Codon-optimized cas12i3 protein-encoding genes and uses thereof

Abstract

The application discloses a codon-optimized Cas12i3 protein coding gene and application thereof. Specifically disclosed is a DNA molecule, and a nucleotide sequence of the DNA molecule is SEQ ID No. 3. The application also discloses a gene editing system containing the DNA molecule and a method for improving the efficiency of Cas12i3 protein-mediated gene editing. In order to better apply the Cas12i3 protein to mammalian gene editing, different codon optimization schemes are designed, and through software evaluation and experimental verification, a codon optimization scheme with good Cas12i3 expression and editing effect is compared and screened. The application screens the codon optimization scheme for efficiently expressing the Cas12i3 protein in mammals, which can greatly improve the editing efficiency of the Cas12i3 protein, lays a foundation for the wide application of the Cas12i3 protein in mammals, and has great practical application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the codon-optimized Cas12i3 protein-encoding gene and its applications. Background Technology

[0002] CRISPR / Cas is an adaptive immune system developed by prokaryotes to defend against viral infection or bacteriophage invasion. Because type II (e.g., CRISPR / Cas9) and type V (e.g., CRISPR / Cas12a) gene editing systems are relatively simple in composition, easy to operate, and highly efficient, they have been widely studied and applied in gene editing. After recognizing a DNA target via a single guide RNA (sgRNA), the CRISPR / Cas9 system typically produces double-strand breaks. The cell then repairs these breaks through non-homologous end joining and homologous recombination mechanisms, resulting in insertions, deletions, or, when a donor template is available, gene insertions and specific base changes. Currently, CRISPR / Cas9 technology is widely used in many species, including microorganisms, plants, and animals. CRISPR / Cas technology has overcome the limitations of traditional breeding, shortened breeding cycles, and accelerated the process of animal genetic improvement.

[0003] Chinese invention patent CN111757889B discloses a Cas protein, Cas12f.4, which is referred to herein as Cas12i3. This Cas protein can be edited in eukaryotes. Cas9 and Cas12a proteins are approximately 1300 amino acids in size, while Cas12i3 is only 1045 amino acids in size. Further optimization of the Cas12i3 protein-encoding gene to increase Cas12i3 protein expression and development of a more efficient Cas12i3-based gene editing system will better enable the application of Cas12i3 protein in mammalian gene editing, laying the foundation for its widespread use in mammals and possessing broad application value. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to increase the expression level of Cas12i3 protein and / or improve the efficiency of Cas12i3 protein-mediated gene editing. The technical problem to be solved is not limited to the technical subject matter described herein; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, the present invention first provides a DNA molecule, wherein the nucleotide sequence of the DNA molecule may be SEQ ID No. 3.

[0006] The DNA molecule may be a codon-optimized Cas12i3 protein gene, which is a codon-optimized Cas12i3 protein encoding gene based on mammalian preferences.

[0007] The present invention also provides a biomaterial, which may be any of the following:

[0008] A1) An expression cassette containing the DNA molecule;

[0009] A2) A recombinant vector containing the DNA molecule or a recombinant vector containing the expression cassette of A1);

[0010] A3) Recombinant microorganisms containing the DNA molecule, or recombinant microorganisms containing the expression cassette of A1), or recombinant microorganisms containing the recombinant vector of A2);

[0011] A4) A recombinant host cell containing the DNA molecule, or a recombinant host cell containing the expression cassette of A1), or a recombinant host cell containing the recombinant vector of A2).

[0012] Furthermore, the expression cassette described in A1), the recombinant vector described in A2), the recombinant microorganism described in A3), and the recombinant host cell described in A4) can all express the DNA molecule.

[0013] In the above-mentioned biological materials, the host cell may be a mammalian cell.

[0014] In the aforementioned biological materials, the carrier may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0015] The microorganisms described herein may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Specifically, the bacteria may originate from genera such as *Escherichia* sp., *Erwinia* sp., *Agrobacterium* sp., and *Flavobacterium* sp., but are not limited to these. The fungi may be yeasts, and may originate from genera such as *Saccharomyces*, *Kluyveromyces*, and *Pichia pastoris*, but are not limited to these. The actinomycetes may originate from genera such as *Streptomyces* sp., *Nocardia* sp., and *Micromonospora* sp., but are not limited to these. The algae may originate from genera such as *Fucus* sp. and *Achnanthes* sp., but are not limited to these. The viruses may be rotavirus, herpesvirus, influenza virus, adenovirus, etc., but are not limited to these.

[0016] The host cell (also called the recipient cell) described herein may be an animal cell. The term "host cell" can be understood not only to the specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, need not be completely identical to the original parent cell but are still included within the scope of the host cell. Suitable host cells are those known in the art, and the animal cell may be a mammalian cell. In one or more embodiments of the invention, the mammalian cell is a sheep fibroblast.

[0017] The present invention also provides any of the following applications of the DNA molecule and / or the biological material:

[0018] B1) Application in increasing Cas12i3 protein expression;

[0019] B2) Application in improving the efficiency of Cas12i3 protein gene editing;

[0020] Application of B3 in Cas12i3 protein-mediated gene editing;

[0021] B4) Application in the preparation of Cas12i3 protein-mediated gene editing systems;

[0022] Application of B5 in the preparation of Cas12i3 protein-mediated gene editing products.

[0023] The gene editing described in this article may refer to gene editing targeting mammals or gene editing targeting mammalian cells.

[0024] The gene-editing products described in this article may include, but are not limited to, cell models, animal models, and new animal breeds.

[0025] The present invention also provides a gene editing system, which may include the DNA molecule and / or the recombinant vector (A2).

