Application of sequence preference cytosine deaminase in base editing

Abstract

The invention discloses an application of sequence preference cytosine deaminase in base editing, and the amino acid sequence of the sequence preference cytosine deaminase comprises one of SEQ ID No: 3 to SEQ ID No: 80. The invention discloses a series of cytosine deaminase with activity, wherein the cytosine deaminase comprises a plurality of sequence preference cytosine deaminase. Experimental results show that a cytosine base editing system constructed on the basis of the cytosine deaminase can only efficiently edit cytosine with specific sequence preference in a genome, so that editing of non-target cytosine is reduced. The method can provide important technical support for research of point mutation in a genome, creation of a special site disease model and correction of genetic mutation sites, and has a wide application prospect in the fields of precision medicine and agricultural breeding.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and more specifically, to the application of a sequence-biased cytosine deaminase in base editing. Background Technology

[0002] Gene editing technology rapidly and efficiently induces single-nucleotide mutations in organisms by knocking out, knocking in, replacing, screening large fragments, and rearranging specific target gene sequences. This technology provides strong support for the comprehensive analysis of genome structure and function, precision medicine, and even precision breeding of plants and animals. The CRISPR / Cas system uses sgRNA and Cas nucleases to target genome sequences for multi-site gene editing. Base editing technology, leveraging the CRISPR / Cas system's ability to target genome sequences, inactivates Cas nickase by single-strand cutting and combines it with deaminases, achieving precise gene editing through single-base substitution of DNA.

[0003] Sequence preferences of deaminases enable the editing of specific bases within a window, significantly reducing bystander editing. Therefore, developing base editing systems with specific sequence preferences is crucial for precision medicine and breeding. Currently, the number of deaminases used in this field with sequence preference selection is limited, restricting the application of gene editing to treat many important disease sites. Developing novel base editing systems with new sequence preferences will provide more possibilities for expanding the applications of gene editing. Summary of the Invention

[0004] This application addresses the shortcomings of existing technologies by proposing the application of a sequence-biased cytosine deaminase in base editing, thereby solving the technical problem of limited selectable editing sites in related technologies.

[0005] This application provides an application of a sequence-biased cytosine deaminase in base editing, wherein the amino acid sequence of the sequence-biased cytosine deaminase includes one of SEQ ID No:3 to SEQ ID No:80.

[0006] Alternatively, the nucleotide sequence of the sequence-preferred cytosine deaminase includes one of the following: SEQ ID No:4, SEQ ID No:6, SEQ ID No:9, SEQ ID No:13, SEQ ID No:14, SEQ ID No:19, SEQ ID No:22, SEQ ID No:23, SEQ ID No:28, SEQ ID No:36, SEQ ID No:50, SEQ ID No:52, SEQ ID No:53, SEQ ID No:55, SEQ ID No:56, SEQ ID No:57, SEQ ID No:59, SEQ ID No:61, SEQ ID No:65, SEQ ID No:70, SEQ ID No:73, SEQ ID No:80.

[0007] Furthermore, the sequence-preferred cytosine deaminase recognizes a cytosine base located one position after the 3' end of a specific base, said specific base including at least one of adenine, thymine, cytosine, and guanine.

[0008] Furthermore, the nucleic acid sequence used to construct the expression vector of the sequence-biased cytosine deaminase is as shown in SEQ ID No:1 or SEQ ID No:2.

[0009] Alternatively, cells that can accept the expression vector include rice protoplasts or human HEK293T cells.

[0010] Alternatively, the human HEK293T cells can be used to construct a base-editing library cell line to verify the characteristics of the sequence-biased cytosine deaminase.

[0011] Alternatively, the base editing is performed using a gene editing system comprising a base editor component and a base editing guide RNA component, the base editor component comprising the sequence-preferred cytosine deaminase.

[0012] Furthermore, the base editing is used to correct pathogenic sites in the human genome and / or to achieve precise editing of the plant genome.

[0013] The beneficial technical effects of the technical solutions provided in this application include: This application identifies a series of active cytosine deaminases, including several sequence-biased cytosine deaminases. Experimental results show that a cytosine base editing system built based on these cytosine deaminases can efficiently edit only specific sequence-biased cytosines in the genome, thereby reducing the editing of non-target cytosines. This invention can provide important technical support for the study of point mutations in the genome, the creation of disease models at specific sites, and the correction of genetic mutation sites, showing broad application prospects in precision medicine and agricultural breeding.

