Codon optimization method

Abstract

The invention relates to the technical field of molecular biology, in particular to a codon optimization method. The invention provides a codon optimization method, the codon is replaced according to parameters such as codon type and quantity, local GC content, local repetitive sequence, mRNA secondary structure, mRNA free energy and the like, and the optimized sequence can be subjected to experimental screening to obtain a DNA sequence of high-expression protein. The downstream of the method is mainly the application in the IVT mRNA direction: the optimized DNA sequence is constructed into a vector, the plasmid is linearized to serve as a template for in-vitro mRNA transcription, the mRNA is encapsulated into LNP or LNP coupled with other molecules, and the protein expression in 293T cells (not limited to 293T cells) or mice is obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a method for codon optimization. Background Art

[0002] Codons are specific sequences of three adjacent nucleotides on messenger RNA (mRNA) that determine the type of amino acids and, in turn, the sequence of proteins. Codon optimization, also known as codon engineering or codon adaptation, is a technique that optimizes the expression efficiency of genes in specific hosts by changing the codon usage in gene sequences.

[0003] The methods of codon optimization mainly include computer simulation optimization, experimental screening optimization and deep learning methods based on big data. Among them, computer simulation optimization predicts the optimal codon combination through algorithms, experimental screening optimization compares the expression efficiency of different codon combinations through experimental means, and deep learning methods based on big data use large-scale bioinformatics data to train models and predict optimal codons. In terms of optimization strategy, local or global codon replacements can be performed on gene sequences according to the codon preference of host cells to improve expression efficiency. At present, many companies have posted automatic codon optimization tools on their websites.

[0004] Existing codon optimization tools mainly use IT algorithms to optimize the GC content, codon usage frequency, mRNA secondary structure, RNA enzyme splicing sites, and RNA stable trans-acting elements of a specific cell, thereby increasing protein expression at the plasmid level. Its downstream applications are mainly for recombinant proteins: 1) Plasmids are transfected into cells, and the expressed proteins are subjected to WB, ELISA and other identification experiments; 2) Plasmids are transformed into hosts (such as E. coli, yeast, insects or mammalian cells), and proteins are induced to express in large quantities, purified, and obtained as protein products. In addition, IT algorithms often provide unique codon-optimized sequences, which are not convenient for customers to screen.

[0005] It can be seen that the existing codon optimization technology has achieved remarkable results in improving the efficiency of gene expression. However, this technology also has certain limitations. For example, the IT algorithm only inputs a unique sequence and cannot provide more options; different host cells have different preferences for codons, so the optimization results may vary depending on the host cell; and the sequences output by the IT algorithm mostly rely only on the CAI value to judge the quality of the sequence, but sequences with high CAI values ​​often do not show good expression effects in actual operations. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for codon optimization in order to further increase the expression level.

[0007] In the present invention, the codon optimization method comprises:

[0008] Obtaining a protein encoding nucleic acid sequence according to the codon preference of the host, and then adjusting the GC content, repeat sequence, secondary structure and / or free energy in the obtained sequence to obtain an optimized encoding nucleic acid;

[0009] The GC content includes: the GC content of the entire length of the encoding nucleic acid and the GC content within a unit length.

[0010] The method of the present invention replaces codons according to set parameters, which can reduce a large number of codon sequencing schemes to only dozens or even a few codon sequencing schemes. The optimized sequence can be screened experimentally to obtain a DNA sequence of a highly expressed protein.

[0011] The GC content in a nucleic acid sequence refers to the ratio of guanine (G) to cytosine (C). The method of the present invention not only adjusts the GC content within the full length, but also controls the local GC content in the sequence, and replaces the codons in that part when the local GC content is too high. By dual control of the local GC content and the full-length GC content, the energy required for the target gene during DNA replication can be further reduced, and the energy demand generated by high GC or low GC sequences can be reduced, thereby improving the stability of mRNA and then increasing the yield of protein.

[0012] In the present invention, the local GC content, i.e., the GC content within a unit length, refers to the proportion of G and C in a nucleic acid sequence fragment of a specific length. For example, the GC content within a unit length in the present invention is the GC content within a length of 10bp to 100bp. Preferably, it is the GC content within a length of 20bp to 50bp. More preferably, it is the GC content within a length of 30bp. Taking a unit length of 10bp as an example, the GC content within the unit length refers to the GC content within the range of 1 to 10bp, 2 to 11bp, 3 to 12bp...n to n+9bp (and so on) in the sequence. In a sequence to be optimized, the unit length is at least one of 10bp to 100bp, for example, its length can be at least one of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100bp.

[0013] In some embodiments, the GC content per unit length is 20% to 95%. Preferably, the GC content is 26% to 63% or 40% to 83%. In some specific embodiments, the GC content per unit length is 26% to 63% or 43% to 80%, or 46% to 83%, or 40% to 80%.

[0014] In some embodiments, the full-length GC content is higher than 0% to 15% of the total GC content of the host species.

[0015] In some specific embodiments, taking human cells as the host, the full-length GC content is adjusted to 52.27% to 67.27%. Preferably, taking human cells as the host, the codon optimization method adjusts the full-length GC content to 56% to 63%.

[0016] In some other specific embodiments, taking the host as Macaca fascicularis cells as an example, the full-length GC content is adjusted to 49.64% to 64.64%. Preferably, taking Macaca fascicularis cells as the host, the full-length GC content is 54% to 61%.

[0017] The repetitive sequence in the nucleic acid sequence refers to the same or symmetrical sequence fragments that appear in different positions in the nucleic acid sequence of the coding protein. If there are repetitive fragments in the amino acid sequence of the protein, the coding nucleic acid is likely to also have corresponding repetitive sequences. However, the previous optimization method paid less attention to the repetitive sequences in the optimized nucleic acid sequence. In the present invention, the longest repetitive sequence is controlled within 30bp, which can improve the stability of the target gene during DNA replication and reduce the loss of fragments caused by homologous recombination or other reasons that occur during replication of the target gene. In an embodiment of the present invention, the length of the repetitive sequence is not more than 30bp, and preferably, the length of the repetitive sequence is not more than 20bp.

[0018] Nucleic acid secondary structure involves DNA double helix and RNA folding. The secondary structure of RNA refers to the three-dimensional spatial structure formed by the spatial folding of single-stranded RNA molecules, which is usually manifested as a hairpin-type single-stranded structure, and the single-stranded structure folds back to form a local small double helix, also known as a stem-loop structure or a ball-loop structure. Avoiding the generation of RNA secondary structure is conducive to more accurate and efficient protein translation, and is also more conducive to mRNA to exert its physiological activity more accurately and efficiently. In the present invention, the adjustment of the secondary structure includes reducing the continuous base pairing region.

[0019] mRNA free energy refers to the energy absorbed or released when an mRNA molecule changes from one conformation to another under specific conditions. It reflects the stability of the mRNA structure and is an important factor in evaluating the folding state and function of mRNA. The codon-optimized nucleic acid sequence is used for protein expression or the preparation of mRNA transfection reagents. Therefore, the present invention adjusts the free energy of the optimized sequence. The adjustment of the free energy includes making the minimum free energy of the sequence 80% to 100% of the minimum free energy. For example, the minimum free energy of the sequence is 85% to 95% of the minimum free energy. In a specific embodiment, the minimum free energy of the sequence is 80% to 85%, 85% to 90%, 90% to 95% or 95% to 100% of the minimum free energy.

[0020] In the present invention, the adjustment includes: using the codons with the second, third, and fourth codon preferences to replace the codons with the first percentage, and the percentages of the codons with the first, second, third, and fourth usage frequencies in the full length of the encoding nucleic acid are: 40% to 100%, 0% to 50%, 0% to 25%, and 0% to 15%, respectively.

[0021] In some specific embodiments, the host is a human cell, and the adjustment comprises:

[0022] Among Phe codons, UUU is used at a frequency of 1% to 25%, and UUC is used at a frequency of 75% to 99%;

[0023] Among the codons for Leu, the usage frequency of CUG was 86% to 99%, the usage frequency of CUC was 1% to 10%, the usage frequency of UUA was 0% to 1%, the usage frequency of UUG was 0% to 1%, the usage frequency of CUU was 0% to 1%, and the usage frequency of CUA was 0% to 1%;

[0024] Among the codons of Ile, the usage frequency of AUU is 5% to 25%, the usage frequency of AUC is 75% to 94%, and the usage frequency of AUA is 0% to 1%;

[0025] Among the codons of Val, the usage frequency of GUU is 0% to 5%, the usage frequency of GUC is 5% to 15%, the usage frequency of GUA is 0% to 1%, and the usage frequency of GUG is 79% to 95%;

[0026] Among the codons for Ser, the usage frequency of UCU is 0% to 20%, the usage frequency of UCC is 10% to 25%, the usage frequency of UCA is 0% to 1%, and the usage frequency of UCG is 0% to 1%; the usage frequency of AGU is 0% to 1%, and the usage frequency of AGC is 52% to 90%;

[0027] Among the codons of Pro, the usage frequency of CCU is 20% to 50%, the usage frequency of CCC is 44% to 72%, the usage frequency of CCA is 0% to 8%, and the usage frequency of CCG is 0% to 6%;

[0028] Among Thr codons, ACU is used at a frequency of 0% to 1%, ACC at a frequency of 50% to 78%, ACA at a frequency of 20% to 48%, and ACG at a frequency of 0% to 1%;

[0029] Among Ala codons, GCU is used at a frequency of 3% to 20%, GCC at a frequency of 73% to 90%, GCA at a frequency of 0% to 6%, and GCG at a frequency of 0% to 1%;

[0030] Among Tyr codons, UAU is used at a frequency of 1% to 25%, and UAC is used at a frequency of 75% to 99%;

[0031] Among His codons, CAU is used at a frequency of 1% to 15%, and CAC is used at a frequency of 85% to 99%;

[0032] Among Gln codons, CAA is used at a frequency of 1% to 15%, and CAG is used at a frequency of 85% to 99%;

[0033] Among the codons for Asn, the usage frequency of AAU is 5% to 30%, and the usage frequency of AAC is 70% to 95%;

[0034] Among Lys codons, AAA is used at a frequency of 1% to 25%, and AAG is used at a frequency of 75% to 99%;

[0035] Among the codons of Asp, the usage frequency of GAU is 3% to 35%, and the usage frequency of GAC is 65% to 97%;

[0036] Among the codons for Glu, the usage frequency of GAA is 10% to 35%, and the usage frequency of GAG is 65% to 90%;

[0037] Among Cys codons, UGU is used at a frequency of 15% to 40%, and UGC is used at a frequency of 60% to 85%;

[0038] Among the codons for Arg, the usage frequency of CGU is 0% to 1%, the usage frequency of CGC is 1% to 8%, the usage frequency of CGA is 0% to 1%, the usage frequency of CGG is 44% to 72%, the usage frequency of AGA is 15% to 50%, and the usage frequency of AGG is 1% to 8%;

[0039] Among the codons for Gly, the usage frequency of GGU is 0% to 5%, the usage frequency of GGC is 65% to 98%, the usage frequency of GGA is 1% to 20%, and the usage frequency of GGG is 1% to 10%.

