A PBase protein, fusion protein, nucleic acid and gene integration system and application

Abstract

The present invention provides a PBase protein, a fusion protein, a nucleic acid, a gene integration system, a gene delivery system and an application. The PBase protein retains the ability to bind to the ITR sequence of the Piggybac transposon and cut to separate the Piggybac transposon from the donor plasmid in a linear manner, and the PBase protein has a lower ability to bind to the DNA double strand within 10 bp upstream and downstream of "TTAA". When the PBase protein is connected with a site-specific DNA nuclease to prepare a fusion protein, the PBase protein can efficiently integrate a long fragment of exogenous genes at the double-strand break generated by the site-specific DNA nuclease, thereby achieving the effect of site-specific insertion of long fragment genes.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing, and specifically relates to a PBase protein, a fusion protein, a nucleic acid and a gene integration system and applications. Background Art

[0002] The loss of protein function caused by endogenous gene mutations causes many human diseases. One of the main strategies for current gene therapy is to introduce a foreign gene (gene of interest, GOI) with the correct sequence and stably express it to compensate for the loss of endogenous protein function. A major strategy to achieve stable expression of foreign genes is to integrate foreign gene sequences into genes through methods such as viral integration, transposase-transposon system or homologous recombination (HR) mediated by CRISPR-Cas system. Viral integration and transposase-transposon system can efficiently integrate foreign genes into the genome, but their random integration characteristics lead to their high genotoxicity. CRISPR-Cas-mediated DSB repair through homologous recombination (HR) pathway can achieve site-specific integration of foreign genes, but the integrated gene fragments are short in length and the integration efficiency is low. In addition, the preparation of linear donor DNA containing homology arms is time-consuming and expensive. Therefore, the development of new gene editing tools that can efficiently integrate foreign DNA sequences (especially long fragments) into specific sites of the genome has become a major challenge in the current field of gene therapy.

[0003] The PB transposase-transposon system can efficiently carry out gene transposition integration in mammalian cells, providing a good molecular basis for the development of efficient new tools for site-specific integration through molecular evolution. PB transposase (PBase) can cut transposon DNA carrying GOI and efficiently integrate it into the "TTAA" target site in the mammalian genome in a seamless pasting manner. Previous studies have shown that by mutating some sites on the PBase enzyme that bind to the genomic target site DNA, the PBase enzyme can retain the cleavage activity and ITR binding ability of the PB transposon DNA (Transposon DNA), but almost completely lose the activity of integrating transposon DNA into the genomic "TTAA" site, which can change the transposon integration efficiency ("Exc+Int-" mutant). And studies have found that combining the corresponding "Exc+Int-" mutant with Cas9-gRNA can integrate transposon DNA into the specific site guided by gRNA. However, it still faces the problem of low site-specific integration efficiency and high off-target rate.

[0004] In 2013, a US laboratory published a PBase paper (doi:10.1073 / pnas.1305987110), which stated that the R372AK375A D450N mutant had transposon donor DNA cleavage activity, but its "TTAA" integration ability was very low. Therefore, patent WO2020250181 used the R372A K375A D450N mutant of US researchers in combination with nucleases such as Cas9, which has targeted integration function, and made some expansions on the PB mutant. However, the existing technology still lacks PB mutants with higher integration effect and lower "TTAA" integration ability. Summary of the invention

[0005] In view of the technical defects of the prior art that the PBase site-specific integration efficiency is low and the off-target rate is high, the present invention provides a PBase protein, fusion protein, nucleic acid, gene integration system, gene delivery system and application, wherein the PBase protein retains the ability to bind to the ITR sequence of the Piggybac transposon and cut to separate the Piggybac transposon from the donor plasmid in a linear manner, and the PBase protein has a lower ability to bind to the DNA double strand within 10bp upstream and downstream of "TTAA". When the PBase protein is connected with a site-specific DNA nuclease to prepare a fusion protein, the PBase protein can efficiently integrate a long fragment of exogenous genes at the double-strand break generated by the site-specific DNA nuclease, thereby achieving the effect of site-specific insertion of long fragment genes.