[0014] Additional features and advantages of this application will be set forth in part in the description which follows, and will become apparent from the description or may be learned by practice of this application. Attached Figure Description

[0015] The above and / or additional contents and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 This application provides a schematic diagram of the construction of a preferred cytosine deaminase vector; Figure 2 A schematic diagram illustrating the cell line construction of the base editing library used in this application; Figure 3 This application presents an analysis of the editing window and efficiency of a base editor containing a sequence-biased cytosine deaminase in a base editing library cell line. Figure 4 This application includes an analysis of the editing preferences of a base editor containing sequence-biased cytosine deaminase in a base editing library cell line; Figure 5 This application includes a schematic diagram illustrating the base editing principle of a base editor containing a sequence-biased cytosine deaminase; Figure 6 This application includes a base editor containing sequence-biased cytosine deaminases for preference analysis at five endogenous sites in rice; Figure 7 This application includes a base editor containing sequence-biased cytosine deaminases for endogenous site preference analysis in four human cell lines. Figure 8 Editing of three preferred deaminases at endogenous sites in plants and animals; Figure 9 Statistics on the proportion of human disease loci with different sequence preferences; Figure 10 The base editor in this application can accurately edit statistics on rice loci types. Detailed Implementation

[0016] The embodiments of this application are described below with reference to the accompanying drawings. It should be understood that the embodiments described below with reference to the accompanying drawings are exemplary descriptions for explaining the technical solutions of the embodiments of this application, and do not constitute a limitation on the technical solutions of the embodiments of this application.

[0017] Those skilled in the art will understand that, unless specifically stated otherwise, the terms "described" and "the" as used herein may also include plural forms. It should be further understood that the word "comprising" as used in the specification of this application means the presence of the described features, integers, steps, or operations, but does not exclude other features, information, data, steps, operations, and / or combinations thereof supported by this art.

[0018] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0019] Sequence preferences of deaminases enable the editing of specific bases within a window, significantly reducing bystander editing. Therefore, developing base editing systems with specific sequence preferences is crucial for precision medicine and breeding. Currently, the number of deaminases with sequence preference selection in this field is limited, restricting gene editing for treating many important disease sites. Since the development of cytosine base editing tools in 2016, scientists have discovered hundreds of different types of deaminases, but the number of deaminases with significant preferences is small, and the types of preferences they can achieve are very limited, with only a few concentrated preference types such as TC preference (e.g., eA3A or A3Bctd deaminases), CC preference (e.g., A3G deaminases), and GC / AC preference (e.g., Sdd3), far from meeting the needs of precise editing. Xu et al. systematically tested the editing characteristics of 272 newly identified deaminases, finding that only about a dozen showed significant preferences, mainly concentrated on the previously reported TC preference. Huang et al. analyzed 332 deaminases and identified 45 active single-stranded deaminases. Among them, only three deaminases showed significant sequence bias: CC bias (Sdd59) and GC / AC bias (Sdd3 and Sdd10). Therefore, developing base editing systems with novel sequence biases will provide more possibilities for expanding gene editing applications.

[0020] Materials and Methods 1. Carrier Construction The target fragments optimized from rice and human bicodons were constructed into the vectors SEQ ID NO:1 and SEQ ID NO:2.

[0021] 2. Isolation and transformation of rice protoplasts The protoplasts used in this invention are all derived from the rice variety Zhonghua "11".

[0022] 2.1 Rice seedling cultivation (1) Place the unopened sterile water, sterilized filter paper, tweezers, rolling pin, petri dish, test tube rack, and 50 ml centrifuge tube in the laminar flow hood and sterilize with ultraviolet light for 20 min. (2) Take about 15 mL of rice seeds in a 50 mL centrifuge tube, add 75% ethanol and rinse for 1 min, repeat twice; (3) Remove the supernatant, add 2.5% sodium hypochlorite solution (containing Tween 20) to about 40 mL, place in a mixer and stir at 40 r / min for 20 min; (4) Transfer the glass bottle containing MS medium to the laminar flow hood for later use, turn on the glass bead sterilizer and insert the tweezers upside down into the glass beads for sterilization; (5) Pour out the 2.5% sodium hypochlorite solution in the clean bench, and add an appropriate amount of sterile water to the rice seeds to rinse. Repeat 5-6 times until no obvious turbidity is observed in the liquid in the centrifuge tube. (6) Use the nozzle to draw out the excess liquid from the seeds, then pour the rice seeds evenly onto the filter paper, and then cover them with another layer of filter paper to absorb the moisture. The specific standard is that the seeds are matte in color when the moisture has been absorbed. (7) Use tweezers to pick up rice seeds and spread them evenly on the culture medium. Then use a rolling pin to gently press the seeds into the culture medium, seal them again with a rubber band, and place them in a 26℃ light-proof constant temperature incubator for 2-3 weeks to obtain etiolated seedlings.

