Editing system for accurate knock-in of large-fragment gene and application of editing system

Abstract

The invention discloses an editing system for accurate knock-in of a large-fragment gene and application of the editing system, the editing system can express dCas9-FokI fusion protein and EBNA1 protein, and comprises an oriP element, a region for inserting a double sgRNA coding sequence and a donor template sequence for knock-in of the large-fragment gene. According to the editing system, cut FokI nuclease is generated in a dimerization form, a recognition sequence of sgRNA is not damaged, dCas9-FokI nuclease and a donor template are continuously expressed by mediation of an EBNA1 / oriP attachment, a target sequence in which accurate large-fragment gene knock-in does not occur is continuously cut, DSB frequency is generated as many as possible, and meanwhile, the target sequence in which accurate large-fragment gene knock-in does not occur is continuously cut. Further, HDR is generated as much as possible, and the purpose of improving knock-in of large-fragment genes is achieved. By means of the editing system, large-fragment gene knock-in can be efficiently carried out, and a feasible method and a powerful gene editing tool are provided for gene function research.

Description

Technical Field

[0001] This application relates to the field of gene editing technology, specifically to an editing system for precise knock-in of large gene fragments and its applications. Background Technology

[0002] In recent years, gene editing technology has developed rapidly, especially CRISPR / Cas9-based systems and their derivative tools. Due to their flexibility and high efficiency, they have been widely applied in various fields of life sciences. The CRISPR / Cas system originates from the adaptive immune mechanism developed by bacteria and archaea during evolution, used to defend against viral and exogenous DNA invasion. The CRISPR / Cas9 gene editing system consists of a Cas9 protein with endonuclease activity and a single-stranded guide RNA (sgRNA). The sgRNA binds to the Cas9 protein and guides it to target the Cas9 site for cleavage. The two cleavage domains of Cas9, RuvC and HNH, cleave the DNA double strand, resulting in double-strand breaks (DSBs). The cell then initiates two main repair mechanisms: non-homologous endjoining (NHEJ) and homology-directed repair (HDR). NHEJ is a mismatch repair mechanism; DNA double strand repair via NHEJ results in random insertions and deletions (indels). HDR is a precise repair mechanism in which a foreign gene fragment from a homologous donor integrates into the target site via homologous recombination in the presence of a homologous donor. Currently, the CRISPR / Cas9 gene editing system has demonstrated high efficiency and accuracy in editing single-base changes or small fragments (<100 bp) of genes. However, for the need to knock in large fragments (≥100 bp) of genes, the editing efficiency of existing tools remains significantly insufficient, becoming a bottleneck limiting its further application. Furthermore, existing systems have limitations in the sustained and stable expression of the edited element, affecting the improvement of HDR efficiency.

[0003] To improve the specificity of gene editing and reduce off-target effects, various strategies have been proposed. For example, the dCas9-FokI system (also known as fCas9) utilizes a pair of sgRNAs to guide two catalytically inactivated dCas9 proteins (D10A and H840A mutants) to fusion with FokI endonucleases, targeting different sites on the DNA double strand. DNA can only be cleaved to produce DSBs when the two FokI monomers form a dimer within a suitable interval (e.g., 15-25 bp). This type of system relies on the synergistic effect of the two sgRNAs, which not only improves targeting specificity but also significantly reduces the risk of off-target effects by 50-1500 times, showing a clear advantage over traditional wild-type Cas9. However, there are no reports on constructing large-fragment gene knock-in vectors using the dCas9-FokI system.

[0004] Therefore, there is an urgent need to develop a novel gene editing system that can support efficient knock-in of large gene fragments, while also having low off-target effects and sustained expression capabilities, in order to promote the development of this technology in cutting-edge research and clinical applications. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide an editing system for precise knock-in of large gene fragments and its application. This editing system is a replicative dCas9-FokI editing system, capable of efficient knock-in of large gene fragments while also exhibiting low off-target effects and sustained expression capability.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] In a first aspect, this application provides an editing system for precise knock-in of large gene fragments, the editing system being capable of expressing the following (A1), (A2), and including the following (A3)~(A5):

[0008] (A1) dCas9-FokI fusion protein, the nucleotide sequence of the gene encoding which is shown in SEQ ID NO.1;

[0009] (A2) EBNA1 protein, the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.2;

[0010] (A3)oriP element, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0011] (A4) The region used to insert the coding sequence of the double sgRNA, which contains the insertion site and the promoter located upstream of the insertion site;

[0012] (A5) A donor template sequence for large gene knock-in, the donor template sequence comprising the left homologous arm (Left HA) of the knock-in site, the right homologous arm (Right HA) of the knock-in site, and the large fragment to be knocked in.

[0013] This editing system is a replicative dCas9-FokI editing system, which is a circular expression vector. The system is an attachment plasmid based on oriP / EBNA1, integrating the dCas9 protein with a non-specific FokI restriction endonuclease to achieve efficient and specific DNA double-strand breaks (DSBs), while simultaneously carrying a homologous recombination repair template (HDR template). This replicative dCas9-FokI editing system addresses three major challenges faced by the traditional CRISPR-Cas9 system in large-fragment gene knock-in: (1) low editing efficiency; (2) plasmid loss in dividing cells leading to transient expression; and (3) difficulty in delivering large-fragment HDR templates.

