Method for quantitative regulation of gene translation and use thereof

Abstract

The application discloses a method for quantitatively regulating gene translation and application thereof. The method comprises the following steps: sequencing translation efficiency of Kozak sequences and variants thereof before a start codon of a target gene or before an upstream open reading frame of a non-coding region of the target gene, and performing gene editing on the Kozak sequences according to a sequencing result, so as to realize in-situ manipulation of the Kozak sequences and quantitatively regulate gene translation, and the Kozak sequences and the variants thereof are 3-6 bp in length respectively. The application establishes a highly efficient, flexible and widely applicable gene expression regulation method. By using a precise gene editing tool to customize the Kozak sequences of the target gene in-situ, the expression level of the target gene can be quantitatively controlled at the gene translation stage.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a method for quantitatively regulating gene translation and its application. Background Technology

[0002] Artificial regulation of gene expression plays a crucial role in many fields, such as gene function research, disease treatment, and agricultural breeding. Currently, gene expression regulation methods developed based on gene editing technology can only achieve broad-based suppression or overexpression of gene expression, severely lacking methods for fine-tuning gene expression, especially methods for predictively and quantitatively controlling protein synthesis to target levels. While some studies have reported relatively micro-regulatory effects in plant subjects by using CRISPR / Cas9 and base editors to edit upstream open reading frames (UORFs) of gene non-coding regions, this method is only applicable to genes with uORFs and is often limited to upregulating gene expression, exhibiting poor flexibility and a relatively narrow applicability.

[0003] Numerous studies have demonstrated the significant role of Kozak sequences in the initiation of gene translation. In practical analyses, endogenous genes exhibit diverse Kozak sequences, corresponding to varying translation efficiencies. Therefore, Kozak sequences may be ideal editing targets for regulating gene expression. For example, CN111647625A discloses a method for increasing the expression level of human coagulation factor IX, including optimizing the human coagulation factor IX gene; replacing the Kozak sequence; constructing an adeno-associated virus (AAV) expression plasmid using the optimized human FIX gene and Kozak sequence, and packaging recombinant AAV. Using the optimized gene coding sequence and Kozak sequence significantly increased the expression level of the human coagulation factor FIX gene, but still could not achieve fine-grained regulation of gene expression.

[0004] In summary, existing technologies are insufficient for precise and quantitative regulation of gene expression. Developing methods for precise and quantitative regulation of gene expression is one of the problems that the field of gene editing needs to solve. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention presents a method for quantitatively regulating gene translation and its application. This invention is the first to propose and develop the use of precise gene editing tools to customize Kozak sequences in situ to achieve translation control of endogenous genes. It has bidirectional and predictable quantitative regulatory functions and can be applied to all protein-coding genes in eukaryotes. This will greatly enrich gene expression regulation methods and widely promote the development of application fields such as breeding for superior traits in agriculture.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for quantitatively regulating gene translation, the method comprising:

[0008] The translation efficiency of Kozak sequences and their variants preceding the start codon of the target gene or preceding the open reading frame upstream of the non-coding region of the target gene is ranked.

[0009] Based on the sequencing results, the Kozak sequence is edited as needed to achieve quantitative regulation of gene translation based on in situ manipulation of the Kozak sequence;

[0010] The length of the Kozak sequence and its variants is independently 3 to 6 bp, including but not limited to 3 bp, 4 bp, 5 bp or 6 bp.

[0011] In this invention, bioinformatics analysis was used to statistically analyze the start codon (ATG) and the nucleotides preceding and following it (Kozak sequence). The results showed that the -6 to -1 nucleotides (ATG set to position 0) had a significant bias, indicating their important role in gene translation initiation and thus making them potentially ideal gene editing targets for controlling gene expression. To avoid affecting the structure of the target protein, the -6 to -1 nucleotides (KZ6), especially the -3 to -1 nucleotides (KZ3), were selected as the target editing region. To clarify the specific effects of each KZ3 on gene translation, it was found that there are 64 different base combinations for KZ3. This invention first constructed dual fluorescent reporters for 64 KZ3 variants, and used the average fluorescence intensity of EGFP compared to mCherry as the corresponding KZ3 translation efficiency. Different KZ3 sequence variants showed a wide range of translation efficiencies without changing the transcription level, with a difference of up to 10.15 times. Subsequently, the different KZ3 variants were ranked based on efficiency, and this ranking can serve as an indicator for subsequent protocol design and translation prediction.

