An engineered CRISPR fusion protein and system for precise gene editing and its application

Abstract

The present invention provides an engineered CRISPR enzyme and system for precise gene editing and its application, wherein the CRISPR enzyme has an amino acid sequence as shown in SEQ ID No: 1, and the gene editing system comprises a CRISPR fusion protein and a guide RNA component. The present invention is used for gene editing, gene targeting, gene cutting, preparation of gene-edited animals, establishment of animal disease models, establishment of disease tissues, preservation and transformation of animal genetic species resources of eukaryotic cells, achieving efficient gene editing, with predictable editing products and very few by-products, thus making up for the shortcomings of existing gene editing tools.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to an engineered CRISPR fusion protein and system for precise gene editing and applications thereof. Background Art

[0002] CRISPR is the abbreviation of "Clustered Regularly Interspaced Short Palindromic Repeats". It is a revolutionary gene editing system composed of CRISPR-related proteins and guide RNA. It can accurately cut specific target sites of DNA, causing double-strand breaks, and then triggering the cell's endogenous DNA repair mechanism. This process can lead to the knockout, knock-in or replacement of the target gene, thereby changing the function of the target gene.

[0003] In CRISPR, endogenous repair pathways in cells include non-homologous end joining and homologous recombination repair. Non-homologous end joining is a relatively common repair pathway that directly connects the broken DNA ends, but this connection process is often random and may lead to mutations. In contrast, homologous recombination repair is a precise repair pathway that relies on an exogenous donor template that can provide a sequence that matches the target site. Through homologous recombination repair, precise gene editing can be achieved, such as point mutations, insertion of specific sequences, or deletion of specific fragments. Compared with random mutations, precise gene editing has broader application prospects and lower safety risks.

[0004] However, the incidence of homologous recombination repair is low, which makes it more difficult to use homologous recombination for precise gene editing. In addition, homologous recombination repair also requires the introduction of exogenous donor templates, which further increases the complexity and technical challenges of the operation. Therefore, improving the efficiency and accuracy of homologous recombination repair and simplifying the operation process are one of the important directions of current CRISPR research. Summary of the invention

[0005] The object of the present invention is to provide an engineered CRISPR fusion protein and a gene editing system for precise gene editing and its application, so as to achieve efficient gene editing, predictable editing products and few by-products, and make up for the shortcomings of existing gene editing tools. The protein provided by the present invention is based on a new CRISPR nuclease, which is named RhoCas12a protein, having an amino acid sequence as described in SEQ ID No: 1. After amino acid optimization and addition of multiple new functional domains, RhoCas12a protein has the function of efficient and precise gene editing in eukaryotic cells. The modified protein is named CdtCasRT fusion protein, having an amino acid sequence as shown in SEQ ID No: 2.

[0006] According to the sequence alignment results of Cas proteins, RhoCas12a protein has a low sequence consistency with the reported Cas proteins. Further analysis of the evolutionary tree of Cas proteins found that RhoCas12a protein belongs to the Cas12a protein branch and has certain amino acid differences with other Cas12a proteins discovered so far, and can be regarded as a new Cas12a protein.

[0007] An obvious fact is that changes in some amino acid residues do not affect the functional activity of the protein. Therefore, changes to some amino acid residues of the RhoCas12a protein are also within the scope of protection of the present invention as long as these changes do not affect the desired function and activity of the RhoCas12a protein.

[0008] The biological functions of the RhoCas12a protein include, but are not limited to, the activity of binding to the guide RNA, the endonuclease activity, and the activity of binding to and cutting a specific site of the target sequence under the guidance of the guide RNA.

[0009] At the same time, the present invention provides an engineered CdtCasRT fusion protein based on RhoCas12a protein construction, specifically, the fusion protein includes amino acid replacement and optimized RhoCas12a protein, and adds a mouse-derived nuclear import protein Cdt's nuclear import-related functional domain at its N-terminus, the two are connected by a linker peptide sequence, and the reverse transcription protein RT is added to the C-terminus of the RhoCas12a protein, as well as multiple nuclear localization signals (NLS) and protein detection tags (HA Tag). The CdtCasRT fusion protein can bind to the corresponding guide RNA, and after reverse transcription of its exposed 3' end, it can be used as a donor template to trigger precise gene editing of the target site.