[0014] The functions of each component in this editing system are described below:

[0015] (A1) dCas9-FokI fusion protein

[0016] dCas9 is an abbreviation for "dead Cas9." Its key feature is that point mutations (D10A and H840A for SpCas9) inactivate the original DNA-cutting activity of Cas9, while retaining its ability to precisely bind to specific DNA sequences guided by sgRNA. FokI is a restriction endonuclease. FokI monomers form a dimer between two sgRNAs, generating cleavage activity and producing DSBs by cutting the DNA double strand. The dCas9-FokI fusion protein combines the precise localization ability of dCas9 with the cleavage ability of FokI. Only when the binding sites of the dCas9-FokI fusion protein and DNA are appropriately spaced (typically 15-25 bp apart) and oriented oppositely can the two FokI domains aggregate into an active dimer, performing cleavage in the middle region.

[0017] (A2) EBNA1 protein and (A3) oriP element

[0018] The EBNA1 protein specifically binds to the oriP element. This binding initiates replication, maintaining the plasmid copy number at 5-100 per cell during the S phase of cell division. Simultaneously, it physically anchors the plasmid carrying oriP to the host cell's chromosome. Thus, when the cell divides, the plasmid can be efficiently distributed to the two daughter cells like a miniature chromosome, thereby preventing dilution and loss of the plasmid during cell proliferation.

[0019] (A4) Region used for inserting double sgRNA coding sequences

[0020] Dual sgRNAs are essential for activating the dCas9-FokI system. By designing two distinct sgRNAs, each targeting a suitable upstream or downstream sequence from the target gene site, a pair of sgRNAs can simultaneously recruit two inactive dCas9 proteins. This allows the two FokI nucleases to form a dimer, cleaving the target DNA without damaging the sgRNAs, thus preserving dCas9's recognition of the target sequence. The promoter region used for inserting the dual sgRNA coding sequence ensures efficient transcription of both sgRNAs.

[0021] (A5) Donor template sequence for large gene knock-in

[0022] The (A5) element comprises the left homologous arm (Left HA) of the knock-in site, the right homologous arm (Right HA) of the knock-in site, and the large fragment to be knocked in between (such as a reporter gene, therapeutic gene, etc.). The homologous arm is a sequence that is completely homologous to the upstream and downstream genomic sequences of the cleavage site, serving as a reference sequence for HDR repair. The fragment to be knocked in is the sequence that needs to be integrated into the genome. (A5) serves as a repair template. When dCas9-FokI causes a DSB in the genome, the cell initiates the repair mechanism. If the HDR pathway is followed, the cell uses this donor template provided on the plasmid as a reference to copy the entire "left homologous arm-fragment to be knocked in-right homologous arm" structure to the genomic cleavage site, thereby achieving precise knock-in of a large fragment.

[0023] In some implementations, the promoter upstream of the insertion site in the region used to insert the dual sgRNA coding sequence is the U6 promoter.

[0024] In some embodiments, the replicative dCas9-FokI editing system further includes a drug screening gene and / or a reporter gene. The drug screening gene and the reporter gene reside in the same expression cassette, linked by a self-cleaving oligopeptide-coding gene. This self-cleaving oligopeptide can be a 2A self-cleaving oligopeptide derived from a viral genome, such as the F2A peptide from foot-and-mouth disease virus (FMDV), the E2A peptide from equine rhinitis virus type A (ERAV), the T2A peptide from thoseoasigna virus, the P2A peptide from porcine swine varicella-virus-1 (PTV-1), or a 2A peptide from Theylvirus or a myocarditis virus.

[0025] In some preferred embodiments, the drug screening gene is a puromycin resistance gene, and the reporter gene is an mcherry reporter gene.

[0026] In some preferred embodiments, the full nucleotide sequence of the replicative dCas9-FokI editing system is shown in SEQ ID NO. 6 + SEQ ID NO. 7.

[0027] The vector shown in SEQ ID NO.6+SEQ ID NO.7 is named “epi-dCas9-FokI-GAPDH-double sgRNA-KI”. It is formed by sequentially inserting a Cas9 fragment, a double sgRNA fragment, sequences containing left and right homologous arms, and the fragment to be knocked in (P2A-CopGFP) into a universal vector (named “epi-dCas9-FokI-sgRNA”, the nucleotide sequence of which is shown in SEQ ID NO.4).

[0028] Secondly, this application provides recombinant bacteria or transgenic cell lines comprising the editing system described in the first aspect.

[0029] Thirdly, this application provides the application of the editing system described in the first aspect in any of the following:

[0030] (1) Knocking in large gene fragments into organisms or biological cells;

[0031] (2) Prepare products for large-fragment gene knock-in of organisms or biological cells.

[0032] Fourthly, this application provides a method for gene editing of an organism or biological cells using the editing system described in the first aspect, comprising the following steps:

[0033] To construct the editing system described in the first aspect, a scaffold sequence is added to the 3' end of the first sgRNA coding sequence targeting the target gene, and a U6 promoter coding sequence is added to the 5' end of the second sgRNA. The two sgRNAs are then linked end-to-end and inserted into the insertion site of the aforementioned vector.