[0026] Furthermore, the gene editing system may also include guide RNA (gRNA) or a gRNA expression vector.

[0027] The gRNA expression vector may be a recombinant vector containing a DNA molecule encoding the gRNA.

[0028] The gRNA guides the Cas12i3 protein to perform gene editing on the target gene in the target cell.

[0029] The gene editing system described in this article can be a Cas12i3 protein-mediated gene editing system (CRISPR / Cas12i3 gene editing system), which can precisely target the target gene, produce a cut, and cause a double-strand break in the target gene DNA.

[0030] This invention also provides the application of the gene editing system in gene editing, preparation of gene editing products, or improvement of gene editing efficiency.

[0031] The present invention also provides a method for improving the efficiency of Cas12i3 protein-mediated gene editing, the method comprising the steps of gene editing using the DNA molecule, the recombinant vector (A2), and / or the gene editing system.

[0032] Furthermore, the method may include constructing the DNA molecule and the DNA molecule encoding gRNA into a vector to obtain a gene editing vector, and using the gene editing vector to perform gene editing on a target gene in a mammal.

[0033] The gene editing described in this article includes in vitro gene editing, in vivo gene editing, or a combination thereof.

[0034] The gene editing described in this article may include gene knockout, gene knock-in, gene mutation, gene fragment replacement, or gene modification.

[0035] Furthermore, the gRNA can target the ZFX gene or the tdTomato gene.

[0036] In one embodiment of the present invention, the target sequence of the gRNA may be SEQ ID No. 7.

[0037] The ZFX gene is an X-linked zinc finger protein gene, a single-copy gene located on the X chromosome. The nucleotide sequence of the ZFX gene (sheep ZFX gene) can be found at positions 22500545-22537460 in GenBank Accession No. NC_056080.1 (Update Date 4-Nov-2022).

[0038] Furthermore, the gRNA can target the tdTomato gene.

[0039] In one embodiment of the present invention, the target sequence of the gRNA may be SEQ ID No. 14.

[0040] The nucleotide sequence of the tdTomato gene can be positions 2529-3959 of GenBank Accession No. KT878736.1 (Update Date 06-OCT-2015).

[0041] The present invention also provides a method for increasing the expression level of Cas12i3 protein, the method comprising the steps of constructing a recombinant expression vector containing the DNA molecule, introducing the recombinant expression vector into a host cell to obtain a recombinant host cell, and culturing the recombinant host cell.

[0042] Furthermore, the host cell may be a sheep fibroblast.

[0043] The amino acid sequence of the Cas12i3 protein described in this article may be SEQ ID No. 1.

[0044] To better apply the Cas12i3 protein to mammalian gene editing, this invention designed different codon optimization schemes. Through software evaluation and experimental verification, the codon optimization schemes with good Cas12i3 expression levels and editing effects were selected. This invention has identified codon optimization schemes for efficient Cas12i3 protein expression in mammals, which can significantly improve the editing efficiency of Cas12i3 protein, laying the foundation for the widespread application of Cas12i3 protein in mammals and possessing significant practical application value. Attached Figure Description

[0045] Figure 1 Codon usage frequency distribution and GC content distribution for Cas12i3 codons without mammalian codon optimization and those optimized for four mammalian codons.

[0046] Figure 2 To observe the expression of EGFP in sheep fibroblasts 24 h after transfection with plasmids without mammalian codon optimization and with four mammalian codons under a fluorescence microscope.

[0047] Figure 3 Flow cytometry scatter plots of EGFP expression in sheep fibroblasts 24h, 48h, and 72h after transfection with plasmids without mammalian codon optimization and with four mammalian codons.

[0048] Figure 4 The bar graphs show the expression of EGFP in sheep fibroblasts 24h, 48h, and 72h after transfection with plasmids that were not optimized for mammalian codons and those optimized for four mammalian codons, for flow cytometry analysis.

[0049] Figure 5 Western blot was used to detect the expression of Cas12i3 protein in sheep fibroblasts 48 h after transfection with plasmids that had not undergone mammalian codon optimization and those that had undergone four mammalian codon optimizations.

[0050] Figure 6To assess the editing efficiency of Cas12i3 in sheep fibroblasts (48 h post-transfection) with and without mammalian codon optimization for T7E1.

[0051] Figure 7 To analyze the proportion of tdTomato mean fluorescence intensity quenched in tdTomato-labeled sheep fibroblasts by Cas12i3 without mammalian codon optimization and with four mammalian codons (48 h post-transfection), flow cytometry was used.

[0052] Figure 8 Flow cytometry analysis was performed to determine the weak tdTomato fluorescence intensity (<10) in tdTomato-labeled sheep fibroblasts resulting from Cas12i3 without mammalian codon optimization and with four mammalian codons optimization. 3 The proportion of cells (48h post-transfection).

[0053] Figure 9 The fluorescence distribution peaks of tdTomato-labeled sheep fibroblasts after transfection with the Cas12i3 plasmid (which was not optimized for mammalian codons and Codon OptimWiz codons) (48 h post-transfection). Detailed Implementation

[0054] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0056] definition

[0057] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0058] gRNA

[0059] "Guide RNA (gRNA)" and "mature crRNA" are used interchangeably and have the meanings commonly understood by those skilled in the art. Generally, guide RNA may comprise a direct repeat sequence and a guide sequence, or essentially consist of a direct repeat sequence and a guide sequence (also called a spacer sequence in the context of an endogenous CRISPR system). In some cases, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize with it and guide the specific binding of the CRISPR / Cas complex to the target sequence.