[0010] The production method of the CdtCasRT fusion protein of the present invention is not limited to any specific method. For example, a genetic engineering method, such as recombinant protein prokaryotic expression technology, can be used to produce the CdtCasRT fusion protein.

[0011] At the same time, the present invention provides a DNA fragment, the sequence of which is shown in SEQ ID No: 3, specifically comprising:

[0012] (1) A DNA sequence encoding the CdtCasRT fusion protein of the present invention.

[0013] (2) A sequence having one, multiple or all codons optimized compared to the DNA sequence of the CdtCasRT fusion protein of the present invention.

[0014] (3) A sequence having one, multiple deleted or added functional domain sequences compared to the DNA sequence of the CdtCasRT fusion protein of the present invention.

[0015] (4) A DNA sequence that is complementary, reverse or reverse complementary to any of the above-mentioned DNA sequences.

[0016] At the same time, the present invention provides a guide RNA, which includes a backbone region, a targeting sequence for targeting nucleic acid, and a reverse transcription sequence. The backbone sequence of the guide RNA is shown in SEQ ID No: 4, and can be encoded by the DNA fragment shown in SEQ ID No: 5.

[0017] The framework region may also be referred to as a protein binding segment or a protein binding sequence, and the targeting sequence of the targeting nucleic acid may also be referred to as a targeting segment of the targeting nucleic acid or a guide sequence of the targeting target sequence. In the present invention, the framework region sequence is fixed, and the targeting sequence and the reverse transcription sequence are designed according to the target region. The framework region of the guide RNA can be combined with the CdtCasRT fusion protein of the present invention to form a nucleic acid protein complex, which has the function of binding to the target gene and realizing precise gene editing on the target gene.

[0018] The targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the present invention contains a nucleotide sequence that is reverse complementary to the target nucleic acid sequence, that is, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid can interact with the target nucleic acid in a specific manner by base pairing. Therefore, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid can be changed or modified to adapt to hybridization with any desired sequence in the target nucleic acid.

[0019] At the same time, the present invention also provides a plasmid vector, which contains a DNA sequence that can express the above-mentioned CdtCasRT fusion protein and guide RNA, and related regulatory sequences. The regulatory sequences of the vector include but are not limited to: a promoter, a terminator, a reporter gene expression cassette, a resistance gene expression cassette, and a vector backbone; the vector form includes but is not limited to plasmids, viruses, cosmids, phages, etc.

[0020] At the same time, the present invention provides an engineered non-natural CRISPR / Cas system precision gene editing system, which is composed of a DNA sequence encoding a CdtCasRT fusion protein and one or more guide RNAs or DNA sequences encoding guide RNAs. The guide RNA includes a backbone region that binds to the CdtCasRT fusion protein, a targeting sequence that targets a target gene, and a reverse transcription sequence, wherein the targeting sequence and the reverse transcription sequence are variable depending on the target gene sequence. It is worth noting that the guide RNA can also be chemically synthesized.

[0021] The CRISPR / Cas system can hybridize with the target gene and perform precise gene editing on the target gene sequence, i.e., deletion, knock-in and replacement of the expected DNA sequence, with little or no unexpected byproducts. After the above precise gene editing, the expression product of the target gene can be changed.

[0022] At the same time, the present invention also provides an activated CRISPR complex, which includes the following three components:

[0023] (1) Protein component, namely CdtCasRT fusion protein.

[0024] (2) Nucleic acid component, namely the guide RNA mentioned above.

[0025] (3) The target gene component binds to the target sequence on the guide RNA. The target gene forms a reverse complement with the targeting sequence of the guide RNA, which can trigger the CdtCasRT fusion protein to accurately edit the target gene.

[0026] At the same time, the present invention also provides an engineered eukaryotic cell cultured in vitro, wherein the eukaryotic cell comprises one or more of the CdtCasRT fusion protein, guide RNA, CRISPR composition, activated CRISPR complex, and vector described in the present invention.

