Protein complex for efficiently and accurately knocking in long-fragment DNA (Deoxyribose Nucleic Acid) and application thereof

Abstract

The invention relates to a protein complex for efficiently and accurately knocking in long-fragment DNA (Deoxyribose Nucleic Acid) and application of the protein complex. Specifically, the invention relates to a protein complex which sequentially comprises Cas9, a flexible linker 1, Red alpha, a flexible linker 2 and Red beta from an N end to a C end. The RED-CRISPR system formed by the protein can obviously improve the knock-in efficiency of long-fragment genes in various cells, the HDR efficiency is improved, the generation of byproducts in the gene knock-in process is reduced, the homozygous gene knock-in ratio is improved, the off-target and chromosome translocation risks in the gene editing process are reduced, and efficient and accurate knock-in of long-fragment DNA is realized. The method has wide application prospect and practical value in research in the field of gene editing.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and specifically relates to a protein complex for efficiently and accurately knocking in long-fragment DNA and its application. Background Art

[0002] Gene knock-in technology refers to the technology of inserting and stably integrating exogenous genes or DNA sequence fragments into the target genome, so that they are stably inherited and expressed in animal tissues or cells. It is an important tool in the fields of studying gene function, building animal models, and developing gene therapy, and has extremely broad application prospects.

[0003] Traditional gene knock-in technology mainly relies on the spontaneous homologous recombination (HR) mechanism in cells, or is mediated by zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which are costly and cannot be precisely controlled. In recent years, the emergence of CRISPR-Cas9 gene editing technology derived from Streptococcus pyogenes has brought new breakthroughs in gene knock-in technology. The CRISPR-Cas9 system has simple components, including Cas9 protein with endonuclease activity and a sgRNA targeting a specific sequence of the genome. Under the guidance of sgRNA, the Cas9 enzyme can accurately cut DNA and generate double-stranded DNA breaks (DSBs), and finally repair the broken DNA through the non-homologous end joining (NHEJ) or homology-dependent repair (HDR) pathway in cells, thereby generating gene knockout (KO) or knock-in (KI), achieving specific gene editing.

[0004] Currently, efficient gene knockout can be achieved using CRISPR-Cas9, but precise gene knock-in through the HDR repair pathway is still inefficient. This is because NHEJ occurs throughout the cell cycle, while HDR only occurs during DNA replication (S / G2), using sister chromatids as repair templates, so the frequency of HDR in cells is much lower than NHEJ. Although recently developed base editors (BEs), lead editors (PEs) and their derivative technologies can bypass the HDR pathway to achieve efficient and precise single-base replacement or knock-in of small fragments (<400bp), the knock-in of long DNA fragments (>1kb) still mainly relies on the HDR repair method. Therefore, in terms of long-fragment gene knock-in, whether it is the construction of gene knock-in cell lines, gene knock-in animal models or clinical correction of genetic diseases, the inefficiency of the HDR repair method has hindered the widespread application of this technology. Summary of the invention

[0005] The purpose of the present invention is to establish an efficient and accurate long-fragment DNA knock-in method based on the CRISPR-Cas system to solve the technical problems of low efficiency, poor accuracy, high cost and long cycle of long-fragment DNA knock-in in gene editing technology.

[0006] To achieve the above object, the present invention relates to the following projects:

[0007] On the one hand, the present invention relates to a protein complex, whose constituent elements from N-terminus to C-terminus are Cas9, flexible linker 1, Redα, flexible linker 2 and Redβ.

[0008] in,

[0009] (1) The flexible linkers 1 and 2 respectively include SGGSXTENSGGS, (GGGGS)5, (GGGGS)3-GSGGGS, preferably SGGSXTENSGGS;

[0010] Preferably, the amino acid sequences of the flexible linkers 1 and 2 are shown in SEQ ID NO.3, respectively, and preferably, the nucleotide sequences are shown in SEQ ID NO.4;

[0011] (2) The amino acid sequence of the Redα protein is shown in SEQ ID NO.5, and preferably the nucleotide sequence encoding it is shown in SEQ ID NO.6;

[0012] (3) The amino acid sequence of the Redβ protein is shown in SEQ ID NO.7, and the nucleotide sequence encoding it is shown in SEQ ID NO.8;

[0013] (4) The amino acid sequence of the above-mentioned Cas9 protein is shown in SEQ ID NO.9, and the nucleotide sequence encoding it is shown in SEQ ID NO.10.

[0014] The amino acid sequence of the protein complex is shown in SEQ ID NO.1, and optionally, the nucleotide sequence encoding the protein complex is shown in SEQ ID NO.2.

[0015] In another aspect, the present invention relates to a method for constructing the above-mentioned protein complex, comprising the following steps:

[0016] (1) Mammalian codon optimization of Redα and Redβ;

[0017] (2) connecting the mammalian codon-optimized Redα and Redβ via flexible linker 2 to construct a Redα-flexible linker 2-Redβ fusion protein;

[0018] (3) The prepared Redα-flexible linker 1-Redβ fusion protein is connected to Cas9 via flexible linker 1 to form Cas9-flexible linker 1-Redα-flexible linker 2-Redβ fusion protein, i.e., the protein complex.

[0019] On the other hand, the present invention also relates to a plasmid comprising a nucleotide sequence encoding the above-mentioned protein complex.

[0020] Wherein, the DNA sequence of the above plasmid is shown as SEQ ID NO.11.

[0021] The plasmid further comprises hU6, gRNA scaffold, CBh and T2A, and an element encoding a fluorescent protein; preferably, the plasmid comprises hU6, gRNA scaffold, CBh, a portion encoding a protein complex, T2A, and a portion encoding a fluorescent protein in order from 5' to 3'.

[0022] The fluorescent proteins include EGFP, EBFP and mCherry, preferably EGFP and EBFP.

[0023] On the other hand, the present invention also relates to the above-mentioned plasmid construction method, which comprises the following steps:

[0024] (1) Synthesizing a flexible linker 1-Redα-flexible linker 2-Redβ-EGFP DNA sequence, the sequence being shown in SEQ ID NO.12;

[0025] (2) Amplify flexible linker 1-Redα-flexible linker 2-Redβ-EGFP by PCR;

[0026] (3) The PX458 vector was linearized using a rapid restriction endonuclease, and the DNA sequence of flexible linker 1-Redα-flexible linker 2-Redβ-EGFP was inserted into the Cas9 sequence of the PX458 vector by homologous recombination to obtain;

[0027] (4) The DNA sequence of PX458-flexible linker 1-Redα-flexible linker 2-Redβ-EGFP was introduced into Escherichia coli DH5α;

[0028] (5) Extract, identify and obtain the recombinant plasmid, namely RED-CRISPR.