[0060] Cas protein

[0061] A nuclease protein, "Cas protein," is interchangeable with "Cas nuclease," "Cas enzyme," "CRISPR / Cas protein," and "Cas effector protein." The Cas protein described in this invention is selected from the Cas12i3 protein of the V-type Cas family. The Cas protein, once bound to gRNA or mature crRNA, forms a ribonucleoprotein complex containing a guide sequence that hybridizes to the target sequence and binds to the Cas protein. This ribonucleoprotein complex can recognize and cleave polynucleotides that hybridize with the guide RNA or mature crRNA.

[0062] target sequence

[0063] The target sequence is interchangeable with "target site sequence" and "target recognition sequence," with the target site sequence corresponding to the spacer. The target sequence determines the location of gene editing and its specificity.

[0064] Codon optimization

[0065] Codon optimization refers to the process of optimizing a target gene by primarily utilizing preferred codons and avoiding rare codons with low utilization, without altering the protein's amino acid composition. After codon optimization, the gene exhibits sporadic mutations compared to the original sequence. In protein synthesis, codons play a crucial role in translating gene information into protein sequence information. Different species may use different codons to translate the same amino acid, exhibiting codon biases due to species differences. Although the natural causes of codon biases are not yet fully understood, this phenomenon significantly impacts protein expression efficiency. For recombinant protein expression, sequence optimization based on species-specific codon biases is typically necessary to achieve optimal expression results. This optimization is particularly important when using heterologous protein expression systems, as the target gene from another species needs to be expressed as a recombinant protein in a host that does not naturally express the gene. Codon optimization is also applied to improve mRNA stability and enhance transcription and translation efficiency.

[0066] Codon fitness index

[0067] The codon adaptation index (CAI) refers to the degree of similarity between the frequency of synonymous codons in a coding region and the frequency of use of the optimal codon, with a value between 0 and 1. CAI can be used to assess the expression level of a foreign gene in the host; a higher CAI indicates a higher expression level of the foreign gene in the host. The working principle of codon adaptation index analysis tools is as follows: using the sequence of a highly expressed gene as a reference (reference sequence), the codon usage frequency of the target gene matches that of the reference sequence. If the analyzed CAI is low, the gene expression level in the host cell is low.

[0068] Rare codons

[0069] Rare codons: There are 64 genetic codons, but most organisms exhibit codon bias, tending to utilize only a subset of them. The most frequently used codons are called optimal codons, while those less frequently used are called rare codons. The frequency of rare codon usage is a crucial factor influencing recombinant protein expression levels. Rare codon calculation tools can be used to calculate the codon usage frequency of a target sequence and display its distribution. Optimizing the sequence based on the analysis results can effectively improve the expression level of recombinant proteins. Therefore, understanding the frequency and distribution of rare codons in the sequence and optimizing the sequence before recombinant protein expression is essential.

[0070] carrier

[0071] A recombinant DNA vector is a self-replicating DNA molecule used in genetic engineering to transfer DNA fragments (target genes) into recipient cells. The three most commonly used vectors are bacterial plasmids, bacteriophages, and plant and animal viruses. Vectors can be classified into cloning vectors and expression vectors according to their function. Cloning vectors are the simplest vectors and are mainly used to clone and amplify DNA fragments. They mainly include plasmid vectors, bacteriophage vectors, and viral vectors. Expression vectors, in addition to the basic elements of cloning vectors, also contain DNA elements necessary for transcription and translation, such as promoters and terminators. The promoters involved in this invention are the U6 and CBh promoters. The U6 promoter belongs to the pol III type promoter, and the sequence it drives is very short. Currently, common expression vectors using the U6 promoter include gRNA and siRNA. The sequence driven by the U6 promoter terminates upon encountering pol(U). The CBh promoter is an artificially constructed combinatorial promoter composed of a mixed sequence of the cytomegalovirus (CMV), early enhancer, chicken beta-actin promoter, and chicken beta-actin (CBA) and mouse minute virus (MMV) introns, used to drive high-level gene expression in mammalian vectors.

[0072] T7E1 enzyme digestion

[0073] T7E1, short for T7 Endonuclease I, is a unique DNA endonuclease that can recognize and cleave incompletely paired DNA, cross-shaped DNA structures, Holliday structures, and other DNA editing techniques. T7E1 is commonly used for detecting mutants created by CRISPR / Cas, TALEN, and other editing tools.

[0074] The primary sheep fibroblasts used in the following examples were isolated in the laboratory. The preparation method is as follows: A small amount of ear tissue from sheep within 2 weeks of birth was taken and placed in PBS. In a clean bench, the ear tissue was sterilized in 75% alcohol for 1 min, washed three times with PBS, and then minced to 1 mm³ using sterile scissors. 200 μL of fetal bovine serum was added, and the mixture was transferred to a cell culture dish and incubated upside down at 37°C with 5% CO₂ for 1 hour. Complete culture medium was carefully added, taking care not to dislodge the tissue fragments. After approximately one week of culture, fibroblasts emerged from the tissue fragments. Once confluent, the cells were digested with trypsin, expanded, and then cryopreserved for later use.

[0075] The PX458 vector in the following examples is derived from the Addgene plasmid sharing information database (number 48138).

[0076] Example 1: Design and Evaluation of Cas12i3 Codon Optimization Scheme

[0077] 1. Design different Cas12i3 codon optimization schemes

[0078] For the Cas12i3 amino acid sequence (SEQ ID No.1), in addition to the unoptimized mammalian codon sequence, four mammalian codon optimization schemes (Codon OptimWiz, GeneOptimizer, Jcat, and General Biol) were designed.

[0079] Unoptimized indicates the lack of mammalian codon optimization. The nucleotide sequence of the unoptimized Cas12i3 protein coding sequence is shown in SEQ ID No. 2.

