IscB / ωRNA-based adeno-associated virus delivery system and its applications

Abstract

This invention belongs to the field of gene editing technology, specifically relating to an adeno-associated virus (AAV) delivery system based on IscB / ωRNA and its applications. This invention engineered IscB and its corresponding ωRNA to develop smaller, more efficient IscB protein and ωRNA backbones. Gene editing elements containing truncated IscB and ωRNA backbones are inserted between two inverted terminal repeat sequences to construct an IscB / ωRNA-based gene editing expression cassette. In the IscB / ωRNA-based gene editing expression cassette provided by this invention, a first promoter drives the expression of ωRNA, and a second promoter drives the expression of the IscB protein-encoding gene. The expressed ωRNA and IscB recognize and cleave the target gene, performing precise gene editing through self-cleaving homologous directed repair of nucleic acid donors, resulting in higher gene editing and delivery efficiency, and better safety.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, specifically relating to an adeno-associated virus delivery system based on IscB / ωRNA and its applications. Background Technology

[0002] Gene editing delivery tools are crucial for the rapid preparation of edited animals. Among numerous delivery tools, virus-mediated delivery vectors have been widely used in various fields of biology. The unique life cycle and virus-cell interaction characteristics of adeno-associated viruses (AAVs) have driven the development of recombinant AAVs as an ideal gene editing delivery tool. AAVs are notable for their ability to precisely edit the genome, maintaining genome integrity (avoiding the introduction of insertion / deletion mutations or viral sequences at the target site) while mitigating the risk of off-target genotoxicity.

[0003] IscB, a miniature RNA-guided endonuclease, is considered an ancestor of Cas9 and possesses similar functions, targeting and cutting the genome by assembling with ωRNA. IscB is less than half the size of Cas9, making it more suitable for in vivo delivery. However, IscB's low editing efficiency in eukaryotic cells limits its in vivo application. Summary of the Invention

[0004] The purpose of this invention is to provide an adeno-associated virus delivery system based on IscB / ωRNA and its application, which efficiently delivers IscB / ωRNA to target organisms for gene editing, improves gene editing efficiency, and has good safety.

[0005] This invention provides an expression cassette based on IscB / ωRNA gene editing, comprising two inverted terminal repeat sequences and a gene editing element located between the two inverted terminal repeat sequences;

[0006] The gene editing element comprises, in sequence, ωRNA, a first promoter, a self-cleaving homology-directed repair nucleic acid donor, a second promoter, and an IscB protein-encoding gene;

[0007] The ωRNA includes a targeting segment that specifically binds to the target gene and an ωRNA backbone;

[0008] The first promoter drives the expression of ωRNA;

[0009] The self-cleaving homology-directed repair nucleic acid donor performs gene editing on the target gene;

[0010] The second promoter drives the expression of the IscB protein-coding gene;

[0011] The amino acid sequence of the IscB protein is shown in SEQ ID NO:3; the nucleotide sequence of the ωRNA backbone is shown in SEQ ID NO:2.

[0012] Preferably, the self-cleaving homology-directed repair nucleic acid donor includes two cleavage sites and a repair template sequence located between the two cleavage sites; the repair template sequence performs gene editing on the target gene.

[0013] The present invention provides a delivery system for the expression box described in the above technical solution.

[0014] Preferably, the delivery system comprises a recombinant adeno-associated virus vector and / or recombinant adeno-associated virus;

[0015] The recombinant adeno-associated virus vector includes the expression cassette described in the above technical solution;

[0016] The recombinant adeno-associated virus includes the recombinant adeno-associated virus vector or the expression cassette described in the above technical solutions.

[0017] This invention provides a kit for IscB / ωRNA gene editing, comprising the expression cassette or delivery system described in the above technical solution.

[0018] This invention provides the application of the expression cassette, delivery system, or reagent kit described in the above-described technical solutions in gene editing; the gene editing is for non-diagnostic and non-therapeutic purposes.

[0019] The present invention provides a method for gene editing for non-diagnostic and non-therapeutic purposes, comprising the following steps: delivering the expression cassette or the delivery system described in the above technical solution to the object to be edited.

[0020] Preferably, the object to be edited includes cells.