[0023] 2.2 Protoplast Preparation (1) Preliminary preparation: Wipe the experimental table clean, cover the table surface with garbage bags or plastic wrap, clean the scissors and tweezers, prepare the Flying Eagle blade, plastic petri dishes, aluminum foil, sterilized 100 mL conical flasks and 0.45 μm filter membranes, prepare the water bath to 55℃ in advance, prepare the enzymatic hydrolysate and 0.6 mol / L mannitol (after filtration), and confirm that W5 is sufficient and free from contamination.

[0024] (2) Cut off the roots and leaves of the seedlings, leaving the stems and leaf sheaths. Wipe off the moisture on the surface of the stems and cut them into strips about 0.5 mm wide with a new blade. Immediately after cutting, transfer them to a 0.6 mol / L mannitol solution and place them in the dark for 10 min. Cut off a suitable amount of Magic Filter Cloth, filter out the mannitol solution, transfer the stem segments to the enzymatic hydrolysate in the dark, vacuum pump (value displayed as 0.6) for 30 min, and enzymatically hydrolyze them in a shaker at 26℃ at 40 r / min in the dark for 4 h.

[0025] (3) Filter out the enzyme hydrolysate, add W5 solution (less than 40 mL each time) to the conical flask, gently shake horizontally for 10 s, filter the protoplasts into a 50 mL round-bottom centrifuge tube using a 40 μm nylon membrane, release the protoplasts, and repeat the above operation to wash 4 times. (4) Centrifuge horizontally at 250 r / min for 3 min to precipitate protoplasts, and aspirate the supernatant with 5 mL and 1 mL pipettes; resuspend the protoplasts in 10 mL of W5 solution, centrifuge at 250 r / min for 3 min at room temperature, and discard the supernatant; resuspend in an appropriate amount of MMG solution to achieve a protoplast concentration of 2 × 10⁻⁶. 6 / mL, blood cell counter count.

[0026] 2.3 Protoplast Transformation (1) Add 5 μg of plasmid to a 2 mL round-bottom centrifuge tube (if there are multiple plasmids, add 5 μg of each plasmid), and add 200 μL of protoplasts (approximately 4 × 10⁻⁶). 5 Add 220 μL of PEG solution to the cells, quickly invert to mix, and incubate at room temperature in the dark for 25 min to induce transformation; (2) After the induction and transformation is complete, slowly add 1 mL of W5 solution, gently invert to mix, centrifuge horizontally at 250 r / min for 3 min at room temperature, and discard the supernatant; (3) Resuspend in 1 mL W5 solution. If fluorescence needs to be observed, transfer to a 24 or 48-well plate. If used to extract protoplast genomic DNA, place the round-bottom centrifuge tube in an EP tube rack and incubate in the dark. (4) Extract DNA after culturing for 60-72 h.

[0027] 3. Construction of cell lines for base editing libraries (SG libraries) (1) HEK293T cells were plated one day before transfection using 10 cm dishes, with 5 million cells per dish. Transfection was performed 24 hours after plating, when the cells reached approximately 80% confluence. Each well contained approximately 17 μg of plasmid DNA, 8.5 μg of sg library DNA, and 8.5 μg of transposase DNA, along with 340 ng of fluorescent protein (GFP) as a marker. Transfection was performed using Lipoo 8000 lipid nanoparticles. One hour before transfection, the serum-containing medium was replaced with serum-free medium for starvation treatment, and 4-6 hours after transfection, the medium was replaced back with serum-containing medium. Cells were then cultured at 37°C in a 5% CO2 incubator.

[0028] (2) 48 h after transfection, the culture medium was replaced with (1-2 ug / mg) puromycin medium; (3) Change the culture medium every 2-3 days and continue screening for 12-14 days until the cells are stable and then freeze them.

[0029] 4. Cell plating and transfection For endogenous target detection, HEK293T cells were plated one day before transfection using 48-well plates, with 45,000 cells per well. 24 hours after plate formation, transfection was performed when cell confluence reached approximately 80%, with approximately 1 μg plasmid DNA, 750 ng editor + 250 ng guide RNA, and 20 ng GFP as a marker per well. For SG library cell line detection, SG library cells were plated one day before transfection using 12-well plates, with 300,000 cells per well. 24 hours after plate formation, transfection was performed when cell confluence reached approximately 80%, with approximately 3 μg plasmid DNA, and 80 ng GFP as a marker per well.