[0027] The eukaryotic cells described in the present invention generally include all mammals, including primary cells cultured in vitro of humans, mice, monkeys, cattle, etc., or commonly used cell lines (such as HEK293, HELA, CHO, Cos-7, etc.). In addition, some plant cells are also included, such as Arabidopsis, corn, rice, etc.

[0028] The CdtCasRT fusion protein, guide RNA, CRISPR composition, activated CRISPR complex, vector, and engineered eukaryotic cells described in the present invention can be used for any one or more of the following purposes: gene editing, gene targeting, gene cutting, preparation of gene-edited animals, construction of disease model animals or tissues, and preservation and transformation of animal genetic germplasm resources of eukaryotic cells.

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

[0030] 1. The present invention provides an engineered CRISPR fusion protein that can perform efficient and precise gene editing on the target site without the need for additional donor DNA fragments. The editing products are predictable, with no obvious off-target phenomenon and very few by-products, and it has better safety than conventional gene editing.

[0031] 2. The CRISPR nuclease of the present invention belongs to the Cas12a protein family. Different from the commonly used Cas9 enzyme and various gene editing tools derived from it, the engineered CRISPR fusion protein constructed by the present invention can perform efficient gene editing in the target site area with high AT content, making up for the shortcomings of existing gene editing tools.

[0032] 2. The engineered CRISPR fusion protein provided by the present invention contains a functional domain that can efficiently enter the nucleus. Compared with conventional CRISPR nucleases that only rely on polypeptide nuclear import signals, it has a higher nuclear import efficiency, thereby having a higher gene editing efficiency in eukaryotic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the gene editing result of CdtCasRT fusion protein in the β-thalassemia-28 (A>G) site in HEK293T cells.

[0034] Figure 2 This is the gene editing result of CdtCasRT fusion protein in the β-thalassemia CD17 (A>T) locus in HEK293T cells.

[0035] Figure 3 The results of gene editing of CdtCasRT fusion protein in the β-thalassemia CD41 / 42 (-TCTT) locus in HEK293T cells. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings. In the embodiments, if no special instructions are given, the technical means used are the conventional technical means in this area. These technical means can refer to "Molecular Cloning Laboratory Manual" (MOLECULAR CLONING: A LABORATORY MANUAL), "Molecular Biology Experiment Design" (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY), "Animal Cell Culture" (ANIMAL CELL CULTURE) etc. In addition, in the embodiments, when not particularly specified, the experimental conditions and the reagents and equipment used all meet the standards recommended by conventional standards or manufacturers. If the reagents or equipment involved do not specify their sources, they are conventional commodities that are generally available on the market. It should be clear to those skilled in the art that the embodiments are only for the purpose of illustrating this research, and do not mean to limit the scope of protection claimed in this research.

[0037] Example 1: Preparation of CdtCasRT fusion protein

[0038] 1. Acquisition of CdtCasRT fusion protein sequence information

[0039] By sequencing the culture metagene and combining protein cluster analysis, a new type of Cas nuclease was obtained. The Cas protein is an effector protein in the CRISPR / Cas system. The present invention named it RhoCas12a protein, which has an amino acid sequence as described in SEQ ID No: 1. After amino acid optimization and the addition of multiple new functional domains, the RhoCas12a protein has the function of efficient and accurate gene editing in eukaryotic cells. The modified protein is named CdtCasRT fusion protein, which has an amino acid sequence as shown in SEQ ID No: 2. The DNA sequence information of the human codon preference of the CdtCasRT fusion protein is determined based on the amino acid sequence, and the full-length DNA sequence is chemically synthesized, and the sequence is shown in SEQ ID No: 3.

[0040] 2. Obtaining the amplified fragment of CdtCasRT fusion protein sequence

[0041] The polymerase chain reaction (PCR) was used to obtain the target fragment. The reaction system is shown in Table 1 and the reaction procedure is shown in Table 2.