[0006] To solve the above technical problems, the present invention provides a technical solution: a PBase protein, which is based on the amino acid sequence shown in SEQ ID NO: 3, and contains amino acid mutations at at least three of the following amino acid sites: positions 166, 286, 289, 302, 315, 347, 375, 376, 441, 450, 498, 520, 521, 523 and 557, and the PBase protein retains or enhances gene integration ability, and the target site and recognition ability of the PBase protein are reduced.

[0007] The gene integration ability is reflected as a combination of the ITR binding ability of PBase and the transposon donor cutting activity.

[0008] In the present invention, the ITR binding ability refers to the affinity of the PBase protein for binding to the sequence-specific ITR of the Piggybac transposon.

[0009] In the present invention, the cleavage transposon donor activity is the ability of the PBase protein to cleave the Piggybac transposon from the corresponding donor DNA double strand and release it.

[0010] In the present invention, the target site recognition ability refers to the ability of the PBase protein to bind to the double-stranded DNA within 10 bp upstream and downstream of the target site "TTAA" by relying on its own DNA binding region.

[0011] Preferably, the PBase protein comprises an amino acid mutation at at least one of the following amino acid sites: R315, N347, K375 and R376, and further comprises an amino acid mutation at at least one of the following amino acid sites: A166, N286, S289, G302, Q441, D450, S498, E520, A521, T523 and T557, based on the amino acid sequence shown in SEQ ID NO: 3. Among them, amino acid mutations at R315, N347, K375 and R376 can reduce the target site recognition ability of the PBase protein, and amino acid mutations at A166, N286, S289, G302, Q441, D450, S498, E520, A521, T523 and T557 can improve the ITR binding ability or transposon donor cleavage activity of the PBase protein.

[0012] Preferably, the PBase protein comprises at least the following amino acid mutation combinations based on the amino acid sequence shown in SEQ ID NO: 3:

[0013] (1) R315, N347 and D450; or

[0014] (2) N347, R376 and D450.

[0015] Preferably, the amino acid mutation is an amino acid substitution, preferably an amino acid substitution as shown in Table 1.

[0016] Table 1 Amino acid substitutions at PBase mutation sites

[0017] Location Wild-type amino acid residues Amino acid residues replaced by mutation 166 A H, R, K 286 N H, R, K 289 S H, R, K 302 G H, R, K 315 R A, E, D 347 N S,T,Y,Q,C,G,A,E,D 375 K A, E, D 376 R A, E, D 441 Q H, R, K 450 D N,S,T,C,Y,A,R 498 S N 520 E H, R, K 521 A H, R, K 523 T R,N 557 T H, R, K

[0018] In a preferred embodiment of the present invention, the PBase protein comprises at least the following amino acid mutation combinations based on the amino acid sequence shown in SEQ ID NO: 3:

[0019] (1) R315A / E / D, N347S / T / Y / Q / C / G / A / E / D and D450N; or

[0020] (2)N347S / T / Y / Q / C / G / A / E / D, R376E and D450N.

[0021] More preferably, the PBase protein comprises at least the following amino acid mutation combinations based on the amino acid sequence shown in SEQ ID NO: 3:

[0022] (1) R315A, N347E and D450N; or

[0023] (2) N347A, R376E and D450N; or

[0024] (3) N347E, R376E and D450N.

[0025] More preferably, the PBase protein includes the amino acid mutation combination N347A, R376E and D450N based on the amino acid sequence shown in SEQ ID NO:3, and also includes one or more of the following amino acid mutations: A166H / R / K, N286H / R / K, S289H / R / K, G302H / R / K, Q441H / R / K, S498N, E520H / R / K, A521H / R / K, T523R / N and T557H / R / K.

[0026] Further preferably, the PBase protein includes the amino acid mutation combination N347A, R376E and D450N based on the amino acid sequence shown in SEQ ID NO:3, and also includes one or more of A166K, N286R, S289R, G302R, Q441R, S498N, E520R, A521R, T523R and T557R.