[0029] On the other hand, the present invention also relates to the use of the above-mentioned plasmid to knock in long-fragment DNA sequences in gene editing.

[0030] On the other hand, the present invention also relates to a method for site-directed gene knock-in, which comprises using the above-mentioned plasmid.

[0031] In one aspect of the present invention, a novel CRISPR-Cas9 and Redα / β coupling system is established, which is named RED-CRISPR system. The RED-CRISPR system is composed of the lambda phage Redα / β recombination system ( Figure 1 ) is fused with Redα and Redβ proteins and Cas9 through a flexible linker. The specific elements of the RED-CRISPR system vector include hU6, gRNA scaffold, CBh, spCas9, SGGSXTENSGGS, Redα, SGGSXTENSGGS, Redβ, T2A and fluorescent protein ( Figure 2 , 3 ). Among them, hU6, gRNA scaffold, CBh, spCas9, T2A and fluorescent protein are widely used CRISPR-Cas9 system vector (such as the PX458 vector used in the present invention) elements, SGGSXTENSGGS, Redα, SGGSXTENSGGS, Redβ are unique elements of the RED-CRISPR system vector of the present invention ( Figure 2 and Figure 3 ).

[0032] Compared with the traditional Cas9 or most other reported optimization strategies, the RED-CRISPR system established by the present invention has the following advantages and positive effects:

[0033] (1) The RED-CRISPR system significantly improves the efficiency of long-fragment gene knock-in in various cell types, including immortalized cell lines ( Figure 5 ), mouse primary neurons ( Figure 6 ) and induced pluripotent stem cells ( Figure 7 )wait.

[0034] Since the RED-CRISPR system integrates the Redα / β recombination system, the Redα and Redβ proteins will play the role of recombinases, effectively promoting the integration of homologous repair templates, thereby significantly improving the efficiency of long-fragment gene knock-in, including dividing cells (immortal cell lines and induced pluripotent stem cells, etc.) and non-dividing cells (mouse primary neurons, etc.). Experiments have shown that the RED-CRISPR system can enhance HDR by up to 5 times in dividing cells and by up to 10 times in non-dividing cells.

[0035] (2) RED-CRISPR improves HDR efficiency while reducing the NHEJ rate, that is, reducing the production of byproducts during gene knock-in ( Figure 8 ).

[0036] This is because the RED-CRISPR system changes more inherent NHEJ repair pathways to HDR repair pathways, thereby increasing the HDR:Indel ratio and reducing the proportion of byproducts during gene knock-in.

[0037] (3) RED-CRISPR increases the homozygous gene knock-in rate ( Fig. 9 ).

[0038] Experiments have shown that RED-CRISPR significantly increased the knock-in rate of homozygous long fragments in HeLa cells. This advantage is another manifestation of the RED-CRISPR system's significant improvement in HDR efficiency.

[0039] (4) RED-CRISPR reduces the risk of off-target and chromosomal translocation during gene editing ( Fig.10 and Fig.11 ).

[0040] The term "coupling" refers to connecting different proteins to each other through a linker to form a fusion protein. In the present invention, the CRISPR-Cas9 system is fused with the Redα / β system to form the RED-CRISPR system.

[0041] The term "hU6" refers to the vector element hU6, ie, the human U6 promoter.

[0042] The term "gRNA scaffold" refers to the vector element gRNA scaffold, which is a specific RNA encoded by a plasmid vector and is used to guide DNA nucleases to edit sequences at specific sites in the genome.

[0043] The term "CBh" refers to the vector element CBh, ie, the CAG promoter.

[0044] The term "spCas9" refers to the nuclease spCas9 widely used in CRISPR-Cas9, and in the present invention specifically refers to the vector element Cas9.

[0045] The term "XTEN" refers to the XTEN linker widely used in the construction of various Cas9 fusion proteins, which encodes a short peptide without a specific structure and can therefore be used as a flexible linker (also referred to as a linker in the text, and the two can be used interchangeably) to connect two functional proteins to form a fusion protein (protein complex). In addition, studies have shown that XTEN can enhance the stability of fusion proteins. In the present invention, in order to increase the flexibility of XTEN, SGGS sequences are added at both ends of its sequence, and its sequence is shown in SEQ ID NO.13. Redα, Redβ and Cas9 are coupled to form a protein complex.

[0046] The term "T2A" refers to the carrier element T2A, ie, the 2A self-cleaving peptide.

[0047] The term "codon optimization" refers to the use of genetic engineering technology to adjust the synonymous codons of genes according to the codon preference of the host, so as to eliminate rare codons, simplify the secondary structure of mRNA, and optimize the repetitive sequence.

[0048] The term "long DNA fragment" refers to a DNA fragment with a base pair length exceeding 1 kb in the protein complex, plasmid vector, etc. involved in the present invention.

[0049] The term "linearization" refers to the use of restriction endonucleases to create gaps in circular plasmid DNA to form chained DNA sequences, which facilitate gene editing operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 :Redα is a 5'-3' exonuclease that binds to the end of double-stranded DNA and digests DNA from the 5' end to the 3' end to produce a 3' overhang; Redβ is a single-stranded DNA annealing protein (SSAP) that binds to the exposed 3' single-stranded DNA to form a DNA-protein heterocomplex, promoting strand annealing and strand exchange.

[0051] Figure 2 : Schematic diagram of the design of Cas9, Cas9-Redα, Cas9-Redβ, Cas9-Redα-Redβ and Redα-Cas9-Redβ plasmids and their products for long-fragment DNA knock-in experiments. Among them, the design highlighted by the black box (Cas9-Redα-Redβ) is the RED-CRISPR system established by the present invention. Other designs (Cas9-Redα, Cas9-Redβ and Redα-Cas9-Redβ) of the present invention were also developed according to the establishment steps of the RED-CRISPR system, but after testing, the HDR enhancement ability was significantly lower than that of the RED-CRISPR system.

[0052] Figure 3 : Schematic diagram of all elements on the RED-CRISPR plasmid map established in the present invention.

[0053] Figure 4 :Schematic diagram of the RED-CRISPR system knocking in long DNA.