[0042] Table 1 PCR reaction system

[0043]

[0044] Table 2 PCR amplification program

[0045]

[0046] 3. PCR product digestion and vector ligation

[0047] The PCR products are separated by electrophoresis in 1% agarose gel. After separation, the target band is cut off and recovered. The recovery process can be performed using a kit. First, the agarose gel block is dissolved by heating with a dissolution buffer, then added to an adsorption column and centrifuged to allow the DNA to adsorb on the column. Finally, sterile water is used for elution. In order to improve the efficiency of the experiment, the PCR product can be digested with a restriction endonuclease. Double digestion can be used to improve efficiency. The specific composition of the double enzyme digestion system can be seen in Table 3.

[0048] Table 3 Enzyme digestion system

[0049]

[0050] In this embodiment, the bacterial expression vector pGEX-6P-1 was used as a plasmid, which has ampicillin resistance and a GST tag at its N-terminus. The cleaved protein can be purified by purifying using glutathione agarose beads. The ligase used was T4 ligase, and the ligation was performed at room temperature for 2-3 hours. The composition of the ligation system can be seen in Table 4.

[0051] Table 4 Connection system

[0052]

[0053] 4. Transformation of ligation products

[0054] In order to make the in vitro recombined DNA enter the host bacteria and express in large quantities, transformation technology is needed. Transformation is actually a natural phenomenon, and many bacteria will undergo transformation. However, under experimental conditions, high-efficiency transformation is required. Therefore, competent cells are generally used as carriers. Competent cells are a special type of cell with a high transformation efficiency, and Escherichia coli is a commonly used host. This experiment uses the heat shock transformation method, and the transformation system is shown in Table 5. Add the ligation product to DE5α competent cells, place on ice for 30 minutes, then heat shock in a 42°C water bath for 90 seconds, and then place on ice for 150 seconds. Next, shake on a shaker at 37°C for 40-60 minutes, and add 400μL of antibiotic-free LB medium. Then, aspirate 200-400μL of culture solution, add ampicillin-resistant LB solid medium, spread evenly, and finally culture overnight in a 37°C incubator.

[0055] Table 5 Transformation system

[0056]

[0057] 5. Screening of transformants

[0058] Screen the positive clones, observe the colonies on the plate, and select single colonies of uniform size. Place the selected colony gun tip or toothpick into the culture medium of the corresponding resistance, and culture in a 37°C incubator until the bacterial solution becomes turbid. Then perform bacterial solution PCR. During this process, the bacterial solution can be lysed through the denaturation effect of 95°C, so that the DNA in the bacteria is released as a template, and then the target fragment is amplified according to the principle of PCR. After the amplification is completed, agarose gel electrophoresis is performed, and the system of bacterial solution PCR is shown in Table 6. Observe whether there are positive clones in the bands in the gel. The procedure of bacterial solution PCR is shown in Table 7. Finally, a single clone is selected and the plasmid is amplified in large quantities.

[0059] Table 6 Bacterial liquid PCR system

[0060]

[0061] Table 7 Bacterial liquid PCR program

[0062]

[0063] 6. Extraction and identification of recombinant plasmid

[0064] After selecting positive clones, they were cultured in LB medium with corresponding resistance. This experiment used LB medium and cultured at 37°C for 8-10 hours until the bacterial solution became turbid. Then the plasmid was extracted. This experiment used enzyme digestion to identify the plasmid. The plasmid enzyme digestion identification system is shown in Table 8. Then the plasmid cutting experiment was carried out, and the cut bands were observed in agarose gel electrophoresis. Finally, the plasmid was sequenced to ensure the correctness of the sequence.

[0065] Table 8 Plasmid restriction enzyme digestion identification system

[0066]

[0067] 7. Expression of CdtCasRT fusion protein

[0068] In this experiment, CdtCasRT fusion protein was expressed in Escherichia coli C43 (DE3). First, the positive monoclonal clone was cultured overnight at 37°C and 220rpm in ampicillin-resistant LB medium, and then cultured in 1L of ampicillin-resistant LB medium under the same conditions for 5-10 hours. When the bacterial solution becomes turbid and its OD600 value reaches 0.8-1, the temperature is lowered to 20°C. Subsequently, 0.3 mol / L of IPTG was used to induce the expression of the recombinant protein at 20°C. Since the promoter in the vector used in this experiment is the lac promoter, before the addition of isopropyl β-D-1-thiogalactopyranoside, the lactose operon encoding the repressor protein can be active to close the lac promoter, so that it cannot express protein, but after induction, the promoter is open, so that the target protein can be expressed.