[0027] In a specific embodiment of the present invention, the amino acid sequence of the PBase protein is shown in SEQ ID NO: 5, 7 or 9.

[0028] To solve the above technical problems, the present invention provides a technical solution: a fusion protein comprising the PBase protein as described herein fused to a site-specific DNA nuclease, and the fusion does not change the original functions of the PBase protein and the specific DNA nuclease.

[0029] In the present invention, the site-specific DNA nuclease is a nuclease that has the function of specifically recognizing and binding to a nucleic acid sequence and can cut the recognized nucleic acid sequence to produce a double-strand break (DSB), such as meganuclease, Cas, ZFN and TALEN.

[0030] In the present invention, the Cas is a Cas enzyme in the CRISPR system that can produce site-specific DNA double-strand breaks, such as Cas9 and Cas12, which need to be mediated by guide RNA (sgRNA) to recognize the target site.

[0031] In the present invention, the ZFN is a zinc finger structure recognition sequence fused DNA endonuclease, such as Zinc-Finger-FokI.

[0032] In the present invention, the TALEN is a transcription activator-like effector recognition sequence fusion endonuclease, such as TALE-FokI.

[0033] In the present invention, the meganuclease is a homing endonuclease with a longer recognition sequence, such as I-SceI.

[0034] In the present invention, the PBase protein has the following function: inserting the target gene in the donor into the double-strand break generated by the site-specific DNA nuclease.

[0035] Preferably, in the fusion protein, the site-specific DNA nuclease and the PBase protein are directly connected by a chemical bond or connected through a linker, the linker is preferably 3*G4S, and the chemical bond is preferably a peptide bond. More preferably, the structure connected by the linker is: site-specific DNA nuclease-linker-PBase protein from the N-terminus to the C-terminus.

[0036] More preferably, in the fusion protein, a Flag sequence is further included between the site-specific DNA nuclease and the PBase protein, and the Flag sequence is only used to detect the fusion protein and does not affect the function of the fusion protein.

[0037] Preferably, the specific DNA nuclease includes an NLS sequence, the NLS sequence refers to a nuclear localization sequence, and the amino acid sequence of the NLS sequence may be as shown in SEQ ID NO: 1, positions 1373-1379 of the amino acid sequence. When the specific DNA nuclease includes an NLS sequence, the structure of the fusion protein from the N-terminus to the C-terminus is: specific DNA nuclease-NLS-linker-PBase protein.

[0038] In a specific embodiment of the present invention, the PBase protein in the fusion protein lacks methionine (M) at position 1 in the amino acid sequence.

[0039] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 6, 8 or 10.

[0040] In order to solve the above technical problems, the present invention provides a technical solution: an isolated nucleic acid encoding the PBase protein as described herein and / or the fusion protein as described herein.

[0041] In a specific example of the present invention, the isolated nucleic acid comprises the nucleotide sequence shown at positions 4183-5964 of SEQ ID NOs: 135-137, or the isolated nucleic acid comprises the nucleotide sequence shown in SEQ ID NOs: 135-137.

[0042] The M encoded by the start codon ATG will be removed during the maturation of the PBase protein, and the removal of the start amino acid M does not affect the function of the PBase protein; and the PBase coding sequence located downstream is often not required to include the start codon ATG in the fusion protein. Therefore, the isolated nucleic acid may include the start codon ATG upstream of the coding region of PBase, or may not include the start codon ATG.

[0043] To solve the above technical problems, the present invention provides a technical solution: a gene integration system, which includes the PBase protein as described herein or the fusion protein as described herein, or the isolated nucleic acid as described herein; and a Piggybac transposon donor nucleic acid carrying an exogenous target gene.

[0044] Preferably, the fusion protein comprises a PBase protein and a Cas system as described herein, wherein the Cas system comprises a Cas protease and a guide RNA (sgRNA), and the Cas protease is, for example, a Cas9 and Cas12 protease.