[0021] This invention provides the application of the expression cassette, delivery system, or kit described in the above-described technical solutions in the preparation of gene-edited animals; wherein the gene editing is for non-diagnostic and non-therapeutic purposes.

[0022] This invention provides a method for preparing gene-edited animals, comprising the following steps: introducing the expression cassette or delivery system described in the above technical solution into an animal embryo; wherein the gene editing is for non-diagnostic and non-therapeutic purposes.

[0023] Beneficial effects:

[0024] This invention, through engineered modification of IscB and its corresponding ωRNA, develops smaller and more efficient IscB protein and ωRNA backbones. The invention constructs an IscB / ωRNA-based gene editing expression cassette by inserting gene editing elements between two inverted terminal repeat sequences. The gene editing element comprises, in sequence, ωRNA, a first promoter, a self-cleaving homology-directed repair nucleic acid donor, a second promoter, and an IscB protein-coding gene. The ωRNA includes a target segment that specifically binds to the target gene and a ωRNA backbone. The first promoter drives ωRNA expression; the self-cleaving homology-directed repair nucleic acid donor performs gene editing on the target gene; and the second promoter drives the expression of the IscB protein-coding gene. In the IscB / ωRNA-based gene editing expression cassette provided by this invention, the first promoter drives ωRNA expression, and the second promoter drives IscB protein-coding gene expression. The expressed ωRNA and IscB recognize and cleave the target gene, and the self-cleaving homology-directed repair nucleic acid donor performs precise gene editing, resulting in higher gene editing and delivery efficiency, and better safety. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0026] Figure 1 Different types of recombinant adeno-associated virus vectors and their editing efficiencies are represented; where A represents the structure of different types of recombinant adeno-associated virus vectors, and B represents the percentage of green and red fluorescent cells.

[0027] Figure 2 for Tyr A photograph of a mutated mouse expressing green fluorescence;

[0028] Figure 3 for Tyr The detection results of PCR products from mutated mice expressing green fluorescence are shown in the figure; where A is the 5' end PCR amplification result and B is the 3' end PCR amplification result.

[0029] Figure 4 for Tyr Sequencing results of PCR products from mutated mice expressing green fluorescence; where A is the sequencing result of the 5' end PCR product and B is the sequencing result of the 3' end PCR product.

[0030] Figure 5 The results show the blastocyst rate and green fluorescence intensity of bovine embryos infected with recombinant adeno-associated virus (AAV); where A represents the blastocyst rate and B represents the green fluorescence detection result. express P <0.05; express P <0.01; express P <0.001;

[0031] Figure 6 The image shows a cow; where A is... SLICK Gene-edited cattle, B represents wild cattle;

[0032] Figure 7 For wild cattle and SLICK Gene identification diagram of the gene-edited cow. Detailed Implementation

[0033] This invention provides an expression cassette based on IscB / ωRNA gene editing, comprising two inverted terminal repeat sequences and a gene editing element located between the two inverted terminal repeat sequences; the gene editing element comprises, in sequence, ωRNA, a first promoter, a self-cleaving homology-directed repair nucleic acid donor, a second promoter, and an IscB protein-coding gene; the ωRNA comprises a targeting segment that specifically binds to the target gene and an ωRNA backbone; the first promoter drives the expression of the ωRNA; the self-cleaving homology-directed repair nucleic acid donor performs gene editing on the target gene; and the second promoter drives the expression of the IscB protein-coding gene.

[0034] In one embodiment, the first promoter of the present invention includes the U6 promoter. In one embodiment, the second promoter of the present invention includes the U1a promoter. In one embodiment, the nucleotide sequence pairs of the inverted terminal repeat (ITR) of the present invention are as shown in SEQ ID NO:18 and SEQ ID NO:19, specifically: CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGCACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCGCAGCTGCCTGCAGG.

[0035] As one embodiment, the amino acid sequence of the IscB protein of the present invention is shown in SEQ ID NO:3, specifically: MMAVVYVISKSGKPLMPTTRCGHVRILLKEGKARVVERKPFTIQLTYESAEETQPLVLGIDPGRTNIGMSVVTESGESVFNAQIRTRNKDVPKLMKDRKQYRMAHRRLKRRCKRRRRAKAAGTAFEEGEKQRLLPGCFKPITCKSIRNKEARFNNRKRPVGWLTPTANHLLVTHLNVVKKVQKILPVAKVVLELNRFSFMAMNNPKVQRWQYQRGPLY GKGSVEEAVSMQQDGHCLFCKHGIDHYHHVVPRRKNGSETLENRVGLCEEHHRLVHTDKEWEANLASKKSGMNKKYHALSVLNQIIPYLADQLADMFPGNFCVTSGQDTYLFREEHGIPKDHYLDAYCIACSALTDAKKVSSPKGRPYMVRQFRRHDRQACHKANLNRRYYMGGKLVATNRHKAMDQKTDSLEEYRAAHSAADVSKLTVKHPSAQYKDMSRIMPGSIL. As one embodiment, the nucleotide sequence of the IscB protein encoding gene of this invention is shown in SEQ ID NO:1. The TAM sequence recognized by the IscB protein shown in SEQ ID NO:3 of this invention is NWRRNA, where N represents A, T, C, or G, W represents A or T, and R represents A or G. This invention does not have strict requirements on the specific sequence of the target segment that specifically binds to the target gene; conventional design based on the target segment of the target gene is sufficient. The IscB protein shown in SEQ ID NO:3 of this invention is obtained by truncating the C-terminus of the wild-type IscB protein (GeneID:65280600) by 50 aa, and the editing efficiency is significantly improved compared with the wild-type IscB protein.

[0036] As one embodiment, the nucleotide sequence of the ωRNA backbone of the present invention is shown in SEQ ID NO:2. The ωRNA backbone shown in SEQ ID NO:2 of the present invention is obtained by truncating the wild-type ωRNA backbone, and the editing efficiency is significantly improved compared with the wild-type ωRNA backbone.

[0037] In one embodiment, the self-cutting homology-directed repair nucleic acid donor of the present invention includes two cleavage sites and a repair template sequence located between the two cleavage sites; the repair template sequence performs gene editing on the target gene.

[0038] In one embodiment, the nucleotide sequence of the cleavage site described in this invention is ATTTGAGTGTCTCCGAAAAGAA (SEQ ID NO:20).

[0039] The present invention provides a delivery system for the expression box described in the above technical solution.

[0040] In one embodiment, the delivery system of the present invention includes a recombinant adeno-associated virus vector and / or a recombinant adeno-associated virus. In another embodiment, the recombinant adeno-associated virus vector of the present invention includes the expression cassette described in the above-described technical solutions; the recombinant adeno-associated virus includes the recombinant adeno-associated virus vector or the expression cassette described in the above-described technical solutions.

[0041] In one embodiment, the recombinant adeno-associated virus vector of the present invention includes a basic adeno-associated virus vector and the aforementioned expression cassette inserted into the basic adeno-associated virus vector. In another embodiment, the basic adeno-associated virus vector of the present invention includes the pEJS1089: mini-AAV.sgRNA.Nme2Cas9 plasmid (purchased from Addgene, catalog number 159536).

[0042] This invention provides a kit for IscB / ωRNA gene editing, comprising the expression cassette or delivery system described in the above technical solution.

[0043] This invention provides the application of the expression cassette, delivery system, or reagent kit described in the above-described technical solutions in gene editing; the gene editing is for non-diagnostic and non-therapeutic purposes.

[0044] The present invention provides a method for gene editing for non-diagnostic and non-therapeutic purposes, comprising the following steps: delivering the expression cassette or the delivery system described in the above technical solution to the object to be edited.

[0045] In one embodiment, the object to be edited in this invention includes cells. In another embodiment, the cells in this invention include fertilized egg cells. This invention does not have strict requirements regarding the delivery method; conventional methods in the art, such as infection, are acceptable.

[0046] The present invention provides the application of the expression cassette, delivery system, or kit described in the above-described technical solutions in the preparation of gene-edited animals.

[0047] In one embodiment, the animals described in this invention include rats and / or cattle.

[0048] This invention provides a method for preparing gene-edited animals, comprising the following steps: introducing the expression cassette or the delivery system described in the above technical solution into an animal embryo.