[0012] This invention is the first to utilize precise gene editing technology to perform in situ customization of the Kozak sequence, which significantly affects gene translation initiation. This enables efficient and flexible control of gene expression during the translation stage, allowing for bidirectional regulation of specific gene expression, with both upregulation and downregulation effects. Furthermore, the results can be predicted based on in vitro reporter systems and KZ3 translation efficiency rankings. Secondly, the gene modification occurs at the genomic level, and the regulatory effects are long-term and heritable. In addition, this method does not affect the transcriptional level of genes, nor does it alter gene transcripts or protein structures, thus avoiding changes in the spatiotemporal expression of target genes and offering higher safety.

[0013] It is understood that the variants described in this invention include sequences obtained by base deletion, addition, or substitution based on the Kozak sequence.

[0014] In one specific embodiment of the present invention, modifying KZ3(CGT) of the EGFP gene in HEK293-EGFP cells to TGT, GGT, and AGT can achieve the effect of upregulating or downregulating the expression level of green fluorescent protein.

[0015] In another specific embodiment of the present invention, deleting the T base of KZ3(CGT) of the EGFP gene in HEK293-EGFP cells, or deleting GT and inserting it into AAC, can achieve the effect of upregulating or downregulating the expression level of green fluorescent protein.

[0016] Preferably, the Kozak sequence and its variants are 3 bp in length.

[0017] Preferably, the Kozak sequences and their variants, arranged in descending order of translation efficiency, are ATC, AAC, ACC, ACA, AGC, ATA, GAC, GCC, AGA, AAG, GAA, GTC, ACG, GAG, ATG, AAA, ACT, GCA, ATT, AAT, AGT, GTA, AGG, GGG, GGA, GCT, GAT, GCG, GGC, GGT, GTG, GTT, CAA, CAG, TAA, CTA, CCA, TCA, CTC, TAG, CTG, TAC, TCG, CAC, CCC, TTC, TTA, TCC, CCG, CGA, CAT, CTT, TCT, CGG, TTG, CCT, TGA, TAT, TGG, CGC, TGC, TTT, CGT, and TGT.

[0018] This invention reveals that the KZ3 translation efficiency characteristics remain relatively consistent across different mammalian cell types, without affecting gene transcription levels. Furthermore, these 64 KZ3 reporter systems can be re-ranked and reshaped to better reflect the characteristics of different cell types.

[0019] It is understood that all gene editing methods commonly used in the field are applicable to this invention. Optionally, the gene editing methods include single-base editing methods and / or prime editor methods. Specifically, single-base editing methods may include cytosine base editors (CBE) that can induce the conversion of base C to T and adenine base editors (ABE) that can induce the conversion of base A to G; prime editor methods include prime editors (PE), etc.

[0020] Optionally, the start codon of the target gene is ATG.

[0021] It is understood that the method of this invention, which edits the Kozak sequence of the target gene, is universal and can achieve translation control of the vast majority of genes. It is not only applicable to all protein-coding genes in mammals, but can also be extended to the entire eukaryotic world, with extremely broad application potential, such as in the construction of animal models and breeding. Taking agricultural breeding as an example, the application of this invention can quickly obtain agricultural varieties with different expression levels of genes of interest or dose-response to traits, which will enrich agricultural breeding technology and expand the abundance and progress of breeding products with superior traits.

[0022] Optionally, the target gene includes, but is not limited to, any one or a combination of at least two of EGFP, P53, SOD1, SMN, HR, or U2HR.

[0023] Secondly, the present invention provides a method for constructing a biological model, the method comprising:

[0024] The biological model is obtained by regulating the translation level of the target gene in the experimental organism using the quantitative gene translation regulation method described in the first aspect.

[0025] Preferably, the experimental organism includes, but is not limited to, any one of the following: rabbit, human cell line HEK293, Hacat cells, mouse NIH3T3 cell line, porcine fibroblasts, or rabbit fibroblasts.

[0026] Preferably, the target gene includes the HR gene.

[0027] Preferably, the biological model includes the Mary Una rabbit model of hypotrichosis.

