OpenCRISPR fusion protein for gene editing of cyprinid fishes and application of OpenCRISPR fusion protein

Abstract

The invention discloses an OpenCRISPR fusion protein for gene editing of cyprinid fishes and application of the OpenCRISPR fusion protein, and belongs to the technical field of fish gene editing. In order to solve the technical problem that a gene editor of cyprinid fishes is low in efficiency and the like, OpenCRISPR protein is designed by utilizing artificial intelligence, c-myc-NLS nuclear signal peptide is fused on the basis, the OpenCRISPR fusion protein is obtained, the editing efficiency of the OpenCRISPR fusion protein in the cyprinid fishes (such as zebra fishes) can be improved, and the OpenCRISPR fusion protein has an application prospect in gene editing of the cyprinid fishes.

Description

Technical Field

[0001] This invention belongs to the field of fish gene editing technology, specifically relating to an OpenCRISPR fusion protein for gene editing in cyprinid fish and its applications. Background Technology

[0002] Gene editing technology, as a core tool of modern molecular breeding, has demonstrated enormous potential in the genetic improvement of aquatic animals. However, current gene editing practices in fish, particularly in cyprinid fish (such as zebrafish, carp, and crucian carp), still face severe technical bottlenecks. Currently, mainstream high-efficiency gene editors, such as SpCas9 (Streptococcus pyogenes Cas9, the core tool protein first developed and widely used by American researchers) derived from Streptococcus pyogenes, and its derivatives such as single-base editors (BE) and lead editors (PE), are primarily designed and optimized based on mammalian cell models. These editors generally exhibit low editing efficiency and poor stability in cyprinid fish cells and embryos, resulting in gene knockout and knock-in success rates that fail to meet the demands of high-efficiency breeding, severely restricting their large-scale application in precision breeding of cyprinid fish.

[0003] The core reason for the aforementioned predicament lies in the lack of deep adaptation of existing editors to the biological characteristics of cyprinid fishes. Cyprinid fishes possess unique genomic structures, chromatin states, cell cycle characteristics, and physiological temperature ranges. Currently widely used Cas proteins (represented by SpCas9) and their accompanying guide RNA systems have not been systematically optimized in terms of expression levels, protein stability, nuclear localization efficiency, and synergy with endogenous repair mechanisms in cyprinid cells. Specifically, exogenous Cas proteins may face problems such as insufficient expression levels, misfolding, and rapid degradation rates in fish cells, and their nuclear transport efficiency is low, making it difficult to accumulate sufficient concentrations of active proteins in the nucleus to effectively perform editing functions. Therefore, simply using editors designed for mammals cannot overcome the technical obstacles in cyprinid fishes.

[0004] While some existing technologies have attempted to improve Cas protein expression in fish through conventional methods such as codon optimization and promoter replacement, these improvements are conventional techniques in the field, offering limited enhancements and lacking universality. They fail to fundamentally address the issue of insufficient editor activity in cyprinid fishes. Finding or designing novel Cas protein or editor architectures that can naturally adapt or be deeply modified to function efficiently in cyprinid fishes has become a critical technical challenge urgently requiring breakthroughs in this field.

[0005] In recent years, breakthroughs in artificial intelligence technology, particularly deep learning-based protein structure prediction and design methods (such as AlphaFold2 and RFdiffusion), have provided powerful new tools for de novo design or deep modification of functional proteins. AI models can predict and optimize key properties of proteins, such as folding stability, active site conformation, and substrate binding affinity, opening up unprecedented possibilities for developing novel gene-editing tools with customized capabilities.

[0006] In summary, given the current inefficiency of gene editors in cyprinid fish, an innovative solution is urgently needed in this field. This solution should not be limited to fine-tuning existing tools, but should focus on revolutionary design concepts—for example, using artificial intelligence to design a novel Cas protein architecture from scratch or through deep learning, coupled with precise cell biology modifications (such as optimizing nuclear localization signals)—to create a new gene editing tool with ultra-high expression, efficient nuclear entry, excellent stability, and strong editing activity in cyprinid fish cells. This will be key to advancing gene editing breeding technology in cyprinid fish and other aquatic animals to a new level. Summary of the Invention

[0007] To address the technical problems of low efficiency in gene editors for cyprinid fish, this invention utilizes artificial intelligence to design an OpenCRISPR protein and fuses it with a c-myc-NLS nuclear signal peptide. The resulting OpenCRISPR fusion protein can improve its editing efficiency in zebrafish.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide an OpenCRISPR fusion protein for gene editing in cyprinid fishes, the gene encoding the OpenCRISPR fusion protein being shown in SEQ ID NO.5.