[0080] Codon OptimWiz, GeneOptimizer, Jcat, and General Biol represent four mammalian codon optimization schemes, wherein: the nucleotide sequence of the Cas12i3 protein-coding sequence optimized according to the Codon OptimWiz scheme is shown in SEQ ID No. 3; the nucleotide sequence of the Cas12i3 protein-coding sequence optimized according to the GeneOptimizer scheme is shown in SEQ ID No. 4; the nucleotide sequence of the Cas12i3 protein-coding sequence optimized according to the Jcat scheme is shown in SEQ ID No. 5; and the nucleotide sequence of the Cas12i3 protein-coding sequence optimized according to the General Biol scheme is shown in SEQ ID No. 6.

[0081] 2. Software evaluation of different codon optimization schemes

[0082] The codon adaptation index (CAI) refers to the degree of similarity between the frequency of codon usage in a foreign sequence and the optimal frequency of codon usage in the host cell. Theoretically, the closer this value is to 1, the higher the protein expression of the foreign mRNA in the host cell. Table 1 lists the CAI of the Cas12i3 codon in humans and sheep under four mammalian codon optimization schemes. Compared with Unoptimized, the CAI of the four codon-optimized Cas12i3 codons in both humans and sheep is improved, especially the Jcat and Codon OptimWiz schemes, which have a CAI of 1 or close to 1 (the CAI of the Jcat scheme in humans and sheep are 0.99 and 1.00, respectively, and the CAI of the Codon OptimWiz scheme in humans and sheep are 0.98 and 0.96, respectively). The codon usage frequency plot shows that Unoptimized has more rare codons, while the other four optimization schemes show significant improvement, especially the Jcat and Codon OptimWiz schemes. Figure 1 ).

[0083] GC content is a crucial factor affecting protein expression. Compared to the two hydrogen bonds between AT and GC, there are three hydrogen bonds between GC, so GC content directly impacts DNA stability. High GC content indirectly reduces protein expression. Table 1 lists the GC content of five codons, ranging from 53.67% to 63.22%. The overall GC content distribution of the five codons was analyzed using GenScript's rare codon analysis tool (https: / / www.genscript.com / tools / rare-codon-analysis). The GC content distribution shows that Jcat has many regions with GC content exceeding 70% (GC content below 30% or above 70% may reduce protein expression levels), while Unoptimized and Codon OptimWiz have a few regions with GC content exceeding 70%, which may affect Cas12i3 expression. Figure 1 ).

[0084] Table 1. CAI values ​​of Cas12i3 codons

[0085]

[0086] Although the Jcat and Codon OptimWiz schemes show superior performance in CAI evaluations, further experimental verification is needed to select the optimal mammalian codon optimization scheme, as both schemes are also affected by GC content.

[0087] Example 2: Cas12i3 expression levels under different codon optimization schemes

[0088] 1. Carrier Construction

[0089] PX458 (U6-sgRNA-CBh-Cas9-T2A-EGFP-bGH polyA) was double-digested with restriction endonucleases BbsI (NEB (Beijing) Co., Ltd.) and XbaI (NEB (Beijing) Co., Ltd.) to remove the sgRNA scaffold sequence. The digestion system consisted of 5 μg PX458, 25 units of BbsI, 25 units of XbaI, 10 μL of Cutsmart, and ddH2O to a final volume of 100 μL. The reaction conditions were: incubation at 37°C for 6 h. The DNA was recovered and its concentration determined using a recovery kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D2111-02). Primers 5'-CACCACTAGTT-3' and 5'-CTAGAACTAGT-3' were synthesized and annealed to generate a DNA double strand complementary to the digested linear PX458 vector. Annealing system: 2.5 μL of 5'-CACCACTAGTT-3' (100 μM), 2.5 μL of 5'-CTAGAACTAGT-3' (100 μM), 1 μL of T4 ligase buffer, and ddH2O to a final volume of 10 μL. Annealing procedure: 95℃ for 5 min in a metal bath, then open the metal bath lid, close the metal bath, and allow to cool to room temperature. The recovered linear PX458 vector (double-digested with BbsI and XbaI) was ligated with the annealing product using a T4 ligase kit (Baori Biotechnology Co., Ltd.) to form U6-CBh-Cas9-T2A-EGFP-bGHpolyA.

[0090] The plasmid U6-CBh-Cas9-T2A-EGFP-bGH polyA was digested with the restriction endonucleases AgeI (NEB (Beijing) Co., Ltd.) and FseI (NEB (Beijing) Co., Ltd.) to remove the Cas9 coding sequence. The digestion system consisted of 5 μg of the plasmid (U6-CBh-Cas9-T2A-EGFP-bGH polyA), 20 units of AgeI, 20 units of FseI, 10 μL of cutsmart, and ddH2O to a final volume of 100 μL. The reaction was carried out at 37°C for 6 h. The plasmid was recovered and its concentration determined using a recovery kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D2111-02). The recombinant vectors obtained by assembling the enzyme digestion product (AgeI and FseI double-digested plasmid U6-CBh-Cas9-T2A-EGFP-bGH polyA) and Cas12i3 protein-encoding DNA optimized with different codons (SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6) using a seamless cloning kit were U6-CBh-Cas12i3(Unoptimized)-T2A-EGFP-bGH polyA, CBh-Cas12i3(CodonOptimWiz)-T2A-EGFP-bGH polyA, U6-CBh-Cas12i3(GeneOptimizer)-T2A-EGFP-bGH polyA, U6-CBh-Cas12i3(Jcat)-T2A-EGFP-bGH polyA and U6-CBh-Cas12i3(General Biol)-T2A-EGFP-bGH polyA. The vector links the fluorescent reporter gene EGFP to the Cas12i3 sequence via T2A. Cas12i3 and EGFP are transcribed and translated in the same open reading frame (ORF), so the expression of EGFP can indirectly reflect the expression of Cas12i3.