[0028] Thirdly, the present invention provides the application of biological models prepared by the method for constructing biological models described in the second aspect in drug screening.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) This invention is the first to use precise gene editing technology to perform in situ customization of Kozak sequences that significantly affect the initiation of gene translation. It can achieve efficient and flexible control of gene expression during the translation stage, and can bidirectionally regulate the expression of specific genes, with both up-regulation and down-regulation effects. Moreover, its regulatory effect is predictable and has good micro-regulation properties.

[0031] (2) The method for quantitatively regulating gene translation in this invention has extremely wide applicability and can be applied to all protein-coding genes in eukaryotes;

[0032] (3) The gene modification of the quantitative gene translation regulation method of the present invention occurs at the genome level, and the regulatory effect is long-term and heritable. In addition, the method does not affect the transcription level of the gene, does not change the transcript and protein structure of the gene, and therefore does not cause changes to the spatiotemporal expression of the target gene, thus having higher safety. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the principle of the method for quantitatively regulating gene translation according to the present invention;

[0034] Figure 2 A diagram illustrating the sequence characteristics of translation start sites in human transcripts;

[0035] Figure 3 A ranking plot of translation efficiency for 64 KZ3 sequence variants;

[0036] Figure 4 The translation effect diagram of the KZ3 variant in NIH3T3, RFF, and PFF;

[0037] Figure 5A A design diagram for customizing the KZ3 sequence of the GFP gene using base editing;

[0038] Figure 5B Illustration of cell clone genotypes for obtaining different KZ3 variants of the GFP gene through base editing;

[0039] Figure 5C Fluorescence images of cell clones of different KZ3 variants of the GFP gene obtained through base editing;

[0040] Figure 5D A statistical graph of green fluorescence intensity for cell clones of different KZ3 variants of the GFP gene obtained through base editing;

[0041] Figure 5E Graph showing the fold change in green fluorescence intensity of cell clones of different KZ3 variants of the GFP gene obtained through base editing;

[0042] Figure 5F This image shows the transcriptional level detection of different KZ3 variants of the GFP gene obtained through base editing in cell clones.

[0043] Figure 6A A design diagram for customizing the KZ3 sequence of the GFP gene using guided editing;

[0044] Figure 6B Genotypes of HEK293-GFP cell clones obtained from different KZ3 editing modes;

[0045] Figure 6C Fluorescence images of HEK293-GFP cell clones obtained from different KZ3 editing modes;

[0046] Figure 6D A statistical graph of the average green fluorescence intensity of HEK293-GFP cell clones with different KZ3 sequences;

[0047] Figure 6E Transcriptional level analysis of HEK293-GFP cell clones with different KZ3 sequences, designed to guide editing;

[0048] Figure 7A A schematic diagram illustrating the KZ3 editing of the human TP53 gene;

[0049] Figure 7B Cell clones and genotypes obtained from KZ3 editing of the TP53 gene;

[0050] Figure 7C Transcriptional analysis of cell clones that performed KZ3 editing of the TP53 gene;

[0051] Figure 7D Protein expression analysis of cell clones that performed KZ3 editing of the TP53 gene;

[0052] Figure 7E Quantitative analysis of protein expression in cell clones that have undergone KZ3 editing of the TP53 gene;

[0053] Figure 8A A schematic diagram of KZ3 editing in the human SMN gene;

[0054] Figure 8B Cell clones and genotypes obtained from SMN gene KZ3 editing;

[0055] Figure 8C Transcriptional analysis of cell clones that edited the SMN gene KZ3;

[0056] Figure 8D Protein expression analysis of cell clones that were edited for the SMN gene KZ3;

[0057] Figure 8E Quantitative analysis of protein expression in cell clones that have undergone SMN gene KZ3 editing;

[0058] Figure 9A Here is a schematic diagram illustrating the KZ-edit strategy of uORF, using the HR gene as an example;

[0059] Figure 9B A schematic diagram of the design of the dual-luciferase in vitro reporter system for U2HR;

[0060] Figure 9C Transcriptional analysis for the U2HR dual-luciferase in vitro reporter system;

[0061] Figure 9DFluorescence ratio analysis for the U2HR dual-luciferase in vitro reporter system;

[0062] Figure 10A A schematic diagram illustrating the regulation of uORF expression in the rabbit HR gene using base editing;

[0063] Figure 10B A diagram illustrating a rabbit model modified with U2HRKZ3 obtained by embryonic RNA injection.