[0009] In one embodiment of the present invention, the OpenCRISPR fusion protein is composed of the CRISPR effector protein OpenCRISPR-1, which is designed and predicted by an artificial intelligence algorithm, and the nuclear signal peptide c-myc-NLS.

[0010] In one embodiment of the present invention, the amino acid sequence of the CRISPR effector protein OpenCRISPR-1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the nucleotide sequence of the nuclear signal peptide c-myc-NLS is shown in SEQ ID NO.3.

[0011] The second objective of this invention is to provide the application of the aforementioned OpenCRISPR fusion protein in gene editing of cyprinid fishes.

[0012] A third objective of this invention is to provide a recombinant expression vector containing the OpenCRISPR fusion protein encoding gene described above.

[0013] A fourth objective of this invention is to provide the application of the above-mentioned recombinant expression vector in gene editing of cyprinid fishes.

[0014] A fifth object of the present invention is to provide a gene editing system, the system comprising: a) The OpenCRISPR fusion protein described above, or its mRNA, or the nucleotide sequence encoding the OpenCRISPR fusion protein described above; b) At least one guide RNA, said guide RNA being able to guide the OpenCRISPR fusion protein to target a DNA sequence.

[0015] The sixth objective of this invention is to provide the application of the above-described gene editing system in gene editing of cyprinid fish.

[0016] A seventh object of the present invention is to provide a kit comprising the above-described gene editing system.

[0017] The eighth object of the present invention is to provide the application of the above-described kit in gene editing of cyprinid fish.

[0018] The beneficial effects of this invention are: This invention utilizes artificial intelligence to design OpenCRISPR proteins, and then fuses c-myc-NLS and H2B nuclear entry signal peptides to obtain two OpenCRISPR fusion proteins (OP-c-myc and OP-H2B). The editing efficiency of these OpenCRISPR fusion proteins ranges from 8.3% to 31.25%, with an INDEL mutation rate of 51.3% in a single sample. Further analysis of the effects of the two nuclear entry signal peptides on the editing efficiency of the OpenCRISPR proteins revealed that OP-H2B had an editing efficiency of 0% to 8.3%, while OP-c-myc had an editing efficiency of 25% to 31.25%, indicating that the c-myc-NLS nuclear entry signal peptide is more suitable for gene editing in zebrafish embryos. The OpenCRISPR fusion proteins provided by this invention can improve their editing efficiency in cyprinid fish (such as zebrafish), and have promising applications in gene editing of cyprinid fish. Attached Figure Description

[0019] Figure 1 Observational image of body color changes in zebrafish embryos after injection; Figure 2Image showing the PAGE gel assay results for gene editing in zebrafish; the red box represents mutant individuals, and the arrows represent controls; Figure 3 This is a diagram showing the multiple sequence alignment results of the mutant. Figure 4 This is a graph showing the analysis of Sanger sequencing results; in which, Figure 4 In the figure, A represents the analysis results of SPCas9 gene editing. Figure 4 B in the figure represents the analysis results of OpenCRISPR-1 gene editing. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0022] The AB strain zebrafish used in this study were purchased from the National Zebrafish Resource Center (http: / / www.zfish.cn) and raised at the Zebrafish Breeding Center of the Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences. The rearing conditions were 28.5°C, with feeding twice daily of Artemia. The light-dark cycle ratio was 14:10 (14 h light: 10 h dark). Zebrafish fertilized eggs were cultured in a 28.5°C constant-temperature incubator. After 4-5 days post-fertilization (dpf), the eggs were able to swim horizontally and were then transferred to 10 L rearing tanks, fed Paramecium. After 15 dpf, they were transferred to a recirculating aquaculture system and fed Artemia again.