[0030] Transfection was performed using Lipoo 8000 lipid nanoparticles. One hour before transfection, the serum-containing medium was replaced with serum-free medium for starvation treatment. Four to six hours after transfection, the medium was replaced with serum-containing medium. Cells were cultured at 37°C in a 5% CO2 incubator for 72 hours, after which DNA was lysed and collected.

[0031] Example 1: The base editing system of this application exhibits bias when tested on a base editing library. This invention constructs a base editing tool for testing 78 deaminases of the SCP1.201 family whose activity has not been verified (as shown in SEQ ID No:3~SEQ ID No:80). Figure 1 To accurately evaluate the characteristics of base editing tools based on these deaminases, 400 sgRNAs and their corresponding target sequences were inserted into the genome of the HEK293T cell line based on the principle of transposition. Figure 2 By delivering base editing tools to sgRNA library cell lines, 22 active deaminases were identified. Figure 3 The names and sequence listing IDs of these 22 deaminases are shown in Table 1. Among them, the SCPB31 deaminase base editing system, based on SCPF12, exhibits a strong TC preference; SCPF4 exhibits a very strong CC preference; SCPB4 exhibits a strong CC and AC preference; and SCPB24 exhibits a strong AC and TC preference. Figure 4 Among them, deaminases with strong CC and AC preference, as well as those with strong AC and TC preference, are reported for the first time.

[0032] Based on the construction of cell lines for base editing libraries, this application enables high-throughput mining and testing of cytosine deaminases, overcoming the difficulty of finding cytosine deaminases with specific base preferences in existing technologies. Following this concept, other types of base-preferenced deaminases can also be mined in high throughput, which is beneficial for constructing new gene editing systems.

[0033] Table 1. Deaminases identified with editing activity

[0034] Example 2: The base editing system of this application showed preference in tests on human or plant endogenous sites. This discovery of sequence-biased cytosine deaminases allows for the selection of appropriate deaminases based on the sequence preference of the target editing site to construct a base editing system. This system can then replace cytosine with thymine at any position, achieving precise editing. Figure 5 ).

[0035] To further confirm the editing preference of the aforementioned base editing system at endogenous sites, this embodiment tested the editing characteristics at endogenous sites in humans and rice. In this embodiment, sgRNAs were constructed at 5 endogenous sites in rice and 4 endogenous sites in humans (endogenous site information is shown in Table 2), and co-transformed with the aforementioned base editing system into rice protoplasts or co-transfected into human cell lines. The results at the endogenous sites showed (…). Figure 6 and Figure 7 In the statistical analysis of editing at five endogenous sites in rice, SCPF12 and SCPB31 showed a clear TC preference, while SCPB4 showed both AC and CC preferences, consistent with the results presented in the transfected sg library. In the statistical analysis of editing at four endogenous sites in humans, SCPF4 showed a clear CC preference, SCPB4 showed both AC and CC preferences, and SCPF12 showed a TC preference, consistent with the results presented in the transfected sg library. At these endogenous sites, the aforementioned deaminases all exhibited significant preferences and high editing efficiency. For example, SCPF4 (CC preference) at the OsCDC48-T2 site only edited the CC at positions 4 and 8, while the editing efficiency of AC, GC, and TC around these positions was very low. SCPF12 (TC / CC preference) at HsFANCF-T1 only edited the TC / CC at positions 6, 7, 8, and 11, while almost no editing occurred at the nearby GC. SCPB4 (CC / AC bias) at the HsHEK3-T1 site only involves editing of CC and AC at positions 4, 5, and 9, while GC and TC around these positions are almost unedited. Figure 8 ).

[0036] Table 2. Rice and human endogenous site sequences used for testing sequence-preferred cytosine deaminase characteristics.