[0069] After overnight induction, E. coli was collected by centrifugation, and ReqCas9 was resuspended in a resuspension buffer (25mmol / L Tris-HCl (pH8.0), 1mol / L NaCl, 3mmol / L DTT) supplemented with 1mmol / L protease inhibitor PMSF (phenylmethanesulfonyl fluoride). The lysate was obtained by ultrasonic lysis, and the bacterial fragments were removed by centrifugation at 16000rpm for 40 minutes at 4°C. The lysate was first purified using glutathione agarose 4B (GS4B) beads, and then the beads were washed with 20mL wash buffer 1 and 20mL wash buffer 2. The bound protein was cut overnight at 4°C using protein phosphatase in wash buffer 1 (25mmol / L Tris-HCl (pH8.0), 300mmol / L NaCl, 3mmol / LDTT) to remove the GST tag. After this step, the cut CdtCasRT fusion protein was eluted from the GS4B resin and further purified.

[0070] 8. Purification of CdtCasRT fusion protein

[0071] In this example, affinity chromatography and ion exchange chromatography were used to purify the CdtCasRT fusion protein. The purification method used is based on GST affinity chromatography technology, using glutathione transferase as an affinity tag. The molecular weight of GST is about 26kD, and its principle is to achieve separation by specifically binding to GSH and GET. Glutathione agarose 4B (GS4B) beads (GE Healthcare) can adsorb recombinant proteins with GST tags. Finally, protein phosphatase is used to remove the GST tag. According to the isoelectric point of the protein, different ion exchange columns are selected. The isoelectric point of CdtCasRT is about 9, and it can bind nucleic acids, so a cation exchange column was selected in this experiment. Then, a heparin column was used to remove nucleic acids. Buffer A used for the cation exchange column is 25mmol / L Tris-HCl (pH7.5), 3mmol / L DTT; buffer B is 25mmol / L Tris-HCl (pH7.5), 1mmol / L NaCl, 3mmol / L DTT. The buffer used for the heparin column was 25mmol / L Tris-HCl (pH8.0), 3mmol / L DTT, and the buffer A was 25mmol / L Tris-HCl (pH8.0), 1mmol / L NaCl, 3mmol / L DTT.

[0072] The amino acid sequence of the CdtCasRT protein purified in this example is shown in SEQ ID No: 2.

[0073] Example 2: Preparation and purification of guide RNA

[0074] 1. Preparation and purification of guide RNA in vitro transcription template

[0075] In order to synthesize the sgRNA transcription template in vitro, a specific DNA sequence with a T7 promoter was used as the upstream primer, and the backbone sequence of the sgRNA was used as the downstream primer. A 17-base complementary pairing was designed between the upstream and downstream primers so that they could be used as reaction templates for PCR amplification.

[0076] In the PCR reaction, the amplified product can be preliminarily detected by 2% agarose gel electrophoresis. During the detection, the PCR product is loaded into the agarose gel tank together with the appropriate molecular weight marker for electrophoresis. By comparing with the size of the expected sgRNA, it is determined whether the band position in the electrophoresis result is consistent with the expectation.

[0077] If the band position is consistent with the expected size, it indicates that the PCR amplification is successful and further recovery and purification can be performed. The recovery and purification method can be performed using an agarose gel recovery kit (such as Omega's agarose gel recovery kit) according to the steps in the instructions. The recovered and purified DNA fragment is the in vitro transcription template of the sgRNA and can be used for subsequent in vitro transcription reactions.

[0078] The purified DNA fragment has a nucleotide sequence as shown in SEQ ID No: 5, and after in vitro transcription, an sgRNA as shown in SEQ ID No: 4 can be obtained.