[0091] 2. Cell electroporation

[0092] Healthy sheep fibroblasts were transferred to 10cm culture dishes and cultured until the cell confluence reached approximately 80%. Cells were collected by trypsin digestion into EP tubes. The cells were resuspended in 100μL of electroporation buffer (Beijing Enggen Biotechnology Co., Ltd., catalog number 98668-20) and 7μg of plasmids (the five plasmids constructed above) were added and mixed thoroughly. The cells were then placed in a Lonza Amaxa Nucleofector 2B transfection instrument, programmed A-033, and electroporated. Immediately after electroporation, 500μL of DMEM high-glucose medium was added, and the cells were incubated at 37℃ for 10 min. Cells were then seeded into 6-well plates with complete medium containing 20% ​​FBS. After 6 h, the medium was replaced with complete medium containing 15% FBS.

[0093] 3. Data Analysis

[0094] 24 hours after transfection, fluorescence microscopy revealed that among the four codon optimization schemes, the CodonOptimWiz scheme exhibited the strongest EGFP fluorescence, followed by GeneOptimizer, Jcat, and General Biol schemes, which also showed strong EGFP fluorescence. Unoptimized showed the weakest EGFP fluorescence. Figure 2 ).

[0095] Cells were digested and analyzed by flow cytometry at 24h, 48h, and 72h after transfection, and the EGFP fluorescence intensity of EGFP-positive cells was analyzed. The results showed that the Condon OptimWiz protocol resulted in the highest EGFP expression at 24h, 48h, and 72h. The Jcat protocol also showed high EGFP expression, followed by the GeneOptimizer and General Biol protocols. Unoptimized cells showed the weakest EGFP expression. Figure 3 and Figure 4 ).

[0096] After 48 hours of transfection with unoptimized and four codon-optimized plasmids, total cellular protein was extracted, and Cas12i3 protein expression was detected by Western blot. Western blot results showed that Codon OptimWiz and Jcat showed the highest Cas12i3 expression, followed by GeneOptimizer, while General Biol and Unoptimized showed weak expression. Figure 5 ).

[0097] Example 3: Detection of Cas12i3 editing effect of different codon optimization schemes using T7E1 enzyme digestion method

[0098] 1. Design target sequence

[0099] The five recombinant vectors constructed in Example 2 were digested and recovered using both KpnI (NEB (Beijing) Co., Ltd.) and SpeI (NEB (Beijing) Co., Ltd.) enzymes (the U6 promoter has SpeI and KpnI restriction sites). The digestion system consisted of 5 μg of the plasmid, 50 units of SpeI, 50 units of KpnI, 10 μL of cutsmart, and ddH2O to a final volume of 100 μL. The reaction conditions were: incubation at 37°C for 6 h. Then, 5 μL of BeyoAP alkaline phosphatase (Beyotime Biotechnology Co., Ltd., catalog number D7027) was added, and incubation continued at 37°C for 10 min. The recombinant vectors were recovered and their concentrations determined using a recovery kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D2111-02).

[0100] This invention selects the sheep endogenous gene ZFX (GenBank Accession No. NC_056080.1, positions 22500545-22537460 (Update Date 4-Nov-2022)) and designs a target sequence for gRNA (gRNA1) targeting the ZFX gene. The target sequence is 5'-CAGTACAGCAAGAGTGGATGAAT-3' (SEQ ID No. 7). The DNA fragment expressing gRNA (5'-aaaggacgaaacaccGCTCTGACCACCTGAGAG AATGTGTGCATAGTCACACCAGTACAGCAAGAGTGGATGAATTTTTTTTgtacccgttacataa-3' (SEQ ID No. 8) was amplified using the primers listed in Table 2. Uppercase letters indicate the direct repeat sequence, target sequence, and transcription termination signal, while lowercase letters indicate the vector homologous sequence. The PCR amplification system consisted of: F 1 μL, R 1 μL, PrimeSTAR 15 μL, and ddH2O 13 μL. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, and 72℃ extension for 5 s (33 cycles); 72℃ extension for 5 min. After PCR, the PCR product was recovered and its concentration determined using a product recovery kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D2111-02).

[0101] Table 2. Primers used to amplify DNA fragments expressing gRNA (targeting the ZFX gene)

[0102]

[0103] Using a seamless cloning kit, the DNA fragment expressing gRNA (SEQ ID No. 8) was homologously recombined with five recombinant vectors that had been digested with SpeI and KpnI to form five recombinant vectors with the ZFX target, which are the gene editing vectors.

[0104] The gene editing vectors mentioned above contain the ZFX gene editing target and different Cas12i3 codons (Unoptimized, Codon OptimWiz, GeneOptimizer, Jcat, and General Biol). After being introduced into recipient cells, the transcribed guide RNA can target the ZFX gene through base complementarity, causing DNA double-strand breaks upstream and downstream of the ZFX gene target. Different codon optimization schemes result in different Cas12i3 expression levels, leading to different gene editing efficiencies.