[0037] Example 3: The preferred base editing system of this application can be used for pathogenic site correction and precise editing of plant genomes. In the medical field, according to the ClinVar database of pathogenic sites in the human genome (December 1, 2024 version), there are 598,742 single-base diseases caused by T-to-C mutations in DNA. These diseases can be treated by correcting the C-to-T mutation using a cytosine base editor. The cytosine deaminases mined in the above examples—SCPF12 with a TC preference, SCPF4 with a CC preference, SCPB4 with an AC / CC preference, and SCPB24 with an AC / TC preference—can effectively solve the problem of flanking editing in the following situations. Taking SCPF4 with a CC preference as an example, when the target cytosine disease site is 5'-CC-3', and if the five positions before and after it in the genome are other preferred cytosines (AC, GC, TC), using SCPF4 with a CC preference to treat the disease site can achieve precise CC editing without editing other preferred C cytosines, thus achieving precise editing. Therefore, this embodiment evaluated the number of sites in the database that can achieve precise treatment without side-side editing using the aforementioned biased base editing system compared to an unbiased base editing system. By taking 5 bp sequences before and after the C sequence requiring correction, it was statistically determined that 57,317 pathogenic sites can be precisely corrected and treated using the TC biased base editing system developed in this study without side-side editing; 62,517 diseases can be precisely corrected and treated without side-side editing using the CC biased base editing system of this application; 114,995 diseases can be precisely corrected and treated without side-side editing using the CC / AC biased base editing system of this application; and 105,283 diseases can be precisely corrected and treated without side-side editing using the AC and TC biased base editing systems of this application. Figure 9 These data fully demonstrate the irreplaceable and significant value of the deaminase preference mechanism in this application for precision gene therapy. This application further evaluates the ability of preference-based deaminases to achieve precise single-base substitution without flanking editing in plants. Plant genome-wide statistical analysis revealed that when the editing target is CC, compared to conventional deaminase systems, the SCPF4 preference-based deaminase can overcome 48.47% of flanking editing, achieving precise single-base editing. Similarly, when the editing targets are TC, AC / CC, and AC / TC, the preference-based deaminase can overcome 45.23%, 24.3%, and 24.83% of flanking editing, respectively. Figure 10These results indicate that the aforementioned preferred deaminases provide highly precise and pure editing tools for plant genetic improvement, enabling more efficient and accurate regulation of traits such as plant growth and development, stress resistance, yield, and quality, thus providing strong support for the sustainable development of agricultural production.

[0038] In summary, this application provides an application of a sequence-preferred cytosine deaminase in base editing, wherein the amino acid sequence of the sequence-preferred cytosine deaminase includes one of SEQ ID No:3 to SEQ ID No:80. This application has identified a series of active cytosine deaminases, including several sequence-preferred cytosine deaminases. Experimental results show that a cytosine base editing system constructed based on this cytosine deaminase can efficiently edit only specific sequence-preferred cytosines in the genome, thereby reducing the editing of non-target cytosines. This invention can provide important technical support for point mutation research in the genome, the creation of disease models at specific sites, and the correction of genetic mutation sites, showing broad application prospects in precision medicine and agricultural breeding.

[0039] Those skilled in the art will understand that the steps, measures, and solutions in the various operations, methods, and processes discussed in this application can be alternated, modified, combined, or deleted. Furthermore, other steps, measures, and solutions in the various operations, methods, and processes discussed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and solutions in related technologies that are similar to those disclosed in this application can also be alternated, modified, rearranged, decomposed, combined, or deleted.

[0040] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0041] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0042] The above description is only a partial implementation of this application. It should be noted that for those skilled in the art, other similar implementation methods based on the technical concept of this application, without departing from the technical concept of this application, also fall within the protection scope of the embodiments of this application.

Claims

1. The application of a sequence-biased cytosine deaminase in base editing, characterized in that, The amino acid sequence of the sequence-biased cytosine deaminase includes one of SEQ ID No:3 to SEQ ID No:

80.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the sequence-preferred cytosine deaminase includes one of the following: SEQ ID No:4, SEQ ID No:6, SEQ ID No:9, SEQ ID No:13, SEQ ID No:14, SEQ ID No:19, SEQ ID No:22, SEQ ID No:23, SEQ ID No:28, SEQ ID No:36, SEQ ID No:50, SEQ ID No:52, SEQ ID No:53, SEQ ID No:55, SEQ ID No:56, SEQ ID No:57, SEQ ID No:59, SEQ ID No:61, SEQ ID No:65, SEQ ID No:70, SEQ ID No:73, SEQ ID No:

80.

3. The application as described in claim 1, characterized in that, The sequence-preferred cytosine deaminase recognizes a specific base located one position after the 3' end of a particular base, the specific base including at least one of adenine, thymine, cytosine, and guanine.

4. The application as described in claim 1, characterized in that, The nucleic acid sequence used to construct the expression vector of the sequence-biased cytosine deaminase is as shown in SEQ ID No:1 or SEQ ID No:

2.

5. The application as described in claim 4, characterized in that, Cells that can accept the expression vector include rice protoplasts or human HEK293T cells.

6. The application as described in claim 5, characterized in that, A base-editing library cell line was constructed using the human HEK293T cells to verify the characteristics of the sequence-biased cytosine deaminase.

7. The application as described in claim 1, characterized in that, The base editing is performed using a gene editing system, which includes a base editor component and a base editing guide RNA component, wherein the base editor component includes the sequence-preferred cytosine deaminase.

8. The application as described in claim 1, characterized in that, The base editing is used to correct pathogenic sites in the human genome and / or to achieve precise editing of the plant genome.

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