[0040] Furthermore, the start codon is AUG; the stop codon is UAA, UAG or UGA, and the codon for Trp is GU U.

[0041] In the scheme of the present invention, the codon usage frequency suitable for the host can be obtained by trying the protein expression amount under different codon usage. As mentioned above, the codon usage frequency is obtained after a lot of optimization. Studies have shown that, compared with the scheme of codon optimization based on the natural usage frequency of human codons in the prior art, the codon usage frequency in the encoding nucleic acid is adjusted to meet the above percentage, which can more effectively improve the expression effect of the protein and obtain more protein. When the host is a cell from other biological sources, the codon usage frequency is different from the above data and needs to be optimized for a specific species. In the method described in the present invention, the step of obtaining the coding nucleic acid sequence of the protein according to the codon preference of the host includes: obtaining the amino acid sequence to be optimized, and determining the species direction to be optimized; obtaining the codon usage frequency table of the corresponding species in nature; according to the adjusted codon table, the amino acid sequence to be optimized is randomly distributed and optimized to obtain the coding nucleic acid sequence.

[0042] In the method of the present invention, if the GC content, repeat sequence, secondary structure and / or free energy still cannot reach the above-mentioned standards after the adjustment of codons, the protein encoding nucleic acid sequence obtained according to the codon preference of the host is adjusted. The adjustment described herein includes lowering the screening criteria and relaxing the frequency range of codon usage to ensure that more codons can be selected in order to obtain appropriate analysis results in the subsequent analysis.

[0043] The method of the present invention also includes the step of translating the optimized sequence and comparing the translated protein sequence with the target protein sequence to see whether they are consistent.

[0044] Furthermore, the present invention provides a codon optimization system, which includes a module for implementing the above-mentioned method.

[0045] Furthermore, the present invention also provides a computer-readable storage medium storing a computer program, wherein the computer program implements the steps of the above-mentioned method when executed by a processor.

[0046] Furthermore, the present invention also provides a codon-optimized electronic device, which includes a storage medium, a processor, and a computer program stored in the storage medium, and the processor executes the computer program to implement the method as described above.

[0047] Furthermore, the present invention also provides a biomaterial comprising at least one of the following I) to V):

[0048] 1), optimizing the nucleic acid obtained by the method described above;

[0049] II), an expression unit containing the nucleic acid described in I);

[0050] III), a plasmid vector containing the nucleic acid described in I), or containing the expression unit described in II);

[0051] IV), mRNA, which contains a 5'-terminal cap structure and the nucleic acid described in I), or contains a 5'-terminal cap structure and the expression unit described in II);

[0052] V), a host cell, which comprises a nucleic acid described in I) integrated into its genome, or an expression unit described in II) integrated into its genome, or a plasmid vector described in III) transformed or transfected therewith.

[0053] In the present invention,

[0054] The nucleic acid encoding chicken ovalbumin has a sequence as shown in any one of SEQ ID NOs: 1 to 4;

[0055] or a nucleic acid sequence encoding chicken ovalbumin in which one or more bases are substituted, deleted, added and / or replaced in the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 4;

[0056] Or a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence as described above.

[0057] The present invention optimizes the codons of the nucleic acid encoding chicken ovalbumin and obtains four versions of optimized sequences. The obtained nucleic acid sequence is that the expression level of OVA in the host is significantly improved compared with the wild type and other control ratios, and the mRNA prepared from these nucleic acids can have a good transfection efficiency, and when transferred into cells, especially 293T cells, a higher expression level can be obtained to better simulate the immune response of the human immune system.

[0058] In the present invention,

[0059] The nucleic acid encoding the Cre recombinase has a sequence as shown in any one of SEQ ID NOs: 10 to 13;

[0060] or a nucleic acid sequence encoding a Cre recombinase in which one or more bases are substituted, deleted, added and / or replaced in the nucleic acid sequence shown in any one of SEQ ID NOs: 10 to 13;

[0061] Or a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence as described above.

[0062] The present invention performs codon optimization on four versions of Cre recombinase, and the expression level of the obtained Cre recombinase protein is significantly improved compared with the wild type and other control ratios. The mRNA transfection reagent prepared from these nucleic acids can have a good transfection efficiency, and the mRNA transfection reagent can be transferred into cells, especially 293T cells, to more effectively exert the function of the Cre recombinase system.

[0063] In the present invention,

[0064] The nucleic acid encoding Gaussia luciferase has a sequence as shown in any one of SEQ ID NOs: 15 to 18;

[0065] or a nucleic acid sequence encoding Gaussia luciferase in which one or more bases are substituted, deleted, added and / or replaced in the nucleic acid sequence shown in any one of SEQ ID NOs: 15 to 18;

[0066] Or a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence as described above.

[0067] When Gaussia luciferase is applied to human HEK293T cells, the expression efficiency is not high. The inventors independently developed an artificial codon optimization method to optimize the proportion of different codons for the same amino acid in the wild-type Gaussia luciferase DNA sequence, GC content, sequence repetitiveness, etc., to highly express the DNA sequence of the Gaussia luciferase version. The expression effect after codon optimization is 1.4-16.2 times higher than that of the wild-type sequence.

[0068] In the present invention,

[0069] The nucleic acid encoding the firefly luciferase has a sequence as shown in any one of SEQ ID NOs: 23 to 24;

[0070] or a nucleic acid sequence encoding a firefly luciferase in which one or more bases are substituted, deleted, added and / or replaced in the nucleic acid sequence shown in any one of SEQ ID NOs: 23 to 24;

[0071] Or a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence as described above.

[0072] The firefly luciferase protein of the embodiment of the present invention has an expression level increased by 2 to 201 times in cell experiments compared with the wild type and other control examples, and has a significant increase in fluorescence brightness in mouse experiments, which greatly enhances the application value of firefly luciferase as a reporter gene.

[0073] In the present invention,

[0074] The nucleic acid encoding SpCas9 has a sequence as shown in any one of SEQ ID NOs: 33 to 37.

[0075] or a nucleic acid sequence encoding SpCas9 in which one or more bases are substituted, deleted, added and / or replaced in the nucleic acid sequence shown in any one of SEQ ID NOs: 33 to 37;

[0076] Or a nucleic acid sequence that is at least 80% identical to a nucleic acid sequence as described above.

[0077] The expression level of the CRISPR / Cas9 protein provided in the technical solution of the present invention is increased by 4-8 times compared with the wild type or other control groups, and has a higher gene editing efficiency, thus greatly improving the application value of Cas9 protein in gene editing.

[0078] In the present invention, the at least 80% identity includes: at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity or at least 99.9% identity.

[0079] Compared with the wild type, the expression of the sequence optimized in the embodiment of the present invention is significantly increased. Compared with other optimization schemes in the prior art, the expression of the sequence optimized in the embodiment of the present invention is also significantly increased.

[0080] The expression unit provided by the present invention comprises the nucleic acid and the promoter as described above.

[0081] For different expression vectors, the expression unit of the present invention may further include other elements, such as enhancers, terminators and / or nuclear localization signals.

[0082] In some embodiments, the promoter is selected from the group consisting of a eukaryotic promoter and a prokaryotic promoter, which is not limited in the present invention. For example, the promoter is a T7 promoter, a CMV promoter, a CAG promoter, an EF1a promoter, a PGK promoter, a U6 and H1 promoter, an EFS promoter, a CBh promoter, a SFFV promoter, a MSCV promoter, a SV40 promoter, a UBC promoter, or a TRE promoter.

[0083] In some embodiments, the enhancer is selected from SV40 enhancer, CMV enhancer, SV-1 enhancer, ROSA26 enhancer, EF1α enhancer, HARE5 enhancer, UBC enhancer, EF1A enhancer, PGK enhancer, CAGG enhancer, COPIA enhancer, or ACT5C enhancer.

[0084] In some embodiments, the terminator is selected from T7 phage terminator, TO phage terminator, λ phage terminator, SV40 terminator, CMV terminator, rrnB terminator, bGH terminator, hGH terminator, or rbGlob terminator.

[0085] In some embodiments, the nuclear localization signal is SV40 NLS, whose amino acid sequence is PKKKRKV, and the nuclear localization signal is located between the promoter and the nucleic acid as described above.

[0086] In some embodiments, the expression unit includes a promoter, a 5'UTR, a Kozak, a nucleic acid as described above, a 3'UTR, a polyA tail and a restriction enzyme site from the 5' end to the 3' end. In some specific embodiments, the polyA tail is 110 bp long.

[0087] The plasmid vector provided by the present invention comprises a backbone vector and at least one of the following i) to ii):

[0088] i), a nucleic acid as described above;

[0089] ii) An expression unit as described above.

[0090] The plasmid vector described in the present invention is used for amplification, preservation, transformation and / or transfection of nucleic acid, and the present invention does not limit this. The plasmid vector described in the present invention may be circular or linear, and the present invention does not limit this. The present invention does not limit the insertion site of the nucleic acid and / or expression unit in the plasmid vector. Preferably, it can be inserted at the multiple cloning site of the plasmid vector, or it can be inserted into other regions. In some embodiments, it is a cloning vector, an expression vector, a herpes simplex virus vector, a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated virus vector. For example, the cloning vector is a pUC series plasmid vector, a pBR322 plasmid vector, a pGEM series plasmid vector, a pET series plasmid vector, a Yest series plasmid vector or a Gateway plasmid vector, etc. For example, the adeno-associated virus vector is an rAAV vector, and its serotype includes AAV1, AAV2, AAV5, AAV6, AAV8 or AAV9. In some embodiments, the retroviral vector pmRVac vector is used as an example for expression verification, and its effect is better than other vectors.

[0091] Furthermore, the present invention provides an mRNA comprising a 5' end cap structure and the aforementioned nucleic acid, or comprising a 5' end cap structure and the aforementioned expression unit.

[0092] In the present invention, the mRNA includes 5'UTR, Kozak, the aforementioned nucleic acid, 3'UTR, polyA tail and restriction enzyme cutting site which are connected in sequence.

[0093] In the present invention, the sequences of the 5'UTR, 3'UTR or Kozak are not limited.

[0094] The length of the polyA tail is 50-200 bp, for example, it can be 50 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp or 200 bp. Alternatively, the length of the polyA tail can also be any intermediate value between the first two point values.