[0045] In a specific embodiment of the present invention, in the piggybac transposon donor nucleic acid, the exogenous target gene has RE ITR and LE ITR sequences on both sides, which can be both RE ITR, both LE ITR, or both RE ITR and LE ITR.

[0046] To solve the above technical problems, the present invention provides a technical solution: a gene delivery system, the gene delivery system comprising one or more vectors, the vector comprising one or more of the PBase protein as described herein, the fusion protein as described herein, the isolated nucleic acid as described herein and the gene integration system as described herein, the vector further comprising a piggybac transposon donor nucleic acid carrying an exogenous target gene, the vector being, for example, an adenovirus vector, a lipid nanoparticle, a lentivirus vector, a ribonucleoprotein complex and a virus-like particle.

[0047] A gene integration method comprises the following steps: using the gene integration system as described herein to integrate an exogenous target gene into the genome of a host cell.

[0048] Preferably, the gene integration system enters the host cell via a gene delivery system as described herein.

[0049] Preferably, the method is for non-diagnostic and / or therapeutic purposes.

[0050] To solve the above technical problems, the present invention provides a technical solution: a pharmaceutical composition comprising one or more of the PBase protein described herein, the fusion protein described herein, the isolated nucleic acid described herein, the gene integration system described herein, and the gene delivery system described herein.

[0051] To solve the above technical problems, the present invention provides a technical solution: a kit comprising one or more of the PBase protein as described herein, the fusion protein as described herein, the isolated nucleic acid as described herein, the gene integration system as described herein, the gene delivery system as described herein, and the pharmaceutical composition as described herein.

[0052] To solve the above technical problems, the present invention provides a technical solution: a set of medicine kits, the set of medicine kits comprising medicine kit A, and the medicine kit A includes one or more of the PBase protein described herein, the fusion protein described herein, the isolated nucleic acid described herein, the gene integration system described herein, the gene delivery system described herein, the pharmaceutical composition described herein, and the kit described herein.

[0053] Preferably, the kit also includes kit B, which includes reagents for detecting or screening whether gene integration is successful, such as detection reagents that specifically bind to the integrated gene, such as probes and antibodies.

[0054] To solve the above technical problems, the present invention provides a technical solution: use of one or more of the PBase protein described herein, the fusion protein described herein, the isolated nucleic acid described herein, the gene integration system described herein, the gene delivery system described herein, the pharmaceutical composition described herein, and the kit described herein in the preparation of reagents for integrating genes or drugs for treating genetic diseases.

[0055] Preferably, the use is for non-diagnostic or therapeutic purposes.

[0056] Preferably, the genetic disease is such as epidermolysis bullosa.

[0057] To solve the above technical problems, the present invention provides a technical solution: a method for integrating genes or treating genetic diseases, the method comprising administering one or more of the PBase protein as described herein, the fusion protein as described herein, the gene integration system as described herein, the gene delivery system as described herein, the pharmaceutical composition as described herein, and the kit as described herein to a subject in need thereof. The genetic disease is, for example, epidermolysis bullosa.

[0058] To solve the above technical problems, the present invention provides a technical solution: one or more of the PBase protein as described herein, the fusion protein as described herein, the gene integration system as described herein, the gene delivery system as described herein, the pharmaceutical composition as described herein, and the kit as described herein for integrating genes or treating gene diseases. The gene disease is, for example, epidermolysis bullosa.

[0059] In the present invention, the mutant is named after the mutation it contains, and the mutant template is the wild-type PBase (SEQ ID NO: 3). For example, PBase N347A_R376E_D450N is based on SEQ ID NO: 3 and includes the following three amino acid mutations: N at position 347 is mutated to A, R at position 376 is mutated to E, and D at position 450 is mutated to N.

[0060] The PBase protein of the present invention can be prepared according to conventional methods for preparing proteins in the art.

[0061] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0062] The reagents and raw materials used in the present invention are commercially available.

