Engineered isymu1 tnpb protein variants and uses

Abstract

The application discloses an engineered ISYmu1 TnpB protein variant, which comprises E260D and E45R amino acid mutations relative to a wild-type ISYmu1 TnpB, and provides a gene editing system, an adenine base editor and a cytosine base editor based on the protein variant. The enISYmu1 TnpB protein variant exhibits significantly improved editing efficiency in mammalian cells, and the editing efficiency is increased by more than 30 times at some sites; the base editor developed based on the enISYmu1 TnpB realizes high-efficiency base conversion at multiple endogenous loci. In addition, the super-small size of the enISYmu1 TnpB enables in-vivo delivery through a single AAV vector, and the targeting of Vegfa and Hif1a genes realizes a significant anti-neovascularization treatment effect in a laser-induced choroidal neovascularization mouse model. The application provides a new tool with high efficiency and compactness for gene editing and in-vivo gene therapy.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to engineered ISYmu1 TnpB protein variants and their applications. Background Technology

[0002] The CRISPR-Cas system, as an adaptive immune system in prokaryotes, has been developed into a powerful genome editing tool. Nucleases such as Cas9 and Cas12a, guided by guide RNA, can achieve targeted DNA cleavage and are widely used in basic research and gene therapy. However, these proteins are typically large (generally exceeding 1200 amino acids), making in vivo delivery via adeno-associated virus (AAV) vectors difficult due to the limited packaging capacity of AAV (approximately 4.7 kb).

[0003] In recent years, the TnpB protein has attracted widespread attention as an evolutionary precursor to the CRISPR-Cas12 system. The TnpB protein is only about 400 amino acids long, roughly one-third the size of SpCas9, and is associated with the IS200 / IS605 transposon. Guided by reRNA (right-end element RNA), it can introduce DNA double-strand breaks at locations adjacent to the TAM (transposon-associated motif) sequence. This ultra-small size makes TnpB an ideal candidate for single-AAV delivery gene editing.

[0004] ISYmu1 TnpB, derived from Youngiibacter multiovrans (382 amino acids), has been identified as a highly active micro-editor. However, the editing efficiency of wild-type ISYmu1 TnpB remains unsatisfactory for many therapeutic and research applications. Although studies have systematically analyzed ISDra2 TnpB using deep mutational scanning (DMS) and obtained some ISYmu1 variants through cross-species mutational transfer, systematic engineering of ISYmu1 TnpB has not been fully explored.

[0005] Therefore, there is an urgent need in this field to develop novel engineered variants of ISYmu1 TnpB and their gene editing systems that are more efficient in editing and suitable for mammalian cell and in vivo applications. Summary of the Invention

[0006] To address the technical deficiencies of existing technologies, this invention provides an engineered ISYmu1 TnpB protein variant and its applications.

[0007] The technical solution adopted in this invention is: an engineered ISYmu1 TnpB protein variant, wherein the protein variant is an E260D amino acid mutation in the amino acid sequence of wild-type ISYmu1 TnpB, wherein the amino acid sequence of wild-type ISYmu1 TnpB is shown in SEQ ID NO: 1.

[0008] Furthermore, the protein variants also include the E45R amino acid mutation.

[0009] Furthermore, the amino acid sequence of the protein variant is shown in SEQ ID NO: 2 or 3.

[0010] An isolated nucleic acid molecule that encodes the aforementioned protein variant.

[0011] A gene editing system comprising: (a) the protein variants described above, or the nucleic acid molecules described above; and (b) Guide RNA (reRNA), or a nucleic acid molecule encoding the guide RNA.

[0012] Furthermore, the guide RNA is 18 nucleotides in length.

[0013] An adenine base editor, comprising: (a) A nickase variant of the protein variant described above, wherein the nickase variant comprises a nickase variant (containing the E279A mutation); and (b) Adenosine deaminase or its functional variants.

[0014] Furthermore, the adenosine deaminase is TadA8e. V106W .

[0015] Furthermore, the adenosine deaminase is fused to the C-terminus of the protein variant.

[0016] A cytosine base editor comprising: (a) a nickase variant of the protein variant described above, wherein the nickase variant comprises a nickase variant (containing the E279A mutation); and (b) APOBEC3A cytosine deaminase or its functional variants, and uracil glycosylation inhibitors (UGI).

[0017] A recombinant vector containing the aforementioned nucleic acid molecules.

