Low immunogenic ascas12a protein mutant and application thereof
By mutating the AsCas12a protein with mutations such as P635C, P885W, and P1068C, its immunogenicity in cattle was reduced, thus solving the problem of immune response of the AsCas12a protein in cattle and maintaining its stability and gene editing efficiency in cattle cells.
Patent Information
- Application Number
- CN202511269356.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-08
- Publication Date
- 2025-11-18
- Estimated Expiration
- 2045-09-08
AI Technical Summary
The immunogenicity of the AsCas12a protein in cattle may lead to immune system recognition and response, affecting its stability and gene editing efficacy in vivo.
A mutant of the AsCas12a protein was designed. By introducing mutations such as P635C, P885W, and P1068C into the wild-type AsCas12a protein, its immunogenicity in cattle was reduced while maintaining its structural stability and nuclease activity.
It significantly reduced the immunogenicity of AsCas12a protein in cattle, improved its stability and gene editing efficiency in bovine cells, and did not significantly affect the incidence of insertion/deletion mutations.
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Figure CN120738154B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a low-immunogenic AsCas12a protein mutant and its applications. Background Technology
[0002] The CRISPR-Cas system has become a core technology tool for gene editing, widely used in livestock breeding, disease resistance improvement, and biomedical research. AsCas12a is a highly efficient CRISPR effector nuclease with a compact enzyme structure, a broad PAM recognition range, and sticky DNA ends, making it suitable for multi-target editing and complex regulation of large animal genomes. In recent years, AsCas12a has shown good application potential in cattle, and is expected to be used for improving production performance and genetic quality.
[0003] However, AsCas12a, as a heterologous protein derived from bacteria, may trigger immune system recognition and response when expressed in cattle, thus affecting its stability and gene-editing efficacy in vivo. Previous studies have shown that heterologous Cas proteins in humans can be presented by host MHC class I molecules, inducing CD8... + T cell-mediated cellular immune responses. In cattle, although the immune response mechanism of AsCas12a has not been systematically studied, its potential immunogenicity constitutes one of the key safety risks for the application of AsCas12a in bovine gene editing, given that the bovine immune system also has a sensitive ability to recognize foreign proteins. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a low-immunogenic AsCas12a protein mutant and its applications. The AsCas12a protein mutant provided by this invention significantly reduces the immunogenicity of AsCas12a protein in cattle, thereby reducing the recognition and response of the immune system.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] The present invention provides a low immunogenic AsCas12a protein mutant, wherein the AsCas12a protein mutant has one or more mutations among P635C, P885W and P1068C in the wild-type AsCas12a protein; the amino acid sequence of the wild-type AsCas12a protein is shown in SEQ ID NO.14.
[0007] Preferably, the amino acid sequence of the AsCas12a protein mutant is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.
[0008] The present invention provides a nucleic acid molecule that encodes the AsCas12a protein mutant described in the above technical solution.
[0009] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13.
[0010] The present invention provides an expression vector comprising a backbone vector and a nucleic acid molecule recombined onto the backbone vector; the nucleic acid molecule is the nucleic acid molecule described in the above technical solution.
[0011] Preferably, the skeleton carrier includes a pY010 carrier.
[0012] This invention provides the application of the AsCas12a protein mutant, nucleic acid molecule, or expression vector described in the above-described technical solutions in the CRISPR-Cas system.
[0013] Preferably, the application is 1) and / or 2).
[0014] 1) Reduce the immunogenicity of AsCas12a protein mutants in animals;
[0015] 2) Maintain the expression and function of the AsCas12a protein mutant in animal cells.
[0016] Preferably, the animal includes a cow.
[0017] Preferably, the animal cells include bovine fibroblasts.
[0018] Beneficial effects:
[0019] This invention provides a low-immunogenic AsCas12a protein mutant, wherein the AsCas12a protein mutant has one or more mutations among P635C, P885W, and P1068C in the wild-type AsCas12a protein; the amino acid sequence of the wild-type AsCas12a protein is shown in SEQ ID NO.14. The AsCas12a protein mutant provided by this invention has the following advantages:
[0020] 1) Low immunogenicity: The AsCas12a protein mutant designed in this invention significantly reduces the immunogenicity of AsCas12a protein in cattle, thereby reducing the recognition and response of the immune system.
