at the RNA level (Figure 1a).

[0078] To assess the efficiency of RNA base editing, we constructed a Gaussia luciferase reporter carrying a nonsense mutation, abbreviated as Gluc W160X. Targeted editing of UAG on the Gluc W160X transcript can convert the stop codon to UIG, thereby functionally restoring the activity of Gaussia luciferase (Figure 1b). The editing efficiency of PA-rABE can be determined by detecting the intensity of luciferase.

[0079] Four catalytically inactivated Cas13 proteins (for specific information on Cas13 proteins, please refer to PMID:33941935, PMID:29551272, PMID:29070703) were respectively combined with ADAR2dd C -nMag fusion expression, constructing different versions of dCas13-ADAR2dd C -nMag fusion protein and combined with pMag-ADAR2dd N HEK-293T cells were co-transfected with the reporter system (Figure 1c). The results showed that the PA-rABE system constructed using mini dCas13X as an RNA recruitment platform had the highest editing efficiency (Figure 1d).

[0080] Based on this, we tested the efficiency of ADAR2dd protein at different segmentation sites for RNA editing and found that the ADAR2dd protein segmented at E466 / P467 had the highest editing efficiency, showing a ~2.9-fold activation under light conditions (Figure 1e). Therefore, we selected ADAR2dd 316-466 (all ADAR2dd segments in subsequent examples). N Both are ADAR2dd 316-466 and ADAR2dd 467-700 (all ADAR2dd in subsequent embodiments) C Both ADAR2dd (467-700) are used to construct a light-activated RNA base editor.

[0081] Example 2: Obtaining a high-efficiency and low-leakage PA-rABE through molecular optimization

[0082] The relative positions of gene elements and the linker sequence have a significant impact on the activity of fusion proteins. To determine the optimal configuration of PA-rABE, we adjusted the relative positions of the various gene elements and constructed a series of PA-rABE variants.

[0083] The results of luciferase activity assay showed that nMag-ADAR2dd C (nMag at the N end) and mini dCas13X-ADAR2dd N The editing efficiency is highest when using the -pMag (each functional block is sorted from N to C) combination (Figure 2a).

[0084] To further improve the editing performance of this combination, we selected nine different rigid and flexible linkers and cloned them between the segmented ADAR2dd and the light-sensitive protein (unless otherwise specified, nMag links ADAR2dd). C and ADAR2dd N Use the same linker when connecting pMag, mini dCas13X-ADAR2dd N The linker between them is SEQ ID NO.16: GGGGGSGGGGSGGGGSGGGGS. The results show that:

[0085] When the linker sequence between the segmented ADAR2dd and the photosensitive protein is L2 (GSGGGGS), PA-rABE exhibits stronger light dependence, with a photoinduction fold of approximately 13.8-fold (Figure 2b).

[0086] It is noteworthy that the L1-PA-rABE, L2-PA-rABE, and L3-PA-rABE variants all employ flexible GS linkers, and their editing activity increases with the length of the GS linker sequence. Theoretically, the activity of these three variants under dark conditions should also increase or decrease sequentially; however, the L2-PA-rABE variant has the lowest background (baseline noise under dark conditions). Therefore, we hypothesize that adjusting the length of the GS linker can further improve the editing performance of PA-rABE, and constructed eight L2-PA-rABE variants.

[0087] The results of luciferase activity assay showed that:

[0088] L2-4(GSGGGG) PA-rABE editing efficiency was improved by 1.8 times, and the induction fold increased from 13.8 times to 19.2 times (Figure 2c).

[0089] Furthermore, we found that the number of DRs and the Magnets variant also significantly affected the editing activity of PA-rABE. When the crRNA was flanked by double DR sequences, the editing activity of PA-rABE was significantly increased by 3-fold compared to the single DR system (Figure 2d).

[0090] When pMag is combined with nMagHigh, not only is the editing efficiency of PA-rABE improved by 2.2 times, but the light dependence of PA-rABE is also greatly improved, giving us the optimal version of the PA-rABE system with a light induction factor of up to ~47 times (Figure 2e).