[0018] A recombinant host cell comprising the aforementioned nucleic acid molecules, gene editing system, adenine base editor, or cytosine base editor.

[0019] A composition comprising the above-described protein variant, nucleic acid molecule, gene editing system, adenine base editor, or cytosine base editor, and a pharmaceutically acceptable vector or delivery vector.

[0020] The above-mentioned protein variants, gene editing systems, adenine base editors, or cytosine base editors are used in the preparation of reagents for in vitro gene editing.

[0021] The aforementioned protein variants, gene editing systems, adenine base editors, or cytosine base editors are used in the preparation of drugs for treating angiogenesis-related diseases.

[0022] Furthermore, the angiogenesis-related disease is choroidal neovascularization (CNV).

[0023] A method for gene editing of target DNA in vitro or in vivo, comprising the step of introducing the aforementioned protein variant or gene editing system into a cell containing the target DNA.

[0024] The beneficial effects of this invention are as follows: This invention provides an engineered ISYmu1 TnpB protein variant and its application, which has the following beneficial effects: 1. Significantly improved editing efficiency: The enISYmu1TnpB protein variant provided by this invention has an average editing efficiency of about 2.0 times higher than that of wild type at multiple endogenous gene sites, and can be more than 30 times higher at some sites, which is significantly better than the ISYmu1-WFR variant reported in the prior art.

[0025] 2. Supports guide RNA truncation: enISYmu1TnpB can effectively use truncated guide RNA with a sequence length of 18 nucleotides, and the editing efficiency is 1.24 times higher than that of 20 nucleotide guide RNA.

[0026] 3. Suitable for base editing: The SminiABE and SminiCBE base editors, developed based on enISYmu1TnpB nickase, have achieved efficient A-T-to-GC and C-G-to-TA conversions at multiple endogenous gene sites, which are significantly superior to wild-type ISYmu1n-ABE / CBE.

[0027] 4. In vivo delivery of a single AAV: The ultra-small size (382 amino acids) of enISYmu1TnpB enables in vivo delivery via a single AAV vector. In a mouse laser-induced choroidal neovascularization model, it targets the Vegfa and Hif1a genes, achieving indel frequencies of approximately 20% and 38%, respectively, and significantly inhibits pathological angiogenesis (approximately 37% inhibition rate), demonstrating good therapeutic potential. Attached Figure Description

[0028] Figure 1 The engineering strategy and activity screening results of ISYmu1TnpB protein; Figure 1 A in the diagram is a schematic diagram of an engineering strategy based on multiple sequence alignment; Figure 1 Figure B shows the results of fluorescence reporter system screening of 17 single-point mutants. Figure 1 C represents a comparison of the editing activities of combined mutants; Figure 1 D represents the result of guide RNA length optimization.

[0029] Figure 2 This study aimed to assess the editing activity of enISYmu1TnpB at endogenous gene loci in mammalian cells. Figure 2 In the figure, A represents a comparison of the editing efficiency of multiple endogenous sites in wild-type and mutant individuals; Figure 2 In this context, B represents a comparison of the editing efficiency of enISYmu1TnpB with ISDra2TnpB, SaCas9, and enOsCas12f1. Figure 2 C in the equation represents a comparison of the editing activity of the enISYmu1TnpB and ISYmu1-WFR variants.

[0030] Figure 3 Development and characterization of the SminiABE adenine base editor; Figure 3 Comparison of ASminiABE and ISYmu1n-ABE at 10 endogenous sites; Figure 3 B in the figure represents the statistical analysis of average editing efficiency; Figure 3 In the text, C represents the editing window analysis for A-to-G conversion.

[0031] Figure 4 Development and characterization of the SminiCBE cytosine base editor; Figure 4 In the figure, A represents a comparison of SminiCBE and ISYmu1n-CBE at 10 endogenous sites; Figure 4 B in the figure represents the statistical analysis of average editing efficiency; Figure 4 The 'C' in the text refers to the C-to-T conversion editing window analysis.

[0032] Figure 5 The in vivo therapeutic efficacy of enISYmu1TnpB in a laser-induced CNV mouse model; Figure 5 In this context, A represents the in-body editing efficiency; Figure 5 B in the figure represents the result of fluorescein angiography (FFA); Figure 5 In this context, C represents the quantitative analysis of the CNV area; Figure 5 In this context, DF represents the CNV length and thickness measured by OCT.