[0063] The positive and progressive effects of the present invention are:

[0064] Compared with the PBase mutants of the prior art (such as R372A K375A D450N and other mutations further added thereto), the PBase protein provided by the present invention has a higher transposon DNA cutting activity (theoretically, high cutting activity indicates high targeted integration efficiency), and the ability of DNA double-strand binding within 10bp upstream and downstream of "TTAA" decreases, which reduces the "TTAA" dependent integration ability (theoretically, the "TTAA" dependent integration ability is low and indicates lower off-target). When the PBase protein is connected with a site-specific DNA nuclease to prepare the fusion protein of the present invention, the PBase protein can efficiently integrate long fragment exogenous genes at the double-strand breaks produced by the site-specific DNA nuclease, thereby achieving the effect of fixed-point insertion of long fragment genes. In addition, the PBase protein, fusion protein, nucleic acid and gene integration system provided by the present invention can achieve the knock-in of exogenous genes of longer lengths (for example, up to 12kb) compared to conventional homologous recombination, and circular plasmids can be used as donors, simplifying the donor DNA preparation process and reducing costs, thereby improving the value of industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1A The plasmid map of the RFP reporter gene is shown. The map has AAVS1 homology arms, which can be homologously recombined into the AAVS1 site of the human cell genome, and positive cells are selected using puromycin. The CAG promoter-driven RFP gene expression is interrupted by the Piggybac transposon sequence containing the SV40 promoter-bacitracin resistance gene-polyA expression cassette. Excision of the Piggybac transposon sequence can restore RFP expression. The RFP expression efficiency can represent the efficiency of PBase cutting transposon.

[0066] Figure 1B The number of RFP-positive cells in reporter cells after transfection with different PBase mutant plasmids is shown to reflect the transposon cleavage activity. RFP reporter gene ( Figure 1A The Cas9-gRNA homologous recombination method was used to integrate the AAVS1 locus of 293F cells and screen the positive monoclonal cell lines as reporter cells. PBase and its mutants were constructed in the pcDNA3.1 vector and transfected into reporter cells. The number of RFP-positive cells was counted 5 days after transfection.

[0067] Figure 2 The relative integration efficiency of wt Hyperactive PBase and its mutants in HeLa cells is shown. HeLa cells were co-transfected with NeoR-GFP transposon donor DNA plasmid (eg Figure 7) and pcDNA3.1-PBase plasmid. 2 days and 14 days after transfection, the GFP positive ratio was detected by flow cytometry, representing the transfection efficiency and integration efficiency, respectively. The relative integration efficiency was calculated according to the ratio of GFP on day 14 / GFP on day 2.

[0068] Figure 3A The map of the GFP C-terminal receptor plasmid is shown. A gRNA recognition site target region and an mRNA splicing acceptor are added upstream of the GFP C-terminal coding sequence on the plasmid. There is no promoter upstream of the Bxb1 attB-BSD sequence of the receptor plasmid. Only through the specific recombination of the Bxb1 integrase to the Bxb1 attP site downstream of the PGK promoter pre-placed on the genome can BSD expression be driven, so that positive recombinant cells can be selected with bacillusin and a monoclonal cell line can be established.

[0069] Figure 3B The PB transposon donor plasmid map with the GFP N-terminal coding sequence is shown. The transposon donor plasmid has an mTagBFP2-2A-Puro expression cassette, which continuously expresses BFP-2A-Puro in transfected positive cells and can be selected with puromycin. The CAG promoter contains the GFP N-terminal coding sequence downstream, and there is an mRNA splicing donor downstream. If the transposon donor DNA is targeted and integrated into the upstream gRNA target region of the GFP C-terminal coding sequence, the complete green fluorescent protein can be expressed through mRNA maturation and splicing after transcription from the CAG promoter, and the detection of the proportion of GFP-positive cells can represent the efficiency of site-specific integration.

[0070] Figure 3C The plasmid map shows the CMV promoter expressing spCas9-PBase and the U6 promoter driving ROSA26 gRNA transcription. NLS (nuclear localization sequence) refers to the nuclear localization sequence.