[0054] Figure 5: a, Schematic diagram of the design of the 2.5kb long fragment (IRES-mCherry-P2A-EGFP) knock-in experiment at the human GAPDH site; b, Schematic diagram of the design of Cas9 and RED-CRISPR plasmids for the 2.5kb knock-in experiment; c, Representative flow cytometry analysis images showing the comparison of the dual fluorescence gene knock-in efficiency of Cas9 and RED-CRISPR at the GAPDH site in HEK293T cells; d and e, Determination and comparison of the 2.5kb KI efficiency of Cas9 and RED-CRISPR at the GAPDH and HSP90AA1 sites in HEK293T (d) and HeLa (e) cells.

[0055] Figure 6 : a, Schematic diagram of the experimental process of long-fragment knock-in in primary mouse neurons; b, Representative fluorescence images after Cas9 and RED-CRISPR knock-in of P2A-mKate at the Actb site in mouse neurons; c, Analysis and comparison of the knock-in efficiency of P2A-mKate at the Actb site in neurons by Cas9 and RED-CRISPR.

[0056] Figure 7 : a, Analysis and comparison of IRES-mCherry knock-in efficiency of Cas9 and RED-CRISPR at the GAPDH site of iPS cells; b, Representative flow cytometric analysis images of fluorescent protein knock-in at the GAPDH site of iPS cells by Cas9 and RED-CRISPR.

[0057] Figure 8 : a, Schematic diagram of the amplicon sequencing library construction process; b, Quantitative analysis of the HDR or NHEJ efficiency of Cas9 and RED-CRISPR at the GAPDH and HSP90AA1 sites in HEK293T cells.

[0058] Fig. 9:a, Experimental flow of IRES-mCherry KI monoclonal isolation and identification. b, Schematic diagram of primer design for 5'Junction PCR, 3'Junction PCR and Full-length PCR. c and d, PCR analysis of HeLa KI monoclonals. In this study, 30 Cas9 group monoclonals (c) and 32 RED-CRISPR group monoclonals (d) were selected for PCR analysis. After collecting each clone and extracting its genome, PCR identification was performed according to the scheme designed in b. The expected band position of the PCR product is indicated by a red arrow; the PCR product of the unedited wild-type cell is shown in the rightmost lane of each gel as a control. The black asterisk indicates the incorrect PCR product in each gel image, which is manifested as deviation from the expected band position or the absence of the expected band. At the same time, the red asterisk summarizes the monoclonal clones of each group with imperfect repair (referring to the difference between the repaired sequence and the original sequence in individual bases, which is not 100% identical, and the number of differences is the floating amount, and the change in the floating amount is well known to technicians in this field) based on the results of three PCR experiments. Identified homozygous HeLa clones in each group are indicated by green arrows.

[0059] Fig.10 :a and b, compare the specific off-target number of RED-CRISPR and Cas9 at different sites, and compare the mismatch tolerance of the two for off-target sites. Among them, No. mismatches refers to the number of mismatches; rep. refers to the experimental repetition. Different mismatch numbers are displayed in different colors.

[0060] Fig.11 :The pie chart shows the proportion of non-editing (Germline), insertion / deletion (Indel) and chromosomal translocation (Translocation) when Cas9 and RED-CRISPR edit the MYC1 and EMX1 sites in HEK293T (a) and HeLa (b) cells, respectively. DETAILED DESCRIPTION

[0061] The design ideas of the RED-CRISPR system are as follows:

[0062] The idea of ​​the present invention is to find a simple and efficient exogenous recombinase / recombination system to couple with Cas9 to construct a new, efficient and accurate CRISPR-homologous recombination coupling system ( Figure 2 ), combining the precision of the HDR pathway with the high efficiency of the exogenous recombinase / recombination system, effectively enhances the efficiency of CRISPR-Cas9-mediated HDR. In addition, the exogenous recombinase / recombination system can also promote long-fragment gene knock-in in non-dividing cells.

[0063] The Redα / β system is a highly efficient homologous recombination system in λ phage, which encodes a pair of recombinase proteins, Redα and Redβ. Among them, Redα is an exonuclease responsible for digesting the DSB end from 5' to 3' to form a 3' single-stranded overhang structure; Redβ is a single-stranded DNA annealing protein (SSAP) that promotes the completion of recombination through strand annealing and strand exchange ( Figure 1 ). Due to its simplicity and high efficiency, the Redα / β recombination system has been developed by researchers for bacterial genetic engineering (recombineering), such as bacterial artificial chromosome (BAC) cloning construction. However, the prior art does not disclose or teach the use of Redα / β for gene editing, especially site-directed gene knock-in, and it is still unknown whether Redα / β can promote CRISPR-Cas9-mediated homology-dependent repair (HDR) in mammalian cells to achieve efficient gene knock-in.

[0064] Therefore, the present invention couples the CRISPR-Cas9 system with the Redα / β system by constructing a fusion protein, that is, Redα and Redβ of the Redα / β system are directly fused to the C-terminus of Cas9 to form a new fusion protein. When Cas9-Redα-Redβ is used for long-fragment gene knock-in, the Cas9-Redα-Redβ / sgRNA complex first targets and binds to the genomic target site, and forms a DNA double-strand break (DSB) by cutting the genomic DNA through Cas9. Next, the Cas9-coupled Redα exerts exonuclease activity to digest the single strand at the end of the DSB in the 5' to 3' direction to form a 3' single-stranded overhang structure; at the same time, the homologous repair template will also undergo terminal single-strand digestion to form a 3' overhang structure under the action of Redα or other exonucleases in the cell. Next, the Cas9-coupled Redβ binds to the single strand at the end of the DSB and undergoes chain annealing with the homologous repair template. Finally, the gap is repaired and connected by the intracellular polymerase and ligase to achieve traceless insertion of long fragments. On the other hand, since Redβ can promote homologous recombination through the strand invasion mechanism, the DSB single strand bound by Redβ can also directly invade the homologous repair template that has not been digested by the exonuclease, completing the long-fragment gene knock-in by strand invasion ( Figure 4 ). The protein complex of the present invention can shorten the steps and time required in the knock-in process of long gene fragments, improve the efficiency of knockout, and increase the knock-in success rate and accuracy, thus making up for the problems of low knock-in efficiency and low accuracy in the prior art.

[0065] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0066] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0067] Example

[0068] Example 1: Construction of RED-CRISPR (Cas9-Redα-Redβ) vector

[0069] (1) Synthesize the XTEN-Redα-XTEN-Redβ-EGFP DNA sequence (commissioned to Qingke Biotechnology Co., Ltd. for synthesis), the sequence is shown in SEQ ID NO. 12.