[0095] The sequence of the restriction enzyme cleavage site is repeated once in the plasmid vector as described above. In the embodiment of the invention, Sap I is used as the restriction enzyme cleavage site for plasmid linearization. In addition, any other restriction endonuclease site that can linearize the plasmid can be used.

[0096] Furthermore, the mRNA preparation method comprises linearizing the plasmid vector as described above, followed by capping and purification.

[0097] Specifically, the mRNA preparation method includes digesting the plasmid vector as described above with a restriction endonuclease, adding a capping enzyme and other raw materials required for capping, and performing a capping reaction at 37° C. for 1 hour; then obtaining an mRNA sample after chromatography purification, sterilizing and filtering for later use.

[0098] Furthermore, the present invention also provides a host cell, which is transformed or transfected with the plasmid vector as described above, or has the nucleic acid as described above integrated into its genome, or has the expression unit as described above integrated into its genome.

[0099] In the present invention, the host is a human cell or a mammalian cell. In addition, the host can also be a prokaryotic microorganism or a eukaryotic microorganism. The eukaryotic host includes but is not limited to yeast and insect cells, and the prokaryotic host includes but is not limited to Escherichia coli. For example, the host is BL21 (DE3), BL21 (DE3) pL ysS, DH5α, JM109, JM110, TOP10, HB101, XL1-Blue of Escherichia coli. The human cell is a 293T cell.

[0100] Furthermore, the present invention also provides a method for constructing a host cell, which comprises treating the cell with the mRNA and lipid as described above. In the embodiment of the present invention, the treatment comprises transfection and / or transformation.

[0101] The present invention also provides a product obtained by culturing the host cell as described above.

[0102] Furthermore, the present invention also provides a method for preparing a recombinant protein, which comprises:

[0103] After obtaining the nucleic acid molecule optimized by the method described above, the nucleic acid molecule is introduced into a host cell and then cultured to obtain a product containing a recombinant protein.

[0104] Furthermore, the present invention also provides a method for preparing an mRNA transfection preparation or vaccine, which comprises: after obtaining the nucleic acid molecule optimized by the method described above, connecting the nucleic acid molecule to a vector, linearizing it, capping it, purifying it, and encapsulating it to obtain the mRNA transfection preparation or vaccine.

[0105] The present invention provides a method for codon optimization, which replaces codons according to parameters such as codon type and number, local GC content, local repeat sequence, mRNA secondary structure, mRNA free energy, etc. The optimized sequence can be screened experimentally to obtain a DNA sequence of a highly expressed protein. The downstream of the method of the present invention is mainly applied in the direction of IVT mRNA: the optimized DNA sequence is constructed into a vector, the plasmid is linearized and used as a template for in vitro transcription of mRNA, and the mRNA is encapsulated into LNP or LNP coupled to other molecules, and there is a significant increase in protein expression in 293T cells (not limited to 293T cells) or mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The standard codon representation is shown;

[0107] Figure 2 Indicates the codon representation of the species "Homo sapiens";

[0108] Figure 3 GC content analysis diagram of the first version of the DNA sequence in Example 1 is shown;

[0109] Figure 4 A schematic diagram showing the analysis of the repetition of the DNA sequence of the first version in Example 1;

[0110] Figure 5 A schematic diagram of the secondary structure of the DNA sequence RNA of the first version in Example 1 is shown;

[0111] Figure 6 GC content analysis of OVA (1) in Example 1 is shown;

[0112] Figure 7 Schematic diagram showing the sequence duplication analysis of OVA (1) in Example 1;

[0113] Figure 8 A schematic diagram of the RNA secondary structure of OVA (1) in Example 1 is shown;

[0114] Fig. 9 GC content analysis of OVA (2) in Example 1 is shown;

[0115] Fig.10 Schematic diagram showing the sequence duplication analysis of OVA (2) in Example 1;

[0116] Fig.11 A schematic diagram of the RNA secondary structure of OVA (2) in Example 1 is shown;

[0117] Fig.12 GC content analysis of OVA (3) in Example 1 is shown;

[0118] Fig.13 Schematic diagram showing the sequence duplication analysis of OVA (3) in Example 1;

[0119] Fig.14 A schematic diagram of the RNA secondary structure of OVA (3) in Example 1 is shown;

[0120] Fig.15 GC content analysis of OVA (4) in Example 1 is shown;

[0121] Fig.16 Schematic diagram showing the sequence duplication analysis of OVA (4) in Example 1;

[0122] Fig.17 A schematic diagram of the RNA secondary structure of OVA (4) in Example 1 is shown;

[0123] Fig.18 The GC content ratio analysis results of the first version sequence in Example 2 are shown;

[0124] Fig.19 The dot plot analysis results of the first version sequence duplication in Example 2 are shown;

[0125] Fig. 20 The secondary structure diagram of the RNA sequence corresponding to the first version sequence in Example 2 is shown;

[0126] Fig.21 The GC content ratio analysis results of the Co1 sequence in Example 2 are shown;

[0127] Fig. 22 The dot plot analysis results of the Co1 sequence duplication in Example 2 are shown;

[0128] Fig.23 The secondary structure diagram of the RNA sequence corresponding to the Co1 sequence in Example 2 is shown;

[0129] Fig.24 The GC content ratio analysis results of the Co2 sequence in Example 2 are shown;

[0130] Fig.25 The dot plot analysis results of the Co2 sequence duplication in Example 2 are shown;

[0131] Fig.26 The secondary structure diagram of the RNA sequence corresponding to the Co2 sequence in Example 2 is shown;

[0132] Fig. 27 The GC content ratio analysis results of the Co3 sequence in Example 2 are shown;

[0133] Fig.28 The dot plot analysis results of the Co3 sequence duplication in Example 2 are shown;

[0134] Fig.29 The secondary structure diagram of the RNA sequence corresponding to the Co3 sequence in Example 2 is shown;

[0135] Fig.30 The GC content ratio analysis results of the Co4 sequence in Example 2 are shown;

[0136] Fig.31 The dot plot analysis results of the Co4 sequence duplication in Example 2 are shown;

[0137] Fig.32 The secondary structure diagram of the RNA sequence corresponding to the Co4 sequence in Example 2 is shown;

[0138] Fig.33 This is a graph showing the GC content ratio of the GLuc1 DNA sequence in Example 3;

[0139] Fig.34 This is a diagram showing the sequence duplication analysis of the GLuc1 DNA sequence in Example 3;

[0140] Fig.35 Schematic diagram of the RNA secondary structure of the GLuc1 DNA sequence in Example 3;

[0141] Fig.36 This is a graph showing the GC content ratio of the GLuc2 DNA sequence in Example 3;

[0142] Fig.37 This is a diagram showing the sequence duplication analysis of the GLuc2 DNA sequence in Example 3;

[0143] Fig.38 Schematic diagram of the RNA secondary structure of the GLuc2 DNA sequence in Example 3;

[0144] Fig.39 This is a graph showing the GC content ratio of the DNA sequence of GLuc3 in Example 3;

[0145] Fig.40 This is a sequence duplication analysis diagram of the DNA sequence of GLuc3 in Example 3;

[0146] Fig.41 This is a schematic diagram of the RNA secondary structure of the DNA sequence of GLuc3 in Example 3;

[0147] Fig.42This is a graph showing the GC content ratio of the GLuc4 DNA sequence in Example 3;

[0148] Fig.43 This is a sequence duplication analysis diagram of the GLuc4 DNA sequence in Example 3;

[0149] Fig.44 Schematic diagram of the RNA secondary structure of the GLuc4 DNA sequence in Example 3;

[0150] Fig.45 A schematic diagram showing the GC content ratio analysis of the first version DNA sequence of luciferase in Example 4;

[0151] Fig.46 Schematic diagram showing the sequence duplication analysis of the first version of the luciferase DNA sequence in Example 4;

[0152] Fig.47 A schematic diagram showing the RNA secondary structure of the first version of the luciferase DNA sequence in Example 4;

[0153] Fig.48 A schematic diagram showing the GC content ratio analysis of LUC-1 in Example 4 is shown;

[0154] Fig.49 Schematic diagram showing the sequence duplication analysis of LUC-1 in Example 4;

[0155] Fig.50 Schematic diagram of the RNA secondary structure of LUC-1 in Example 4;

[0156] Fig.51 A schematic diagram showing the GC content ratio analysis of LUC-2 in Example 4 is shown;

[0157] Fig.52 Schematic diagram showing the sequence duplication analysis of LUC-2 in Example 4;

[0158] Fig.53 A schematic diagram of the RNA secondary structure of LUC-2 in Example 4 is shown;

[0159] Fig.54 This is a schematic diagram of the DNA sequence analysis of WT in Example 5;

[0160] Fig.55 This is a schematic diagram of the DNA sequence analysis of VB1 in Example 5;

[0161] Fig.56 This is a schematic diagram of the DNA sequence analysis of VB2 in Example 5;

[0162] Fig.57 This is a schematic diagram of the DNA sequence analysis of VB3 in Example 5;

[0163] Fig.58 This is a schematic diagram of the DNA sequence analysis of VB4 in Example 5;

[0164] Fig.59 This is a schematic diagram of the DNA sequence analysis of VB5 in Example 5;

[0165] Fig.60 Shown is the map of pmRVac vector;

[0166] Fig.61 The western blot detection diagram after expression of each group of cells in Example 1 is shown;

[0167] Fig.62 The SDS-PAGE electrophoresis detection diagram of each group of cells after expression in Example 1 is shown;

[0168] Fig.63 Grayscale analysis of the western blot gel image after expression of each group of cells in Example 1 (****P<0.0001);

[0169] Fig.64 In the figure, 64-1 shows the WB experimental results (NC: Negative Control), and 64-2 shows the analysis of the expression levels of Cre Recombinase in different samples (WB image band gray value analysis, ***P<0.001, ****P<0.0001);

[0170] Fig.65 In the figure, 65-1 shows the results of tdTomato fluorescence imaging of liver slices in vivo (magnification 200x, exposure 5ms, gain 5x), and 65-2 shows the results of tdTomato fluorescence imaging of spleen slices in vivo (magnification 200x, exposure 5ms, gain 5x);

[0171] Fig.66 This is a graph showing the expression effect intensity of four Gaussia luciferase genes in HEK293T cells in Example 3;

[0172] Fig.67 Schematic diagram of firefly luciferase gene cell experiment data in Example 4; columns 1 to 9 respectively show the expression levels of nucleic acids of control sequence 1, control sequence 2, control sequence 3, control sequence 4, control sequence 5, control sequence 6, control sequence 7, LUC-1 and LUC-2 in cells;

[0173] Fig.68The in vivo imaging results 6 hours after LNP-mRNA injection in Example 4 are shown in the figure; wherein: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right: control sequence 1, control sequence 3, control sequence 5, LUC-1 and LUC-2;

[0174] Fig.69 The in vivo imaging results after 24 hours of LNP-mRNA injection in Example 4 are shown in the figure; wherein: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right: control sequence 1, control sequence 3, control sequence 5, LUC-1 and LUC-2;

[0175] Fig.70 The in vivo imaging results after 48 hours of LNP-mRNA injection in Example 4 are shown in the figure; wherein: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right: control sequence 1, control sequence 3, control sequence 5, LUC-1 and LUC-2;

[0176] Fig.71 The in vivo imaging results after 72 hours of LNP-mRNA injection in Example 4 are shown in the figure; wherein: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right: control sequence 1, control sequence 3, control sequence 5, LUC-1 and LUC-2;

[0177] Fig.72 This is an immunoblot of the Cas9 protein expressed in Example 5;

[0178] Fig.73 This is a graph showing the relative expression of the Cas9 protein obtained in Example 5;

[0179] Fig.74 Comparison of the functional verification of the Cas9 protein expressed in cells obtained in Example 5;

[0180] Fig.75 This is a visual data analysis diagram comparing the functional verification of Cas9 protein expressed in cells obtained in Example 5. DETAILED DESCRIPTION

[0181] The present invention provides a method for codon optimization, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0182] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0183] In the present application, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone.