[0023] Example 1: This embodiment achieves efficient editing of the zebrafish genome by designing a fusion protein and a corresponding nucleotide construct. The core of this method lies in using an OpenCRISPR-1 sequence designed and optimized by artificial intelligence (AI) and combining it with nuclear localization signals (c-myc-NLS or H2B) for fusion to form an OP-c-myc or OP-H2B fusion protein.

[0024] The specific implementation method includes the following steps: 1. AI design and optimization of OpenCRISPR-1 sequences (1) Dataset preparation and model training The first step in the design process was to construct and organize a large-scale dataset containing sequences of various CRISPR effector proteins (https: / / www.profluent.bio / ). These sequences came from a wide range of genomic and metagenomic data, including various known CRISPR-Cas systems in nature. Through screening and annotation, the research team extracted key functional regions that play a central role in DNA cutting and genome editing. Subsequently, deep learning techniques were used to process these sequence data. The training model was based on large language models (LLMs), which are originally used for natural language processing but were adapted and optimized in this invention for protein sequence generation and optimization. By inputting a large amount of sequence information of CRISPR effector proteins, the model learned the complex relationship between protein sequences and their functions, and was able to predict the impact of sequence variations on protein function.

[0025] (2) Sequence design and generation Based on the trained model, the AI ​​system began generating new CRISPR effector protein sequences. The OpenCRISPR-1 sequence was generated during this process. The AI ​​design focused on ensuring that the newly generated sequences, while preserving core functions, introduced adaptive variations to meet the specific genome editing needs of zebrafish. The AI-designed OpenCRISPR-1 sequence structurally retained the key nuclease activity regions of the CRISPR system, but introduced optimized sequence variations in non-conserved regions. These variations, accurately predicted by the AI ​​model, enhanced protein stability and improved its functional expression in non-natural hosts. Furthermore, the design considered edit specificity, ensuring that the generated OpenCRISPR-1 protein could achieve highly precise targeted editing within the zebrafish genome.

[0026] (3) Sequence optimization and validation After generating the initial OpenCRISPR-1 sequence, further optimization steps ensure its effectiveness in zebrafish. First, an AI model simulates the binding and cleavage process of the OpenCRISPR-1 protein with the target DNA sequence in the zebrafish genome to assess its editing efficiency and off-target risks. Through these simulations, the AI ​​can identify potential off-target effects and adjust the sequence to minimize non-specific cleavage. After determining the optimal sequence, codon optimization is performed. This process adjusts the coding sequence of the OpenCRISPR-1 protein to better suit the translation preferences of zebrafish cells. This optimization improves protein expression levels and stability, thereby ensuring efficient function in practical applications.

[0027] 2. Constructing nucleotide expression vectors (1) Nucleotide sequence design and optimization The amino acid sequence of OpenCRISPR-1, designed using AI, is shown in SEQ ID NO.1. Zebrafish-specific codon optimization was performed on the coding sequence of OpenCRISPR-1 to improve protein expression efficiency in target cells. The coding sequence of OpenCRISPR-1 after codon optimization is shown in SEQ ID NO.2.

[0028] (2) Carrier construction Two OpenCRISPR-1 fusion proteins (OP-c-myc and OP-H2B) were constructed by fusing the OpenCRISPR-1 coding sequence, as shown in SEQ ID NO.2, with the nuclear insertion signal peptides c-myc-NLS and H2B, respectively (the nucleotide sequence of c-myc-NLS is shown in SEQ ID NO.3, and the nucleotide sequence of H2B is shown in SEQ ID NO.4). The nucleotide sequence encoding the fusion protein OP-c-myc is shown in SEQ ID NO.5, and the nucleotide sequence encoding the fusion protein OP-H2B is shown in SEQ ID NO.6.