[0079] 2. sgRNA in vitro transcription and purification

[0080] After obtaining the purified sgRNA in vitro transcription template, use the TranscriptAid T7 High Yield Transcription Kit for in vitro transcription. In this process, all reagents should be thawed on ice and the samples should be added on ice throughout the process. The operation process is as follows:

[0081] (1) Centrifuge all reagents at low speed and recover the residual liquid on the tube wall. Add samples according to Table 9, where the amount of DNA template used for in vitro transcription is not less than 1 μg. After mixing all liquids, incubate the reaction mixture at 37°C. Adjust the incubation time according to the length of the expected transcription product. Transcripts less than 100 nt in length require at least 4 to 8 hours of incubation, while transcripts greater than 100 nt in length require about 2 hours of incubation.

[0082] Table 9 In vitro transcription system

[0083]

[0084] (2) Removal of residual DNA substrate: Add 2 μL of DNase I to the incubated reaction product and gently pipette to mix. Incubate the reaction mixture at 37°C for 15 minutes to remove the DNA substrate.

[0085] (3) Termination of the reaction: Add 4 μL of 0.5 M EDTA to the reaction product and incubate the reaction mixture at 65°C for 10 minutes. EDTA can neutralize Mg2+ in the polymerase reaction to deactivate the reaction.

[0086] The sgRNA obtained by in vitro transcription was manually extracted and purified by the phenol-chloroform method as follows:

[0087] (1) Add 115 μL of DEPC-treated water and 15 μL of 3 M sodium acetate to 20 μL of the 26 μL reaction product obtained by in vitro transcription, mix by gently pipetting, and vortex for 20 seconds.

[0088] (2) Add an equal amount of phenol-chloroform (1:1) mixture to the total volume of 150 μL of the product obtained by the above operation and vortex for 30 seconds.

[0089] (3) After 5 minutes of ice bath, place the sample in a 4°C centrifuge and centrifuge at 12,000 rpm for 1 minute. After centrifugation, the product will present a three-layer interface, and the upper aqueous phase contains the RNA product obtained by in vitro transcription. Carefully aspirate the upper aqueous phase and transfer it to a new centrifuge tube, and discard the middle protein phase and the lower organic phase.

[0090] (4) Add twice the volume of ice-bathed anhydrous ethanol to the removed aqueous phase, vortex for 30 seconds, and then place in an ice bath for 30 minutes. Next, place the sample in a 4°C centrifuge at 12,000 rpm for 10 minutes.

[0091] (5) You should see a white RNA precipitate in the centrifuge tube. Aspirate and discard all the liquid in the tube, then add 500 μL of 70% ethanol that has been cooled to ice, and grind the white precipitate into particles with a pipette. Vortex to clean the RNA precipitate, then centrifuge the sample at 12,000 rpm in a 4°C centrifuge for 5 minutes.

[0092] (6) Aspirate and discard the ethanol in the centrifuge tube, then place the tube on ice and allow it to air dry naturally in a clean bench until the white precipitate turns into transparent flakes.

[0093] (7) Finally, add 20-50 μL of DEPC-treated water for dissolution.

[0094] (8) When testing nucleic acid concentration, a spectrometer can be used to measure the ratio of OD260 to OD280. An RNA OD260 / OD280 ratio between 1.7 and 2.1 indicates that the RNA product is relatively pure. If the ratio is lower than 1.7, it indicates contamination with phenol or protein, which may be caused by improper operation during the purification process. In this case, the phenol-chloroform method can be used for purification again.

[0095] Example 3: Precise gene editing of thalassemia gene-related loci in HEK293T cells by CdtCasRT fusion protein

[0096] 1. Design of guide RNA: According to the pig HBB gene sequence on NCBI, the corresponding three β-thalassemia gene mutations that are highly prevalent in humans, namely -28 (A>G) site, CD17 (A>T) site and CD41 / 42 (-TCTT) site, were searched. According to the design principles of guide RNA and PAM, three editing sites were selected for the HBB gene, and the corresponding guide RNA was designed. The designed guide RNA has a backbone sequence as shown in SEQ ID No.4, which can be obtained by encoding the nucleotide sequence as shown in SEQ ID No:5.

[0097] 2. Prepare the culture medium: Add 10% fetal bovine serum and a mixture of antibiotics such as penicillin-streptomycin to DMEM / F-12 growth medium and filter through a 0.22 μm filter.