[0049] In one embodiment, the animals described in this invention include mice and / or cattle. This invention does not impose strict requirements on the method of introducing the delivery system into animal embryos; conventional methods in the art can be used, such as using the delivery system to infect animal fertilized eggs, obtaining transfected embryos, and then performing maternal transplantation.

[0050] To further illustrate the present invention, the adeno-associated virus delivery system based on IscB / ωRNA and its applications provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, all experimental materials used in the following embodiments are commercially available products. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0052] Example 1

[0053] 1. Synthesize IscB mutants with a 50aa truncated (IscB) # The nucleic acid sequence of ωRNA and the truncated ωRNA backbone mutant (ωRNA) # The nucleic acid sequence of the backbone is as follows:

[0054] IscB #

[0055] ωRNA # Backbone: 5'-CAGGCUGAGACAUUCGUAAAGGCCGAAAGGCCGGACAUAGCGGAUGCCAGUAAGCCAGGGGGAACAAUCACCUCCUGUUAUCAGAGAGUUUUACAAAAGGAGGAACGG-3' (SEQ ID NO: 2).

[0056] 2. Construction of recombinant adenovirus-associated vectors

[0057] (1) Using pEJS1089: mini-AAV.sgRNA.Nme2Cas9 plasmid as backbone, ωRNA was inserted into the HindIII restriction site. # Backbone sequence; double digestion at NcoI and NotI sites, insertion of IscB # The nucleic acid sequence was obtained by inserting a ωRNA targeting sequence (5'-UAGCCUGCAGGUCGAC-3', SEQ ID NO:4) into the BspQI endonuclease site and inserting a non-self-cleaving homologous directed repair nucleic acid donor sequence into the C-terminus of Poly-A, thus obtaining the first recombinant adenovirus-related vector, denoted as type ①;

[0058] The non-self-cleaving homologous directed repair nucleic acid donor sequence is specifically: 5'--3' (SEQ ID NO:15);

[0059] (2) Using pEJS1089: mini-AAV.sgRNA.Nme2Cas9 plasmid as the backbone, ωRNA was inserted into the HindIII restriction site. # Backbone sequence; double digestion at NcoI and NotI sites, insertion of IscB #The nucleic acid sequence was obtained by inserting a ωRNA target sequence (SEQ ID NO:4) into the BspQI endonuclease site and a non-self-cleaving homologous directed repair nucleic acid donor sequence shown in SEQ ID NO:15 into the SalI endonuclease site, thus obtaining the second recombinant adenovirus-related vector, denoted as type ②.

[0060] (3) Using pEJS1089: mini-AAV.sgRNA.Nme2Cas9 plasmid as the backbone, ωRNA was inserted into the HindIII restriction site. # Backbone sequence; double digestion at NcoI and NotI sites, insertion of IscB # The nucleic acid sequence was obtained by inserting the ωRNA targeting sequence into the BspQI endonuclease site (SEQ ID NO:4), and inserting the cleavage site sequence into the 5' and 3' ends of SEQ ID NO:15 (SEQ ID NO:20) to obtain the self-cleavage homologous directed repair nucleic acid donor. The donor was further inserted into the C end of Poly-A to obtain the third recombinant adenovirus-related vector, denoted as type ③.

[0061] (4) Using pEJS1089: mini-AAV.sgRNA.Nme2Cas9 plasmid as the backbone, ωRNA was inserted into the HindIII restriction site. # Backbone sequence; double digestion at NcoI and NotI sites, insertion of IscB # The nucleic acid sequence was obtained by inserting the ωRNA target sequence (SEQ ID NO:4) into the BspQI endonuclease site, and inserting the cleavage site sequence (SEQ ID NO:20) into the 5' and 3' ends of SEQ ID NO:15, respectively, to obtain the self-cleavage homologous directed repair nucleic acid donor. Further insertion of the SalI endonuclease site yielded the fourth recombinant adenovirus-related vector, denoted as type ④.

