Low immunogenicity of lwcas13a protein mutant and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to a low immunogenic LwCas13a protein mutant and application thereof.The LwCas13a protein mutant provided by the present application has one or more mutations of Q436C, K560D and K76C on the wild-type LwCas13a protein.The LwCas13a protein mutant provided by the present application significantly reduces the immunogenicity of the LwCas13a protein in a bovine body, thereby reducing the recognition and reaction of the immune system.The LwCas13a protein mutant provided by the present application not only does not affect the activity of the LwCas13a nuclease, but also maintains the effective expression and function of the LwCas13a nuclease in bovine cells.The gene editing efficiency of the LwCas13a protein mutant provided by the present application is equivalent to that of the wild type, and the interference efficiency is not significantly affected.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of genetic engineering, in particular to a low-immunogenic LwCas13a protein mutant and application thereof. BACKGROUND

[0002] The CRISPR-Cas system has expanded into various types of effector enzyme tools. In addition to DNA-targeted editing, Cas13, as an RNA-targeted nuclease, has shown wide application potential in post-transcriptional regulation, RNA virus resistance, reversible editing and high-fidelity regulation. Cas13 can achieve specific RNA cleavage, degradation or modification, and is suitable for bovine gene function research and molecular breeding improvement.

[0003] With the increasing application of Cas13 in animal models and agricultural organisms, the safety of long-term expression in vivo has also been paid more and more attention. Cas13 protein is derived from bacteria, and as a heterologous expression product, it may be recognized as an attack target of the immune system in large mammals such as cattle. If Cas13 is presented as a T cell antigen peptide by the major histocompatibility complex (MHC) class I molecule after expression, it will likely induce CD8 + T cell-mediated cytotoxicity, leading to the elimination of protein expression, the inability to stably regulate targeted RNA, and even the induction of immune-related side reactions. There is currently no systematic study to reveal the immunogenic epitope distribution, presentation mechanism and feasible protein engineering modification path of Cas13 in cattle. SUMMARY

[0004] To solve the above problems, the application provides a low-immunogenic LwCas13a protein mutant and application thereof. The LwCas13a protein mutant provided by the application significantly reduces the immunogenicity of LwCas13a protein in cattle, thereby reducing the recognition and response of the immune system.

[0005] To achieve the above purpose, the application provides the following technical scheme.

[0006] The application provides a low-immunogenic LwCas13a protein mutant, wherein one or more mutations of Q436C, K560D and K76C occur in the wild-type LwCas13a protein; and the amino acid sequence of the wild-type LwCas13a protein is shown in SEQ ID NO. 14.

[0007] Preferably, the amino acid sequence of the LwCas13a protein mutant is shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0008] The application provides a nucleic acid molecule, which is a nucleic acid molecule encoding the LwCas13a protein mutant.

[0009] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO. 13.

[0010] The application provides an expression vector, which comprises a backbone vector and a nucleic acid molecule recombined to the backbone vector; the nucleic acid molecule is the nucleic acid molecule described in the above technical solution.

[0011] Preferably, the backbone vector comprises a pC014-LwCas13a-msfGFP vector.

[0012] The application provides an application of the LwCas13a protein mutant described in the above technical solution, the nucleic acid molecule described in the above technical solution or the expression vector described in the above technical solution in a CRISPR-Cas system.

[0013] Preferably, the application is 1) and / or 2):

[0014] 1) reducing the immunogenicity of the LwCas13a protein mutant in an animal body;

[0015] 2) maintaining the expression and function of the LwCas13a protein mutant in an animal cell.

[0016] Preferably, the animal comprises a cow.

[0017] Preferably, the animal cell comprises a cow fibroblast.

[0018] Beneficial effects:

[0019] The application provides a low-immunogenic LwCas13a protein mutant, which is subjected to one or more mutations of Q436C, K560D and K76C on a wild-type LwCas13a protein; the amino acid sequence of the wild-type LwCas13a protein is shown in SEQ ID NO. 14. The LwCas13a protein mutant provided by the application has the following advantages:

[0020] 1) low immunogenicity: the LwCas13a protein mutant designed by the application significantly reduces the affinity of LwCas13a combined with the MHC-I class molecule of a cow, thereby reducing the recognition and reaction of the immune system to the LwCas13a protein, ensuring the long-term stable expression of the protein and the targeting RNA regulation ability, and reducing the rejection reaction of the immune system.

[0021] 2) High stability: The present invention ensures the structural stability of LwCas13a when designing mutations in the LwCas13a protein. It not only does not affect the activity of its nuclease, but also maintains its effective expression and function in bovine cells.

[0022] 3) Maintaining gene editing efficiency: The gene editing efficiency of the LwCas13a protein mutant provided by this invention is comparable to that of the wild type, and does not significantly affect the interference efficiency;

[0023] 4) High operability: By optimizing the immunogenicity of the protein, this invention has better safety and efficacy in large-scale production and long-term in vivo application. This advantage further expands the application potential of LwCas13a in large mammals (such as cattle). Attached Figure Description

[0024] 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.

[0025] Figure 1 The predicted MHC-I binding scores of BoLA-6:01301 with wild-type and mutant peptides of LwCas13a are shown.