[0021] 2) Improved stability: The present invention ensures the structural stability of AsCas12a when designing mutations in the AsCas12a protein, which not only does not affect its nuclease activity, but also maintains its effective expression and function in bovine cells.
[0022] 3) Maintaining gene editing efficiency: The gene editing efficiency of the AsCas12a protein mutant provided by this invention is comparable to that of the wild type, and does not significantly affect the incidence of insertion / deletion mutations. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0024] Figure 1 This includes the immunogenic epitope sequence and mutation location;
[0025] Figure 2 The MHC-I binding prediction scores for BoLA-2:01801 with wild-type and mutant peptides of AsCas12a are shown.
[0026] Figure 3 Quantitative analysis results of ELISpot images;
[0027] Figure 4 For wild-type and single-point mutation variants MSTN Gene editing efficiency results. Detailed Implementation
[0028] This invention provides a low-immunogenic AsCas12a protein mutant, wherein the AsCas12a protein mutant has one or more mutations among P635C, P885W, and P1068C in the wild-type AsCas12a protein; the amino acid sequence of the wild-type AsCas12a protein is shown in SEQ ID NO.14, and is as follows:
[0029] WT (SEQ ID NO.14):
[0030]
[0031] The amino acid sequence of the P1068C mutant AsCas12a protein of the present invention is shown in SEQ ID NO. 1, where proline (P) is mutated to cysteine (C) at position 1068 relative to the amino acid sequence of the wild-type AsCas12a protein; the amino acid sequence of the P635C mutant AsCas12a protein of the present invention is shown in SEQ ID NO. 2, where proline (P) is mutated to cysteine (C) at position 635 relative to the amino acid sequence of the wild-type AsCas12a protein; and the amino acid sequence of the P885W mutant AsCas12a protein of the present invention is shown in SEQ ID NO. 3, where proline (P) is mutated to tryptophan (W) at position 885 relative to the amino acid sequence of the wild-type AsCas12a protein. Specifically, the amino acid sequences are as follows:
[0032] P1068C (SEQ ID NO.1):
[0033]
[0034] P635C(SEQ ID NO.2):
[0035]
[0036] P885W(SEQ ID NO.3):
[0037]
[0038] This invention systematically evaluated the potential T-cell epitope composition and immune recognition risk of AsCas12a in the context of bovine immunity. Based on the presentation characteristics of bovine MHC class I molecules, and through immunogenicity prediction algorithms and protein structure analysis, a low-immunogenic AsCas12a mutant was designed. The AsCas12a protein mutant designed in this invention significantly reduced the immunogenicity of AsCas12a protein in cattle, thereby reducing the recognition and response of the immune system. In addition, this invention ensured the structural stability of AsCas12a protein during mutation design, not only without affecting its nuclease activity, but also maintaining its effective expression and function in bovine cells. The gene editing efficiency of the AsCas12a protein mutant provided by this invention is comparable to that of the wild type, and it does not significantly affect the incidence of insertion / deletion mutations.
[0039] This invention provides a nucleic acid molecule that encodes the AsCas12a protein mutant described in the above-mentioned technical solution. As one embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.11, SEQ ID NO.12, or SEQ ID NO.13.
[0040] This invention provides an expression vector comprising a backbone vector and a nucleic acid molecule recombined onto the backbone vector; the nucleic acid molecule is the nucleic acid molecule described in the above-described technical solution. As one embodiment, the backbone vector comprises a pY010 vector.
[0041] Based on the above advantages, the present invention provides the application of the AsCas12a protein mutant, the nucleic acid molecule, or the expression vector described in the above technical solution in the CRISPR-Cas system.
[0042] As one implementation, the application is 1) and / or 2):
[0043] 1) Reduce the immunogenicity of AsCas12a protein mutants in animals;
[0044] 2) Maintain the expression and function of the AsCas12a protein mutant in animal cells.