[0033] Figure 6 This is a guide sequence list in a specific embodiment of the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1: Design and screening of ISYmu1TnpB protein variants Engineering strategies guided by multiple sequence alignment Given the lack of a complete structural model capable of capturing all relevant conformational states, this invention employs a sequence-guided approach to identify beneficial mutations in ISYmu1TnpB. Specifically, ISYmu1TnpB was subjected to multiple sequence alignment (MSA) with five closely related TnpB orthologs. When ISYmu1TnpB carried neutral or negatively charged residues at the alignment position, while the corresponding homolog carried positively charged residues at that position, these residues were replaced with arginine (R) to enhance electrostatic interactions with negatively charged gRNA or target DNA. Furthermore, several conserved alignment positions were identified, with ISYmu1 possessing unique residues; these residues were replaced with evolutionarily conserved amino acids present in the orthologs.

[0036] Fluorescence reporting system Editing activity was assessed using the EGFXP fluorescent reporter system. This system restores EGFP expression after target cleavage and subsequent single-strand annealing-mediated repair. HEK293T cells were co-transfected with a plasmid expressing the TnpB variant and reRNA, and EGFP fluorescence signal was analyzed by flow cytometry 48 hours later.

[0037] Based on MSA-guided design, 17 single-point substitution variants were generated and their activities were evaluated. Among them, six mutations, E8K, W39R, Q97R, Q102R, E216I, and Y251H, significantly reduced activity; while variants such as S87E, D88R, Q217R, A221S, L225K, L235G, and Y261F retained activities comparable to the wild type.

[0038] Combination mutation E260D was combined with other beneficial mutations identified in the initial screening. The E260D single mutant showed a 1.56-fold improvement over the wild type; the E45R / E260D double mutant showed a 2.0-fold improvement.

[0039] Guide RNA optimization Guide sequences of 14 to 20 nucleotides in length were tested, with a 20-nucleotide length as a reference. ISYmu1TnpB showed robust activity under 18-nucleotide guidance, with an efficiency 1.24-fold higher than the 20-nucleotide version. Truncation to 16 nucleotides resulted in a moderate decrease (1.17-fold), while the decrease was more pronounced with 14-nucleotide guidance. U4A modification unexpectedly reduced activity, indicating that the effect of guide RNA modification is context-dependent.

[0040] Example 2: Editing activity of enISYmu1TnpB at endogenous loci Comparison of in vivo editing activities Engineered enISYmu1TnpB paired with an 18-nucleotide guide RNA (enISYmu1TnpB-gl18) exhibited robust activity at multiple targets, including EMX1, VEGFA, and CHR2, generally outperforming the 20-nucleotide guide RNA (enISYmu1TnpB-gl20). In contrast, wild-type ISYmu1TnpB-gl20 showed almost no activity at most tested sites. Statistical analysis confirmed that the 18-nucleotide guide RNA was significantly superior to both the 20-nucleotide guide RNA and the wild-type enzyme.

[0041] Comparison with other small editors EnISYmu1TnpB was compared with three established small CRISPR / Cas editors (ISDra2TnpB, SaCas9, and enOsCas12f1). EnISYmu1TnpB significantly outperformed ISDra2TnpB and enOsCas12f1 at sites such as DNMT3B, PCSK9, and VEGFA. Quantitative comparison of average indel frequencies confirmed that enISYmu1TnpB achieved significantly higher overall editing efficiency than all three editors (P < 0.001). EnISYmu1TnpB was compared with recently reported ISYmu1-WFR variants at 13 endogenous human target sites. EnISYmu1TnpB exhibited consistently high levels of activity at all tested sites. In contrast, ISYmu1-WFR showed significantly reduced activity at most sites, including CBE1, CHR2, PCSK9 (sg1, sg2, sg3, sg7), and DMD (sg2, sg3), with relative editing ratios ranging from 0.4 to 0.8. Only at a few sites, such as DMDsg1 and VEGFAsg5, did ISYmu1-WFR achieve comparable or slightly higher efficiency.

[0042] Example 3: Base Editor Based on enISYmu1TnpB Construction of nuclease inactivation variants By comparing the full-length sequences of ISDra2TnpB and ISYmu1TnpB, the putative catalytic residues D185 and E279 in ISYmu1TnpB were identified. Mutating these residues to alanine, the two single mutants (D185A and E279A) almost completely eliminated the cleavage activity.