[0034] The recombinant vector is introduced into an organism or biological cell to achieve large-fragment gene knock-in of different target genes in the genome of the organism or biological cell.

[0035] The constructed replicative dCas9-FokI editing system simultaneously expresses the nuclease, sgRNA, and large knock-in template used to generate DSBs, and enriches and continuously expresses them, resulting in the same probability of HDR repair after each DSB generation. Figure 1This diagram illustrates the principle of large-fragment gene knock-in using a replicative dCas9-FokI editing system vector. As shown, paired sgRNAs recognize the target site and bind to dCas9. The two FokI nucleases fused with dCas9 form a dimer to cleave the target site without disrupting the sgRNA's recognition sequence. The dCas9-FokI-KI system is then continuously expressed via an attachment element, further editing the target sequence of indels generated solely by NHEJ. This maximizes the number of DSBs while simultaneously and continuously expressing a donor template carrying a 400bp homologous arm within the same vector, maximizing HDR (High-Density Regulator) until the desired editing is achieved.

[0036] During the cleavage and repair process, the indels generated by NHEJ do not disrupt the recognition sequence of dCas9's sgRNA. Therefore, the continuously expressed dCas9-FokI nuclease can continue to cleave these indel-generating sequences as well as sequences that have not been cleaved. The resulting DSB can then use the continuously expressed large fragment knock-in donor template to perform precise large fragment knock-in. Sequences that have undergone precise large fragment knock-in cannot be recognized by dCas9 again. Sequences that only undergo NHEJ and those that have not been cleaved will undergo the next cleavage, ultimately improving the efficiency of precise large fragment knock-in.

[0037] The effect of episome-based sustained expression systems on improving gene knockout efficiency (via the NHEJ pathway) is predictable. This is because knockout does not involve homologous recombination, so it does not damage the integrity of the vector or interfere with replication; its effect is a direct and linear manifestation of sustained cleavage activity. However, integrating a donor template into a replicative vector and performing a successful HDR event means that the vector itself, as a template sequence, is cleaved and integrated into the genome. This could very likely lead to the destruction or loss of the vector structure, thus terminating its "persistence." In contrast, the replicative dCas9-FokI editing system (i.e., epi-dCas9-FokI-GAPDH-double sgRNA-KI vector) provided in this application does not damage the episome vector even after genome knock-in, maintaining its replication and sustained expression functions. This contradicts the theoretical risk (i.e., donor integration leading to vector interruption), demonstrating that this vector design successfully overcomes a potential technical obstacle.

[0038] Compared with existing technologies, it has at least the following beneficial effects:

[0039] This application provides a replicative dCas9-FokI editing system that utilizes the EBNA1 / oriP attachment to continuously cleave DNA sequences recognized by undisturbed sgRNAs, thereby continuously generating DSBs. Simultaneously, a donor template sequence for large fragment knock-in is continuously expressed in the same vector, increasing the likelihood of large fragment HDRs and ultimately improving the efficiency of large fragment gene knock-in. This replicative dCas9-FokI editing system provides a new method for gene editing and offers new technologies and materials for pig genetic improvement and disease-resistant breeding. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the principle of large-fragment gene knock-in using the replicative dCas9-FokI editing system provided in this application embodiment.

[0041] Figure 2 The epi-dCas9-FokI-sgRNA vector element map provided in the embodiments of this application.

[0042] Figure 3 The epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector element map provided in the embodiments of this application.

[0043] Figure 4 The image shows the epi-dCas9-FokI-GAPDH-double sgRNA-KI vector element map provided in the embodiments of this application.

[0044] Figure 5 The image shows the dCas9-FokI-GAPDH-double sgRNA-KI vector element map provided in the embodiments of this application.

[0045] Figure 6 This is a diagram showing the target knockout efficiency of different pairs of sgRNAs of the GAPDH gene in the examples.

[0046] Figure 7 The images show large fragment knock-in fluorescence patterns of the GAPDH gene epi-dCas9-FokI-GAPDH-double sgRNA-KI vector, dCas9-FokI-GAPDH-double sgRNA-KI vector, and epi-Cas9-GAPDH-sgRNA-KI vector in the examples.

[0047] Figure 8 The diagram shows the large fragment knock-in efficiency of the GAPDH gene epi-dCas9-FokI-GAPDH-double sgRNA-KI vector, epi-Cas9-GAPDH-sgRNA-KI vector, and dCas9-FokI-GAPDH-double sgRNA-KI vector in the examples. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0049] The materials used in the following embodiments are not limited to those listed below, and other similar materials may be used instead. Unless otherwise specified, the instruments shall be used under conventional conditions or as recommended by the manufacturer. Those skilled in the art should have relevant knowledge of the use of conventional materials and instruments.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this application pertains. Before a detailed description of this application, the following definitions are provided to better understand it.

[0051] To better understand this teaching and without limiting its scope, all figures and other numerical values ​​used in the specification and claims to express quantities, percentages, or proportions should, in all cases, be understood to be modified by the term "about." Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary depending on the desired properties sought. At a minimum, each numerical parameter should be interpreted based at least on the reported significant figures and by applying common rounding techniques.