[0105] 2. Electroporation followed by T7E1 restriction enzyme digestion

[0106] The five gene-editing vectors from step 1 of this example were transformed into sheep fibroblasts via electroporation (the electroporation steps are the same as in Example 2). After 48 hours, the genome was extracted using a genome extraction kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D3018-02). 100 ng of the extracted sheep genome was used as a template for PCR amplification. The amplification reaction system and procedure were as follows: The total volume of the amplification reaction was 50 μL. Primers listed in Table 3 were used for amplification, with the following components: 100 ng DNA template, 1 μL each of 10 μmol / L forward and reverse primers, 25 μL PrimeSTAR (Baori Biotechnology Co., Ltd.), and the volume was brought to 50 μL with sterile deionized water. The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 72℃ extension for 30 s (33 cycles); and a final extension at 72℃ for 5 min. After PCR, the PCR product was recovered using a product recovery kit, and the concentration was determined.

[0107] Table 3. Primers for obtaining sequences around the target site

[0108]

[0109] Take the PCR product recovered in the previous step and prepare the enzyme digestion system as follows: 500 ng of amplified product, 1.1 μL of Cutsmart, and ddH2O to a final volume of 11.5 μL. After mixing thoroughly, follow the hybridization program: 95℃ for 10 min; -2℃ / s to 85℃; -0.1℃ / s to 25℃. Add 0.5 μL of T7E1 (NEB (Beijing) Co., Ltd.), digest at 37℃ for 15 min, immediately add 2 μL of Loading Buffer, prepare 2% agarose gel for electrophoresis analysis, and observe and analyze the results after enzyme digestion using a gel imaging system.

[0110] Agarose gel electrophoresis revealed that Codon OptimWiz, GeneOptimizer, and Jcat exhibited high editing efficiencies (15.6%, 15.8%, and 14.5%, respectively), while Unoptimized and General Biol showed lower editing efficiencies (13.2% and 8.2%, respectively). Figure 6 ).

[0111] Example 4: Flow cytometry analysis to detect the Cas12i3 editing effect of different codon optimization schemes

[0112] 1. Construction of sheep fibroblasts labeled with tdTomato red fluorescent dye

[0113] 1-1. Construction of CRISPR / Cas9 gene targeting vector

[0114] 1-1-1. Enzyme digestion of PX458 vector

[0115] Enzyme digestion system: 5 μg PX458 vector, 50 units of BbsI, 10 μL Cutsmart, and ddH2O to a final volume of 100 μL. Digestion was performed at 37℃ for 5 h. After digestion, the digestion product was purified using a product purification kit (Guangzhou Meiji Biotechnology Co., Ltd.) to obtain purified PX458 BbsI digestion product. An oligo was designed targeting the goat ZFY gene sequence, and the target sequence was synthesized according to Table 4.

[0116] Table 4. sgRNA target sequences

[0117]

[0118] 1-1-2, Oligo annealing

[0119] The designed oligo was annealed according to the following annealing system and procedure to form an annealed product (double-stranded DNA).

[0120] Annealing system: ZFY-sgRNA-F (100μM) 2.5μL, ZFY-sgRNA-R (100μM) 2.5μL, T4 ligase buffer 1μL, ddH2O to a final volume of 10μL. Annealing procedure: 95℃ for 5 min in a metal bath, then turn off the metal bath, open the lid, and allow it to cool to room temperature before removing it.

[0121] 1-1-3, Connection

[0122] The annealing product was diluted 50 times and ligated with the PX458BbsI digestion product in step 1-1-1 according to the following ligation system and procedure.

[0123] Ligation system: 90 ng PX458 BBSI digestion product, 1 μL annealing product (diluted), 0.5 μL T4 ligase, 1 μL T4 ligase buffer, and ddH2O to a final volume of 10 μL. Ligation procedure: 25℃ for 1 h.

[0124] 10 μL of the above ligation product was used for transformation, bacteria were selected, sequencing was performed, and plasmid was extracted to construct a CRISPR / Cas9 gene targeting vector (i.e., sgRNA expression vector), named PX458-ZFY-sgRNA.

[0125] 1-2. Constructing donor plasmids

[0126] Our laboratory preserves the pCBh-tdTomato-SV40 polyA plasmid. The construction process of this plasmid is as follows: First, pROSA26-promoter (Addgene 21710) is digested with SpeI and XbaI to obtain the digested pROSA26-promoter. The DNA molecule shown in SEQ ID No. 9 (tdTomato-SV40 polyA sequence) is ligated to the digested pROSA26-promoter using seamless cloning assembly technology to obtain pROSA26-tdTomato-SV40 polyA. The CBh promoter was obtained by double digestion of PX458 with KpnI and AgeI. Using pROSA26-tdTomato-SV40 polyA as a template, the sequence other than the ROSA26 promoter was amplified with primer F (5'-tttttttcaggttggaccggTGCCACCATGGACTAGTATGGTGAGCAAGGGCGA-3') and primer R (5'-taccgtaagttatgtaacggggtacCCAGCTTTTGTTCCCTTTAGT-3'). This sequence was then combined with the CBh promoter sequence using seamless cloning assembly technology to construct pCBh-tdTomato-SV40 polyA.

[0127] This plasmid was able to express red fluorescence normally in primary goat fibroblasts. The sequences flanking the site of cleavage by the nuclease at the ZFY target site (3-4 bp upstream of PAM) were used as homologous arms (left homologous arm (HA-L) 925 bp, nucleotide sequence SEQ ID No. 10; right homologous arm (HA-R) 958 bp, nucleotide sequence SEQ ID No. 11). The left and right homologous arms were amplified by PCR using the primers in Table 5, and the products were recovered using a PCR product recovery kit (Meiji Biotechnology Co., Ltd.).