[0070] (2) Phanta Max Super-Fidelity DNA Polymerase (Norvozymes, P505-d2) was used for PCR reaction to amplify the XTEN-Redα-XTEN-Redβ-EGFP DNA fragment.

[0071] Prepare the following components in a PCR tube:

[0072]

[0073] Carry out the following reaction procedure in the PCR instrument:

[0074]

[0075] After the reaction was completed, 1% agarose gel electrophoresis was performed and gel recovery was performed.

[0076] (3) Using FuniCut TM EcoR I rapid restriction endonuclease (manufactured by Yisheng, 15010ES78) was used to linearize the PX458 vector, wherein the PX458 plasmid contains a Cas9 element. By linearizing it, the connecting part of Cas9 is exposed, making it possible to perform subsequent operations to obtain a homologous recombination plasmid.

[0077]

[0078] Incubate at 37°C for 1 h. After the reaction is completed, perform 0.8% agarose gel electrophoresis and gel recovery.

[0079] (4) Homologous recombination was performed using ClonExpress II One Step Cloning Kit (Novagene, C112-02).

[0080] Prepare the following components in a PCR tube on ice:

[0081]

[0082] Use a pipette to gently pipette to mix, centrifuge briefly, place in a PCR instrument at 37°C for 30 minutes, and immediately place on ice after the reaction to transform competent Escherichia coli DH5α.

[0083] (5) Use The plasmid was extracted using Fastfilter Plasmid DNA Mini Kit (manufactured by Omega, D6944-02) (for detailed steps, refer to the instruction manual), and Sanger sequencing was performed.

[0084] Example 2: Knock-in of the P2A-mCherry-T2A-EGFP sequence at the HSP90AA1 locus in HeLa cells using the RED-CRISPR system

[0085] (1) Design of sgRNA sequence for HSP90AA1 locus

[0086] A 20 bp targeting sequence was selected near the stop codon of the HSP90AA1 locus, adjacent to the PAM sequence, and additional bases were added to the selected sgRNA upstream and downstream sequences as shown below to facilitate sgRNA cloning. The two sequences were ordered from Sangon Biotech.

[0087] sgHSP90AA1 F:5'-CACCGTAGACTAATCTCTGGCTGA-3'(SEQ ID NO.16)

[0088] sgHSP90AA1 R:3'-CATCTGATTAGAGACCGACTCAAA-5'(SEQ ID NO.17)

[0089] (2) Annealing the two DNA sequences of sgRNA

[0090] Prepare the following components in a PCR tube:

[0091]

[0092] After thorough shaking and mixing, perform the following reaction in a PCR instrument: 95°C for 5 min, and cool naturally to room temperature.

[0093] (3) Cloning the annealed sgRNA into the RED-CRISPR vector prepared in Example 1

[0094] Prepare the following components in a PCR tube:

[0095]

[0096] Mix thoroughly with a pipette and place in a PCR instrument to perform the following reaction:

[0097]

[0098] After the reaction, the cells were directly transformed and the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0099] (4) Preparation of HDR repair template

[0100] (i) Template synthesis: The HSP90AA1-P2A-mCherry-T2A-EGFP template sequence (SEQ ID NO. 18) was designed, with 500 bp homology arms on both sides, and was ordered from Qingke Biotechnology Co., Ltd.

[0101] (ii) PCR amplification: Prepare the following components in an EP tube:

[0102]

[0103] Divide it equally into 4 PCR tubes and perform the following procedures in a PCR instrument:

[0104]

[0105] After the reaction is completed, all products are recovered by glue.

[0106] (iii) Gel extraction: refer to the instruction manual of GeneJET Gel Extraction Kit (manufactured by ThermoScientific, K0692).

[0107] (5) Deliver the recombinant RED-CRISPR plasmid containing sgRNA and the HSP90AA1-P2A-mCherry-T2A-EGFP template to HeLa cells

[0108] (i) Inoculation: One day before transfection, 1-2×10 5 HeLa cells (ATCC, CCL-2) were seeded into individual wells of a 24-well plate to reach a density of 70-90% on the day of transfection.

[0109] (ii) Transfection: For a single well of a 24-well plate, 600 ng of RED-CRISPR plasmid and 200 ng of homology repair template were mixed with 1.6 μL of jetPRIME in the buffer provided by jetPRIME reagent (Polyplus, 101000046), allowed to stand at room temperature for 10 min, and then added dropwise to the cell culture medium. Fresh culture medium was replaced after 4 h.

[0110] (6) Fluorescent protein knock-in detection

[0111] (i) Digestion: 5 days after transfection, the transfected cells were digested with 0.25% trypsin (Amresco, C0203), collected in a 1.5 mL EP tube, centrifuged at 1,000 rpm for 5 min, and the supernatant was removed. The cells were resuspended in 1 mL PBS, centrifuged at 1,000 rpm for 5 min, and the supernatant was removed.

[0112] (ii) Resuspension: Resuspend the cells in 500 μL DPBS+4% FBS (Gibco, A5669701) solution and place on ice. Sample loading and detection must be completed within 1 hour.

[0113] (iii) Sample loading: Filter the cell suspension through a clean 200-mesh nylon mesh and transfer it to a flow tube or a 2 mL EP tube for sample loading.

[0114] (iiii) Analysis: The samples were detected using a CytoFLEX S flow cytometer (manufactured by Beckman) and analyzed using FlowJo software.

[0115] In order to illustrate that RED-CRISPR has the effect of improving the efficiency of gene knock-in at different endogenous sites in different immortalized cell lines, the present invention uses the RED-CRISPR system to knock in the P2A-mCherry-T2A-EGFP sequence at the GAPDH site in HeLa cells and the HSP90AA1 and GAPDH sites of HEK293T cells (ATCC, CRL-3216) in the same manner as in Example 2.

[0116] The experimental results are as follows Figure 5 As shown, Figure 5 c is a representative flow cytometry analysis image showing the dual fluorescence gene knock-in efficiency of Cas9 and Cas9-Redα-Redβ at the GAPDH locus of HEK293T cells; Figure 5 d and Figure 5 e shows that in the cells and sites tested, the gene knock-in efficiency of RED-CRISPR was increased by 1.5-2.5 times compared to Cas9.

[0117] Example 3: Using RED-CRISPR to knock in the P2A-mKate sequence at the Actb site in primary mouse neurons

[0118] (1) Design of sgRNA sequence for Actb site

[0119] A 20 bp targeting sequence was selected near the stop codon of the Actb site, adjacent to the PAM sequence, and additional bases were added to the sgRNA upstream and downstream sequences shown below to facilitate sgRNA cloning. The two sequences were ordered from Sangon Biotech.