[0052] Those skilled in the art will understand that, unless otherwise stated, the terms "the," "the," and "the foregoing" used in this application may also include plural forms. In the context of this application, many embodiments use the expressions "comprising" or "including." The expressions "comprising" or "including" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, and method steps specifically listed after the expression, but also other elements, components, parts, and method steps. However, in this document, the expressions "comprising" or "including" can also be understood as closed-ended expressions in certain situations, indicating that they only include the elements, components, parts, and method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of...".

[0053] Example 1: Construction of a replica dCas9-FokI editing system carrier

[0054] This embodiment provides a method for constructing a copy-type dCas9-FokI editing system based on EBNA1 and oriP elements. The specific steps are as follows:

[0055] (1) The plasmid pFokI-dCas9 (Miaoling, #P5109) was digested with the restriction endonuclease MluI-HF (NEB, #R3198S) to generate a linearized pFokI-dCas9 vector as the cloning vector backbone.

[0056] (2) Using pCEP4 (invitrogen, v044-50) plasmid as a template, specific amplification primers oriP-F / R and EBNA1-F / R were designed and synthesized. Corresponding 20 bp homologous arm sequences were added to the 5' end of the primers. At the same time, an Asc1 recognition site was added to the EBNA1-R primer to facilitate subsequent vector modification. After amplification using 2×A8 FastHiFi PCR masterMix (Adley, PC8201) high-fidelity enzyme, the oriP and EBNA1 fragments were obtained. The corresponding primer sequences are shown in Table 1.

[0057] Table 1 Primer sequences for oriP and EBNA1 fragments

[0058]

[0059] The PCR reaction system for the oriP fragment is shown in Table 2:

[0060] Table 2. PCR reaction system for oriP fragments

[0061]

[0062] The PCR amplification procedure is as follows:

[0063] 98 ℃ for 3 min; 98 ℃ for 10 s, 58 ℃ for 15 s, 72 ℃ for 100 s, 30 cycles; 72 ℃ for 3 min, 8 ℃ for 2 min.

[0064] The nucleotide sequence of the PCR amplification product fragment is shown in SEQ ID NO.14. The product fragment is 1943 bp in length and includes oriP and the corresponding homologous arm fragment (hereinafter referred to as "oriP fragment").

[0065] The PCR reaction system for the EBNA1 fragment is shown in Table 3:

[0066] Table 3 PCR reaction system for EBNA1 fragment

[0067]

[0068] The PCR amplification procedure is as follows:

[0069] 98 ℃ for 3 min; 98 ℃ for 10 s, 58 ℃ for 15 s, 72 ℃ for 100 s, 30 cycles; 72 ℃ for 3 min, 8 ℃ for 2 min.

[0070] The PCR amplification product fragment, namely the EBNA1 fragment, has the nucleotide sequence shown in SEQ ID NO.15.

[0071] (3) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (Vazyme, C113-01) recombinant cloning kit, the linearized pFokI-dCas9 vector from step (1) and the oriP and EBNA1 fragments amplified in step (2) were mixed and reacted at 37 °C for 30 min. The product after reaction was transformed into chemically competent Fast-T1 (vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing and identification of positive clones. The successfully constructed vector was named pFokI-dCas9-pCEP4.

[0072] (4) The pFokI-dCas9-pCEP4 plasmid was digested with Asc1 (NEB, #R0558S) to generate a linearized pFokI-dCas9-pCEP4 vector as the cloning vector backbone.

[0073] (5) Using pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W plasmid (Addgene, #67975) as a template, specific primers U6-F / R and PGK-F / R were designed, with corresponding 20 bp homologous arm sequences added. At the same time, the Asc1 restriction site was retained on the U6-F primer. Using plenti-CRISPRV2-GFP plasmid as a template, specific primers scaffold-F / R were designed, with corresponding 20 bp homologous arms added to the 5' end of the primer. At the same time, the Aar1 restriction recognition site was added to the scaffold-F primer for later restriction enzyme digestion and ligation of sgRNA. The U6 promoter fragment, scaffold fragment and PGK fragment were obtained by high-fidelity enzyme amplification using KAPA HiFi HotStart ReadyMix PCR Kit (Roche, KK2602). The primer sequences are shown in Table 4.

[0074] Table 4 Primer sequences for the U6 promoter fragment, scaffold fragment, and PGK fragment.

[0075]

[0076] The amplification system and amplification program for the above PCR are shown in Tables 5 and 6, respectively:

[0077] Table 5 PCR amplification system

[0078]

[0079] Table 6 PCR Amplification Procedure

[0080]

[0081] (6) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (vazyme, C113-01) recombinant cloning kit, the linearized pFokI-dCas9-pCEP4 vector in step (4) and the U6 promoter fragment, scaffold fragment and PGK fragment obtained in step (5) were mixed and reacted at 37 ℃ for 30 min. The product after reaction was transformed into chemically competent Fast-T1 (Vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing to identify positive clones. The successfully constructed vector was named epi-dCas9-FokI-sgRNA intermediate vector.