[0064] Figure 10C The results of first-generation sequencing of the genotypes of newborn rabbits;

[0065] Figure 10D The results of deep sequencing analysis of the genotypes of newborn rabbits;

[0066] Figure 10E Transcriptional analysis of the HR gene in U2HRKZ3-modified rabbits;

[0067] Figure 10F Analysis of HR gene expression levels in U2HRKZ3-modified rabbits;

[0068] Figure 10G The results of H&E staining of skin tissue sections;

[0069] Figure 10H For statistical analysis of hair follicle count;

[0070] Figure 11A Genomic sequence analysis of transcripts encoding human proteins;

[0071] Figure 11B Transcript statistics for each KZ3 sequence;

[0072] Figure 11C The proportion of genes with targetable PAM sequences in the 25 bp DNA surrounding the start codon;

[0073] Figure 11D Statistical analysis of gene transcripts with the “ATG” or “ATGG” initiation pattern;

[0074] Figure 11E This is a schematic diagram for a general KZ3 editing system. Detailed Implementation

[0075] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0076] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0077] This invention uses a dual-fluorescence reporter system to rank the translation efficiency of all 64 KZ3 sequence (first three nucleotides of the start codon) variants. This ranking can directly serve as an indicator for KZ3 customization of target genes. For more accurate evaluation, the 5'UTR of the target gene can be cloned into the dual-fluorescence reporter system for in vitro flow cytometry analysis to obtain an ideal Kozak sequence customization scheme. Based on the gene expression regulation target, a suitable Kozak sequence and a corresponding precise gene editing scheme (named KZ-edit) are selected. When the regulation target is to upregulate gene expression, a KZ3 sequence with stronger translation efficiency is selected; otherwise, a weaker KZ3 sequence is selected. (See diagram below.) Figure 1 As shown. Precision gene editing tools mainly employ base editing and guided editing tools.

[0078] This invention enables targeted gene expression control at the cellular level. The Kozak custom editing system can be delivered into cells via electroporation or lipid transfection. Single-clone cell selection yields cell clones with the target genotype, resulting in the desired alteration of target gene expression levels. Quantitative real-time PCR and Western blotting can be used to detect gene transcription and protein expression levels, respectively.

[0079] If this invention is used for screening for superior agricultural traits or for altering the expression of specific genes in adults, the Kozak sequence customization is as described above. To obtain Kozak sequence-edited animals, chimeric animals with the target Kozak sequence can be obtained by injecting RNA encoding a gene-editing tool into the embryo. Subsequent mating and breeding can yield heterozygous or homozygous animals with the target genotype. For screening superior varieties of agricultural crops, the translation level of the target gene can also be altered by customizing the Kozak sequence to obtain varieties with specific traits. If gene expression regulation is to be performed at the adult animal level, adeno-associated viruses or nanoliposomes can be used for in vivo delivery.

[0080] Example 1

[0081] This embodiment utilizes bioinformatics analysis methods to statistically analyze the start codon and the nucleotides preceding and following it in the first 20 bp. The results are as follows: Figure 2As shown, the results indicate a significant preference for the -6 to -1 nucleotide positions (ATG set to position 0), suggesting their important role in gene translation initiation and making them potentially ideal gene editing targets for controlling gene expression. To avoid affecting the structure of the target protein, in this invention, the -6 to -1 positions (KZ6), particularly the -3 to -1 nucleotide positions (KZ3), are selected as the target editing region.

[0082] To clarify the specific effects of each KZ3 variant on gene translation, dual-fluorescent reporter plasmids for 64 KZ3 variants were first constructed. Primers containing KZ3 variant sequences were synthesized, annealed to form double strands, and then ligated with a pre-digested backbone of the dual-fluorescent reporter plasmid to construct reporter plasmids for assessing KZ3 translation efficiency. HEK293 cells were transfected, and flow cytometry analysis was performed two days after transfection. The average fluorescence intensity of EGFP compared to mCherry was used as the corresponding KZ3 translation efficiency. Different KZ3 sequence variants exhibited a wide range of translation efficiencies, with a difference of up to 10.15-fold, without altering the transcriptional level. The different KZ3 variants were then ranked based on efficiency, as follows: Figure 3 As shown, this ranking can serve as an indicator for subsequent scheme design and translation prediction.