[0091] Compared with existing RNA base editing tools, PA-rABE showed the highest editing efficiency when targeting the Gluc W160X transcript (Figure 2f). In summary, through a series of molecular optimizations, we obtained a photoactivated RNA base editor with high editing efficiency and low background.

[0092] Example 3: Characterization of PA-rABE

[0093] Early studies have shown that the length of crRNA and the position of mismatched bases in the RNA-crRNA double-stranded structure affect the efficiency of RNA base editing.

[0094] (1) In order to characterize the features of PA-rABE, we constructed crRNAs of different lengths to target exogenous reporter Gluc W160X and found that the editing efficiency was higher when the target length was 50-60nt, especially at 50nt, PA-rABE could efficiently repair the activity of luciferase (Figure 3a).

[0095] (2) The target length was fixed at 50 nt, and crRNAs with different mismatch positions were constructed. The test results showed that the effect was best when the mismatch base was located at position 25 (Figure 3b).

[0096] (3) Considering that CRISPR-Cas13 has the ability to self-process and cleave RNA, we expressed the targets mCherry W98X and zsGreen W93X in tandem and then co-transfected HEK293T cells with the reporter system and PA-rABE. Flow cytometry analysis results showed that the editing efficiency of PA-rABE using tandem dual targets was comparable to that of single targets, proving that PA-rABE has multi-pathway linkage function (Figure 3c).

[0097] (4) The light dependence of PA-rABE was further characterized by exposing cells transfected with PA-rABE and Gluc W160X to different light conditions. Luciferase assay results showed that the editing efficiency of PA-rABE increased with increasing light duration and intensity (Fig. 3d, e). RNA base editing activity immediately shut down after light exposure ceased, and subsequent light exposure restored the editing activity of PA-rABE, thus achieving reversible RNA editing (Fig. 3f).

[0098] (5) Most importantly, the PA-rABE system exhibits high spatial specificity. We co-transfected HEK-293T cells with it and mCherry W98X. Twelve hours later, the transfected 10cm dishes were placed on a device with a black perforated pattern on the bottom for illumination. Blue light shone through the small slits onto the corresponding areas of HEK-293T cells, restoring the expression of the red fluorescent protein mCherry, ultimately forming a specific pattern (Fig. 3g), demonstrating that PA-rABE has high spatial specificity.

[0099] In summary, PA-rABE can respond sensitively to blue light, rapidly and reversibly regulate gene expression, and has the characteristics of multi-pathway synergy and high spatiotemporal specificity.

[0100] Example 4: Testing the editing efficiency and specificity of PA-rABE in endogenous genes.

[0101] While reporting systems can quickly characterize the features of PA-rABE, their applicability is limited and they cannot accurately reflect the editing efficiency of PA-rABE. To further test the editing efficiency of PA-rABE, we selected 10 endogenous transcripts with different functions and designed targets to systematically compare the editing efficiencies of PA-rABE, mxABE, and REPAIRv2.

[0102] (1) PA-rABE N (CMV-mini dCas13X-ADAR2dd N -pMag-IRES-EGFP, 335ng) and PA-rABE C (U6-gRNA-CMV-nMagH-ADAR2dd C HEK-293T cells were co-transfected with 565 ng of [agent name missing]. Twenty-four hours post-transfection, one group of cells was irradiated with blue light (0.25 mw / cm²). 2 One group was placed in the dark (on for 1 minute, off for 3 minutes), while the other group was placed in the dark to avoid light. The control group was transfected with equimolar amounts of the mxABE (CMV-minid Cas13X-ADAR2dd) or REPAIRv2 (CMV-dCas13b-ADAR2dd) system and their respective target sites.

[0103] (2) Forty-eight hours after transfection, 70,000 EGFP-positive cells were sorted from the transfected samples using a flow cytometer and RNA was extracted. The RNA from each sample was reverse transcribed into cDNA, and then amplified by PCR to construct a library for next-generation sequencing.

[0104] The results showed that PA-rABE outperformed mxABE and REPAIRv2 in editing efficiency across 10 endogenous transcripts, with an average editing rate of 34.7%, compared to 18.3% for mxABE and 23.6% for REPAIRv2 (Figure 4a, b).