[0026] Figure 2 Quantitative analysis results of ELISpot images;

[0027] Figure 3 For wild-type and single-point mutation variants MSTN Gene editing efficiency results. Detailed Implementation

[0028] This invention provides a low-immunogenicity LwCas13a protein mutant, wherein the LwCas13a protein mutant has one or more mutations among Q436C, K560D, and K76C in the wild-type LwCas13a protein; the amino acid sequence of the wild-type LwCas13a protein is shown in SEQ ID NO.14, and is as follows:

[0029] WT (SEQ ID NO.14):

[0030]

[0031] The Q436C mutation is a mutation of glutamine (Q) at position 436 in the amino acid sequence of the LwCas13a protein mutant to cysteine (C) relative to the amino acid sequence of the wild-type LwCas13a protein, and the amino acid sequence is shown in SEQ ID NO. 1; the K560D mutation is a mutation of lysine (K) at position 560 in the amino acid sequence of the LwCas13a protein mutant to aspartic acid (D) relative to the amino acid sequence of the wild-type LwCas13a protein, and the amino acid sequence is shown in SEQ ID NO. 2; the K76C mutation is a mutation of lysine (K) at position 76 in the amino acid sequence of the LwCas13a protein mutant to cysteine (C) relative to the amino acid sequence of the wild-type LwCas13a protein, and the amino acid sequence is shown in SEQ ID NO. 3, specifically as follows:

[0032] Q436C (SEQ ID NO. 1):

[0033]

[0034] K560D (SEQ ID NO. 2):

[0035]

[0036] K76C (SEQ ID NO. 3):

[0037]

[0038] The application is based on a bovine-specific MHC class I allele database and an immunogenicity prediction algorithm, recognizes and analyzes potential immunodominant epitopes in Cas13, and designs LwCas13a mutants with low immunogenicity by combining protein structural conservation and functional domain layout. The LwCas13a protein mutant designed by the application significantly reduces the immunogenicity of LwCas13a protein in bovine, thereby reducing the recognition and response of the immune system; in addition, the application ensures the structural stability of LwCas13a when designing mutations in LwCas13a protein, which not only does not affect the nuclease activity of LwCas13a, but also maintains its effective expression and function in bovine cells; the gene editing efficiency of the LwCas13a protein mutant provided by the application is equivalent to that of the wild type, and does not significantly affect the interference efficiency.

[0039] The application provides a nucleic acid molecule, which is a nucleic acid molecule encoding the LwCas13a protein mutant described in the above technical solution. As an implementation manner, 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] The application provides an expression vector, which comprises a backbone vector and a nucleic acid molecule recombined on the backbone vector; the nucleic acid molecule is the nucleic acid molecule described in the above technical solution. As an implementation manner, the backbone vector comprises a pC014-LwCas13a-msfGFP vector.

[0041] Based on the above advantages, the application provides the application of the LwCas13a protein mutant described in the above technical solution or the nucleic acid molecule described in the above technical solution or the expression vector described in the above technical solution in a CRISPR-Cas system.

[0042] As an implementation manner, the application is 1) and / or 2):

[0043] 1) reducing the immunogenicity of the LwCas13a protein mutant in animals;

[0044] 2) maintaining the expression and function of the LwCas13a protein mutant in animal cells.

[0045] As an implementation manner, the animals include bovine. As an implementation manner, the animal cells include bovine fibroblasts.

[0046] In order to further illustrate the present application, the low immunogenic LwCas13a protein mutant and its application provided by the present application are described in detail below in combination with examples and drawings, but they cannot be understood as limiting the scope of protection of the present application.

[0047] Examples

[0048] 1. Immunogenic epitope prediction

[0049] The binding affinity of 94 MHC alleles of bovine to all 9-mer peptide segments in the LwCas13a protein sequence was predicted. The highest scoring peptide segment was selected as the high immunogenic epitope. It was found from the prediction results that the binding of the BoLA-6:01301 gene peptide segment was the most obvious. Therefore, this allele was selected as the focus of the next study, and finally three immunogenic epitopes with the highest binding capacity to the BoLA-6:01301 gene were screened out: KQNEVKENL (SEQ ID NO. 4), KLYNKIEDL (SEQ ID NO. 5) and VLHLKDSVL (SEQ ID NO. 6).

[0050] 2. Mutation design

[0051] Referring to the LwCas13a protein sequence, three single-point mutant variants were designed for the three immunogenic epitopes screened in step 1. The structural stability after mutation was evaluated to ensure that the mutation would not affect the structure of the protein and its nuclease function. The mutant Q436C (KCNEVKENL, SEQ ID NO. 7), K560D (DLYNKIEDL, SEQ ID NO. 8) and K76C (VLHLCDSVL, SEQ ID NO. 9) with high protein structure stability and low MHC-I binding capacity were selected.