[0120] sgActb F:5'-CACCGCACCGCAAGTGCTTCTAGG-3'(SEQ ID NO.21)

[0121] sgActb R: 3'-CGTGGCGTTCACGAAGATCCCAAA-5' (SEQ ID NO. 22)

[0122] (2) Annealing the two DNA sequences of sgRNA

[0123] Prepare the following components in a PCR tube:

[0124]

[0125] After thorough shaking and mixing, perform the following reaction in a PCR instrument: 95°C for 5 min, and cool naturally to room temperature.

[0126] (3) Cloning the annealed sgRNA into the RED-CRISPR vector prepared in Example 1

[0127] Prepare the following components in a PCR tube:

[0128]

[0129] Mix thoroughly with a pipette and place in a PCR instrument to perform the following reaction:

[0130]

[0131] After the reaction, the cells were directly transformed and the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0132] (4) Preparation of HDR repair template

[0133] (i) Template synthesis: The mActb-P2A-mKate template sequence (SEQ ID NO. 23) was designed, with 500 bp homology arms on both sides, and was ordered from Qingke Biotechnology Co., Ltd.

[0134] (ii) PCR amplification: Prepare the following components in an EP tube:

[0135]

[0136] Divide it equally into 4 PCR tubes and perform the following procedures in a PCR instrument:

[0137]

[0138] After the reaction is completed, all products are recovered by glue.

[0139] (iii) Gel extraction: refer to the instruction manual of GeneJET Gel Extraction Kit (manufactured by ThermoScientific, K0692).

[0140] (5) The RED-CRISPR plasmid containing sgRNA and mActb-P2A-mKate template was delivered to primary mouse neurons (derived from E18 C57BL / 6J mice purchased from Beijing Weitong Lihua) (using Lonza 4D-Nucleofector electroporation).

[0141] (i) The neuronal cells were washed twice with HBSS solution, the supernatant was removed, and 2.5×10 6 cells and mixed with 4.5 μg RED-CRISPR plasmid and 1.5 μg repair template.

[0142] (ii) Transfer the neuron and DNA mixture to an electroporation cell and select Program O-005, install the electroporation cell and run the program.

[0143] (iii) After electroporation, the electroporation cell was immediately removed and 500 μL of RPMI 1640 medium (manufactured by ThermoScientific, 11875093) pre-equilibrated at 37°C was added. All cells were transferred to a pre-prepared 1.5 mL EP tube and incubated for 10 min. The tube was then centrifuged at 1,000 × g for 5 min and the supernatant was removed.

[0144] (iiii) Add 1 mL of Neurobasal plus medium (Gibco, A3582901) (containing 2% B27 supplement, 1× Glutamax, 50 U / mL dual antibody) to resuspend the cells, seed the cells in a poly-lysine-coated 12-well cell culture plate, and culture in a 37°C incubator for 2 h.

[0145] (iiiii) Replace with fresh Neurobasal plus medium (containing 2% B27 supplement, 1×Glutamax, 50 U / mL dual antibody) to remove cell debris.

[0146] (iiiiii) Primary neurons were cultured for 7 days, and half of the culture medium was replaced every three days.

[0147] (6) Fluorescent protein knock-in detection

[0148] (i) 7 days after electroporation, discard the culture medium and wash once with PBS.

[0149] (ii) Add 4% PFA to fix for 20 min and wash twice with PBS.

[0150] (iii) Stain with DAPI diluted in PBS for 2 min and wash twice with PBS.

[0151] (iiii) Microscope observation and counting of the number of red fluorescent neurons.

[0152] The experimental results are as follows Figure 6 As shown in b and c, the fluorescence images show that the RED-CRISPR group can clearly observe the neurons with red fluorescence (mKate) knocked in, while the Cas9 group has difficulty finding the neurons with mKate knocked in. Through statistical analysis, the gene knock-in efficiency of RED-CRISPR is 10 times higher than that of Cas9.

[0153] Example 4: Using RED-CRISPR to knock in the IRES-mCherry sequence at the GAPDH site in human induced pluripotent stem cells (iPSCs)

[0154] (1) Design of sgRNA sequence for GAPDH locus

[0155] After the stop codon of the GAPDH locus, a 20 bp targeting sequence was selected that was adjacent to the PAM sequence, and additional bases were added to facilitate sgRNA cloning according to the sgRNA upstream and downstream sequences shown below. The two sequences were ordered from Sangon Biotech.

[0156] sgGAPDH F: 5'-CACCGAGCCCCAGCAAGAGCACAAG-3' (SEQ ID NO.26)

[0157] sgGAPDH R: 3'-CTCGGGGTCGTTCTCGTGTTCCAAA-5' (SEQ ID NO. 27)

[0158] (2) Annealing the two DNA sequences of sgRNA

[0159] Prepare the following components in a PCR tube:

[0160]

[0161] After thorough shaking and mixing, perform the following reaction in a PCR instrument: 95°C for 5 min, and cool naturally to room temperature.

[0162] (3) Cloning the annealed sgRNA into the RED-CRISPR vector prepared in Example 1

[0163] Prepare the following components in a PCR tube:

[0164]

[0165] Mix thoroughly with a pipette and place in a PCR instrument to perform the following reaction:

[0166]

[0167] After the reaction, the cells were directly transformed and the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0168] (4) Preparation of HDR repair template

[0169] (i) Template synthesis: A GAPDH-IRES-mCherry template sequence (SEQ ID NO. 28) was designed, with 500 bp homology arms on both sides, and was ordered from Qingke Biotechnology Co., Ltd.

[0170] (ii) PCR amplification: Prepare the following components in an EP tube:

[0171]

[0172] Divide it equally into 4 PCR tubes and perform the following procedures in a PCR instrument:

[0173]

[0174] After the reaction is completed, all products are recovered by glue.

[0175] (iii) Gel extraction: refer to the instruction manual of GeneJET Gel Extraction Kit (manufactured by ThermoScientific, K0692).

[0176] (5) Deliver the RED-CRISPR plasmid containing sgRNA and the GAPDH-IRES-mCherry template to iPS cells (prepared by Professor Cheng Linzhao's research group at the University of Science and Technology of China; the cell preparation method can be found in (Chou BK, Mali P, Huang X, et al. Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures[J]. Cell Research: English Edition, 2011(3), BC1) (using Lonza 4D-Nucleofector for electroporation).