[0045] In one embodiment, the animal includes a cow. In another embodiment, the animal cells include bovine fibroblasts.
[0046] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, provides a low-immunogenic AsCas12a protein mutant and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.
[0047] Example 1
[0048] 1. Immunogenic epitope prediction
[0049] The binding affinity of 94 bovine MHC alleles to all 9-mer peptides in the Ascas12a protein sequence was predicted. The peptides with the highest binding affinity were selected as highly immunogenic epitopes. The prediction results showed that the binding affinity to the BoLA-2:01801 gene peptide was the most significant. Therefore, this allele was chosen as the focus of further research, and ultimately, three immunoepochs with the highest binding affinity to the BoLA-2:01801 gene were identified: VPAPYTSKI (SEQ ID NO.4), EPLEITKEI (SEQ ID NO.5), and SPSKFNQRV (SEQ ID NO.6).
[0050] 2. Mutation Design
[0051] Based on the AsCas12a protein structure model (PDB ID: 5b43), three single-point mutation variants were designed for the three immunoepitaxes identified in step 1. The mutation sites did not overlap with DNA and RNA binding or catalytic sites. The structural stability after mutation was assessed to ensure that the mutations did not affect the protein structure or the function of its nucleases. Mutants with high structural stability and low MHC-I binding affinity, P635C (VCAPYTSKI, SEQ ID NO.7), P885W (EWLEITKEI, SEQ ID NO.8), and P1068C (SPSKFNQRV, SEQ ID NO.9), were selected. See [link to relevant documentation]. Figure 1 In this context, triangles represent catalytically active sites in the AsCas12a protein.
[0052] 3. Verification by immunological experiments
[0053] The binding affinity of the mutated peptide to the BoLA-2:01801 gene was predicted using NetMHCpan 4.1 EL. (Example:) Figure 2 As shown, the wild-type AsCas12a peptides all scored higher than the mutant peptides in NetMHCpan, indicating that they have a stronger binding affinity, while the binding affinity of the mutant peptides is significantly reduced.
[0054] This invention uses the ELISpot assay to experimentally verify the immunoreactivity of bovine peripheral blood mononuclear cells from healthy donors to these epitopes and their mutant peptides. Wild-type and mutant peptides were synthesized by Genscript Biotech Inc., with a purity exceeding 98%, and each peptide was dissolved in ddH2O to a concentration of 1 mg / ml according to the instructions. In the ELISpot assay, this invention uses a bovine IFNγ pre-coated ELISpot kit (Mabtech) to detect the reactivity of antigen-specific T cells, with 5 × 10⁶ cells per well. 5 Cells were incubated with a 10 μg / ml peptide at 37°C for 48 hours. This experiment assessed immunogenicity by detecting whether the peptide, after binding to MHC class I molecules, was recognized by T cells and whether it activated CD8. + Decreased T cell activity and reduced number of spots reflect reduced T cell IFNγ secretion. (See results below.) Figure 3 Among them, the values at the upper end of the wild-type and mutant peptides (e.g., 0.0011) are... P value.
[0055] The results showed that the wild-type peptide elicited a strong immune response, while the number of spots produced by the single-point mutant peptide was significantly reduced, indicating a decreased immune response to these variants.
[0056] 4. Validation of gene editing efficiency
[0057] The coding sequences for AsCas12a wild-type and mutants (P635C, P885W, and P1068C) were synthesized by a gene company, as follows:
[0058] WT (SEQ ID NO.10):
[0059]
[0060] P1068C(SEQ ID NO.11):
[0061]
[0062] P635C(SEQ ID NO.12):
[0063]
[0064] P885W(SEQ ID NO.13):
[0065]
[0066] The coding sequences of wild-type AsCas12a and three mutants were cloned into vector pY010 (pcDNA3.1-hAsCpf1, Addgene, #69982) using homologous recombination, resulting in four recombinant vectors.