[0029] (3) Constructing a fusion vector of OpenCRISPR-1 fusion protein and pcDNA-3.1 vector. The synthesized OP-c-myc and OP-H2B nucleotide sequences were inserted into the pcDNA-3.1 vector using molecular cloning techniques to construct the eukaryotic expression plasmids pcDNA3.1-OP-c-myc and pcDNA3.1-OP-H2B. Restriction endonuclease digestion and PCR amplification were used to verify the correct insertion of the OP-c-myc and OP-H2B sequences into the vector. Sequencing was used to confirm the integrity and correctness of the inserted sequences.

[0030] (4) Preparation of OpenCRISPR-1 fusion protein mRNA Using the constructed plasmid as a template, PCR amplification was performed using specific primers OP-Dr-F1 (SEQ ID NO.7) and OP-Dr-R1 (SEQ ID NO.8) to obtain a linearized DNA template containing the T7 promoter. Subsequently, the linearized template was transcribed in vitro using the mMessagemMachine T7 Ultra kit, and after tailing and capping reactions, highly intact OpenCRISPR-1 protein mRNA was finally purified.

[0031] 3. Design and synthesis of sgRNA targeting the zebrafish slc45a2 gene OpenCRISPR-1 protein is a Cas9-like nuclease that is designed or deeply engineered from scratch using artificial intelligence algorithms. Its mechanism of recognizing and cutting DNA is similar to that of the SpCas9 protein derived from Streptococcus pyogenes, both relying on the guidance of guide RNA and using NGG as the adjacent motif of the prototype spacer region.

[0032] To verify the gene editing activity of OpenCRISPR-1 in zebrafish, this study selected the zebrafish slc45a2 gene as the target gene. The loss of function of this gene leads to a skin albinism phenotype, which makes it easy to observe the editing effect directly.

[0033] The specific steps are as follows: The online design tool ChopChop (https: / / chopchop.cbu.uib.no / ) was used to scan and design sgRNA target sites in exon 1 of the zebrafish slc45a2 gene. sgRNA in vitro transcription templates for the selected target sites were designed and synthesized for both OpenCRISPR-1 and SpCas9 systems.

[0034] OpenCRISPR-1 system: DNA transcription templates for sgRNA were prepared by overlap extension PCR using the upstream primer OpenCRISPR-1-F as shown in SEQ ID NO.9 and the downstream primer OpenCRISPR-1-R as shown in SEQ ID NO.10.

[0035] SpCas9 system (control): DNA transcription templates for sgRNA were prepared by overlapping extension PCR using the upstream primer SpCas9-F as shown in SEQ ID NO.11 and the downstream primer SpCas9-R as shown in SEQ ID NO.12.

[0036] The DNA template obtained above was used with HiScribe TMThe T7 Quick High Yield RNA Synthesis Kit was used for in vitro transcription, and the purified sgRNA was obtained.

[0037] The nucleotide sequence of the OpenCRISPR-1 specific sgRNA (OpenCRISPR-1 SgRNA) obtained by this method is shown in SEQ ID NO.13; the nucleotide sequence of the SpCas9 specific sgRNA (SpCas9 SgRNA) obtained by this method is shown in SEQ ID NO.14.

[0038] 4. Microinjection of zebrafish embryos Zebrafish single-cell embryos were collected for microinjection.

[0039] Experimental group: OP-c-myc mRNA or OP-H2B mRNA at a final concentration of 300 ng / μL was mixed with 200 ng / μL of OpenCRISPR-1 sgRNA targeting slc45a2 and injected into the cytoplasm of the embryo.

[0040] Control group: Commercially available SpCas9 protein at a final concentration of 5 pmol / μL was mixed with 200 ng / μL of SpCas9 sgRNA targeting slc45a2 and injected into the cytoplasm of the embryo.

[0041] The injected embryos were placed in a culture dish containing embryo culture medium and incubated in a constant temperature incubator at 28.5°C.

[0042] 5. Gene editing efficiency and phenotypic analysis (1) Phenotypic observation Pigmentation in embryos was observed under a stereomicroscope at 24 hpf, 48 hpf, and 72 hpf. Individuals with successfully edited slc45a2 genes will exhibit varying degrees of albinism. Figure 1 As shown, compared to the wild type, the slc45a2 mutant showed lighter body color at 24 hpf, 48 hpf, and 72 hpf.