[0177] Resuspend 2×10 6 iPS cells were inoculated with 10 μg of RED-CRISPR plasmid and 5 μg of repair template. Electroporation was performed using the CB-150 program. To improve the survival rate of iPS cells, ROCK inhibitor Y-27632 (StemCell Technologies, 72304) was added to the culture medium at a final concentration of 10 μM in the first 24 hours after inoculation. Flow cytometry analysis was performed on the fluorescent protein knock-in efficiency of iPS cells 4 days after electroporation.

[0178] The results are as follows Figure 7 As shown, the gene knock-in efficiency of RED-CRISPR is 5 times higher than that of Cas9.

[0179] Example 5: RED-CRISPR system knock-in gene and single clone isolation and identification

[0180] (1) Using RED-CRISPR, the IRES-mCherry sequence was knocked into the GAPDH site of HeLa cells, and the experiment was carried out in the same manner as in Example 1-4.

[0181] (2) Monoclonal sorting: Single mCherry-positive HeLa cells (ATCC, CCL-2) were sorted into 96-well cell culture plates pre-added with culture medium and cultured in a 37°C cell culture incubator for 2-4 weeks. The culture medium should be replenished at any time during this period.

[0182] (3) Single clone picking: After 10 days of cell sorting, observe under a microscope every 1-2 days to mark the location of the formed cell clones. Remove the culture medium in the well and drip an appropriate amount of 0.25% trypsin (Amresco, C0203) on the cloned cells. After digestion for 30 seconds, transfer them to a 24-well plate pre-added with culture medium and continue dispersed culture.

[0183] (4) Single clone collection and genome extraction: After the cells have grown to full size in the 24-well plate, they are digested and the cells are recovered. The cell genome was extracted using a Genomic DNA Purification Kit (NEB, T3010S), and the experimental steps were performed according to the kit instructions.

[0184] (5) 5'junction, 3'junction and full-length PCR: Different PCR amplification reactions were performed on the monoclonal genome. The 5'junction PCR primers were GAPDH 5'F (SEQ ID NO.31) and GAPDH 5'R (SEQ ID NO.32); the 3'junction PCR primers were GAPDH 3'F (SEQ ID NO.33) and GAPDH 3'R (SEQ ID NO.34). The full-length PCR was performed using primers and primers.

[0185] (6) Sanger sequencing: After the PCR was completed, 1.5% agarose gel electrophoresis was performed, and the crude PCR products of representative clones (different bands of heterozygotes can be cut out and recovered from the gel) were subjected to Sanger sequencing. The sequencing results were visualized and analyzed using SnapGene software.

[0186] The experimental results are as follows Fig. 9 As shown, 13 imperfectly repaired clones and 2 homozygous knock-ins were identified among the 30 clones picked by the Cas9 group ( Fig. 9 c), while only 10 imperfectly repaired clones were found among the 32 clones picked by the Cas9-Redα-Redβ group, but 6 homozygous knock-ins were identified ( Fig. 9 d). The results demonstrate that RED-CRISPR has higher editing accuracy and homozygous knock-in efficiency.

[0187] Example 6: Amplicon sequencing to detect HDR and Indel ratios of the RED-CRISPR system at the GAPDH and HSP90AA1 sites in HEK293T cells

[0188] (1) Cell transfection: HEK293T cells were seeded in 24-well plates 24 h before transfection and allowed to grow to a cell density of 70-90% on the day of transfection. On the day of transfection, 500 ng of RED-CRISPR plasmid and 50 ng of single-stranded oligonucleotide ssODN (GAPDH: SEQ ID NO.39; HSP90AA1: SEQ ID NO.40) were transfected into a single well of the 24-well plate by 1.1 μL jetPRIME (manufactured by Polyplus, 101000046). Fresh culture medium was replaced 5 h after transfection.

[0189] (2) Cell genome extraction: Digest and collect cells 72 hours after transfection. Genomic DNA Purification Kit (NEB, T3010S) was used, and the experimental steps were carried out according to the instructions of the kit.

[0190] (3) Library construction: Prepare the following components in a PCR tube:

[0191]

[0192] Carry out the following reaction procedure in the PCR instrument:

[0193]

[0194] After the reaction is completed, 5 μL of PCR product is taken for 1.5% agarose gel electrophoresis, and samples that can identify the target band are preferably used for subsequent experiments.

[0195] In a new PCR tube, prepare the following components:

[0196]

[0197] Carry out the following reaction procedure in the PCR instrument:

[0198]

[0199] After the reaction, different samples were pooled together and purified by 1.5% agarose gel electrophoresis.

[0200] (4) NGS sequencing: The library concentration was measured using the Qubit dsDNA HS assay kit (manufactured by ThermoScientific, Q32854), and sequencing was performed using the PE150 mode of the Illumina Novaseq6000 platform (the sequencing methods described herein are all conventional methods in the prior art).

[0201] (5) Data analysis: First, the samples were split and the connectors were removed. The data of each group of samples after splitting were imported into the CRISPResso2 website and the relevant sequence information was filled in. The quantification window was set to 10 bp near the sgRNA cutting site. To avoid quantitative errors caused by sequencing errors, substitutions were ignored. In this study, at least 500,000 reads were detected for each amplicon library.

[0202] The experimental results are as follows Figure 8 As shown, the results prove that RED-CRISPR has a higher HDR:Indel ratio, that is, RED-CRISPR improves HDR efficiency while reducing the production of by-products during gene knock-in.

[0203] In order to verify the safety of RED-CRISPR involved in the present invention, the present invention uses GUIDE-seq technology to detect off-target cleavage events when the RED-CRISPR system and traditional Cas9 are edited at the MYC1, EMX1, GAPDH and HSP90AA1 sites of HEK293T cells. And further use PEM-seq technology to detect the chromosome translocation ratio caused by the RED-CRISPR system and traditional Cas9 when editing the MYC1 and EMX1 sites of HEK293T and HeLa cells. For specific methods, refer to Tsai SQ, Zheng Z, Nguyen NT, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases [J]. Nature Biotechnology.

[0204] The results of GUIDE-seq are as follows Fig.10 As shown, Fig.10 The specific off-target numbers of Cas9-Redα-Redβ and Cas9 at different sites were compared, and it was found that the off-target sites of the RED-CRISPR system were reduced by more than half compared with traditional Cas9. Fig.11 As shown, Fig.11 The proportions of non-editing (Germline), insertion / deletion (Indel), and chromosome translocation (Translocation) events caused by Cas9-Redα-Redβ and Cas9 when editing the MYC1 and EMX1 sites in HeLa cells were compared. The results were consistent with the GUIDE-seq results, confirming that the translocation events caused by RED-CRISPR were significantly reduced.