[0052] 3. Immunological experiment verification

[0053] The binding capacity of the mutated peptide segment to the BoLA-2:01801 gene was predicted by NetMHCpan 4.1 EL. As shown in Table 1, the scores of the wild-type peptide segments of LwCas13a predicted in NetMHCpan were higher than those of the mutant, indicating that they had strong binding capacity, and the binding capacity of the mutated peptide segment was significantly reduced. Figure 1

[0054] ​The present application uses ELISpot experiment to verify the immune response degree of bovine peripheral blood mononuclear cells from healthy donors to these epitopes and mutant peptide segments. Wild type and mutant peptide segments are synthesized by Genscript, with a purity of more than 98%, and each peptide segment is dissolved to 1 mg / ml according to the instructions. In the ELISpot experiment, the present application uses bovine IFNγ pre-coated ELISpot kit (Mabtech) to detect the reactivity of antigen-specific T cells, 5×10 5 cells are plated per well, and incubated with 10 μg / ml peptide segments at 37°C for 48 hours. This experiment evaluates immunogenicity by detecting whether the peptide segments are recognized by T cells after binding to MHC class I molecules, and the ability of CD8 + T cells to activate is reduced, the number of spots is reduced, reflecting the reduction of T cell IFNγ secretion, and the results are shown in Figure 2 , wherein the numerical value at the top of the wild type and mutant peptide segments is P value (such as <0.0001, i.e. P <0.0001).

[0055] The results show that the wild type peptide segment triggers a strong immune response, and the number of spots produced by the single point mutant peptide segment is significantly reduced, indicating a reduced immune response to these variants.

[0056] 4. Verification of gene editing efficiency

[0057] The coding sequences of LwCas13a wild type and mutants (Q436C, K560D and K76C) are synthesized by a commissioned gene company, as follows:

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

[0059]

[0060] Q436C (SEQ ID NO. 11):

[0061]

[0062] K560D (SEQ ID NO. 12):

[0063]

[0064] K76C (SEQ ID NO. 13):

[0065]

[0066] The coding sequences of the wild type and three mutants of LwCas13a were cloned into the vector pC014-LwCas13a-msfGFP (Addgene, #91902) using the method of homologous recombination, obtaining four recombinant vectors.

[0067] The bovine fibroblasts were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS), and the cells were seeded into a 6-well plate one day before transfection. When the cells grew to 70%-80% confluence, the recombinant vectors and guide RNA (5'-ATACCTTGTACCGTCTTTCATGGGTTTG-3', SEQ ID NO. 15) were transfected. Lipofectamine 2000 (Thermo Fisher Scienticific) was used for transfection, and 4 μg of DNA was used per well according to the recommended protocol of the manufacturer; a control group was set up: the recombinant vector containing the coding sequence of the wild type LwCas13a was transfected, but the guide RNA was not transfected. Total RNA was extracted 48 hours later, and after reverse transcription to synthesize cDNA, it was used for qPCR to detect the interference efficiency. The primer sequences used for qPCR amplification are as follows:

[0068] F: 5'-GATGGCACACCGTACGCAAG-3', SEQ ID NO. 16;

[0069] R: 5'-CTGATAACATCAGCCATTCAGCCTA-3', SEQ ID NO. 17.

[0070] The results show that the interference efficiency of the mutants in gene editing is not significantly different from that of the wild type, which indicates that these mutations do not affect the nuclease activity of LwCas13a and do not affect its editing effect (P>0.05). Figure 3 , P＞ 0.05).

[0071] In summary, the LwCas13a protein mutant designed in the present application significantly reduces the immunogenicity of LwCas13a protein in bovine, thereby reducing the recognition and response of the immune system; in addition, the LwCas13a protein mutant described in the present application not only does not affect the activity of LwCas13a nuclease, but also maintains its effective expression and function in bovine cells; finally, the gene editing efficiency of the LwCas13a protein mutant provided in the present application is comparable to that of the wild type, and the interference efficiency is not significantly affected.

[0072] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained on the basis of the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A low immunogenic LwCasl3a protein mutant, characterized in that, The LwCas13a protein mutant is mutated at Q436C, K560D or K76C on a wild-type LwCas13a protein; the amino acid sequence of the wild-type LwCas13a protein is shown as SEQ ID NO. 14; and the amino acid sequence of the LwCas13a protein mutant is shown as SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is a nucleic acid molecule encoding the LwCas13a protein mutant of claim 1.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 11, SEQ ID NO. 12 or SEQ ID NO.

13.

4. An expression vector, characterized by, The expression vector comprises a backbone vector and a nucleic acid molecule recombined to the backbone vector; and the nucleic acid molecule is the nucleic acid molecule of claim 2 or 3.

5. The expression vector of claim 4, wherein, The backbone vector comprises a pC014-LwCas13a-msfGFP vector.

6. Use of the LwCas13a protein mutant of claim 1 or the nucleic acid molecule of claim 2 or 3 or the expression vector of claim 4 or 5 in a CRISPR-Cas system.

7. Use according to claim 6, characterized in that, The use is 1) and / or 2): 1) reducing immunogenicity of the LwCas13a protein mutant in an animal body; and / or 2) maintaining expression and function of the LwCas13a protein mutant in an animal cell.

8. Use according to claim 7, characterized in that, The animal comprises a cow.

9. Use according to claim 7, characterized in that, The animal cell comprises a cow fibroblast.

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