[0184] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0185] In this application, "include", "comprising", and "having" are used interchangeably, and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising", and "having" in this article also provides "consisting of" or "as shown in" solutions.

[0186] In the present invention, the "codon" refers to a triplet nucleotide residue sequence on RNA (or DNA), which encodes a specific amino acid. The most common start codon is the methionine or valine codon.

[0187] In the present invention, the "codon table" refers to the fact that the genetic code is traditionally expressed in the form of an RNA codon table; this is because when a cell ribosome makes a protein, it is the messenger RNA that guides the synthesis of the protein. The sequence of the messenger RNA is determined by the genomic DNA. With the rise of computational biology and genomics, most genes can now be found at the DNA level, so the DNA codon table has become increasingly useful.

[0188] In the present application, "identity" can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0189] The percent identity between the two sequences varies with the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced for optimal alignment of the two sequences and the length of each space. In the present invention, the 80% or more identity includes: 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0190] In the present application, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'.

[0191] In the present application, the nucleic acid molecules encompass deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense strands and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone bonding or chemically modified residues.

[0192] In the present application, "plasmid vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.

[0193] In the present application, "host cell" refers to a cell into which exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parental cell in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity as screened or selected in the initially transformed cell.

[0194] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0195] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0196] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0197] The codon table involved in the embodiment is shown in Table 1:

[0198] Table 1

[0199]

[0200] Taking human cells as the host, the frequency of human codon usage is as follows:

[0201] Table 2

[0202]

[0203]

[0204] In the present invention, "CAI value" refers to the CAI value corresponding to a species, which quantifies the frequency of codon types preferred by highly expressed genes in the species. The CAI value ranges from 0 to 1. A high CAI value of the target gene means that it can be more effectively expressed in the species.

[0205] Conventional IT algorithms only adjust parameters, such as increasing the CAI value, or controlling the GC content value to about 60%, etc., and the calculation process is issued by instructions one by one. Artificial intelligence will make a personalized judgment based on the entire sequence, and choose to lower or increase a certain value selectively, allowing the existence of local values ​​that do not meet the IT algorithm parameters. At the same time, artificial intelligence can also determine whether the sequence needs to be optimized, and it does not require further optimization for the optimized sequence. Artificial intelligence can give multiple optimized versions, and then screen out the better version based on the experimental results. The IT algorithm generally only gives a codon-optimized version sequence. The relevant optimization data of the present invention show that for some verified genes, the protein expression level has been increased by several times or even hundreds of times.

[0206] The sequences involved in the embodiments are as follows:

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0223] Example 1 Chicken ovalbumin (OVA) codon optimization

[0224] The optimized sequence is derived from the wild-type CDS region sequence of the species Gallus gallus.

[0225] Related information

[0226] Sequence source: https: / / www.ncbi.nlm.nih.gov / nuccore / 2099367237

[0227] The DNA sequence optimization method in the present invention is as follows.

[0228] (1) Amino acid sequence analysis

[0229] The present invention relates to a wild-type version of chicken ovalbumin amino acid sequence, as shown in Table 3.

[0230] Table 3 Chicken ovalbumin amino acid count

[0231]

[0232] Amino acid sequence 1: MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGA KDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGL FRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP*.

[0233] (2) Conversion of amino acid sequence into DNA sequence

[0234] Convert all amino acid sequences to the most commonly used codon sequence of the species to be used, for example, for the species "Homosapiens", convert all "alanine" to "GCC". The commonly used codon sequence of species can be found at the following website (link http s: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606).

[0235] After following the above method, the first version of the DNA sequence (SEQ ID NO: 7) was obtained:

[0236]

[0237] (3) DNA sequence analysis

[0238] The GC content of the first version of the DNA sequence obtained in step 2 is analyzed. Figure 3 shown.

[0239] GC content analysis tool URL: https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html

[0240] The first version of the DNA sequence obtained in step 2 is analyzed for sequence duplication. Figure 4 shown.

[0241] Sequence duplication analysis tool URL: https: / / www.vectorbuilder.cn / tool / sequence-dot-plot.html

[0242] The DNA sequence of the first version of the DNA sequence was subjected to sequence RNA secondary structure and free energy analysis. The minimum free energy (MFE) of the sequence RNA was -475.50 kcal / mol, and the RNA secondary structure was Figure 5 shown.

[0243] RNA secondary structure and free energy analysis tool URL: http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi

[0244] (4) DNA sequence adjustment

[0245] By combining parameters such as codon preference, GC content ratio, sequence repetition, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0246] The optimization description of each parameter is described below.

[0247] Codon preference: After obtaining the first version of the DNA sequence based on the most commonly used codons, the codons with the second, third, and fourth most frequent codons are used to replace the codons with the first most frequent codons. The principle is that the proportion of the codons with the first, second, third, and fourth most frequent codons in the total sequence of the protein is controlled within the range of: 40-100%, 0-50%, 0-25%, and 0-15%. Figure 1(https: / / zh.wikipedia.org / wiki / DNA%E5%AF%86%E7%A0%81%E5%AD%90%E8%A1%A8) shows that the codons corresponding to amino acids are distinguished according to the standard codon table, and then combined with Figure 2 The codon table of the species Homo sapiens was used to obtain the codon preference. In the species Homo sapiens, GGC of glycine accounts for 40-100%, GGA accounts for 0-50%, GGG accounts for 0-25%, and GGT accounts for 0-15%.

[0248] 1) GC content ratio: higher than the total GC content ratio of the species, which is 0% to 15%. The total GC content of the species "Homo sapiens" is 52.27%, so the total GC content ratio of the optimized DNA sequence should be in the range of "52.27% to 67.27%". Different species should have different GC contents. For example, the total GC content of the species "Macaca fascicularis" is 49.64%, so the total GC content ratio of the optimized DNA sequence should be in the range of "49.64% to 64.64%". The local GC content of the DNA sequence should be controlled between 30% and 95%.

[0249] 2) Sequence repetition: Optimize the number of repeated sequences and sequence length to minimize the number of repeated sequences. For example, replace a codon in a sequence segment with a repeated sequence greater than 20 bases with a synonymous codon to reduce the number of bases in the repeated sequence.

[0250] 3) RNA secondary structure and free energy: Reduce the continuous base pairing region and lower the minimum free energy. If both cannot be achieved at the same time, reducing the continuous base pairing region is the priority.

[0251] According to the above optimization method, optimized sequences 1 to 4 of OVA were obtained, and the free energies were: -446.09 kcal / mol, -444.51 kcal / mol, -450.96 kcal / mol, and -445.90 kcal / mol, respectively.

[0252] The first, second, third and fourth codons of OVA (1) (SEQ ID NO: 1) account for 44-100%, 0-50%, 0-14% and 0-3% of the total sequence of the protein. The GC content analysis is as follows: Figure 6 , the sequence repetition is as follows Figure 7 , RNA secondary structure such as Figure 8 .

[0253] The first, second, third and fourth codons of OVA (2) (SEQ ID NO: 2) account for 44-100%, 0-50%, 0-14% and 0-3% of the total sequence of the protein. The GC content analysis is as follows: Fig. 9 , the sequence repetition is as follows Fig.10 , RNA secondary structure such as Fig.11 .

[0254] The first, second, third and fourth codons of OVA (3) (SEQ ID NO: 3) account for 44-100%, 0-50%, 0-14% and 0-3% of the total sequence of the protein. The GC content analysis is as follows: Fig.12 , the sequence repetition is as follows Fig.13 , RNA secondary structure such as Fig.14 .

[0255] The first, second, third and fourth codons of OVA (4) (SEQ ID NO: 4) account for 44-100%, 0-50%, 0-14% and 0-3% of the total sequence of the protein. The GC content analysis is as follows: Fig.15 , the sequence repetition is as follows Fig.16 , RNA secondary structure such as Fig.17 .

[0256] Table 4 Sequence comparison information of this experiment

[0257]

[0258] Example 2 Codon Optimization of Cre Recombinase (Cre)

[0259] Four versions of artificial codon optimization were performed on the DNA sequence encoding Cre recombinase, where Co1 is shown in SEQ ID NO: 1, Co2 is shown in SEQ ID NO: 2, Co3 is shown in SEQ ID NO: 3, and Co4 is shown in SEQ ID NO: 4. The wild-type (WT) version of the Cre enzyme (with the SV40 NLS amino acid sequence after the promoter, i.e., PKKKRKV, to enter the nucleus to play a role in DNA fragment recombination) is shown in SEQ ID NO: 5, and the Cre recombinase encoding nucleic acid sequence optimized by other companies is shown in SEQ ID NO: 6.

[0260] The DNA sequence optimization method in the present invention is as follows.

[0261] (1) Amino acid sequence analysis

[0262] Table 5 Amino acid sequence analysis

[0263]

[0264] Amino Acid Sequence:

[0265] MGPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIA YNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD

[0266] (2) Convert amino acid sequence to DNA sequence

[0267] The amino acid sequence is fully decoded into the most commonly used codon sequence of the species to be applied (the reference website for the codon usage table is https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606, such as Figure 2 As shown), for example, if the species is "Homo sapiens", all "glycine" is decoded as "GGC", and the codon sequences corresponding to other amino acids are decoded in sequence to obtain the first version of the DNA sequence.