[0071] Figure 4 This is a schematic diagram of the principle of analyzing the efficiency of integration at a specific site. The transposon donor sequence is site-specifically integrated into the sgRNA recognition target site upstream of the GFP C-terminal coding sequence, and forward integration yields BFP + GFP + Cells, reverse integration and non-targeted integration to obtain BFP + GFP - cell.

[0072] Figure 5A Displayed in cells containing the target sequence ( Figure 3A The sequences shown) were co-transfected with different Cas9-PBase-ROSAgRNA plasmids ( Figure 3C ) and the transposon donor plasmid ( Figure 3B The mTagBFP2 and GFP positive rates were detected by flow cytometry 4 days after transfection. Figure 4 Schematic diagram of the principle. The ratio of mTagBFP2 represents the transfection efficiency, and the ratio of GFP represents the efficiency of site-directed integration in the target area.

[0073] Figure 5B The statistical results of the percentage of mTagBFP2 and GFP (according to Figure 5A ).

[0074] Figure 5C The ratio of site-specific integration efficiency to transfection efficiency of different Cas9-PBase mutants in the target region is shown. This ratio reflects the site-specific integration activity of the Cas9-PBase mutant.

[0075] Figure 6 The ratio of target site integration and off-target integration in the stable integration cell population enriched by puromycin selection is shown. Cells co-transfected with Cas9-PBase enzyme and transposon donor plasmid were cultured for 12 consecutive days after transfection, and then the ratio of mTagBFP2 and GFP positive cells was detected by flow cytometry after 10 days of puromycin selection and enrichment. The ratio of target site integration was calculated as 2*GFP positive percentage.

[0076] Figure 7 Shown is another transposon map that can constitutively express the neomycin resistance gene and GFP (NeoR-GFP transposon).

[0077] Figures 8A-8C The efficiency of Cas9-PBase system in integrating NeoR-GFP transposon at three safe sites (ROSA, GSH1, GSH2) in the genome is shown. 8A shows the GFP transfection efficiency detected by flow cytometry 3 days after the co-transfection of Cas-PBase-gRNA plasmid (carrying 3 different gRNAs) and NeoR-GFP transposon plasmid in HeLa cells. 8B shows the GFP stable integration expression efficiency detected by flow cytometry 14 days after transfection. 8C shows the ratio of stable integration efficiency to transfection efficiency.

[0078] Fig. 9The partial sequencing of the junction between the transposon DNA-specific RE-ITR sequence and the site-specific integrated GSH1 site mediated by Cas9-PBase-gRNA is shown. 15 days after HeLa cells were co-transfected with Cas9-PBase(N347A_R376E_D450N)-GSH1 gRNA plasmid and NeoR-GFP transposon, GFP-positive cells were sorted by single cell sorting using a flow cytometer, and single cell clones were cultured and amplified to extract genomic DNA. PCR amplification was performed using a primer combination specific for site-specific integration to screen monoclonal cells with specific integration of the GSH1 site. The PCR amplified bands were sequenced and compared with the theoretical sequence. Compared with the theoretical sequence, the ITR sequence was destroyed in some clones, and most clones contained a small number of base insertions or deletions.

[0079] Fig.10 The percentage of cell clones that integrate the transposon into the GSH1 site by different Cas9-PB mutants was shown. 15 days after HeLa cells were co-transfected with Cas9-PBase-GSH1 gRNA plasmid and NeoR-GFP transposon, GFP-positive cells were sorted by flow cytometry, and single cell clones were cultured and expanded to extract genomic DNA. PCR amplification was performed using a primer combination specific for site-specific integration, and single clones with GSH1 site-specific integration were judged and screened based on the PCR results. The site-specific integration ratio of each Cas9-PB mutant was calculated according to the number of GSH1 site-specific integration clones / total number of clones (R315E_N347E_D450N was 29 / 80; N347A_R376E_D450N was 38 / 86; R372A_K375A_D450N was 40 / 96; N347E_R376E_D450N was 56 / 96).