[0043] The phenotypic mutation rates in embryos injected with OP-1-c-myc, OP-1-H2B, and SpCas9 were 24.3%, 2.1%, and 46%, respectively (Table 1). These results indicate that OP-c-myc, OP-H2B, and SpCas9 can all induce mutations in slc45a2, inhibiting melanin production.

[0044] Table 1. Statistical results of mutation rates observed in different experimental groups.

[0045] (2) Gene editing efficiency analysis Primers near the target site were designed using Primer 3.0 Plus software. The mutation detection efficiency was achieved in embryos aged 24 hpf to 48 hpf. Mutants were detected and screened according to the method described by Tang Guopan et al. (Tang Guopan, Huang Anqun, Sun Zhipeng, et al. Comparative analysis of three CRISPR / Cas9 gene knockout mutation detection methods [J]. Freshwater Fisheries, 2019, 49(02): 20-26.). PCR products from mutant individuals were sequenced and mutation types were analyzed. The detection primers were slc45a2-F (SEQ ID NO.15) and slc45a2-R (SEQ ID NO.16).

[0046] Polyacrylamide gel electrophoresis results showed that the editing efficiency of OpenCRISR protein ranged from 8.3% to 31.25%, with an INDEL mutation rate of 51.3% in a single sample; the editing efficiency of SpCas9 was approximately 50%, with an INDEL mutation rate of 27.8% in a single sample. Further analysis of the effects of two nuclear entry signal peptides on the editing efficiency of OpenCRISR protein revealed that OP-H2B had an editing efficiency of 0% to 8.3%, while OP-c-myc had an efficiency of 25% to 31.25%, indicating that the c-myc-NLS nuclear entry signal peptide is more suitable for gene editing in zebrafish embryos. Figure 2 (and Table 2). Analysis using Sanger sequencing ( Figure 4 The study found that OP-c-myc had an efficiency of 4.3% for deletions exceeding 5 bp at the target site, and 28.8% for deletions of 5 bp or less, while the insertion efficiency was 2.6%, with a single-sample INDEL mutation rate of 51.3%. SpCas9 had an efficiency of 10.3% for deletions of 5 bp or less at the target site, with 8% for deletions of 5 bp or less, and 3.3% for insertions exceeding 5 bp, with a single-sample INDEL mutation rate of 27.8%.

[0047] The results of multiple sequence alignment of the mutant show that ( Figure 3 OP-c-myc and SPCas9 underwent frameshift mutations at the target site. These results demonstrate that OpenCRISPR can serve as a novel gene-editing tool for fish.

[0048] Table 2 Gene editing efficiency in different experimental groups

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An OpenCRISPR fusion protein for gene editing in cyprinid fishes, characterized in that, The gene encoding the OpenCRISPR fusion protein is shown in SEQ ID NO.

5.

2. The OpenCRISPR fusion protein according to claim 1, characterized in that, The OpenCRISPR fusion protein consists of the CRISPR effector protein OpenCRISPR-1 and the nuclear signal peptide c-myc-NLS.

3. The OpenCRISPR fusion protein according to claim 2, characterized in that, The amino acid sequence of the OpenCRISPR-1 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the nucleotide sequence of the nuclear signal peptide c-myc-NLS is shown in SEQ ID NO.

3.

4. The application of the OpenCRISPR fusion protein according to any one of claims 1-3 in gene editing of cyprinid fish.

5. A recombinant expression vector containing the OpenCRISPR fusion protein encoding gene as described in any one of claims 1-3.

6. The application of the recombinant expression vector according to claim 5 in gene editing of cyprinid fish.

7. A gene editing system, characterized in that, The system includes: a) The OpenCRISPR fusion protein of claim 1, or its mRNA, or the nucleotide sequence encoding the OpenCRISPR fusion protein of claim 1; b) At least one guide RNA, said guide RNA being able to guide the OpenCRISPR fusion protein to target a DNA sequence.

8. The application of the gene editing system of claim 7 in gene editing of cyprinid fish.

9. A kit comprising the gene editing system of claim 7.

10. The application of the kit according to claim 9 in gene editing of cyprinid fish.

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