[0268] The DNA sequence of the first version (SEQ ID NO: 14):

[0269]

[0270] (3) DNA sequence analysis

[0271] The first version of the DNA sequence was analyzed for GC content ratio (GC content analysis website is https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html). Fig.18 shown.

[0272] The first version of the DNA sequence was subjected to sequence repetition dot plot analysis (sequence repetition dot plot analysis website is https: / / www.vectorbuilder.cn / tool / sequence-dot-plot.html). Fig.19 shown.

[0273] The first version of the DNA sequence was subjected to the corresponding RNA sequence free energy and secondary structure analysis (the analysis website is the link website: http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi). The minimum free energy (MFE) of the RNA sequence is -490.9 kcal / mol, and the RNA secondary structure is as follows: Fig. 20 shown.

[0274] (4) DNA sequence adjustment

[0275] The DNA sequence is adjusted based on parameters such as codon preference, sequence repetition, GC content ratio, RNA free energy, and RNA secondary structure to obtain an optimized version of the DNA sequence.

[0276] The optimization description of each parameter is described below.

[0277] 1) Codon preference: After obtaining the first version of the DNA sequence based on the most commonly used codons, the codons with the second, third, and fourth most frequently used codons are used to replace the codons with the first most frequently used codons. The general principle is that the proportion of the codons with the first, second, third, and fourth most frequently used codons in the total sequence should be controlled within the range of: 40-100%, 0-50%, 0-25%, and 0-15%. Figure 1 As shown, confirm the codon corresponding to the amino acid according to the standard codon table, and then combine Figure 2 The codon table of the Homo sapiens species was obtained to obtain the codon preference.

[0278] 2) GC content ratio: higher than the total GC content of the species of 0% to 15%. The total GC content of the species "Homo sapiens" is 52.27%, so the total GC content of the optimized DNA sequence should be in the range of "52.27% to 67.27%". Different species have different GC content ratios. In addition, the local GC content of the DNA sequence should be controlled between 30% and 95%.

[0279] 3) Sequence repetition: Optimize the number of repeated sequences and sequence length to minimize the number of repeated sequences. For example, replace a codon in a sequence segment with a repeated sequence greater than 20 bases with a synonymous codon to reduce the number of bases in the repeated sequence.

[0280] 4) RNA secondary structure and free energy: Reduce the continuous base pairing region and lower the minimum free energy. If both cannot be achieved at the same time, reducing the continuous base pairing region is the priority.

[0281] According to the above optimization method, the optimized Cre recombinase coding sequences were obtained and recorded as Co1~4.

[0282] like Figure 21 to Figure 23 As shown, the usage frequency of the codons of Co1 is as follows: the first, second, third, and fourth codons account for 68-100%, 0-29%, 0-7%, and 0-7% in the total sequence. The GC content ratio of the sequence of Co1 is 64.5%. The minimum free energy of the sequence RNA of Co1 is -447.70 kcal / mol.

[0283] like Figure 24 to Figure 26 As shown, the usage frequency of the codons of Co2 is as follows: the first, second, third, and fourth codons account for 44-100%, 0-50%, 0-14%, and 0-3% of the total sequence. The GC content ratio of the sequence of Co2 is 60.4%. The minimum free energy of the sequence RNA of Co2 is -406.70 kcal / mol.

[0284] like Figure 27 to Figure 29 As shown, the usage frequency of the codons of Co3 is as follows: the first, second, third, and fourth codons account for 68-100%, 0-25%, 0-10%, and 0-7% of the total sequence. The GC content ratio of the sequence of Co3 is 65.8%. The minimum free energy of the sequence RNA of Co3 is -461.80 kcal / mol.

[0285] like Figure 30 to Figure 32As shown, the usage frequency of the codons of Co4 is as follows: the first, second, third, and fourth codons account for 50-100%, 0-33%, 0-9%, and 0-3% in the total sequence. The GC content ratio of the sequence of Co4 is 62.5%. The minimum free energy of the sequence RNA of Co4 is -424.50 kcal / mol.

[0286] Table 6. Sequence information comparison of this experiment

[0287]

[0288] Example 3 Codon Optimization of Gaussia Luciferase

[0289] The wild-type CDS sequence from Gaussia princeps was used as the experimental material, Sequence ID: AY015993.1. Four versions of Gaussia luciferase target gene sequences were optimized using an independently developed artificial codon optimization method. The DNA sequence optimization method in the present invention is as follows.

[0290] (1) Amino acid sequence analysis

[0291] The amino acid sequence of the Gaussia luciferase protein involved in the present invention is shown in Table 7.

[0292] Table 7

[0293]

[0294]

[0295] Amino Acid Sequence:

[0296] MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVL

[0297] KEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPE

[0298] IPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD.

[0299] (2) Conversion of amino acid sequence to DNA sequence

[0300] Convert all amino acid sequences into the most commonly used codon sequences for the species to be used, such as Figure 2 The most commonly used codon sequence is available at the following website:

[0301] https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606.

[0302] For the species "Homo sapiens", all "alanine" is changed to "GCC". Obtain the first version of the DNA sequence.

[0303] The first version of the DNA sequence:

[0304] ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCCGAGGCCAAG

[0305] CCCACCGAGAACAACGAGGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCCAC

[0306] CACCGACCTGGACGCCGACCGGGGCAAGCTGCCCGGCAAGAAGCTGCCCCTGGAGG

[0307] TGCTGAAGGAGATGGAGGCCAACGCCCGGAAGGCCGGCTGCACCCGGGGCTGCCTG

[0308] ATCTGCCTGAGCCACATCAAGTGCACCCCCAAGATGAAGAAGTTCATCCCCGGCCGG

[0309] TGCCACACCTACGAGGGCGACAAGGAGAGCGCCCAGGGCGGCATCGGCGAGGCCAT

[0310] CGTGGACATCCCCGAGATCCCCGGCTTCAAGGACCTGGAGCCCATGGAGCAGTTCAT

[0311] CGCCCAGGTGGACCTGTGCGTGGACTGCACCACCGGCTGCCTGAAGGGCCTGGCCA

[0312] ACGTGCAGTGCAGCGACCTGCTGAAGAAGTGGCTGCCCCAGCGGTGCGCCACCTTCGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGCGCCGGCGGCGAC (SEQ ID NO: 21)

[0313] (3) DNA sequence analysis

[0314] The first version of the DNA sequence was analyzed for GC content. Fig.42 shown.

[0315] GC content ratio analysis link:

[0316] https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html

[0317] The first version of the DNA sequence was analyzed for sequence duplication. Fig.43 shown.

[0318] DNA sequence duplication analysis link:

[0319] https: / / www.vectorbuilder.cn / tool / sequence-dot-plot.html

[0320] The first version of the DNA sequence was subjected to sequence RNA secondary structure and free energy analysis. The minimum free energy (MFE) of the sequence RNA was -241.70 kcal / mol. The RNA secondary structure was Fig.44 shown.

[0321] RNA secondary structure and free energy analysis link:

[0322] http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi

[0323] (4) DNA sequence adjustment

[0324] By combining parameters such as codon preference, GC content ratio, sequence repetition, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0325] The optimization description of each parameter is described below.

[0326] 1) Codon preference: After obtaining the first version of the DNA sequence based on the most commonly used codons, the codons with the second, third, and fourth most frequently used codons are used to replace the codons with the first most frequently used codons. As a general rule, the proportion of the codons with the first, second, third, and fourth most frequently used codons in the total sequence of the protein is controlled within the range of: 40-100%, 0-50%, 0-25%, and 0-15%. Figure 1 As shown, the codons corresponding to amino acids are distinguished according to the standard codon table, and then combined with Figure 2 The codon table of the species Homo sapiens was used to obtain the codon preference. In the species Homo sapiens, GGC of glycine accounts for 40-100%, GGA accounts for 0-50%, GGG accounts for 0-25%, and GGT accounts for 0-15%.

[0327] Standard codon table link URL:

[0328] https: / / zh.wikipedia.org / wiki / DNA%E5%AF%86%E7%A0%81%E5%AD%90%E8%A1%A8

[0329] 2) GC content ratio: higher than the total GC content ratio of the species, which is 0% to 15%. The total GC content of the species "Homo sapiens" is 52.27%, so the total GC content ratio of the optimized DNA sequence should be in the range of "52.27% to 67.27%". Different species should have different GC contents. For example, the total GC content of the species "Macaca fascicularis" is 49.64%, so the total GC content ratio of the optimized DNA sequence should be in the range of "49.64% to 64.64%". The local GC content of the DNA sequence should be controlled between 30% and 95%.

[0330] 3) Sequence repetition: Optimize the number of repeated sequences and sequence length as little as possible. For example, replace a codon in the sequence segment with a repeat sequence greater than 20 bases with a synonymous codon to reduce the number of bases in the repeat sequence. For example: before optimization, the repeat sequence "ATGGAGGACGCCAAGAACATCAAG" has a total of 24 bases appearing 3 times. You can choose to mutate the codons at 2 different positions into synonymous codons, and then obtain another 2 different DNA sequences, "ATGGAAGACGCCAAGAACATCAAG" and "ATGGAGGATGCCAAGAACATCAAG", and the amino acid of the three DNA sequences is "MEDAKNIK".

[0331] 4) RNA secondary structure and free energy: Reduce the continuous base pairing region and lower the minimum free energy. If both cannot be achieved at the same time, reducing the continuous base pairing region is the priority.

[0332] According to the above optimization method, the optimized sequences GLuc1, 2, 3, and 4 of the present invention were obtained.

[0333] like Figure 36 to Figure 38 As shown, the usage ratio of GLuc1 is: the first, second, third, and fourth codons in the total sequence of the protein are controlled in the range of: 68-100%, 0-29%, 0-7%, and 0-7%. The minimum free energy (MFE) of the sequence RNA of GLuc1 is -226.96 kcal / mol.

[0334] like Figure 39 to Figure 41 As shown, the usage ratio of GLuc2 is: the first, second, third, and fourth codons in the total sequence of the protein are controlled in the range of: 44-100%, 0-50%, 0-14%, and 0-3%. The minimum free energy of the sequence RNA of GLuc2 is -202.70 kcal / mol.

[0335] like Figure 42 to Figure 44 As shown, the usage ratio of GLuc3 is: the first, second, third, and fourth codons in the total sequence of the protein are controlled in the range of: 70-100%, 0-40%, 0-15%, and 0-5%. The minimum free energy of the sequence RNA of GLuc3 is -241.70 kcal / mol.

[0336] like Figure 45 to Figure 47 As shown, the usage ratio of GLuc4 is: the first, second, third, and fourth codons in the total sequence of the protein are controlled in the range of: 44-100%, 0-40%, 0-12%, and 0%. The minimum free energy of the sequence RNA of GLuc4 is -210.10 kcal / mol.