[0043] Adenine Base Editor (SminiABE) enISYmu1TnpB E279A With TadA8e V106W (TadA*) fused with C-terminus (enISYmu1TnpB) E279A The gene was named SminiABE (-TadA*). Comparison of SminiABE and ISYmu1n-ABE at 10 endogenous loci showed that SminiABE consistently outperformed the wild-type at all tested sites. At the EMX2 site, SminiABE achieved editing efficiencies exceeding 15% at multiple positions (A3-A11), while ISYmu1n-ABE's editing efficiency was below 9%. At the MECP2 site, SminiABE achieved approximately 10% editing at A5 and A8 positions, while ISYmu1n-ABE's editing efficiency was only 1.5%. Pooled analysis showed that SminiABE's average editing efficiency (approximately 10%) was significantly higher than ISYmu1n-ABE's (approximately 6%; **P<0.01). Both editors exhibited typical base editing windows, primarily spanning A2 to A16 positions, with peak activity concentrated in the A3-A14 range.

[0044] Cytosine base editor (SminiCBE) A cytosine base editor was developed by pairwise fusion of APOBEC3A cytosine deaminase and uracil glycosylation inhibitor (UGI). The A3A-enISYmu1TnpB... E279A The UGI editor was named SminiCBE. Comparison of SminiCBE and ISYmu1n-CBE at 10 endogenous loci showed that SminiCBE exhibited higher C-to-T editing frequencies at all tested sites. At the CBE2sg and PGK1 sites, SminiCBE reached peak C-to-T conversion rates of 16.95% and 20.54%, respectively. Pooled statistical comparisons showed that SminiCBE mediated significantly higher overall cytosine base editing efficiency (****P<0.0001). Both CBE variants exhibited an activity editing window centered on C2-C11, with peak activity concentrated in the C3-C8 range.

[0045] Example 4: In vivo treatment of enISYmu1TnpB in a laser-induced CNV mouse model AAV8-mediated in vivo delivery The therapeutic potential of AAV8-delivered enISYmu1TnpB was evaluated in a mouse model of laser-induced choroidal neovascularization (CNV). Mice received subretinal injections of an AAV8 vector encoding enISYmu1TnpB and a guide RNA targeting Vegfa or Hif1a, followed by laser-induced CNV two weeks later. Deep sequencing analysis of retinal tissue on day 21 post-injection confirmed robust in vivo editing activity, with indel frequencies of approximately 20.68% for Vegfa and 37.67% for Hif1a in the treated eye, while the control eye showed negligible background editing.

[0046] Functional evaluation On day 7 post-laser treatment, fluorescein angiography (FFA) showed a significant reduction in CNV lesion area in the treatment group, corresponding to approximately 37% inhibition of neovascularization (****P<0.0001). Optical coherence tomography (OCT) revealed significantly thinner and flatter lesions in the treated eyes: CNV width decreased from 1280.02 μm in the control group to 734.93 μm, and CNV height decreased from 171.21 μm to 105.09 μm (****P<0.0001).

[0047] sequence list SEQ ID NO: 1 (Wild type ISYmu1 TnpB) MLQHKAYEYRIYPDKKQETLIAKTIGSSRYVYNHFLELWNKEYEETGKGLTYYACSKLLTKLKRDPETVWLCEVDKFSLQNSLRNLSDAFSRFFKGQNEHPQFKSKKSPRQSYTTQYTNNNIAVSGNCLKLPKLGLVKFADSREMKGRILNATVRRKSSGKFFVSILCEEEICELPKTDSSVGIDLGIIDF AVMSDGSRHDNNHFTRQMEERLRREQRKLARRALAAEKRGISLSEARNYQKQRRKVARLYEKVANQRKEYLNKLSTEIVKNHDIICIEDLNVKGMMRNHKLAKSISDVSWTSLVSKLQYKASWYGKEVIRISRWFPSSQICSECGHKDRKKPLHVREWTCPVCHAHHDRDVNAARNILAEGLRIRALTPGS.