[0128] The plasmid pCBh-tdTomato-SV40 polyA was digested with enzymes. Using seamless cloning assembly technology, the left and right homologous arms of the ZFY target were cloned to both ends of pCBh-tdTomato-SV40 polyA, constructing the plasmid HA-L-CBh-tdTomato-SV40polyA-HA-R. Subsequently, the ZFY target recognition sequence was added to the outer sides of the left and right homologous arms to construct the donor plasmid type required for HMEJ (homology-mediated end joining). Furthermore, although the left and right homologous arms on the constructed donor plasmid were derived from goats, they showed extremely high homology with their sheep counterparts. NCBIBLAST analysis showed that the homology of the left homologous arm was 96.11%, and that of the right homologous arm was 97.66%. The nucleotide sequence of the sheep left homologous arm is SEQ ID No. 12, and the nucleotide sequence of the sheep right homologous arm is SEQ ID No. 13.

[0129] Table 5. Primers for the left and right homologous arms of ZFY

[0130]

[0131] 1-3. Construction of sheep fibroblasts labeled with tdTomato red fluorescent dye.

[0132] Using the CRISPR / Cas9 gene targeting vector PX458-ZFY-sgRNA and donor plasmid HA-L-CBh-tdTomato-SV40polyA-HA-R (carrying the exogenous tdTomato gene; although its homologous arm originates from goats, it has high homology with the corresponding homologous arm in sheep, and is therefore expected to be used in sheep) constructed in this embodiment, the exogenous gene (tdTomato gene) was site-directedly integrated into the ZFY gene targeting site via HMEJ-mediated recombination, thus constructing a sheep fibroblast cell line with site-directed integration of the exogenous gene into the ZFY gene. The specific steps are as follows:

[0133] 1-3-1. Co-transfection of sheep fibroblasts with gene-editing plasmid and donor plasmid

[0134] 5000 ng of the constructed donor plasmid HA-L-CBh-tdTomato-SV40 polyA-HA-R and 9536 ng of the gene targeting vector PX458 (PX458-ZFY-sgRNA) carrying the ZFY target (molar ratio 1:1.5) were electroporated into primary sheep fibroblasts (the electroporation procedure was the same as step 2 in Example 2, only the plasmids were different). After 24 hours, tdTomato and EGFP-positive primary sheep fibroblasts were sorted by flow cytometry and seeded at approximately 500 cells per 10 cm cell culture dish. After 2 weeks of culture, the cells in the cell culture dish were individually digested into 96-well plates using a cloning loop.

[0135] 1-3-2. Screening of sheep fibroblasts by site-specific integration of tdTomato

[0136] After the cell clones in the 96-well plate have grown to confluence, the cells are digested, half of the wells are left for initial culture, and the other half of the cells are transferred to 1.5 mL centrifuge tubes. Centrifuge at 12000 rpm for 3 min, discard the supernatant, and add 50 μL of cell identification lysis buffer (prepared as follows: 2 mL Tris-HCl (1 M, pH 8.0), 0.45 mL Triton X-100, 0.45 mL NP-40, 0.02 g proteinase K, dissolved in deionized water and brought to a final volume of 50 mL, filtered through a 0.22 μm filter). The cells are thoroughly resuspended and lysed according to the following program: 65℃, 30 min; 95℃, 15 min; 16℃, infinity. The resulting lysate is used as a DNA template. Primers are designed according to Table 6, and PCR identification is performed.

[0137] Table 6. Primers for site-specific integration identification

[0138]

[0139] Amplification reaction system and procedure: The total volume of the amplification reaction was 50 μL, and its components were as follows: 1 μL DNA template, 1 μL each of 10 μmol / L forward and reverse primers, 10 μL PrimeSTAR (Baori Biotechnology Co., Ltd.), and sterile deionized water to a final volume of 20 μL. The PCR reaction procedure was: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 62℃ annealing for 15 s, 72℃ extension for 50 s (33 cycles); and a final extension at 72℃ for 5 min. The results were detected by agarose gel electrophoresis after PCR.

[0140] The results showed that 16 cell clones were lysed, and 3 clones were identified by PCR as ZFY-integrated single-cell clones. Simultaneously, fluorescence microscopy revealed that all three clones emitted red fluorescence. This indicates that the exogenous gene (tdTomato gene) underwent site-specific integration at the target site, successfully constructing tdTomato-labeled sheep fibroblasts.

[0141] 2. Design target sequences and construct gene editing vectors

[0142] In this embodiment, the ZFY-mediated site-specific integration of tdTomato cell clones constructed in step 1 above was selected as the cell line for subsequent evaluation of the impact of different Cas12i3 codon optimization schemes on editing efficiency. In this embodiment, a target sequence for the gRNA (gRNA2) targeting the tdTomato gene was designed based on the tdTomato coding sequence (GenBank Accession No. KT878736.1, positions 2529-3959 (Update Date 06-OCT-2015)). The target sequence is 5'-AAGACCATCTACATGGCCAAGAA-3' (SEQ ID No. 14). The DNA fragment expressing the gRNA sequence (5'-aaaggacgaaacaccGCTCTGACCACCTGAGAGAATGTGTGCATAGTCACACAAGACCATCTACATGGCCAAGAATTTTTTTgtacccgttacataa-3' (SEQ ID No. 15)) was amplified using the primers in Table 7. The uppercase letters indicate the direct repeat sequence, target sequence, and transcription termination signal, while the lowercase letters indicate the vector homologous sequence. The PCR product was recovered and its concentration was determined using a product recovery kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number D2111-02).

[0143] Table 7 Primers used to amplify DNA fragments expressing gRNA (targeting the tdTomato gene)

[0144]

[0145] Using a seamless cloning kit, the DNA fragment expressing gRNA (SEQ ID No. 15) was homologously recombined with the five recombinant vectors digested with SpeI and KpnI in Example 3 to form five recombinant vectors with the tdTomato target, which are the gene editing vectors.

