A low immunogenic sa cas9 protein mutant and application thereof

Abstract

This invention relates to the field of genetic engineering technology, and in particular to a low-immunogenic SaCas9 protein mutant and its applications. The SaCas9 protein mutant of this invention involves one or more mutations among Q433H, K426W, and K297D in the wild-type SaCas9 protein. The SaCas9 protein mutant of this invention significantly reduces the number of IFN-γ spots induced in ELISpot experiments compared to the wild-type, effectively reducing humoral and cellular immune responses, lowering the immunogenicity of SaCas9, making it more suitable for gene editing, and providing technical support for developing safer CRISPR-based gene editing. The gene editing efficiency of the SaCas9 protein mutant provided by this invention is comparable to that of the wild-type, and it does not affect the nucleic acid recognition and cleavage function of SaCas9.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a low-immunogenic SaCas9 protein mutant and its applications. Background Technology

[0002] The CRISPR / Cas9 system, as a highly efficient and programmable gene-editing tool, has been widely used in various fields such as basic research, disease treatment, and agricultural improvement. Among them, the CRISPR / Cas9 system, derived from Staphylococcus aureus (…),… Staphylococcus aureus SaCas9, with its small molecular weight and ease of packaging into vectors such as adeno-associated virus (AAV), exhibits unique advantages in in vivo delivery applications. In recent years, CRISPR technology has gradually demonstrated great potential in bovine genetic breeding, and SaCas9 has become a potential key tool protein.

[0003] However, as a bacterial protein, SaCas9 expression in animals may trigger innate or adaptive immune responses. Previous studies have shown that humans possess humoral immunity (mediated by antibodies) and cellular immunity (mediated by T cells) against proteins derived from Staphylococcus aureus and Streptococcus pyogenes, leading to immune recognition or inflammatory responses during gene editing. While there is some research on the immunogenicity of SaCas9 in humans, its immune response mechanism in cattle remains unclear, and there is a lack of strategies for assessing bovine immune epitopes and optimizing the Cas9 structure. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a low-immunogenic SaCas9 protein mutant and its applications. The SaCas9 protein mutant provided by this invention significantly reduces the immunogenicity of SaCas9 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 SaCas9 protein mutant with low immunogenicity, wherein the SaCas9 protein mutant has one or more mutations in wild-type SaCas9 protein, namely Q433H, K426W and K297D; the amino acid sequence of the wild-type SaCas9 protein is shown in SEQ ID NO.14.

[0007] Preferably, the amino acid sequence of the SaCas9 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 SaCas9 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 pX601 carrier.

[0012] This invention provides the application of the SaCas9 protein mutant, the nucleic acid molecule, or the 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 SaCas9 protein mutants in animals;

[0015] 2) Maintain the expression and function of SaCas9 protein mutants 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 SaCas9 protein mutant with low immunogenicity, wherein the SaCas9 protein mutant has one or more mutations among Q433H, K426W, and K297D in the wild-type SaCas9 protein; the amino acid sequence of the wild-type SaCas9 protein is shown in SEQ ID NO.14. The SaCas9 protein mutant provided by this invention has the following advantages:

[0020] 1) Low immunogenicity: The SaCas9 protein mutant designed in this invention significantly reduced the number of IFN-γ spots induced in the ELISpot experiment compared with the wild type, effectively reducing humoral and cellular immune responses, lowering the immunogenicity of SaCas9, making it more suitable for gene editing, and providing technical support for the development of safer CRISPR-based gene editing.

[0021] 2) Improved stability: The present invention ensures the structural stability of SaCas9 when designing mutations in the SaCas9 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 SaCas9 protein mutant provided by this invention is comparable to that of the wild type, and does not affect the nucleic acid recognition and cleavage function of SaCas9. 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 predicted MHC-I binding scores of BoLA-6:01301 with wild-type and mutant peptides of SaCas9 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 SaCas9 protein mutant with low immunogenicity, wherein the SaCas9 protein mutant has one or more mutations among Q433H, K426W, and K297D in the wild-type SaCas9 protein; the amino acid sequence of the wild-type SaCas9 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 Q433H mutant SaCas9 protein of this invention is shown in SEQ ID NO.1, where glutamine (Q) is mutated to histidine (H) at position 433 relative to the wild-type SaCas9 protein; the amino acid sequence of the K426W mutant SaCas9 protein is shown in SEQ ID NO.2, where lysine (K) is mutated to tryptophan (W) at position 426 relative to the wild-type SaCas9 protein; and the amino acid sequence of the K297D mutant SaCas9 protein is shown in SEQ ID NO.3, where lysine (K) is mutated to aspartic acid (D) at position 297 relative to the wild-type SaCas9 protein. Specifically, the amino acid sequences are as follows:

[0032] Q433H (SEQ ID NO.1):

[0033]

[0034] K426W(SEQ ID NO.2):

[0035]

[0036] K297D(SEQ ID NO.3):

[0037]

[0038] This invention, based on the bovine MHC-I molecule presentation mechanism, predicts and identifies cellular epitopes in SaCas9 that may trigger immune responses. Through structural conservation analysis and computational mutation design, a SaCas9 mutant with low immunogenicity is designed. The SaCas9 protein mutant designed in this invention significantly reduces the number of IFN-γ spots induced in ELISpot experiments compared to the wild type, effectively reducing humoral and cellular immune responses, lowering the immunogenicity of SaCas9, making it more suitable for gene editing, and providing technical support for developing safer CRISPR-based gene editing. Furthermore, this invention ensures the structural stability of SaCas9 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 SaCas9 protein mutant provided by this invention is comparable to that of the wild type, without affecting the nucleic acid recognition and cleavage functions of SaCas9.

[0039] This invention provides a nucleic acid molecule that encodes the SaCas9 protein mutant described in the above-described 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-mentioned technical solution. As one embodiment, the backbone vector comprises the pX601 vector (pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA).

[0041] Based on the above advantages, the present invention provides the application of the SaCas9 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 SaCas9 protein mutants in animals;

[0044] 2) Maintain the expression and function of SaCas9 protein mutants 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 SaCas9 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 Cas9 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-6:01301 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-6:01301 gene were identified: QQKEIPTTL (SEQ ID NO.4), KLVPKKVD (SEQ ID NO.5), and KQIAKEILV (SEQ ID NO.6).

[0050] 2. Mutation Design

[0051] Based on the SaCas9 protein structure model (PDB ID: 5AXW), 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, Q433H (QHKEIPTTL, SEQ ID NO.7), K426W (KLVPWKVD, SEQ ID NO.8), and K297D (DQIAKEILV, SEQ ID NO.9), were selected. See [link to relevant documentation]. Figure 1 In this context, triangles represent catalytically active sites in the SaCas9 protein.

[0052] 3. Verification by immunological experiments

[0053] The binding affinity of the mutated peptide to the BoLA-6:01301 gene was predicted using NetMHCpan 4.1 EL. (Example: ...) Figure 2 As shown, the wild-type peptides of SaCas9 have higher predicted scores than the mutants in NetMHCpan, indicating that they have a stronger binding ability, while the binding ability 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 are P Values ​​(e.g., <0.0001, i.e.) P <0.0001).

[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, which is consistent with NetMHCpan's predictions.

[0056] 4. Validation of gene editing efficiency

[0057] The coding sequences for SaCas9 wild-type and mutants (Q433H, K426W, and K297D) were synthesized by a genetics company, as follows:

[0058] WT (SEQ ID NO.10):

[0059]

[0060] Q433H(SEQ ID NO.11):

[0061]

[0062] K426W(SEQ ID NO.12):

[0063]

[0064] K297D(SEQ ID NO.13):

[0065]

[0066] The coding sequences of wild-type and three mutant SaCas9 were cloned into vector pX601 (pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA, Addgene, #61591) 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% confluency, the recombinant vector and guide RNA (5'-TCGAACTGATCAATCAGTTCC-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 SaCas9 and did not affect its editing effect. Figure 4 ns is P＞ 0.05).

[0071] In summary, the SaCas9 protein mutant designed in this invention significantly reduces the immunogenicity of SaCas9 protein in cattle, thereby reducing the recognition and response of the immune system. In addition, the SaCas9 protein mutant described in this invention not only does not affect the activity of SaCas9 nuclease, but also maintains its effective expression and function in bovine cells. Finally, the gene editing efficiency of the SaCas9 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 SaCas9 protein mutant with low immunogenicity, characterized in that, The SaCas9 protein mutants are derived from the wild-type SaCas9 protein by mutations of Q433H, K426W, or K297D; the amino acid sequence of the wild-type SaCas9 protein is shown in SEQ ID NO.

14.

2. The SaCas9 protein mutant according to claim 1, characterized in that, The amino acid sequence of the SaCas9 protein mutant is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule is a nucleic acid molecule encoding the SaCas9 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, SEQ ID NO.12 or SEQ ID NO.

13.

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 pX601 carrier.

7. The use of the SaCas9 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 preparation of a CRISPR-Cas9 system.

8. The application according to claim 7, characterized in that, The application is 1) and / or 2): 1) Reduce the immunogenicity of SaCas9 protein mutants in animals; 2) Maintain the expression and function of SaCas9 protein mutants 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.

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