[0062] 3. The different recombinant adenovirus-associated vectors obtained in step 2 were introduced into 293T cells, and after incubation at 37°C for 72 hours, the number of green and red fluorescent cells was analyzed by flow cytometry. The results are as follows: Figure 1 As shown, the autocleavage mode of the fourth recombinant adenovirus-associated vector (type ④) has the highest precise insertion efficiency, and it is named AAV::IscB. # / ωRNA # -HDR.

[0063] Example 2

[0064] Tyr Preparation of mutant and green fluorescent knock-in mice

[0065] 1. First, collect the mouse fertilized eggs. Shave the fur on the back near the tail of the mouse and thoroughly apply 70% ethanol to the surgical site. Pinch the skin along the midline with ophthalmic forceps and make a transverse incision with surgical scissors. Bluntly separate the skin from the muscle, fully exposing the dorsal flank muscles. Lift the peritoneum of the fat pad with ophthalmic forceps, make a small incision with ophthalmic scissors at this point, and gently pull the uterus, fallopian tubes, and ovaries out of the body cavity by grasping the fat pad with forceps. Cut between the fallopian tubes and ovaries with ophthalmic scissors, and then cut the uterus near the fallopian tubes. Place the severed fallopian tubes into a drop of M2 solution in a pre-prepared small glass dish. Tear open the dilated ampulla of the fallopian tube with ophthalmic forceps to reveal the cumulus cell cluster surrounding the fertilized egg. The free fertilized egg will slowly flow out; alternatively, the fertilized egg can be gently pushed out of the opening by squeezing the fallopian tube with forceps.

[0066] 2. Mice Tyr Gene mutations can cause albino mice, resulting in white fur. Constructing targeted mice. Tyr ωRNA of genes # The specific sequence is as follows: 5'-ATTTGAGTGTCTCCGA-3' (SEQ ID NO:5).

[0067]

[0068] 4. Take the ωRNA obtained in step 2. # Replace AAV::IscB obtained in Example 1 # / ωRNA # ωRNA in HDR # Replace the AAV::IscB obtained in Example 1 with the homology-directed repair nucleic acid donor sequence obtained in step 3. # / ωRNA # The coding sequence of the homologous directed repair nucleic acid donor in -HDR was preserved, and the cleavage site was retained to obtain the recombinant adeno-associated virus vector, which was sent to Gemma Biotechnology for AAV packaging to obtain recombinant adeno-associated virus.

[0069] 5. 127 mouse fertilized eggs were mixed with 5×10⁻⁶ eggs. 9 Recombinant adeno-associated virus at VG / mL was incubated at 37°C for 24 hours to obtain infected embryos.

[0070] 6. At a density of 20 embryos per mouse, 2-cell stage embryos were transferred into the oviducts of mice with pseudopregnancy of 0.5 days. Changes in coat color and the expression of green fluorescence in the newborn mice were observed. Results are as follows: Figure 2 As shown, the embryos infected with recombinant adeno-associated virus ultimately resulted in mice changing from black fur to white fur, while also expressing green fluorescent protein, indicating successful editing.

[0071] 7. Safety testing was performed on newborn mice. 5' and 3' amplification primers were designed targeting the insertion site of the homology-directed repair nucleic acid donor sequence. DNA from the edited mouse tail was extracted as a template, and amplification was performed using both the 5' and 3' amplification primers. Results are as follows: Figure 3 As shown, the 5' and 3' amplification products from the edited mouse are of the same size. The amplification primer sequences are as follows:

[0072] 5'arm-F: 5'-TATCCTTCTGTCCAGTGCACCATC-3' (SEQ ID NO:7);

[0073] 5'arm-R: 5'-AAAGTCCCTATTGGCGTTACTATGG-3' (SEQ ID NO:8);

[0074] 3'arm-F: 5'-TGGTCCTGCTGGAGTTCGT-3' (SEQ ID NO:9);

[0075] 3'arm-R: 5'-CTCCATATTTCAGAGCCCCCA-3' (SEQ ID NO: 10);

[0076] 8. The amplification product obtained in step 7 was further purified using a DNA purification kit (Novizan, China). The purified product was then sent to Sanger sequencing by Sangon Biotech. The results are as follows: Figure 4 As shown, the results revealed that the insertion position of the homology-directed repair nucleic acid donor sequence was consistent with the expected result, indicating that AAV::IscB # / ωRNA # -HDR tools can achieve precise insertion of clips.