[0146] The gene editing vectors mentioned above contain the tdTomato gene editing target and different Cas12i3 codons (Unoptimized, Codon OptimWiz, GeneOptimizer, Jcat, and General Biol). After being introduced into recipient cells, the transcribed guide RNA can target the tdTomato gene through base complementarity pairing, causing DNA double-strand breaks upstream and downstream of the tdTomato gene target. Different codon optimization schemes result in different Cas12i3 expression levels, leading to different gene editing efficiencies.

[0147] 2. Electroporation and concurrent flow cytometry analysis of tdTomato fluorescence changes

[0148] The five plasmids from step 2 of this example were transfected into sheep fibroblasts labeled with tdTomato red fluorescence constructed in step 1 via electroporation (the electroporation steps were the same as in Example 2). After 48 hours of electroporation, flow cytometry was used to analyze the effects of different Cas12i3 codon optimization schemes on the changes in tdTomato red fluorescence intensity in EGFP-positive cells (only EGFP-positive cells were considered because all five gene editing vectors carried the EGFP expression sequence, which originated from the PX458 vector; EGFP-positive cells represent cells successfully transfected with the plasmids, helping to reduce errors caused by cell transfection). Specifically, the proportion of quenched average tdTomato fluorescence intensity and the proportion of cells with weak fluorescence intensity (tdTomato fluorescence intensity less than 10 in EGFP-positive cells) were calculated. 3 The percentage of cells with positive EGFP-positive tdTomato fluorescence intensity was calculated. The proportion of quenched tdTomato fluorescence intensity in EGFP-positive cells was shown to be highest in the Codon OptimWiz group among the four codon optimization schemes, and significantly higher than in the Unoptimized group. Figure 7 The results indicate that the Codon OptimWiz protocol has high editing efficiency on the tdTomato gene, resulting in the inactivation of tdTomato protein in a large number of cells. This ultimately leads to weakened or even quenched red fluorescence in many cells, reducing the overall cellular tdTomato fluorescence intensity; that is, the proportion of quenched average tdTomato fluorescence intensity is relatively large. Similarly, by calculating the weak tdTomato fluorescence intensity in EGFP-positive cells (<10... 3 The cell number percentage showed that, compared with Unoptimzed, the CodonOptimiWiz protocol improved the fluorescence intensity of weak tdTomato (<10). 3 The percentage of cells in the cell count () Figure 8Meanwhile, flow cytometry analysis showed that, compared to Unoptimized, the Codon OptimWiz scheme shifted the tdTomato fluorescence peak plot to the left (the coordinate axes from left to right represent tdTomato fluorescence intensity from weak to strong). Figure 9 The results indicate that the Codon OptimWiz protocol has a high editing efficiency on the tdTomato gene, causing the tdTomato protein to become inactive in many cells, which ultimately leads to a decrease or even quenching of red fluorescence, resulting in a decrease in the overall tdTomato fluorescence intensity of the cell population.

[0149] The results above show that, through both indirect and direct detection of Cas12i3 expression methods—observing EGFP fluorescence intensity using fluorescence microscopy, analyzing EGFP fluorescence intensity by flow cytometry, and detecting Cas12i3 expression using Western blot—the Codon OptimWiz codon optimization scheme demonstrated efficient expression of Cas12i3 among the four mammalian codon optimization schemes. Furthermore, the editing efficiency of the unoptimized and four optimized codon schemes at the sheep endogenous gene ZFX site was detected by T7E1 restriction enzyme digestion, and the editing effect on the exogenous gene tdTomato was analyzed by flow cytometry. The results showed that Codon OptimWiz had relatively better editing efficiency compared to other codon optimization schemes. This invention designed four Cas12i3 codon optimization schemes, and through software evaluation and experimental verification, it has been demonstrated that the Codon OptimWiz codon optimization scheme can efficiently express Cas12i3 protein and effectively perform its editing function in mammalian cells, laying the foundation for the widespread application of Cas12i3 in mammalian cells.

[0150] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of DNA molecules in improving the efficiency of Cas12i3 protein gene editing, wherein the nucleotide sequence of the DNA molecule is SEQ ID No. 3, and the gene editing is gene editing targeting sheep endogenous genes.

2. The application of biomaterials in improving the efficiency of Cas12i3 protein gene editing, wherein the biomaterial is any one of the following: A1) An expression cassette containing the DNA molecule described in claim 1; A2) A recombinant vector containing the DNA molecule of claim 1, or a recombinant vector containing the expression cassette of claim A1; A3) A recombinant host cell containing the DNA molecule of claim 1, or a recombinant host cell containing the expression cassette of claim 1, or a recombinant host cell containing the recombinant vector of claim 2; the gene editing is gene editing targeting sheep endogenous genes; the host cell is a mammalian cell.

3. The application of a gene editing system in improving the efficiency of Cas12i3 protein gene editing, wherein the gene editing system comprises the DNA molecule of claim 1 and / or the recombinant vector of claim 2, and the gene editing is gene editing targeting an endogenous sheep gene.

4. A method for improving the efficiency of Cas12i3 protein-mediated gene editing, characterized in that, The method includes the step of gene editing using the DNA molecule of claim 1, the recombinant vector of claim 2, and / or the gene editing system of claim 3, wherein the gene editing is gene editing targeting endogenous genes in sheep.

5. The method according to claim 4, characterized in that, The method includes constructing the DNA molecule described in claim 1 and the DNA molecule encoding gRNA into a vector to obtain a gene editing vector, and using the gene editing vector to perform gene editing on endogenous genes in sheep.

6. The method according to claim 5, characterized in that, The gRNA targets the sheep ZFX gene.

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