[0337] Four codon-optimized versions of DNA sequences were used as examples, and wild-type DNA sequences and DNA sequences from vectors of other companies were used as comparison examples to conduct cell experiments. They are named as shown in Table 8:

[0338] Table 8 Comparative analysis of Gaussia luciferase protein and DNA sequences

[0339]

[0340]

[0341] Example 4 Codon Optimization of Firefly Luciferase

[0342] (1) Amino acid sequence analysis

[0343] The present invention involves two versions of the amino acid sequences of the firefly luciferase, as shown in Table 9.

[0344] Table 9

[0345]

[0346] Amino acid sequence 1: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH IEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLP PGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTL IDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWD EDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL***.

[0347] Amino acid sequence 2:

[0348] ***.

[0349] (2) Convert amino acid sequence to DNA sequence

[0350] Convert all amino acid sequences to the most commonly used codon sequence of the species to be used. For example, for the species "Homosapiens", change all "alanine" to "GCC". Obtain the first version of the DNA sequence. Figure 2 shown.

[0351] Link URL:

[0352] https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606

[0353] https: / / www.kazusa.or.jp / codon /

[0354] The first version of the DNA sequence:

[0355]

[0356] (3) DNA sequence analysis

[0357] The step (2) is performed using amino acid sequence 2 to obtain a DNA sequence that is a first version of the DNA sequence.

[0358] The first version of the DNA sequence was analyzed for GC content. Fig.48 Shown

[0359] Link: https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html

[0360] The first version of the DNA sequence was analyzed for sequence duplication, such as Fig.49 shown.

[0361] Link: https: / / www.vectorbuilder.cn / tool / sequence-dot-plot.html

[0362] The first version of the DNA sequence was subjected to sequence RNA secondary structure and free energy analysis. The minimum free energy (MFE) of the sequence RNA was -687.5 kcal / mol. The RNA secondary structure was Fig.50 shown.

[0363] Link: http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi

[0364] (4) DNA sequence adjustment

[0365] By combining parameters such as codon preference, GC content ratio, sequence repetition, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0366] The optimization description of each parameter is described below.

[0367] 1) Codon preference: After obtaining the first version of the DNA sequence based on the most commonly used codons, the codons with the second, third, and fourth most frequently used codons are used to replace the codons with the first most frequently used codons. As a general rule, the proportion of the codons with the first, second, third, and fourth most frequently used codons in the total sequence of the protein is controlled within the range of: 40-100%, 0-50%, 0-25%, and 0-15%. Figure 5 As shown, the codons corresponding to amino acids are distinguished according to the standard codon table, and then combined with Figure 1The codon table of the species Homo sapiens was used to obtain the codon preference. In the species Homo sapiens, GGC of glycine accounts for 40-100%, GGA accounts for 0-50%, GGG accounts for 0-25%, and GGT accounts for 0-15%.

[0368] Link URL: https: / / zh.wikipedia.org / wiki / DNA%E5%AF%86%E7%A0%81%E5%AD%90%E8%A1%A8

[0369] 2) GC content ratio: higher than the total GC content ratio of the species, which is 0% to 15%. The total GC content of the species "Homo sapiens" is 52.27%, so the total GC content ratio of the optimized DNA sequence should be in the range of "52.27% to 67.27%". Different species should have different GC contents. For example, the total GC content of the species "Macaca fascicularis" is 49.64%, so the total GC content ratio of the optimized DNA sequence should be in the range of "49.64% to 64.64%". The local GC content of the DNA sequence should be controlled between 30% and 95%.

[0370] 3) Sequence repetition: Optimize the number of repeated sequences and sequence length as little as possible. For example, replace a codon in the sequence segment with a repeat sequence greater than 20 bases with a synonymous codon to reduce the number of bases in the repeat sequence. Example: Before optimization, the repeat sequence "ATGGAGGACGCCAAGAACATCAAG" has a total of 24 bases appearing 3 times. You can choose to mutate the codons at 2 different positions into synonymous codons, and then obtain another 2 different DNA sequences, "ATGGAAGACGCCAAGAACATCAAG" and "ATGGAGGATGCCAAGAACATCAAG". The amino acids of the three DNA sequences are all "MEDAKNIK".

[0371] 4) RNA secondary structure and free energy: Reduce the continuous base pairing region and lower the minimum free energy. If both cannot be achieved at the same time, reducing the continuous base pairing region is the priority.

[0372] According to the above optimization method, No. 8 and No. 9 of the present invention were obtained.

[0373] like Figure 51 to Figure 53As shown, the usage ratio of LUC-1 is: the first, second, third, and fourth codons in the total sequence of the protein are controlled in the range of: 68-100%, 0-29%, 0-7%, and 0-7%. The minimum free energy (MFE) of the sequence RNA of No. 8 is -639.90 kcal / mol.

[0374] like Figure 54 to Figure 56 As shown, the usage ratio of LUC-2 is: the first, second, third, and fourth codons in the total sequence of the protein are controlled in the range of: 44-100%, 0-50%, 0-14%, and 0-3%. The minimum free energy of the sequence RNA of No. 9 is -655.40 kcal / mol.

[0375] Table 10

[0376]

[0377] Example 5 SpCas9 codon optimization

[0378] (1) Analysis of amino acid sequence

[0379] The present invention involves only one amino acid sequence (SpCas9):

[0380]

[0381] (2) DNA sequence conversion

[0382] According to the codon preference table, all amino acid sequences are converted into the most commonly used codon sequences of the species to be applied. The frequency of human codon usage is shown in the following table:

[0383] Table 11

[0384] UUU 17.6(714298) UCU 15.2(618711) UAU 12.2(495699) UGU 10.6(430311) UUC 20.3(824692) UCC 17.7(718892) UAC 15.3(622407) UGC 12.6(513028) UUA 7.7(311881) UCA 12.2(496448) UAA 1.0(40285) UGA 1.6(63237) UUG 12.9(525688) UCG 4.4(179419) UAG 0.8(32109) UGG 13.2(535595) CUU 13.2(536515) CCU 17.5(713233) CAU 10.9(441711) CGU 4.5(184609) CUC 19.6(796638) CCC 19.8(804620) CAC 15.1(613713) CGC 10.4(423516) CUA 7.2(290751) CCA 16.9(688038) CAA 12.3(501911) CGA 6.2(250760) CUG 39.6(1611801) CCG 6.9(281570) CAG 34.2(1391973) CGG 11.4(464485) AUU 16.0(650473) ACU 13.1(533609) AAU 17.0(689701) AGU 12.1(493429) AUC 20.8(846466) ACC 18.9(768147) AAC 19.1(776603) AGC 19.5(791383) AUA 7.5(304565) ACA 15.1(614523) AAA 24.4(993621) AGA 12.2(494682) AUG 22.0(896005) ACG 6.1(246105) AAG 31.9(1295568) AGG 12.0(486463) GUU 11.0(448607) GCU 18.4(750096) GAU 21.8(885429) GGU 10.8(437126) GUC 14.5(588138) GCC 27.7(1127679) GAC 25.1(1020595) GGC 22.2(903565) GUA 7.1(287712) GCA 15.8(643471) GAA 29.0(1177632) GGA 16.5(669873) GUG 28.1(1143534) GCG 7.4(299495) GAG 39.6(1609975) GGG 16.5(669768)

[0385] For example, for the species "Homo sapiens", all "alanine" is changed to "GCC". The first version of the DNA sequence, hSpCas9-WT, is obtained.

[0386]

[0387] (3) DNA sequence analysis

[0388] DNA sequence analysis of hSpCas9-WT was performed, including GC content ratio analysis (https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html), sequence repetition analysis (https: / / www.vectorbuilder.cn / too l / sequence-dot-plot.html), and sequence RNA secondary structure and free energy analysis (http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi).

[0389] (4) DNA sequence adjustment

[0390] By combining parameters such as codon preference, GC content ratio, sequence repetition, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0391] The optimization description of each parameter is described below.

[0392] 1) Codon preference: After obtaining the first version of the DNA sequence based on the most commonly used codons, the codons with the second, third, and fourth percentages are used in turn to replace the codons with the first percentage. In general, the proportion of the codons with the first, second, third, and fourth percentages in the total sequence of the protein is controlled within the range of: 40-100%, 0-50%, 0-25%, and 0-15%.

[0393] 2) GC content ratio: higher than the total GC content ratio of the species (0% to 15%). The total GC content ratio of the species “Homo sapiens” is 52.27%, so the total GC content ratio of the optimized DNA sequence should be “52.27% to 67.27%”.

[0394] 3) Sequence repetition: Optimize the number of repeated sequences and sequence length to minimize the number of repeated sequences.

[0395] 4) RNA secondary structure and free energy: Reduce the continuous base pairing region and lower the minimum free energy. If both cannot be achieved at the same time, reducing the continuous base pairing region is the priority.

[0396] According to the above optimization method, a total of 5 versions of nucleic acid sequences were formed, including hSpCas9 in the prior art as a comparative example, which are named as follows:

[0397] Table 11

[0398] Serial number name sequence 1 WT SEQ ID NO:38, DNA sequence analysis schematic diagram as shown Fig.57 2 VB1 SEQ ID NO:33, DNA sequence analysis schematic diagram as shown Fig.58 3 VB2 SEQ ID NO:34, DNA sequence analysis schematic diagram as shown Fig.59 4 VB3 SEQ ID NO:35, DNA sequence analysis schematic diagram as shown Fig.60 5 VB4 SEQ ID NO:36, DNA sequence analysis schematic diagram as shown Fig.61 6 VB5 SEQ ID NO: 37, DNA sequence analysis schematic diagram as shown Fig.62

[0399] Effect verification

[0400] 1. Expression verification of the sequences of Examples 1 to 5 was performed. Taking OVA involved in Example 1 as an example, the construction of other protein expression vectors is similar to this step:

[0401] 1. Vector construction

[0402] The DNA sequence of chicken ovalbumin was used as the Antigen coding region and inserted into the pmRVac backbone by the Gibson method (the DNA sequences of the other elements except the target sequence were consistent with those of the comparative example and the example, and the pmRVac vector was transformed from the pUC19k vector, as shown in the figure Fig.63 ) to obtain pmRVac-OVA ( Fig.63 ) vector Gibson reaction buffer. Then the reaction buffer was transferred to VB UltraStable competent medium by electroporation, SOC culture medium was added, and the cells were cultured on a shaker for 1 hour. After that, part of the culture medium was taken out and spread and streaked on LB plates containing Kana antibiotics, and then cultured in a 37°C incubator overnight.