[0077] Example 3

[0078] PRLR Preparation of gene-edited cattle

[0079] 1. Oocyte collection and in vitro maturation

[0080] Angus bovine ovaries were collected from local slaughterhouses and preserved in physiological saline containing penicillin and streptomycin at 20–25°C. Cumulus-oocyte complexes (COCs) were collected from anterior follicles with a diameter of 3–8 mm using a needle. Under a stereomicroscope, the COCs were washed three times with buffer (M199 + 1% FBS) and then placed in in vitro maturation medium (M199, containing 10% FBS, 20 ng / mL epidermal growth factor, 1 μg / mL β-estradiol, 1 μg / mL gonadotropin, and 0.1 IU / mL luteinizing hormone) for oocyte maturation at 38.5°C and 5% CO2 saturated humidity. After 22 hours of in vitro maturation, cumulus cells were removed with 1 mg / mL hyaluronidase, and mature oocytes that had extruded the first polar body were selected.

[0081] Frozen semen from the same breed of bovine was used for in vitro fertilization (IVF) in an IVF culture medium, with a semen concentration of approximately 2 × 10⁻⁶. 6 Sperm / mL, fertilized eggs were obtained by culturing at 38.5℃ and 5% CO2 for 15 hours.

[0082] 2. Constructing targeted cattle PRLR ωRNA of genes # The specific sequence is as follows: 5'-ATTGAAACTGGCAGGG-3' (SEQ ID NO:11).

[0083]

[0084] 4. Take the ωRNA obtained in step 2. # Replace AAV::IscB obtained in Example 1 # / ωRNA # ωRNA in HDR # Replace the AAV::IscB obtained in Example 1 with the homology-directed repair nucleic acid donor sequence obtained in step 3. # / ωRNA # The coding sequence of the homologous directed repair nucleic acid donor in -HDR was preserved, and the cleavage site was retained to obtain the recombinant adeno-associated virus vector, which was sent to Gemma Biotechnology for AAV packaging to obtain recombinant adeno-associated virus.

[0085] 5. 221 bovine fertilized eggs were mixed with 5 × 10⁻⁶ eggs. 8 VG / mL or 5×10 10 Recombinant adeno-associated virus (AAV) at VG / mL was incubated at 37°C for 12 or 36 hours to obtain infected embryos. The blastocyst rate and fluorescence intensity of green fluorescent embryos were statistically analyzed, and the results are as follows: Figure 5 As shown, 5×10 10 Recombinant adeno-associated virus at VG / mL showed the greatest knock-in effect when incubated with embryos for 12 hours.

[0086] Example 4

[0087] SLICK Preparation of gene-edited (C base deletion) cattle

[0088] 1. Constructing targeted cattle SLICK ωRNA of genes # The specific sequence is as follows: 5'-ATTGAAACTGGCAGGG-3' (SEQ ID NO:12).

[0089] 2. Construct a homologous directed repair nucleic acid donor sequence. The homologous directed repair nucleic acid donor sequence includes a template for the deletion of the C base. The specific sequence is as follows: 5'-ATTTGAGTGTCTCCGAAAAGAAACGTGTGTGAGCTGGCCCTGGGCATGGCCGGCACCACAGCCACTTCGCTGGACCAAACAGACCAACATGTTTAAAAGCCTCAAAAACCATTGAAACTGGCAGGGAAGGAAAGGCAACCAAGCAGAGGGAGTCAGAAGGCTGCAGTTCCAAGCCTGACCAAGACACGGTGTGGCCACGACCCCAAGACAAAACCCCCTTGAATTTGAGTGTCTCCGAAAAGAA-3' (SEQ ID NO:17).

[0090] 3. Take the ωRNA obtained in step 1 # Replace AAV::IscB obtained in Example 1 # / ωRNA # ωRNA in HDR # Replace the AAV::IscB obtained in Example 1 with the homology-directed repair nucleic acid donor sequence obtained in step 2. # / ωRNA # The coding sequence of the homologous directed repair nucleic acid donor in -HDR was preserved, and the cleavage site was retained to obtain the recombinant adeno-associated virus vector, which was sent to Gemma Biotechnology for AAV packaging to obtain recombinant adeno-associated virus.