[0098] 3. Thawing of cells: Place the newly received frozen cells in a 37°C preheated water bath, shake quickly for 2-3 seconds until the graupel is completely thawed, and then take it out immediately. Use 1 ml of growth medium to transfer the cells to a 15 ml tube and shake gently to mix. Centrifuge the cells, aspirate the supernatant, and suspend the cells in a culture dish with new growth medium. Let the cells stand overnight in a constant temperature incubator at 37°C and 5% CO2.

[0099] 4. Prepare cell suspension: Mix CdtCasRT fusion protein and guide RNA suspension in DPBS at a mass ratio of 1:3 and a final concentration of 1 μg / μl. After mixing, transfer the mixture to a sterile microcentrifuge tube, wash the cells twice with DPBS, remove the supernatant and add 0.05% trypsin / 0.5mM EDTA digestion solution, incubate at room temperature for 2-3 minutes, shake gently, and centrifuge the cells at 200×g for 5 minutes.

[0100] The cells were prepared into a suspension, mixed with 1 mL of culture medium, and washed once with 1 ml of DPBS, and then the cells were centrifuged.

[0101] 5. Cell electroporation: Mix the cell suspension and conversion mixture, add them to the electroporation solution, and then transfer the mixture to the electroporator and perform transfection according to the appropriate setting parameters. After transfection, transfer the cell suspension to a CO2 culture chamber at 37°C and place it in a 35mm culture dish for 24-48 hours.

[0102] 6. Total DNA was extracted from cells after electroporation, and the PCR amplification products were cloned by TA and then single clone strains were selected for first-generation sequencing. The results of gene editing were evaluated by comparing with the standard sequence. The sequencing results of the -28 (A>G) site are as follows Figure 1 As shown, the sequencing results of CD17 (A>T) locus are as follows Figure 2As shown, the sequencing results of CD41 / 42 (-TCTT) loci are as follows Figure 3 The results showed that the gene editing was successful, the edited site was precise, and there were no byproducts.

[0103] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. An engineered CRISPR fusion protein for precision gene editing, characterized in that: Starting from the amino acid sequence shown in SEQ ID No: 1, after amino acid optimization and adding multiple new functional domains, the amino acid sequence is shown in SEQ ID No:

2.

2. A DNA fragment, characterized in that: The DNA fragment encodes the CRISPR fusion protein according to claim 1, and its sequence is shown in SEQ ID No:

3.

3. A nucleic acid-protein complex comprising the engineered CRISPR fusion protein for precise gene editing according to claim 1, characterized in that: It also includes a guide RNA, which combines with the CRISPR fusion protein of claim 1 to form a nucleic acid-protein complex to achieve the function of precise gene editing; The guide RNA has a backbone sequence as shown in SEQ ID No: 4; The guide RNA is encoded by the DNA fragment shown in SEQ ID No:

5.

4. A plasmid vector, characterized in that: The plasmid vector comprises the DNA fragment shown in SEQ ID No: 3 and the DNA fragment shown in SEQ ID No: 5, and the regulatory sequences related to their expression.

5. A gene editing system, characterized in that: Comprising the nucleic acid-protein complex according to claim 3.

6. A CRISPR complex in an activated state, characterized in that Contains the following substances: (1) The nucleic acid-protein complex according to claim 3; (2) a target genomic component, i.e., a target sequence bound to the guide RNA; The nucleic acid-protein complex and the target gene component combine with each other to form an activated CRISPR complex.

7. An engineered eukaryotic cell, characterized in that: The eukaryotic cell comprises the CRISPR fusion protein of claim 1, or the DNA fragment of claim 2, or the nucleic acid-protein complex of claim 3, or the plasmid vector of claim 4, or the CRISPR complex in an activated state of claim 6.

8. Use of the fusion protein according to claim 1, the DNA fragment according to claim 2, the nucleic acid-protein complex according to claim 3, the plasmid vector according to claim 4, the gene editing system according to claim 5, the CRISPR complex according to claim 6, or the engineered eukaryotic cell according to claim 7, characterized in that: Used for gene editing, gene targeting, gene cutting of eukaryotic cells, preparation of gene-edited animals, establishment of animal disease models, establishment of disease tissues, preservation and transformation of animal genetic species resources; the described applications are not used for the treatment of diseases.

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