[0083] Furthermore, we analyzed the translation efficiency characteristics of KZ3 in different mammalian cell lines. We selected five KZ3 sequences with different translation efficiencies—ATC, GAA, GTT, TAC, and TGT—and analyzed them in mouse embryonic fibroblast cell line NIH3T3, rabbit fibroblast RFF, and porcine fibroblast PFF, respectively. The corresponding reporter plasmids were transfected into the cells via PEI transfection. Two days later, samples were collected for flow cytometry and transcriptional analysis. The results are as follows: Figure 4 As shown, the KZ3 translation efficiency characteristics remain relatively consistent across different mammalian cell types and do not affect the gene transcription level.

[0084] Example 2

[0085] This embodiment uses HEK293-EGFP cells as an example to verify the method of quantitatively regulating gene translation of the present invention using base editing and guided editing tools.

[0086] Base editing experimental protocol as follows Figure 5AAs shown, the first 3 nucleotides (KZ3) of the EGFP gene start codon are CGT, which are mutated to TGT, GGT, and AGT, respectively. The specific steps include: transfecting HEK293-EGFP cells with the corresponding plasmid of the base editing system by electroporation; collecting a small number of cell samples two days later to test the editing effect; after the target site has shown good editing effect, the remaining cells are diluted and seeded in 10cm dishes for single-clone growth; when the clones grow to a suitable size, they are picked and genotyped to obtain the target clones; the target cell clones are expanded and cultured for flow cytometry analysis and transcriptional analysis.

[0087] The results are as follows Figure 5B As shown, HEK293-EGFP cell clones with KZ3 sequence-3C mutations to T, G, and A were successfully obtained. Fluorescence images reveal that, compared to CGT (WT) cell clones, the fluorescence intensity of cell clones with KZ3 as TGT was weaker; conversely, the fluorescence intensity of cell clones with KZ3 as GGT and AGT was significantly enhanced. Figure 5C Flow cytometry analysis showed that, compared to the original CGT (WT) cell clone, the green fluorescence intensity of the TGT cell clone decreased by 0.57-fold, while the green fluorescence of the GGT and AGT cell clones increased by 5.58-fold and 8.86-fold, respectively. Figure 5D (E), but their GFP gene transcription levels remain essentially unchanged ( Figure 5F ).

[0088] Guided editing of experimental protocols, such as Figure 6A As shown, the steps are as follows: deleting T from CGT and replacing G with T, replacing GT with CC, deleting GT and inserting AAC, and deleting GT and inserting CACC. The specific steps include: transfecting cells with the guide editor and the corresponding guide RNA; preliminarily identifying cells with the target editing effect and performing single-clone screening to obtain target genotype clones; and performing fluorescence observation, cell flow cytometry analysis, and transcription analysis on the target cell clones.

[0089] result Figure 6B The study successfully obtained a series of edited cell clones; fluorescence observation showed that the fluorescence intensity of the delT, G>T, GT>CC, delGT&insAAC, and delGT&insCACC cell clones obtained after KZ3 editing was increased to varying degrees. Figure 6C Further flow cytometry analysis revealed that, compared to the original cell clone CGT(WT), the fluorescence intensities of the delT, G>T, GT>CC, delGT&insAAC, and delGT&insCACC series cell clones increased by 12.53, 1.23, 1.72, 15.94, and 7.05 times, respectively. Figure 6D Similarly, the transcriptional levels of each cell clone did not change significantly. Figure 6E ).

[0090] Example 3

[0091] This embodiment directly edits the Kozak sequence (TP53) of endogenous genes at the genomic level. Figure 7A Using a bootloader editor, the KZ3 sequence GCC in the TP53 gene was replaced with TTT, and the result is as follows: Figures 7B-7E As shown, cell clones P53-TTT-14, P53-TTT-23, and P53-TTT-35 (with the target KZ3 sequence TTT) Figure 7B Its transcription level remains unchanged. Figure 7C However, protein expression levels were significantly reduced. Figure 7D The levels of these three types were reduced to 0.31, 0.55, and 0.60 times that of the wild type, respectively. Figure 7E ).