[0080] Figures 11A-11C 11A shows the integration efficiency of Cas9-PBase triple mutants with different N347 mutation forms and R376E_D450N combinations in specific target regions. Figure 3B ) co-transfection efficiency (mTagBFP2 positive rate). 11B shows the efficiency of site-directed integration in the target region (2*GFP percentage). 11C shows the ratio of on-target integration efficiency / transfection efficiency.

[0081] Figures 12A-12C 12A shows the integration efficiency of Cas9-PBase triple mutants with different D450 mutation forms and N347A_R376E combinations in specific target regions. Figure 3B12B shows the efficiency of site-directed integration in the target region (2*GFP percentage). 12C shows the ratio of on-target integration efficiency to transfection efficiency.

[0082] Fig.13 The site-specific integration efficiency of the Cas9-PBase N347A_R376E_D450N_MutX quadruple mutants is shown. The site-specific integration efficiency of different Cas9-PBase N347A_R376E_D450N_MutX quadruple mutants is improved to varying degrees compared to Cas9-PBase N347A_R376E_D450N.

[0083] Fig.14 This is a schematic diagram of the mechanism of site-specific integration of transposon DNA by the Cas9-PB system. Step 1 is that the PBase part of the fusion protein cuts the Piggybac transposon containing the target gene (GOI) from the donor, steps 2 and 3 are that the fusion protein pulls the target gene to the target site on the target genome through the targeting action of Cas9 and sgRNA, step 4 is that the Cas9 in the fusion protein cuts at the target site to produce a double-strand break (DSB), step 5 is that the PBase part of the fusion protein integrates the target gene into the double-strand break at the target site, and step 6 is the target genome with the target gene integrated. DETAILED DESCRIPTION

[0084] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0085] The reagents, instruments, consumables and sequence information used in the examples of the present invention are shown in Tables 2-4.

[0086] Table 2 Reagent names, suppliers and product numbers

[0087]

[0088] Table 3. Suppliers and product numbers of consumables and instruments used

[0089] name supplier Part Number 1.5ml centrifuge tube Axygen MCT-150-C 15ml centrifuge tube Themo 339650 50ml centrifuge tube Themo 339652 0.2ml flat top PCR tube Axygen PCR-02-C Electrophoresis Apparatus Bio-Rad 1645050 Gel Imager Shanghai Qinxiang Scientific Instrument Co., Ltd. 185068-8B PCR instrument BIO-RAD 621BR44541 5mL pipette Themo 170355N 10mL pipette Themo 170356N 25mL pipette Themo 170357N Biological Safety Cabinets Thermo 1374 Inverted fluorescence microscope Olympus BX53 Shaker SHTIPS TIPSOS-019 Cell culture incubator Themo Forma 371 6cm dish Themo 150462 10cm dish Themo 150466 6-well plate Themo 140675 12-well plate Themo 150628 NC250 counter Chemometec NC-250 Electric constant temperature water bath Shanghai Jinghong Testing Equipment Co., Ltd. DK-S22 Balance Sartorius GL822I-1SCN Ultra-micro-spectrophotometer Themo AZY1810420 Attune NxT Flow Cytometer Themo A24860 Electric pipette Themo S1-9501 Centrifuge Themo ST16R

[0090] Table 4 Primers and sequences involved in the construction of related molecules

[0091]

[0092]

[0093] The molecular construction method used in the present invention is as follows:

[0094] Molecular construction methods1

[0095] 1) First, the wt Hyperactive PBase sequence was optimized according to human codons and synthesized at Sangon Biotechnology Co., Ltd. (wt Hyperactive PBase sequence is shown in SEQ ID NO: 138). After synthesis, it was subcloned into the multiple cloning restriction site of the conventional expression vector pcDNA3.1 vector using HindIII and BamHI to obtain the pcDNA3.1-wt PB plasmid. Using this as a template, the primers CMV-PF-N1 / PBm PR for amplifying the upstream fragment of the PB mutant (PBm), the primers pcDNA3.1-PR-N1 / PBm PF for the downstream fragment, and the primers HindⅢ-PB-PF / BamHI-PB-PR for the fusion of the upstream and downstream fragments were designed using Snapgene3.2.1 software. The primers were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.;