[0403] 2. Clone identification

[0404] Pick multiple single clones from the Kana plate cultured overnight in step 1, dissolve in sterile water to obtain bacterial suspension, take the bacterial suspension for PCR amplification, and identify the correct clone by gel electrophoresis. Take the bacterial suspension of the correct clone and add it to LB medium containing Kana antibiotics, and culture it overnight at 37°C on a shaker.

[0405] 3. Plasmid extraction

[0406] Take the bacterial solution cultured overnight in step 2 and use a plasmid extraction kit to extract the plasmid. Perform restriction digestion, sequencing and other identification on the obtained plasmid to obtain the correct plasmid.

[0407] 4. Plasmid linearization

[0408] The plasmid in step 3 was digested with SapI and purified using a PCR product purification kit to obtain a purified linearized plasmid.

[0409] 5. In vitro transcription of mRNA

[0410] Take the linearized plasmid in step 4 and add it to the transcription system, react at 37°C to obtain a crude mRNA sample, purify it, heat and denature it, add it to the capping system, cap it at 37°C, and obtain the mRNA sample after magnetic bead purification.

[0411] 6. Cell transfection

[0412] mRNA transfection dose: 1ug / well.

[0413] Take the mRNA in step 5 and add it to the 293T cells in a 12-well plate (the cell density during transfection is 80%). After culturing at 37° C. for 24 hours, collect the supernatant and place it on ice for labeling.

[0414] 2. Expression effect detection:

[0415] 2.1. Determination of protein concentration by BCA method

[0416] Dilute the BCA protein standard according to a certain gradient concentration, and add an appropriate amount to a 96-well ELISA plate. Mix BCA working solution reagents A and B in a ratio of 50:1, prepare and use immediately, and add BCA working solution to each well of the ELISA plate. Place the ELISA plate on an oscillator and incubate at 37°C. Use a microplate spectrophotometer to measure the absorbance value at a wavelength of 562nm. With protein content (ug) as the horizontal axis and absorbance value as the vertical axis, draw a standard curve and fit a linear equation.

[0417] Dilute the sample to be tested to an appropriate concentration, add BCA working solution, mix thoroughly, incubate at 37°C, and measure the absorbance. Calculate the protein concentration from the absorbance according to the standard curve, and multiply it by the sample dilution factor to obtain the sample concentration.

[0418] 2.2. WB experiment and SDS-PAGE gel staining and destaining

[0419] Mix the supernatant obtained in step 2.1 evenly, add 4x SDS loading, mix thoroughly, heat to denature, and place on ice. Assemble two precast gels and place them in the electrophoresis tank, add buffer, and spot samples in the same order and sample volume. Perform electrophoresis at 150mV constant voltage for 1h. Cut a 0.45um PVDF membrane of appropriate size and transfer one precast gel immediately after the electrophoresis. Perform transfer at 250mA constant current for 45min. After the transfer, use tweezers to remove the membrane and place it in a container. After rinsing with TBST, add blocking solution and block at room temperature for 1h. Rinse twice with TBST and incubate with primary antibody overnight. Rinse three times with TBST and incubate with secondary antibody at room temperature.

[0420] After washing with TBST for 3 times, drop ECL Western blotting substrate on the membrane and use a gel-shooter to perform luminescence imaging to observe the protein expression results.

[0421] For another precast gel, remove the gel and soak it in a glass dish containing an appropriate amount of Coomassie Brilliant Blue dye solution, place the glass dish on a circular decolorization shaker, and incubate at room temperature for 1 hour. Pour out the dye solution, add an appropriate amount of decolorization solution, and decolorize at room temperature for 4-24 hours, changing the decolorization solution 2-4 times in the middle.

[0422] Take 100ug / well sample for WB and SDS-PAGE experiments.

[0423] 2.3 In vivo experiments

[0424] According to the results of the WB experiment, Co1, Co3, Cre and Cre T in the Cre recombinase obtained in Example 2 were used for the experiment. The mRNA sample obtained in step 6 was encapsulated by LNP and injected into Ai9 mice via the tail vein. The injection dose was low dose (group marked as L, 0.1 mg / kg mouse body weight) and high dose (group marked as H, 0.4 mg / kg mouse body weight). The sample was injected with heart perfusion 48 hours after injection, and then the liver and spleen were dissected for fixation, dehydration, and frozen sections to observe the tdTomato red fluorescence intensity, so as to compare the effect. The negative control group was the PBS injection group.

[0425] 3. Test conclusion

[0426] 3.1 In Example 1, the test results after expression of each group of cells are as follows Figures 64-66 The results showed that in Example 1, at the same dose, OVA (1) had the strongest protein expression, OVA (G) was weaker, OVA (3) was the worst, and no obvious protein expression was observed in other samples. The strength of each sample band in SDS-PAGE gel was basically consistent, indicating that the WB loading amount was consistent.

[0427] 3.2 In Example 2, the test results after expression of each group of cells are as follows Fig.67 ,like Fig.67 In the WB experiment, the size of the Cre recombinase band was consistent; and at different doses (the mRNA transfection dosage was 1 ug / well and 2 ug / well respectively), similar trends were observed; and the expression levels of the internal reference protein Vinculin in different groups were similar, proving that the experimental results are credible. Fig.67 In the WB experiment, at low doses, the expression of Co3 was relatively the highest, followed by Co1, both significantly better than the wild type or other companies' control groups. At high doses, the expression of Co1 was relatively the highest, followed by Co3, both significantly better than the wild type or other companies' control groups. Fig.68 ,In the in vivo experiment, the tdTomato red fluorescence intensity of Co3 was relatively the highest, slightly better than the control group.

[0428] 3.3 In Example 3, 20 ul of cell culture supernatant was mixed with 0.5 ul of substrate coelenterazine and 50 ul of reaction buffer at 4 time points after transfection, 6 h, 24 h, 48 h, and 72 h. The expression product Gaussia luciferase and its substrate coelenterazine will contact with an enzymatic reaction and chemiluminescence. The intensity of chemiluminescence is measured using a microplate chemiluminescence analyzer, and the intensity is converted into a numerical value to show the expression effect of the product. According to the intensity of the chemiluminescence reaction of the expression product, the expression efficiency of mRNA with different codon optimization can be corresponded. The experimental results are shown in Tables 12 and Fig.69 shown.

[0429] Table 12 Comparison of expression effect intensity between the embodiment and the comparative example cell experiment

[0430]

[0431] The results show that GLuc2 and GLuc3 are significantly better than GLuc-wt and slightly better than GLuc-other. GLuc1 and GLuc4 are slightly better than GLuc-wt. The mRNA expression vector constructed by the Gaussia luciferase DNA sequence of the present invention has a higher expression efficiency in HEK293T cells than the mRNA expression vector constructed by the wild-type Gaussia luciferase DNA sequence.

[0432] 3.4 In Example 4, cell experiments and animal experiments were conducted respectively:

[0433] In the cell experiment, two groups of experiments were set up for dose comparison, and the mRNA amounts were 0.25 μg / well and 0.5 μg / well, respectively. The mRNA obtained by expression was added to the 293T cells in a 12-well plate (293T cell dosage 106 / well), cultured at 37°C, and the samples were processed at time points of 6 h, 24 h, 48 h, and 72 h. The cells transfected with mRNA were taken out of the incubator, the cell waste liquid was removed on the clean bench, and the cell lysis solution was added. After reacting at room temperature for 1 min, the cells were gently shaken to fall off, and the cell lysis solution was aspirated into a clean 1.5 mL centrifuge tube, and centrifuged at 4°C 12000 rpm for 3 min. Take an equal amount of the supernatant of the centrifuged cell lysate and add it to the 96-well plate filled with D-Luciferin solution, gently blow and mix, put it in the microplate reader away from light, and read the fluorescence intensity value. The results showed that the expression of Luciferase reached its peak at 24 h, and the corresponding chemiluminescence intensity of LUC-1 and LUC-2 samples was higher ( Fig.70 ).

[0434] In animal experiments,

[0435] The corresponding mRNAs of control sequence 1, control sequence 3, control sequence 5, LUC-1 and LUC-2 were selected for LNP encapsulation, and then the in vivo mouse experiment was performed. 6-8 week old c57BL / 6J female mice were selected. The total injection dose was 10μg and 30μg (the total dose of both legs). The sample concentration and injection volume are shown in the following table. If the injection volume is less than 50μL, use 1×PBS solution to make up to 50μL total injection volume.

[0436] Table 13

[0437]

[0438] Before sample injection, the mice were marked, captured and fixed. After disinfecting the injection area with 70% alcohol, the samples were injected into the muscles of both hind legs. After injection, the mice were returned to the original cage for normal feeding, and live imaging was performed at 6 h, 24 h, 48 h, and 72 h after injection. Before live imaging, 15 mg / mL D-Luciferin solution (substrate) was prepared with D-PBS, filtered through a 0.22 μm filter membrane for sterilization, and kept away from light for later use. Each mouse was weighed and recorded before substrate injection. D-Luciferin solution was injected intraperitoneally into the mice at 150 mg / kg body weight. After substrate injection, the mice were placed in an induced anesthesia chamber (isoflurane gas anesthesia) to fully anesthetize them. After injecting the luciferin substrate, wait for about 15 minutes before live imaging.

[0439] from Figures 71-74 ,It can be seen from the results in Tables 14 to 17 that the in vivo expression ,effects of LUC-1 and LUC-2 samples are relatively strong.

[0440] Table 14 Chemiluminescence detection data of Firefly Luciferase transfection for 6 h

[0441]

[0442]

[0443] Table 15 Firefly Luciferase transfection 24h chemiluminescence detection data

[0444]

[0445] Table 16 Firefly Luciferase transfection 48h chemiluminescence detection data

[0446]

[0447]

[0448] Table 17 Firefly Luciferase transfection 72h chemiluminescence detection data

[0449]

[0450] 3.5 In Example 5,

[0451] 3.5.1 Western Blot (WB) experimental results are as follows Fig.72 The results showed that the expression of VB1-5 proteins was stronger than WT. After luminescence imaging, grayscale scanning analysis was performed using Image J software. After grayscale scanning, all samples were normalized using GAPDH as a reference, and then the fold change of each group relative to the control group was calculated. The data processing results are shown in Fig.74 As shown, VB5 increased 4-fold relative to WT, VB1 and VB2 increased 5-fold relative to WT, and VB3 and VB4 increased 8-fold relative to WT.