[0092] Furthermore, this embodiment directly edits the Kozak sequence of endogenous genes (SMN) at the genome level. Figure 8A Similarly, using a bootloader editor, the KZ3 sequence GCT of the SMN gene was replaced with AAC, and the result was... Figures 8B-8E As shown, cell clones SMN1 / 2-AAC-33, SMN1 / 2-AAC-40, and SMN1 / 2-AAC-54, which possess the target KZ3 sequence AAC, are shown. Figure 8B Its transcription level remains unchanged. Figure 8C However, protein expression levels were significantly increased. Figure 8D These figures represent increases of 2.95, 4.10, and 4.33 times that of the wild type, respectively. Figure 8E ).

[0093] Example 4

[0094] This embodiment uses the upstream open reading frame (uORF) U2HR of the HR gene non-coding region as an example to verify KZ-edit regulation of uORF.

[0095] KZ-edit regulation of human HR gene uORF, the protocol is as follows: Figure 9A and Figure 9BAs shown, increasing and decreasing the KZ translation efficiency of uORF indirectly downregulates and upregulates the expression levels of the major reading frames of downstream HR genes, respectively. For the human HR gene U2HR, three variants (AAC, TTC, and TTT) and a variant with a mutated start codon were designed, and corresponding dual-luciferase reporter plasmids U2HR-WT, U2HR-AAC, U2HR-TTC, U2HR-TTT, and U2HR-dATG were constructed. The ratio of firefly luciferin to kidney luciferin activity (F-luc / R-luc) was used to evaluate translation efficiency. Specific validation steps included transfecting HEK293 cells with the reporter plasmid PEI, and harvesting cells 48 hours later for luciferase and Q-PCR detection. The results are as follows... Figure 9C and Figure 9D As shown, the transcription levels of different reporter plasmids remained essentially unchanged, but the ratios of luciferase changed significantly. The ratios of U2HR-AAC, U2HR-TTC, U2HR-TTT, and U2HR-dATG became 0.53, 1.33, 2.69, and 3.49 times that of U2HR-WT, respectively.

[0096] KZ-edit regulation of the HR gene uORF in rabbits was used to construct a Mariunia oligohair disease (MUHH) rabbit model. The protocol is as follows: Figure 10A As shown, KZ3(CCC) was modified to TTT using base editing technology to increase the expression of the target HR gene, thereby inducing hypotrichosis. The specific steps included: in vitro transcription of the mRNA and sgRNA encoding the gene editing system; embryo microinjection of RNA (concentration: mRNA: 150 ng / μL; sgRNA: 50 ng / μL) and embryo transfer to surrogate rabbits; and obtaining and identifying the gene-edited rabbit pups. Results are as follows... Figures 10B-10H As shown, two U2HR rabbits with the target mutation in the KZ3 sequence were successfully obtained. Compared with age-matched wild-type rabbits, their hair growth was slower, and U2-1 even showed localized bald patches, which better reflected the characteristics related to MUHH disease. Figure 10B Using first-generation Sanger sequencing and amplicon deep sequencing, it was observed that the two gene-modified rabbits exhibited target mutations at the editing sites. U2-1 primarily included three mutant types: TTC, TTT, and del-5bp, accounting for 45%, 32.64%, and 16.73%, respectively. U2-2 mainly showed CCC>TTC mutations, with the target TTC genotype accounting for 93.83%. Figure 10C Further analysis of the transcription and expression levels of the HR gene revealed that the HR transcription levels in the two edited rabbits, U2-1 and U2, remained essentially unchanged. Figure 10E However, the protein expression level achieved the expected increase. Figure 10FThis demonstrates that base editing-mediated KZ3 mutations in uORFr can regulate the translation level of the target gene. Furthermore, staining results from skin tissue sections showed a significant reduction in hair follicles in the U2-1 and U2 edited rabbits. Figure 10G H).