[0105] To assess the transcriptome specificity of PA-rABE, we compared it with the previously reported high-fidelity mxABE.

[0106] (1) Approximately 200,000 positive cells transfected with PA-rABE and mxABE were collected by flow cytometry and RNA was extracted for transcriptional library sequencing.

[0107] (2) Samples transfected only with EGFP were used as the control group, and the editing events generated in this group were used as the background. After subtracting the background from the editing events in each experimental group, the total number of off-target events of PA-rABE in the transcriptome could be obtained.

[0108] Whole transcriptome RNA sequencing showed that the off-target events induced by PA-rABE were similar to those of mxABE (Figure 4c, d), demonstrating that PA-rABE exhibits high-fidelity activity across the transcriptome.

[0109] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A photoactivatable RNA base editor (PA-rABE), wherein the photoactivatable RNA base editor can cause adenosine (A) in the target double-stranded RNA to deaminate into inosine (I), thereby achieving the editing of A into G; The light-sensitive RNA adenosine base editor includes: (1)dCas13-ADAR2dd C -nMag fusion protein, a C-terminal truncated fragment of the ADAR2dd catalytic domain of dCas13 protein and adenosine deaminase acting on RNA (ADAR). C The composition of nMag monomers in the Magnets system, which is induced by blue light dimerization; (2)pMag-ADAR2dd N The fusion protein consists of a pMag monomer of the Magnets system, dimerized by blue light, and an N-terminal truncated fragment of the adenosine deaminase acting on RNA (ADAR) catalytic domain ADAR2dd. N )constitute.

2. The photosensitive RNA adenosine base editor according to claim 1, characterized in that, The dCas13 protein includes, but is not limited to, mini dCas13X, dCas13b, dCas13X or dCas13d proteins, preferably the mini dCas13X protein with a smaller molecular weight.

3. The photosensitive RNA adenosine base editor according to claim 1 or 2, characterized in that, The sequence of ADAR2dd is shown in SEQ ID NO.1; The ADAR2dd mentioned C The sequence is shown in SEQ ID NO.2; The ADAR2dd mentioned N The sequence is shown in SEQ ID NO.3; The sequence of the pMag is shown in SEQ ID NO.4; The sequence of nMag is shown in SEQ ID NO.5; The sequence of the mini dCas13X is shown in SEQ ID NO.

6.

4. The photosensitive RNA adenosine base editor according to any one of claims 1-3, characterized in that, The various functional blocks of the fusion protein are linked by a linker; the linker includes, but is not limited to: L1, the sequence is shown in SEQ ID NO.7; L2, the sequence is shown in SEQ ID NO.8; L3, the sequence is shown in SEQ ID NO.9; L4, the sequence is shown in SEQ ID NO.10; L5, the sequence is shown in SEQ ID NO.11; L6, the sequence is shown in SEQ ID NO.12; L7, the sequence is shown in SEQ ID NO.13; L8, the sequence is shown in SEQ ID NO.14; L9, the sequence is shown in SEQ ID NO.15; Preferably, the Linker is L2.

5. The photosensitive RNA adenosine base editor according to claim 1, characterized in that, The conditions for blue light-induced dimerization are: 0.25 mW / cm². 2 Turn on in 1 minute and turn off in 3 minutes. Blue light wavelength is 470nm.

6. [Amended according to Rule 26, 15.11.2024] The photosensitive RNA adenosine base editor according to any one of claims 1-5 is characterized in that, The target double-stranded RNA is 10-100 nt in length, more preferably 50-60 nt, and most preferably 50 nt.

7. The photosensitive RNA adenosine base editor according to any one of claims 1-6, characterized in that, The editing site is located at position 25 of the target double-stranded RNA.

8. The following applications of the photosensitive RNA adenosine base editor according to any one of claims 1-7: (1) Targeted editing of eukaryotic or prokaryotic cells; (2) To prepare drugs for treating diseases caused by gene mutations; (3) Used for crop genetic breeding; (4) Used to prepare animal models.

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