[0205] In summary, the RED-CRISPR system provided by the present invention has higher editing safety. The above experiments prove that the editing safety of the RED-CRISPR system is greatly improved, with higher accuracy and important application value.

[0206] Sequence Listing:

[0207] SEQ ID NO.1: RED-CRISPR protein complex amino acid sequence

[0208]

[0209] SEQ ID NO.2: Nucleotide sequence of RED-CRISPR protein complex

[0210]

[0211] SEQ ID NO.3: SGGSXTENSGGS amino acid sequence

[0212] SGGSSGGSSGSETPGTSESATPESSGGSSGGS

[0213] SEQ ID NO.4: SGGSXTENSGGS nucleotide sequence

[0214] TCTGGTGGTTCTTCTGGTGGTTCTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCCGAAAGTTCTGGTGGTTCTTCTGGTGGTTCT

[0215] SEQ ID NO.5: Redα amino acid sequence

[0216] MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPRSGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESPIIYRDESMR TACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMWVTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFGEQWR

[0217] SEQ ID NO.6: Redα nucleotide sequence

[0218] ATGACTCCCGATATTATTCTGCAGAGGACTGGAATAGATGTGAGGGCTGTGGAGCAAGGCGATGACGCATGGCACAAACTCAGGCTGGGTGTCATCACCGCCTCAGAGGTTCATAACGTGATAGCCAAGCCTAGATCCGGAAAGAAGTGGCCTGATATGAAGATGAGTTACTTCCATACTCTCCTTGCCGAGGTCTGCACTGGAGTGGCACCAGAGGTCAATGCCAAAGCACTTGCTTGGGGAAAACAATATGAAAACGATGCCCGCACACTGTTCGAGTTCACTTCAGGCGTAAATGTTACAGAGTCCCCCATAATATATAGGGATGAAAGTATGCGCACTGCTTGCAGCCCCGATGGTCTCTGTAGTGATGGGAACGGCCTGGAGCTCAAATGCCCTTTTACCAGCCGGGACTTTATGAAATTTAGGTTGGGGGGTTTTGAGGCTATTAAATCTGCATATATGGCACAAGTTCAATACAGTATGTGGGTCACCCGGAAGAATGCTTGGTATTTTGCTAACTACGATCCCCGAATGAAACGGGAAGGGTTGCACTACGTGGTCATTGAGCGGGATGAGAAATATATGGCCAGTTTTGACGAGATAGTTCCTGAGTTCATAGAAAAAATGGACGAAGCACTGGCAGAAATAGGGTTCGTATTCGGCGAGCAGTGGAGA

[0219] SEQ ID NO.7: Amino acid sequence of Redβ

[0220] MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFIALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESCTCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLAFGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLPLCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAASGGSPKKKRKV

[0221] SEQ ID NO.8: Redβ nucleotide sequence

[0222] ATGTCAACAGCCCTTGCTACTCTCGCTGGAAAGTTGGCCGAACGAGTCGGGATGGACTCTGTTGATCCACAAGAATTGATTACTACCCTTCGACAAACCGCCTTCAAGGGAGATGCAAGTGACGCTCAGTTTATCGCTTTGCTCATCGTTGCTAATCAGTACGGCCTTAATCCATGGACCAAGGAAATCTATGCTTTTCCCGATAAGCAGAACGGCATTGTTCCCGTAGTTGGTGTCGACGGTTGGAGTCGGATTATCAACGAAAACCAACAATTCGACGGAATGGACTTCGAACAAGATAACGAATCATGTACATGCAGGATTTATAGAAAGGACCGGAATCACCCAATCTGTGTGACCGAGTGGATGGATGAATGTCGCAGAGAACCTTTCAAAACCCGCGAGGGAAGAGAAATAACCGGTCCATGGCAATCCCACCCAAAGAGAATGCTGCGACATAAGGCCATGATACAGTGCGCCCGGCTGGCATTTGGGTTCGCAGGTATTTACGATAAGGATGAAGCCGAACGCATAGTCGAGAATACAGCCTACACCGCTGAGCGACAGCCAGAGAGAGATATAACACCAGTGAACGACGAAACAATGCAAGAGATAAATACTTTGCTGATTGCACTGGATAAAACATGGGATGACGATCTGCTGCCTCTCTGTAGTCAAATTTTCAGGCGGGATATTCGGGCTTCATCCGAGCTGACCCAGGCAGAAGCAGTAAAAGCTCTCGGCTTCTTGAAACAAAAGGCCGCAGAACAAAAGGTCGCCGCTTCTGGTGGTTCTCCCAAGAAGAAGAGGAAAGTC

[0223] SEQ ID NO.9: Amino acid sequence of Cas9

[0224]

[0225] SEQ ID NO.10: Cas9 nucleotide sequence

[0226]

[0227] SEQ ID NO.11: RED-CRISPR plasmid DNA sequence

[0228]

[0229] SEQ ID NO.12: XTEN-Redα-XTEN-Redβ-EGFP DNA sequence

[0230]

[0231] SEQ ID NO.13: DNA sequence of upstream primer 1

[0232] GCCAGGCAAAAAAGAAAAAGG

[0233] SEQ ID NO.14: DNA sequence of downstream primer 1

[0234] GATCAGCGAGCTCTAGTTAG

[0235] SEQ ID NO.16: DNA sequence of sgHSP90AA1 F

[0236] CACCGTAGACTAATCTCTGGCTGA

[0237] SEQ ID NO.17: DNA sequence of sgHSP90AA1 R

[0238] CATCTGATTAGAGACCGACTCAAA

[0239] SEQ ID NO.18: HSP90AA1-P2A-mCherry-T2A-EGFP homology repair template DNA sequence

[0240]

[0241] SEQ ID NO.19: DNA sequence of HSP90AA1 Left HA F

[0242] GGCAGAGGCTGATAAGAACGAC

[0243] SEQ ID NO.20: DNA sequence of HSP90AA1 Right HA R

[0244] GGAAAACAAGCCCTGTGGAGAG

[0245] SEQ ID NO.21: DNA sequence of sgActb F

[0246] CACCGCACCGCAAGTGCTTCTAGG

[0247] SEQ ID NO.22: DNA sequence of sgActb R

[0248] CGTGGCGTTCACGAAGATCCCAAA

[0249] SEQ ID NO.23: mActb-P2A-mKate homology repair template DNA sequence

[0250]