[0097] Example 5

[0098] This embodiment evaluates the accessibility of the KZ-edit strategy of the present invention in the genome. First, at the DNA level, the characteristics of sequences near the start codon of all human protein-coding genes were analyzed. Statistical analysis of the sequences 25 bp before and after the start codon revealed a characteristic consistent with the transcript sequence, i.e., a high preference for sequences near the start codon. Figure 11A Specifically, the number of human genes present in all 64 KZ3 variants was listed, with the largest number (3500) being KZ3 with the GCC sequence, and the smallest number (20) being KZ3 with the ATG sequence. Based on translation efficiency obtained from in vitro reporter assays, the first half was defined as highly efficient translation and the second half as inefficient translation, with the transcript percentages being 65% and 35%, respectively. Therefore, theoretically, 35% of gene transcripts can undergo significant upregulation of translation, while 65% can undergo significant downregulation of translation. Figure 11B Of course, each gene transcript can be micro-regulated for quantitative translation using the KZ-edit strategy based on its specific sequence characteristics. Next, the accessibility of each gene was analyzed using commonly used base editors and guide editors, with a focus on commonly used gene editors that recognize four PAM sequences: NG, NGG, TTN, and TTTN. The results showed that within a 25bp range before and after the start codon, functional proteins using NG-PAM can find suitable target sites in all genes. Furthermore, the gene editors recognizing NGG, TTN, and TTTN-PAM could target and edit 34,922, 27,822, and 14,057 gene transcripts, respectively, representing 90%, 75%, and 30%, indicating that existing tools can be used to manipulate the KZ3 sequence of the vast majority of gene transcripts to regulate translation. Figure 11C Because in eukaryotes, protein-coding genes are often initiated by ATG, 100% of protein-coding genes inherently possess a suitable "TG" PAM. Furthermore, statistics show that over 50% of genes have a G base following their start codon, forming TGG-PAM. These genes are suitable for use with the most commonly used gene-editing tools that recognize NGG-PAM. Figure 11D Based on the sequence of the protein gene to be edited and the characteristics of the KZ-edit strategy, this embodiment proposes a general editing design. Figure 11EFor any target gene or upstream open reading frame that starts translation with ATG, a guided editing system that recognizes NG-PAM can be used for targeted editing. This system can recognize the common TG-PAM sequence, with the cut position being 2 positions upstream of ATG. The two bases before ATG can be replaced with any target Kozak sequence using guided editing technology, thereby achieving customized translation regulation.

[0099] In summary, by utilizing precise gene editing tools to in situ customize the Kozak sequence of target genes, quantitative control of gene expression levels can be achieved during the gene translation stage. Compared with previously reported gene knockout, overexpression, and micro-regulation methods, this invention simultaneously possesses the function of upregulating and downregulating gene expression, and its regulatory effect is predictable and exhibits good micro-regulation properties. Previous methods often only have a single regulatory function, and the regulatory effect is difficult to quantify and predict. Furthermore, this invention has extremely broad applicability, applicable to all protein-coding genes in eukaryotes, while previous methods based on manipulating uORF are only applicable to a small number of genes. Taking agricultural breeding as an example, this invention can rapidly obtain agricultural varieties with different expression levels of genes of interest or dose-response effects on traits, enriching agricultural breeding technology and expanding the abundance and progress of breeding superior trait products.

[0100] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a biological model, characterized in that, The method includes: The biological model was obtained by regulating the translation level of the target gene in the experimental subjects using a quantitative gene translation regulation method. The experimental subjects were selected from domestic rabbits; The methods for quantitatively regulating gene translation include: The translation efficiency of Kozak sequences and their variants preceding the start codon ATG of the target gene or preceding the open reading frame upstream of the non-coding region of the target gene is ranked. Based on the sequencing results, the Kozak sequence is edited as needed to achieve quantitative regulation of gene translation based on in situ manipulation of the Kozak sequence; The Kozak sequence and its variants are each 3 bp in length independently; The target gene is HR Gene; The variant is a sequence obtained by base deletion, addition, or substitution based on the Kozak sequence; The Kozak sequences and their variants, ranked from highest to lowest translation efficiency, are ATC, AAC, ACC, ACA, AGC, ATA, GAC, GCC, AGA, AAG, GAA, GTC, ACG, GAG, ATG, AAA, ACT, GCA, ATT, AAT, AGT, GTA, AGG, GGG, GGA, GCT, GAT, GCG, GGC, GGT, GTG, GTT, CAA, CAG, TAA, CTA, CCA, TCA, CTC, TAG, CTG, TAC, TCG, CAC, CCC, TTC, TTA, TCC, CCG, CGA, CAT, CTT, TCT, CGG, TTG, CCT, TGA, TAT, TGG, CGC, TGC, TTT, CGT, and TGT. The gene editing method is a single-base editing method and / or a guided editing method; The biological model is the Mary Una rabbit model of hypotrichosis.

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