[0082] (7) The epi-dCas9-FokI-sgRNA intermediate vector was digested with XhoI (NEB, R0146) to obtain a linearized epi-dCas9-FokI-sgRNA intermediate vector; the specific primer PuroR-F / R was designed using pKLV2-U6gRNA5(BbsI)-PGKpuro2AZsG-W plasmid (Addgene, #67975) as a template; the specific primer SV40 ployA-F / R was designed using plenti-CRISPRV2-GFP plasmid (Addgene, #86153) as a template; and the specific primer mcherry-F / R was designed using pLV3-CMV-HNRNPA1(human)-3×FLAG-mCherry-Puro plasmid (Miaoling, P49072) as a template, with a stop codon added at the 3' end. The PuroR, SV40 ployA, and mcherry fragments were obtained by high-fidelity enzyme amplification using the KAPA HiFi HotStart ReadyMix PCR Kit. The primer sequences are shown in Table 7.

[0083] Table 7 Primers for amplification of PuroR fragment, SV40 ployA fragment, and mcherry fragment

[0084]

[0085] (8) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (vazyme, C113-01), the linearized epi-dCas9-FokI-sgRNA intermediate vector from step (7) and the PuroR fragment, SV40 ployA fragment, and mcherry fragment obtained from step (7) were mixed and reacted at 37 ℃ for 30 min. The product was then transformed into chemically competent Fast-T1 cells (Vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing identification of positive clones. The successfully constructed vector was named epi-dCas9-FokI-sgRNA vector, and its sequence is shown in SEQ ID NO.4. The map of epi-dCas9-FokI-sgRNA vector is shown in [image missing]. Figure 2 .

[0086] (9) The epi-dCas9-Fok1-sgRNA vector plasmid obtained in step (8) was obtained by digesting it with KpnI (NEB, #R3142S) and PmeI (NEB, #R0560S) to obtain a linearized epi-dCas9-Fok1-sgRNA vector plasmid; using plenti-CRISPRV2-GFP plasmid (Addgene, #86153) as a template, specific primers Cas9-F / R were designed, and corresponding homologous arm sequences were added. At the same time, a stop codon was added to the 3' end of the fragment; the Cas9 fragment was obtained by high-fidelity enzyme amplification using KAPA HiFi HotStart ReadyMix PCR Kit. The primer sequences are shown in Table 8 below:

[0087] Table 8 Primer sequences for Cas9 fragment amplification

[0088]

[0089] (10) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (vazyme, C113-01) recombinant cloning kit, the linearized epi-dCas9-FokI-sgRNA vector in step (9) and the Cas9 fragment amplified in step (9) were mixed and reacted at 37 ℃ for 30 min. The product after reaction was transformed into chemically competent Fast-T1 (Vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing and identification of positive clones. The successfully constructed vector was named epi-Cas9-sgRNA vector.

[0090] (11) The epi-dCas9-FokI-sgRNA vector was digested with Aar1 (thermoscient, ER1581) to obtain a linearized epi-dCas9-FokI-sgRNA vector. Specific primers sgRNA-F / R were designed according to different pairs of sgRNAs (the pairs of sgRNA primers are shown in Table 9). A 20 bp homologous arm was added. Using the synthetic vector T-scaffold-U6 (sequence shown in SEQ ID NO.9) as a template, the double sgRNA fragment was amplified using the KAPA HiFi Hot Start Ready Mix PCR Kit (Roche, KK2602) with high fidelity enzyme.

[0091] Table 9. GAPDH site sgRNA sequence

[0092]

[0093] (12) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (vazyme, C112-01), the linearized epi-dCas9-FokI-sgRNA vector from step (11) and the double sgRNA fragment amplified in step (11) were mixed and reacted at 37 ℃ for 30 min. The product after reaction was transformed into chemically competent Fast-T1 (Vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing and identification of positive clones. The successfully constructed vectors were named epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2, epi-dCas9-FokI-GAPDH-double sgRNA3+sgRNA4 and epi-dCas9-FokI-GAPDH-double sgRNA5+sgRNA6, respectively. The sequence of epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 is shown in SEQ ID. As shown in NO.5, the vector spectrum is shown below. Figure 3 .

[0094] (13) The epi-Cas9-sgRNA vector was digested with Aar1 (thermoscient, ER1581) to synthesize oligonucleotide sequences with complementary sticky ends. The primer sequences are shown in Table 10. Take 5 μL of each, vortex and centrifuge to mix, and then place it in a PCR instrument for annealing. The annealing program is: 95 ℃ for 10 min, 65 ℃ for 30 min. Add 90 μL of sterile water to the annealed product for dilution.

[0095] Table 10 sgRNA annealing primers for GAPDH sites

[0096]

[0097] (14) Using a DNA Ligation Kit (Takara, 6023Q), the linearized epi-Cas9-sgRNA vector from step (13) was mixed with the GAPDH-sgRNA1 fragment and GAPDH-sgRNA2 fragment obtained by annealing and dilution in step (13), and the mixture was reacted at 16°C for 30 min. The reaction product was transformed into chemically competent Fast-T1 cells (Vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing and identification of positive clones. The successfully constructed vectors were named epi-Cas9-GAPDH-sgRNA1 vector and epi-Cas9-GAPDH-sgRNA2 vector.