[0048] SEQ ID NO: 2 (ISYmu1 TnpB E260D replacement) MLQHKAYEYRIYPDKKQETLIAKTIGSSRYVYNHFLELWNKEYEETGKGLTYYACSKLLTKLKRDPETVWLCEVDKFSLQNSLRNLSDAFSRFFKGQNEHPQFKSKKSPRQSYTTQYTNNNIAVSGNCLKLPKLGLVKFADSREMKGRILNATVRRKSSGKFFVSILCEEEICELPKTDSSVGIDLGIIDF AVMSDGSRHDNNHFTRQMEERLRREQRKLARRALAAEKRGISLSEARNYQKQRRKVARLYEKVANQRKDYLNKLSTEIVKNHDIICIEDLNVKGMMRNHKLAKSISDVSWTSLVSKLQYKASWYGKEVIRISRWFPSSQICSECGHKDRKKPLHVREWTCPVCHAHHDRDVNAARNILAEGLRIRALTPGS.

[0049] SEQ ID NO: 3(ISYmu1 TnpB E45R / E260D double replacement) MLQHKAYEYRIYPDKKQETLIAKTIGSSRYVYNHFLELWNKEYERTGKGLTYYACSKLLTKLKRDPETVWLCEVDKFSLQNSLRNLSDAFSRFFKGQNEHPQFKSKKSPRQSYTTQYTNNNIAVSGNCLKLPKLGLVKFADSREMKGRILNATVRRKSSGKFFVSILCEEEICELPKTDSSVGIDLGIIDF AVMSDGSRHDNNHFTRQMEERLRREQRKLARRALAAEKRGISLSEARNYQKQRRKVARLYEKVANQRKDYLNKLSTEIVKNHDIICIEDLNVKGMMRNHKLAKSISDVSWTSLVSKLQYKASWYGKEVIRISRWFPSSQICSECGHKDRKKPLHVREWTCPVCHAHHDRDVNAARNILAEGLRIRALTPGS.

[0050] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An engineered ISYmu1 TnpB protein variant, characterized in that, The protein variant is an E260D amino acid mutation in the wild-type ISYmu1TnpB amino acid sequence, wherein the wild-type ISYmu1 TnpB amino acid sequence is shown in SEQ ID NO:

1.

2. The protein variant according to claim 1, characterized in that, The protein variants also include the E45R amino acid mutation.

3. The protein variant according to claim 1 or 2, characterized in that, The amino acid sequence of the protein variant is shown in SEQ ID NO: 2 or 3.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the protein variant described in claim 1 or 2.

5. A gene editing system, characterized in that, Include: (a) the protein variant of claim 1 or 2, or the nucleic acid molecule of claim 4; and (b) Guide RNA, or a nucleic acid molecule encoding the guide RNA.

6. An adenine base editor, characterized in that, Include: (a) A nickase variant of the protein variant of claim 1 or 2, wherein the nickase variant comprises an E279A amino acid mutation; and (b) Adenosine deaminase or its functional variants.

7. The adenine base editor according to claim 6, characterized in that, The adenosine deaminase mentioned is TadA8e. V106W .

8. The adenine base editor according to claim 6, characterized in that, The adenosine deaminase is fused to the C-terminus of the protein variant.

9. A cytosine base editor, characterized in that, Include: (a) A nickase variant of the protein variant of claim 1 or 2, wherein the nickase variant comprises an E279A amino acid mutation; and (b) APOBEC3A cytosine deaminase or its functional variants, and uracil glycosylation inhibitors (UGI).

10. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 4.

11. A recombinant host cell, characterized in that, It comprises the nucleic acid molecule of claim 4, the gene editing system of claim 5, the adenine base editor of any one of claims 6 to 8, or the cytosine base editor of claim 9.

12. A composition, characterized in that, It comprises the protein variant of claim 1 or 2, the nucleic acid molecule of claim 4, the gene editing system of claim 5, the adenine base editor of any one of claims 6 to 8, or the cytosine base editor of claim 9, and a pharmaceutically acceptable vector or delivery vector.

13. The use of the protein variant of claim 1 or 2, the gene editing system of claim 5, the adenine base editor of any one of claims 6 to 8, or the cytosine base editor of claim 9 in the preparation of reagents for in vitro gene editing.

14. Use of the protein variant of claim 1 or 2, the gene editing system of claim 5, the adenine base editor of any one of claims 6 to 8, or the cytosine base editor of claim 9 in the preparation of a medicament for treating angiogenesis-related diseases.

15. The application according to claim 14, characterized in that, The angiogenesis-related disease mentioned is choroidal neovascularization (CNV).

16. A method for gene editing of target DNA in vitro, characterized in that, The step includes introducing the protein variant of claim 1 or 2 or the gene editing system of claim 5 into a cell containing target DNA.

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