[0067] Bovine fibroblasts were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS). One day prior to transfection, cells were seeded into 6-well plates. When cells reached 70%–80% confluence, the recombinant vector and guide RNA (5'-CGTCCTGGCGTGGTAGTCATCGT-3', SEQ ID NO. 15) were transfected. Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific), following the manufacturer's recommended protocol, using 4 µg of DNA per well. After transfection, cellular DNA was extracted and subjected to PCR amplification, followed by sequencing analysis. The primer sequences used for PCR amplification are as follows:
[0068] F: 5'-AGAACAGCGAGCAGAAGGAA-3', SEQ ID NO.16;
[0069] R: 5'-TAGAACAGCAGTCAGCAGAGT-3', SEQ ID NO. 17.
[0070] The results showed that the indel efficiency (insertion / deletion mutation rate) of the mutants in gene editing was not significantly different from that of the wild type, indicating that these mutations did not affect the nuclease activity of AsCas12a and did not affect its editing effect. Figure 4 ns is P＞ 0.05).
[0071] In summary, the AsCas12a protein mutant designed in this invention significantly reduces the immunogenicity of AsCas12a protein in cattle, thereby reducing the recognition and response of the immune system. In addition, the AsCas12a protein mutant described in this invention not only does not affect the activity of AsCas12a nuclease, but also maintains its effective expression and function in bovine cells. Finally, the gene editing efficiency of the AsCas12a protein mutant provided by this invention is comparable to that of the wild type, and does not significantly affect the incidence of insertion / deletion mutations.
[0072] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A low-immunogenic AsCas12a protein mutant, characterized in that, The AsCas12a protein mutants are derived from the wild-type AsCas12a protein by a P635C or P1068C mutation; the amino acid sequence of the wild-type AsCas12a protein is shown in SEQ ID NO.
14.
2. The AsCas12a protein mutant according to claim 1, characterized in that, The amino acid sequence of the AsCas12a protein mutant is shown in SEQ ID NO.1 or SEQ ID NO.
2.
3. A nucleic acid molecule, characterized in that, The nucleic acid molecule is a nucleic acid molecule encoding the AsCas12a protein mutant of claim 1 or 2.
4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 11 or SEQ ID NO.
12.
5. An expression carrier, characterized in that, The expression vector includes a backbone vector and a nucleic acid molecule recombined onto the backbone vector; the nucleic acid molecule is the nucleic acid molecule as described in claim 3 or 4.
6. The expression vector according to claim 5, characterized in that, The skeletal carrier includes the pY010 carrier.
7. The application of the AsCas12a protein mutant of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, or the expression vector of claim 5 or 6 in the CRISPR-Cas system.
8. The application according to claim 7, characterized in that, The application is 1) and / or 2): 1) Reduce the immunogenicity of AsCas12a protein mutants in animals; 2) Maintain the expression and function of the AsCas12a protein mutant in animal cells.
9. The application according to claim 8, characterized in that, The animals mentioned include cattle.
10. The application according to claim 8, characterized in that, The animal cells include bovine fibroblasts.
11. The application of an AsCas12a protein mutant or a nucleic acid molecule or expression vector encoding the AsCas12a protein mutant in a CRISPR-Cas system; wherein the AsCas12a protein mutant is a P885W mutation in wild-type AsCas12a protein; the amino acid sequence of the wild-type AsCas12a protein is shown in SEQ ID NO.14; the expression vector comprises a backbone vector and the nucleic acid molecule recombined onto the backbone vector; the application is to reduce the immunogenicity of the AsCas12a protein mutant in animals.
12. The application according to claim 11, characterized in that, The amino acid sequence of the AsCas12a protein mutant is shown in SEQ ID NO.
3.
13. The application according to claim 11, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.
13.
14. The application according to claim 11, characterized in that, The skeletal carrier includes the pY010 carrier.
15. The application according to claim 11, characterized in that, The animals mentioned include cattle.
Citation Information
Patent Citations
Novel mutations that enhance the DNA cleavage activity of acidaminococcus sp. cpf1
CN112912496A