[0098] (15) Using Asc1 (NEB, #R0558S), the epi-dCas9-FokI-GAPDH-double sgRNA vector, epi-Cas9-GAPDH-sgRNA1 vector, and epi-Cas9-GAPDH-sgRNA2 vector plasmids were digested to generate linearized epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector and epi-Cas9-GAPDH-sgRNA2 vector as cloning vector backbones; using P2A-copGFP(+HA) plasmid as template, specific primers P2A-CopGFP-F / R were designed, and corresponding 20bp homologous sequences were added; using HEK293T genomic DNA as template, specific primers Left HA-F / R and Right HA-F / R were designed, and corresponding homologous arms were added to the 5' end of the primers; using KAPA HiFi Hot Start Ready Mix PCR Kit high-fidelity enzyme amplification, the left homologous arm (Left) of the knock-in site was obtained. The primer sequences for the knock-in site are shown in Table 11. The knock-in site contains the HA fragment, the right homologous arm (Right HA) fragment, and the P2A-CopGFP knock-in fragment.

[0099] Table 11 Primers for Knock-in Amplification Fragments

[0100]

[0101] (16) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (vazyme, C113-01), the linearized epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector and epi-Cas9-GAPDH-sgRNA2 vector, as well as the left homologous arm (Left HA) fragment, right homologous arm (Right HA) fragment and P2A-CopGFP knock-in fragment amplified in step (15) were mixed and reacted at 37 ℃ for 30 min. The reaction product was transformed into chemically competent Fast-T1 (Vazyme, C505-02). Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing identification of positive clones. The successfully constructed vector was named epi-dCas9-FokI-GAPDH-double sgRNA-KI vector (17519 bp), and its sequence is as shown in SEQ ID NO.6+SEQ ID. Sequence NO.7 (the sequence of the epi-dCas9-FokI-GAPDH-double sgRNA-KI vector exceeds the sequence length limit of the patent submission system, therefore it is split into two sequences. SEQ ID NO.6 is named "pre-epi-dCas9-FokI-GAPDH-double sgRNA-KI vector" in the sequence file, and SEQ ID NO.7 is named "post-epi-dCas9-FokI-GAPDH-double sgRNA-KI vector" in the sequence file) is shown below. The vector map can be found in [link to vector map]. Figure 4 The epi-Cas9-GAPDH-sgRNA2-KI vector was also obtained.

[0102] (17) The epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector was digested with SgrDI (thermoscient, ER2031) and AscI (thermoscient, ER1892). Primers were designed to ligate the nicks. The ligation primers are shown in Table 12.

[0103] Table 12 Ligation Primer Sequences

[0104]

[0105] After centrifuging the synthesized oligonucleotide powder containing the ligation primer sequences, it was dissolved and diluted with ddH2O to a final concentration of 10 µM. Then, 5 µL of each of the corresponding upstream and downstream sgRNA primers were added to a PCR tube, vortexed, briefly centrifuged, and annealed to form double-stranded oligos. The annealing program was 95 °C for 10 min; 65 °C for 30 min. Using a DNA Ligation Kit (Takara, 6023Q), the annealed product was reacted in a metal bath at 16 °C for 1 h and ligated into the epi-dCas9-FokI-GAPDH-double sgRNA vector digested with SgrDI and AscI to obtain the control expression vector dCas9-FokI-GAPDH-double sgRNA1+sgRNA2.

[0106] (18) The dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector plasmid was digested with Asc1 (NEB, #R0558S) to generate a linearized dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector as the cloning vector backbone; the specific primers P2A-copGFP(+HA) plasmid were designed using P2A-CopGFP-F / R as template, and corresponding 20 bp homologous sequences were added; the specific primers Left HA-F / R and Right HA-F / R2 were designed using HEK293T genomic DNA as template, and corresponding homologous arms were added to the 5' end of the primers; the left homologous arm (Left HA) fragment, the right homologous arm (Right HA2) fragment, and the P2A-CopGFP knock-in fragment were obtained by high-fidelity enzyme amplification using KAPA HiFi Hot Start Ready Mix PCR Kit. The corresponding primer sequences are shown in Table 13.

[0107] Table 13 Primers for Knock-in Amplification Fragments

[0108]

[0109] (19) According to the instructions of the ClonExpressMultiS One Step Cloning Kit (vazyme, C113-01) recombinant cloning kit, the linearized dCas9-FokI-GAPDH-double sgRNA1+sgRNA2 vector from step (18) was amplified by mixing the left homologous arm (Left HA) fragment, the right homologous arm (Right HA2) fragment, and the P2A-CopGFP knock-in fragment. The mixture was then incubated at 37 ℃ for 30 min. The resulting product was transformed into chemically competent Fast-T1 (Vazyme, C505-02) cells. Single clones were picked and sent to Wuhan Aoke Dingsheng Biotechnology Co., Ltd. for sequencing identification of positive clones. The successfully constructed vector was named dCas9-FokI-GAPDH-double sgRNA-KI vector. The diagram is shown in [reference needed]. Figure 5 Its sequence is shown in SEQ ID NO.8.