[0251] SEQ ID NO.24: DNA sequence of Actb Left HA F

[0252] TAAGGGGAATCCCAGCACC

[0253] SEQ ID NO.25: DNA sequence of Actb Right HA R

[0254] CCTCCCAGGGAGACCAAAG

[0255] SEQ ID NO.26: DNA sequence of sgGAPDH F

[0256] CACCGAGCCCCAGCAAGAGCACAAG

[0257] SEQ ID NO.27: DNA sequence of sgGAPDH R

[0258] CTCGGGGTCGTTCTCGTGTTCCAAA

[0259] SEQ ID NO.28: GAPDH-IRES-mCherry homologous repair template DNA sequence

[0260]

[0261] SEQ ID NO.29: DNA sequence of GAPDH Left HAF

[0262] GGCTGTGGGCAAGGTCATCC

[0263] SEQ ID NO.30: DNA sequence of GAPDH Right HA R

[0264] GCGAGCCCTGACTTCCTCCA

[0265] SEQ ID NO.31: DNA sequence of GAPDH 5'F

[0266] TCACTGCCACCCAGAAGAC

[0267] SEQ ID NO.32: DNA sequence of GAPDH 5'R

[0268] GGAACTGCTTCCTTCACGAC

[0269] SEQ ID NO33: DNA sequence of GAPDH 3'F

[0270] GACCACCTACAAGGCCAAG

[0271] SEQ ID NO.34: DNA sequence of GAPDH 3'R

[0272] TCAAGACCTCACAGCTGCG

[0273] SEQ ID NO.35: DNA sequence of GAPDH_NGS_PCR1 F

[0274] CCCTACACGACGCTCTTCCGATCTCCCTGACAACTCTTTTCATCTTC

[0275] SEQ ID NO.36: DNA sequence of GAPDH_NGS_PCR1 R

[0276] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAAGGGGTCTACATGGCAA

[0277] SEQ ID NO.37: DNA sequence of HSP90AA1_NGS_PCR1 F

[0278] CCCTACACGACGCTCTTCCGATCTGAAGATGACCCTACTGCTGAT

[0279] SEQ ID NO.38: DNA sequence of HSP90AA1_NGS_PCR1 R

[0280] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCCCCTTAAAGTAGTTGTCATGC

[0281] SEQ ID NO.39: GAPDH ssODN sequence

[0282] ATGGCCTCCAAGGAGTAAGACCCCTGGACCACCAGCCCCAGCAAGAGCACGTTTAAACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGT

[0283] SEQ ID NO.40: HSP90AA1 ssODN sequence

[0284] GAAGGAGATGACGACACATCACGCATGGAAGAAGTAGACTAATCTCTGGCGTTTAAACTGAGGGATGACTTACCTGTTCAGTACTCTACAATTCCTCTGATAATATAT

[0285] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A protein complex, which includes Cas9, flexible linker 1, Redα, flexible linker 2 and Redβ from N-terminus to C-terminus.

2. The protein complex according to claim 1, wherein (1) The flexible linkers 1 and 2 respectively include SGGSXTENSGGS, (GGGGS)5, (GGGGS)3-GSGGGS, preferably SGGSXTENSGGS; Preferably, the amino acid sequences of the flexible linkers 1 and 2 are respectively as shown in SEQ ID NO.3, preferably, the nucleotide sequences are as shown in SEQ ID NO.4; (2) The amino acid sequence of the Redα protein is shown in SEQ ID NO.5, and preferably the nucleotide sequence encoding it is shown in SEQ ID NO.6; (3) The amino acid sequence of the Redβ protein is shown in SEQ ID NO.7, and the nucleotide sequence encoding it is shown in SEQ ID NO.8; (4) The amino acid sequence of the Cas9 protein is shown in SEQ ID NO.9, and the nucleotide sequence encoding it is shown in SEQ ID NO.

10.

3. The protein complex according to claim 1, wherein The amino acid sequence of the protein complex is shown in SEQ ID NO.1, and optionally, the nucleotide sequence encoding it is shown in SEQ ID NO.

2.

4. A method for constructing the protein complex according to any one of claims 1 to 3, comprising the following steps: (1) Mammalian codon optimization of Redα and Redβ; (2) connecting the mammalian codon-optimized Redα and Redβ via flexible linker 2 to construct a Redα-flexible linker 2-Redβ fusion protein; (3) The prepared Redα-flexible linker 2-Redβ fusion protein is connected to Cas9 via the flexible linker 1 to form the Cas9-flexible linker 1-Redα-flexible linker 2-Redβ fusion protein, i.e., the protein complex.

5. A plasmid comprising a nucleotide sequence encoding the protein complex according to any one of claims 1 to 3.

6. The plasmid according to claim 5, wherein The DNA sequence of the plasmid is shown in SEQ ID NO.

11.

7. The plasmid according to claim 5, wherein The plasmid further comprises hU6, gRNA scaffold, CBh and T2A, and an element encoding a fluorescent protein; preferably, the plasmid comprises hU6, gRNA scaffold, CBh, a portion encoding a protein complex, T2A, and a portion encoding a fluorescent protein in order from 5' to 3'.

8. The plasmid according to claim 7, wherein The fluorescent proteins include EGFP, EBFP and mCherry, preferably EGFP and EBFP.

9. The method for constructing a plasmid according to claim 5, comprising the following steps: (1) Synthesizing a flexible linker 1-Redα-flexible linker 2-Redβ-EGFP DNA sequence, the sequence being shown in SEQ ID NO.12; (2) Amplify flexible linker 1-Redα-flexible linker 2-Redβ-EGFP by PCR; (3) The PX458 vector was linearized using a rapid restriction endonuclease, and the DNA sequence of flexible linker 1-Redα-flexible linker 2-Redβ-EGFP was inserted into the Cas9 sequence of the PX458 vector by homologous recombination to obtain; (4) Introducing the DNA sequence of PX458-flexible linker 1-Redα-flexible linker 2-Redβ-EGFP into Escherichia coli DH5α; (5) Extract, identify and obtain the recombinant plasmid, namely RED-CRISPR.

10. Use of the plasmid described in any one of claims 5 to 8 for knocking in long DNA sequences in gene editing.

11. A method for site-directed gene knock-in, comprising using the plasmid according to any one of claims 5 to 8.

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