[0091] 5. Mix 236 bovine fertilized eggs with 5×10 10 Recombinant adeno-associated virus (AAV) at a concentration of VG / mL was incubated at 37°C for 12 hours to obtain infected embryos. Estrus synchronization was performed on recipient cows. A progesterone vaginal suppository (CIDR) was inserted into the recipient cow, removed after 7 days, and prostaglandin (PG) was injected. Estrus status was observed. Infected blastocysts were transferred into the recipient cows, and pregnancy was confirmed on day 35 post-transfer using a transrectal ultrasound (EVO) scanner.

[0092] 6. Observation of the appearance of the cattle revealed a significant reduction in coat color, with the cattle being hairless or having only short hair. Figure 6 ),conform to SLICK Edit the phenotypic characteristics of cattle.

[0093] 7. Further extract bovine blood genomic DNA as a template, design amplification primers for the edited site, and perform PCR amplification. The primer sequences are as follows: SLICK -F: 5'-TCCCGAAACAAACCTTACATG-3' (SEQ ID NO: 13); SLICK -R: 5'-AACAGAGCCAGCACTCCATC-3' (SEQ ID NO: 14). The amplified product was purified using a DNA purification kit (Novizan, China). The purified product was then sent to Sanger sequencing by Sangon Biotech. The results are as follows: Figure 7 As shown, cow SLICK The gene C base deletion indicates that the above results suggest the use of AAV::IscB # / ωRNA # -HDR delivery tool successfully achieved accurate editing SLICK Gene-edited cows.

[0094] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An expression cassette based on IscB / ωRNA gene editing, characterized in that, It includes two inverted terminal repeat sequences and a gene editing element located between the two inverted terminal repeat sequences; The gene editing element comprises, in sequence, ωRNA, a first promoter, a self-cleaving homology-directed repair nucleic acid donor, a second promoter, and an IscB protein-encoding gene; The ωRNA includes a targeting segment that specifically binds to the target gene and an ωRNA backbone; The first promoter drives the expression of ωRNA; The self-cleaving homology-directed repair nucleic acid donor performs gene editing on the target gene; The second promoter drives the expression of the IscB protein-coding gene; The amino acid sequence of the IscB protein is shown in SEQ ID NO:3; the nucleotide sequence of the ωRNA backbone is shown in SEQ ID NO:2; The self-cutting homology-directed repair nucleic acid donor includes two cleavage sites and a repair template sequence located between the two cleavage sites; the repair template sequence performs gene editing on the target gene. The nucleotide sequence of the cleavage site is shown in SEQ ID NO:

20.

2. A delivery system comprising the expression box as described in claim 1.

3. The delivery system according to claim 2, characterized in that, The delivery system includes a recombinant adeno-associated virus vector and / or recombinant adeno-associated virus; The recombinant adeno-associated virus vector includes the expression cassette as described in claim 1; The recombinant adeno-associated virus includes the recombinant adeno-associated virus vector or the expression cassette of claim 1.

4. A kit for IscB / ωRNA-based gene editing, characterized in that, Includes the expression box as described in claim 1 or the delivery system as described in claim 2 or 3.

5. The application of the expression cassette of claim 1, the delivery system of claim 2 or 3, or the kit of claim 4 in gene editing; wherein the gene editing is for non-diagnostic and non-therapeutic purposes.

6. A method for gene editing for non-diagnostic and non-therapeutic purposes, characterized in that, The process includes the following steps: delivering the expression box as described in claim 1 or the delivery system as described in claim 2 or 3 to the object to be edited.

7. The method according to claim 6, characterized in that, The object to be edited includes cells.

8. The use of the expression cassette of claim 1, the delivery system of claim 2 or 3, or the kit of claim 4 in the preparation of gene-edited animals; wherein the gene editing is for non-diagnostic and non-therapeutic purposes.

9. A method for preparing a gene-edited animal, characterized in that, The procedure includes the following steps: introducing the expression cassette of claim 1 or the delivery system of claim 2 or 3 into an animal embryo; wherein the gene editing is for non-diagnostic and non-therapeutic purposes.

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