[0110] Example 2: Application of epi-dCas9-FokI and epi-Cas9 editing systems mediated by attachment elements in HEK293T cells.

[0111] This embodiment verifies the application of epi-dCas9-FokI and the epi-Cas9 editing system in gene editing of HEK293T cells. The specific method is as follows:

[0112] (1) One day before transfection, healthy HEK293T cells (Pronosai, CL-0005) were plated in 6-well plates and cultured in fresh medium until the cell confluence was 80%~90% before transfection.

[0113] (2) Transfect according to the polyplus JetPrime transfection instructions. Transfect each six-well plate cell with 2.5 μg of the epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2, epi-dCas9-FokI-GAPDH-double sgRNA3+sgRNA4 and epi-dCas9-FokI-GAPDH-double sgRNA5+sgRNA6 vector plasmids (different paired sgRNAs) from step (12) of Example 1 or the epi-Cas9-GAPDH-sgRNA1 and epi-Cas9-GAPDH-sgRNA2 vector plasmids from step (13) of Example 1. Use 2% FBS / DMEM antibiotic-free medium for transfection, and replace it with 10% FBS / DMEM complete medium 4-6 h after transfection.

[0114] (3) 24 h after transfection, the expression of mcherry fluorescent protein was observed by fluorescence microscopy to test the transfection efficiency. Cells were then cultured in 10% FBS medium containing 2 μg / mL Puromycin to enrich successfully transfected cells and apply pressure to the attachment elements to prevent plasmid loss. On day 3, cells with different paired sgRNAs were collected, and specific primers were designed using the NCBI online website to amplify the editing sites. The primers used were GAPDH-KO-F / R, and the primer sequences are shown in Table 14.

[0115] Table 14 GAPDH Primer Sequences

[0116]

[0117] The cellular genome was amplified by PCR using the KAPA HiFi Hot Start Ready Mix PCR Kit. The amplification system and procedure are described in Example 1. The PCR amplification products were purified and sent to Wuhan Synthego Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were evaluated for efficiency using the online website https: / / ice.synthego.com / # / , and the knockout efficiency was determined through comparative analysis.

[0118] Three vectors with different pairs of sgRNAs, namely epi-dCas9-FokI-GAPDH-double sgRNA1+sgRNA2, epi-dCas9-FokI-GAPDH-double sgRNA3+sgRNA4, and epi-dCas9-FokI-GAPDH-double sgRNA5+sgRNA6, or two vectors with two different sgRNAs, epi-Cas9-GAPDH-sgRNA1 and epi-Cas9-GAPDH-sgRNA2, were designed and enriched with Puromycin for 3 days. The Sanger sequencing results were then analyzed using Synthego online software.

[0119] Figure 6 The diagram shows the target knockout efficiency of different pairs of sgRNAs for the GAPDH gene. The results show that the gene knockout efficiency of GAPDH-dsgRNA-1+2 is the highest, at 47%. Therefore, the GAPDH-dsgRNA-1+sgRNA-2 pair of sgRNAs was selected to construct the epi-dCas9-FokI-GAPDH-dsgRNA-KI system to achieve precise gene knock-in. The epi-Cas9 system was constructed using GAPDH-sgRNA2 to achieve precise gene knock-in.

[0120] Example 3: Application of the dCas9-FokI-KI and Cas9-KI editing system mediated by attachment elements in HEK293T cells.

[0121] This embodiment verifies the gene knock-in effect of the epi-dCas9-FokI-GAPDH-double sgRNA-KI and epi-Cas9-GAPDH-sgRNA-KI editing systems in HEK293T cells. The specific method is as follows:

[0122] (1) Fluorescence microscopy observation: 24 h after the editing system was transfected into HEK293T cells, HEK293T cells were enriched with 2 μg / mL puromycin medium. 3 days after transfection, the dCas9-FokI-GAPDH-double sgRNA-KI plasmid was cultured in normal complete medium for 15 days. The epi-dCas9-FokI-GAPDH-double sgRNA-KI and epi-Cas9-GAPDH-sgRNA-KI plasmids were continuously cultured in 2 μg / mL puromycin medium for 15 days. During the culture, the luminescence of cell samples was observed under an inverted fluorescence microscope on day 0, day 3, day 6, day 10 and day 15 to evaluate the long fragment knock-in efficiency.

[0123] Figure 7 The image shows a comparison of cellular fluorescence after large gene knock-in using the epi-dCas9-FokI-GAPDH-double sgRNA-KI, epi-Cas9-GAPDH-sgRNA-KI, and dCas9-FokI-GAPDH-double sgRNA-KI systems. The image shows that the number of cells expressing green fluorescence in the epi-dCas9-FokI-KI system increases with editing time, and the presence of red fluorescent protein indicating the presence of the replication plasmid further demonstrates the stable and continuous expression of the plasmid mediated by the EBNA1 / oriP attachome element. In contrast, the dCas9-FokI-KI system shows fewer cells expressing green fluorescent protein, and the number of cells expressing green fluorescent protein tends to decrease over time due to the inability of the transfected plasmid to maintain continuous expression.