[0452] 3.5.2 Functional verification of the coding nucleic acid of Example 5 was also performed

[0453] 3.5.2.1 Cell transfection

[0454] Take the mRNA prepared above, and adjust the input amount of mRNA as shown in the following table according to the relative protein expression fold change results obtained in the WB experiment. To ensure the same total amount of input mRNA, use nonsense RNA to supplement the input amount to make the total amount 1μg. Use 293T cells in a 12-well plate (293T cell volume 10E6 cells / well), and add a total of 1μg mRNA and 1μg gRNA (such as gEMX1 mentioned above) to each well. Collect cells 48h after transfection, remove the supernatant as much as possible, and store at -80℃.

[0455] Table 18

[0456] Experimental Group SpCas9 mRNA Nonsense RNA 1 WT mRNA: 1.000 μg 0.000μg 2 VB1 mRNA: 0.250 μg 0.750 μg 3 VB2 mRNA: 0.250 μg 0.750 μg 4 VB3 mRNA: 0.125 μg 0.875 μg 5 VB3 mRNA: 0.250 μg 0.875 μg 6 VB3 mRNA: 0.500 μg 0.500 μg 7 VB4 mRNA: 0.125 μg 0.875 μg 8 VB4 mRNA: 0.250 μg 0.750 μg 9 VB4 mRNA: 0.500 μg 0.500 μg 10 VB5 mRNA: 0.250 μg 0.750 μg

[0457] 3.5.2.2 Cell genome extraction

[0458] Use a cell genome DNA extraction kit to extract the cell genome as described in the kit instructions. After the extraction, take 2 μL to measure the concentration and set aside.

[0459] 3.5.2.3 PCR amplification of the desired fragment

[0460] The target fragment was amplified by PCR using the cell genome extracted above based on the following components and corresponding volumes:

[0461] Table 19

[0462] Components volume Taq enzyme (5U / μl) 1μL 10×Taq Buffer 10μL dNTP Mixture (2.5 mM each) 16μL Template (cell genome, obtained from the previous extraction step) 1 μg Primer 1 4μL Primer 2 4μL Deionized water Make up to 100 μL

[0463] 3.5.2.4 PCR amplification procedures include:

[0464] 94℃, 1min;→[98℃, 10s→68℃, 60s]×30 cycles→72℃, 10min→4℃, temporarily store.

[0465] 3.5.2.5 PCR product purification and recovery

[0466] A common DNA product purification kit was used to purify and recover the PCR product obtained by the PCR amplification described above according to the instructions. After completion, 2 μL was taken to measure the concentration and set aside.

[0467] 3.5.2.6 T7E1 digestion

[0468] The PCR purified product obtained in the above PCR product purification and recovery step was used to perform T7E1 restriction enzyme digestion to identify its gene editing efficiency. The specific operation is as follows.

[0469] The annealing system for PCR purification products is as follows:

[0470] Table 20

[0471] Components volume PCR purified product (obtained from the previous step) 400ng 10×Buffer 2μL Nuclease-free water Make up to 19 μL

[0472] The procedure is as follows:

[0473] 95℃, 5min→95-85℃, -2℃ / s→85-25℃, -0.1℃ / s→4℃, temporarily store.

[0474] Add 1 μL T7 Endonuclease I to the annealing reaction system, digest at 37°C for 15 min, and analyze by 2% agarose gel electrophoresis.

[0475] The results are as follows Fig.74 As shown, in the functional verification, reducing the mRNA input ratio of the disclosed example can still achieve an editing efficiency similar to that of the comparative example.

[0476] The agarose gel electrophoresis images were grayscale scanned and analyzed using Image J, and the gene editing efficiency of each experimental group was calculated according to a publicly available calculation method (see: Guschin, DY, et.al. (2010) Arapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 649, 247–256.).

[0477] The data processing results are as follows Fig.75As shown, when the input amount of mRNA of VB3 in the embodiment of the present disclosure is 1 / 8 of that of the comparative example, it can still achieve similar editing efficiency. The input amount of mRNA is greatly reduced. When the input amount of VB1, VB2, and VB5 is 1 / 4 of that of the comparative example, the editing efficiency is close to that of the comparative example.

[0478] In summary, the expression of hSpCas9 protein provided in the embodiments of the present disclosure is improved compared with the control ratio, which is helpful for better application in in vivo experiments and can also obtain products with market competitiveness.

[0479] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for codon optimization, comprising: Obtaining a protein encoding nucleic acid sequence according to the codon preference of the host, and then adjusting the GC content, repeat sequence, secondary structure and / or free energy in the obtained sequence to obtain an optimized encoding nucleic acid; The GC content includes: the GC content of the entire length of the encoding nucleic acid and the GC content within a unit length.

2. The method according to claim 1, characterized in that The GC content within the unit length range is the GC content within 10 bp to 100 bp; preferably, it is the GC content within 20 bp to 50 bp.

3. The method according to claim 2, characterized in that The GC content within the unit length is 20% to 95%; preferably, the GC content is 26% to 63% or 40% to 83%.

4. The method according to claim 1, characterized in that: The full-length GC content is higher than 0% to 15% of the total GC content of the host species.

5. The method according to claim 4, characterized in that The host is a human cell, and the full-length GC content is 52.27% to 67.27%, and preferably, the full-length GC content is 56% to 63%; The host is a cynomolgus macaque cell, and the full-length GC content is 49.64% to 64.64%. Preferably, the full-length GC content is 54% to 61%.

6. The method according to claim 1, characterized in that The length of the repetitive sequence is no greater than 30 bp, and preferably, the length of the repetitive sequence is no greater than 20 bp.

7. The method according to claim 1, characterized in that The adjustment of the secondary structure includes reducing the continuous base pairing region.

8. The method according to claim 1, characterized in that The adjustment of the free energy includes making the minimum free energy of the sequence 80% to 100% of the lowest free energy.

9. The method according to any one of claims 1 to 8, characterized in that: The adjustment includes: using the codons with the second, third, and fourth codon preferences to replace the codons with the first percentage, and the percentages of the codons with the first, second, third, and fourth usage frequencies in the full length of the encoding nucleic acid are: 40% to 100%, 0% to 50%, 0% to 25%, and 0% to 15%, respectively.

10. The method according to claim 9, characterized in that The adjustments include: Among Phe codons, UUU is used at a frequency of 1% to 25%, and UUC is used at a frequency of 75% to 99%; Among the codons for Leu, the usage frequency of CUG was 86% to 99%, the usage frequency of CUC was 1% to 10%, the usage frequency of UUA was 0% to 1%, the usage frequency of UUG was 0% to 1%, the usage frequency of CUU was 0% to 1%, and the usage frequency of CUA was 0% to 1%; Among the codons of Ile, the usage frequency of AUU is 5% to 25%, the usage frequency of AUC is 75% to 94%, and the usage frequency of AUA is 0% to 1%; The codon for Met is AUG; Among the codons of Val, the usage frequency of GUU is 0% to 5%, the usage frequency of GUC is 5% to 15%, the usage frequency of GUA is 0% to 1%, and the usage frequency of GUG is 79% to 95%; Among the codons for Ser, the usage frequency of UCU is 0% to 20%, the usage frequency of UCC is 10% to 25%, the usage frequency of UCA is 0% to 1%, and the usage frequency of UCG is 0% to 1%; the usage frequency of AGU is 0% to 1%, and the usage frequency of AGC is 52% to 90%; Among the codons of Pro, the usage frequency of CCU is 20% to 50%, the usage frequency of CCC is 44% to 72%, the usage frequency of CCA is 0% to 8%, and the usage frequency of CCG is 0% to 6%; Among Thr codons, ACU is used at a frequency of 0% to 1%, ACC at a frequency of 50% to 78%, ACA at a frequency of 20% to 48%, and ACG at a frequency of 0% to 1%; Among Ala codons, GCU is used at a frequency of 3% to 20%, GCC at a frequency of 73% to 90%, GCA at a frequency of 0% to 6%, and GCG at a frequency of 0% to 1%; Among Tyr codons, UAU is used at a frequency of 1% to 25%, and UAC is used at a frequency of 75% to 99%; Among His codons, CAU is used at a frequency of 1% to 15%, and CAC is used at a frequency of 85% to 99%; Among Gln codons, CAA is used at a frequency of 1% to 15%, and CAG is used at a frequency of 85% to 99%; Among the codons for Asn, the usage frequency of AAU is 5% to 30%, and the usage frequency of AAC is 70% to 95%; Among Lys codons, AAA is used at a frequency of 1% to 25%, and AAG is used at a frequency of 75% to 99%; Among the codons of Asp, the usage frequency of GAU is 3% to 35%, and the usage frequency of GAC is 65% to 97%; Among the codons for Glu, the usage frequency of GAA is 10% to 35%, and the usage frequency of GAG is 65% to 90%; Among Cys codons, UGU is used at a frequency of 15% to 40%, and UGC is used at a frequency of 60% to 85%; The codon for Trp is GUU; Among the codons for Arg, the usage frequency of CGU is 0% to 1%, the usage frequency of CGC is 1% to 8%, the usage frequency of CGA is 0% to 1%, the usage frequency of CGG is 44% to 72%, the usage frequency of AGA is 15% to 50%, and the usage frequency of AGG is 1% to 8%; Among the codons for Gly, the usage frequency of GGU is 0% to 5%, the usage frequency of GGC is 65% to 98%, the usage frequency of GGA is 1% to 20%, and the usage frequency of GGG is 1% to 10%.

11. A codon optimized system, characterized in that: The invention comprises a module for implementing the method according to any one of claims 1 to 10.

12. A codon-optimized electronic device, characterized in that: The invention comprises a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to implement the method according to any one of claims 1 to 10.

13. A computer-readable storage medium storing a computer program, wherein the computer program, when executed by a processor, implements the steps of the method according to any one of claims 1 to 10.

14. A biological material comprising at least one of the following I) to V): I), a nucleic acid optimized by the method according to any one of claims 1 to 10; II), an expression unit containing the nucleic acid described in I); III), a plasmid vector containing the nucleic acid described in I), or containing the expression unit described in II); IV), mRNA, which contains a 5'-terminal cap structure and the nucleic acid described in I), or contains a 5'-terminal cap structure and the expression unit described in II); V), a host cell, which comprises a nucleic acid described in I) integrated into its genome, or an expression unit described in II) integrated into its genome, or a plasmid vector described in III) transformed or transfected therewith.

15. A method for preparing a recombinant protein, comprising: After obtaining the nucleic acid molecule optimized by the method according to any one of claims 1 to 10, the nucleic acid molecule is introduced into a host cell and then cultured to obtain a product containing a recombinant protein.

16. A method for preparing an mRNA transfection preparation or vaccine, comprising: After obtaining the nucleic acid molecule optimized by the method according to any one of claims 1 to 10, the nucleic acid molecule is connected to a vector, linearized, capped, purified and encapsulated to obtain an mRNA transfection preparation or vaccine.

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