[0124] (2) Flow cytometry analysis: HEK293T cells were transfected with the epi-dCas9-FokI-GAPDH-double sgRNA-KI, epi-Cas9-GAPDH-sgRNA-KI, and dCas9-FokI-GAPDH-double sgRNA-KI editing systems. 24 h after transfection, HEK293T cells were enriched with 2 μg / mL puromycin medium. Three days after transfection, conventional plasmids were cultured in normal complete medium for 15 days, while replicative plasmids were continuously cultured in 2 μg / mL puromycin medium for 15 days. Cell samples were collected on days 0, 3, 6, 10, and 15 to evaluate the long fragment knock-in efficiency. In this study, full-length green fluorescent protein (CopGFP) was knocked in, and the FTIC value of the cells was detected using a Beckman CytoFLEX flow cytometer.

[0125] Figure 8 The figure shows a comparison of the efficiency of long-fragment gene knock-in using the epi-dCas9-FokI-GAPDH-double sgRNA-KI, epi-Cas9-GAPDH-sgRNA-KI, and dCas9-FokI-GAPDH-double sgRNA-KI editing systems. Figure A compares the efficiency of epi-dCas9-FokI-GAPDH-double sgRNA-KI with that of dCas9-FokI-GAPDH-double sgRNA-KI, while figure B compares the efficiency of epi-dCas9-FokI-GAPDH-double sgRNA-KI with that of epi-Cas9-GAPDH-sgRNA-KI. As can be seen from the figure, the long-fragment knock-in efficiency of epi-dCas9-FokI-KI at 15 days of editing is 9.55%, which is significantly higher than the highest knock-in efficiency of 2.35% achievable with dCas9-FokI, and also significantly higher than the 6.24% knock-in efficiency of epi-Cas9 at 15 days of editing. The above results demonstrate that the epi-dCas9-FokI-KI system can maximize the chance of DSB generation by continuously and stably expressing dCas9-FokI protein, its corresponding sgRNA, and donor template, thereby increasing the frequency of HDR and improving the accuracy of long fragment knock-in. Furthermore, because the cleavage produced by the dimer FokI endonuclease in the dCas9-Fok1 system has sticky ends, which are more conducive to subsequent HDR repair, the knock-in efficiency of the epi-dCas9-FokI system is higher than that of the epi-Cas9 system.

[0126] In summary, by using the EBNA1 / oriP attachor-mediated dCas9-FokI-KI system, combining the EBNA1 / oriP attachor with dCas9-FokI, the efficiency of large fragment knock-in can be significantly improved. Since the cleavage of the dCas9-FokI system is generated by a dimerized form of the FokI nuclease, the indels generated by NHEJ do not disrupt the sgRNA recognition sequence. Furthermore, by continuously expressing the dCas9-FokI nuclease and the large fragment knock-in donor template using the EBNA1 / oriP attachor, the target sequence that has not undergone targeted editing can be continuously cleaved. This increases the number of DSBs generated and the likelihood of HDR, thereby improving the efficiency of precise large fragment knock-in.

[0127] The present application has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present application. The descriptions of the embodiments above are only for the purpose of helping to understand the present application and its core ideas. It should be noted that those skilled in the art can make several improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. An editing system for precise knock-in of large gene fragments, capable of expressing the following (A1), (A2), and including the following (A3)~(A5): (A1) dCas9-FokI fusion protein, the nucleotide sequence of the gene encoding which is shown in SEQ ID NO.1; (A2) EBNA1 protein, the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.2; (A3)oriP element, the nucleotide sequence of which is shown in SEQ ID NO.3; (A4) The region used to insert the coding sequence of the double sgRNA, which contains the insertion site and the promoter located upstream of the insertion site; (A5) A donor template sequence for large gene knock-in, the donor template sequence comprising the left homologous arm (Left HA) of the knock-in site, the right homologous arm (Right HA) of the knock-in site, and the large fragment to be knocked in.

2. The editing system according to claim 1, wherein it is a ring-shaped expression carrier.

3. In the editing system according to claim 1, the promoter located upstream of the insertion site in the region for inserting the double sgRNA coding sequence is the U6 promoter.

4. The editing system according to claim 1, further comprising a drug screening gene and / or a reporter gene.

5. The editing system according to claim 4, wherein the drug screening gene is a puromycin resistance gene and the reporter gene is an mcherry reporter gene.

6. The editing system according to claim 1, wherein the full nucleotide sequence is shown in SEQ ID NO.6+SEQ ID NO.

7.

7. A recombinant bacterial or transgenic cell line comprising the editing system described in any one of claims 1 to 6.

8. The application of the editing system according to any one of claims 1 to 6 in any of the following: (1) Knocking in large gene fragments into organisms or biological cells; (2) Prepare products for large-fragment gene knock-in of organisms or biological cells.

9. A method for gene editing in an organism or its cells, comprising the following steps: Construct the editing system according to any one of claims 1 to 6, wherein a scaffold sequence is added to the 3' end of the coding sequence of the first sgRNA targeting the target gene, a sequence coding for the U6 promoter is added to the 5' end of the second sgRNA, and the two sgRNAs are joined end to end and then inserted into the insertion site of the above vector. The recombinant vector is introduced into an organism or biological cell to achieve large-fragment gene knock-in of different target genes in the genome of the organism or biological cell.

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