[0096] 2) Use PCR instrument to amplify the upstream and downstream fragments of PBm, 50μL reaction system: 2×Phanta Max Master Mix (Dye Plus) 25μL, ddH2O 20μL, 1.0μL 10μg / μL plasmid template, upstream primer CMV-PF-N1 / PBmPR, downstream primer pcDNA3.1-PR-N1 / PBm PF 2μL each. PCR reaction conditions: 95℃ pre-denaturation for 2min; 95℃ denaturation for 20s, 58℃ annealing for 30s, 72℃ extension for 1min, 30 cycles; 72℃ extension for 5min;

[0097] 3) Ordinary agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.) was used to purify and recover the upstream and downstream fragments of PBm;

[0098] 4) Amplify the full-length fragment of PBm: Use the purified and recovered upstream and downstream fragments of PBm as templates, and the primers are HindⅢ-PB-PF / BamHI-PB-PR. 50μL reaction system: 2×Phanta Max Master Mix (Dye Plus) 25μL, ddH2O 20μL, 10μg / μL PBm upstream and downstream fragments 0.5μL each, forward and reverse primers 2μL each. PCR reaction conditions: 95℃ pre-denaturation for 2min; 95℃ denaturation for 20s, 58℃ annealing for 30s, 72℃ extension for 1.5min, 30 cycles; 72℃ extension for another 5min;

[0099] 5) Purification and recovery of the full-length PBm fragment using a conventional agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.);

[0100] 6) After the PCR product of the full-length PBm fragment was recovered and purified, it was digested with restriction enzymes HindⅢ-HF and BamHI-HF. The reaction system included 0.2μg of PBm fragment, 1uL of restriction enzymes HindⅢ-HF and BamHI-HF, 5μL of 10×rCutSmart buffer, and the mixture was rehydrated to 50μL. The reaction was carried out at 37℃ for 30min. The plasmid pcDNA3.1 was digested with the same enzymes as a vector. The reaction system included 2μg of plasmid, 1μL of restriction enzymes HindⅢ-HF and BamHI-HF, 5μL of 10×rCutSmart buffer, and the mixture was rehydrated to 50μL.

[0101] Incubate at 37°C for 1 hour.

[0102] 7) A common agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.) was used to purify and recover the PBm full-length fragment and plasmid vector after enzyme digestion;

[0103] 8) The PBm full-length fragment after enzyme digestion was ligated with the plasmid vector using T4 DNA Ligase purchased from Novigene. The reaction system was referred to the T4 DNA Ligase instruction manual. The reaction conditions were: 25°C for 30 min.

[0104] 9) Take 5 μL of the ligation product and gently add it to 50 μL of DH5α cloning competent cells, flick to mix, and place on ice for 30 minutes; heat shock in a 42°C water bath for 30 seconds and immediately place on ice for 2 minutes; add 200 μL of antibiotic-free LB medium equilibrated to room temperature. Oscillate at 37°C and 180 rpm for 1 hour; evenly spread 200 μL of bacterial solution on a solid LB plate (containing antibiotics: ampicillin working concentration 50 μg / ml); invert the LB plate and culture it in an incubator at 37°C overnight;

[0105] 10) Select positive clones and send them to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing;

[0106] 11) After the bacterial solution with correct sequencing was expanded, pcDNA3.1-PBm was extracted using the endotoxin-free plasmid mini-extraction kit from Tiangen Biochemical Technology Co., Ltd. After the extraction, the concentration and quality of the plasmid were detected by ultra-micro spectrophotometer, and the pcDNA3.1-PBm plasmid was double-digested with endonucleases HindⅢ and BamHI-HF to verify the size of the plasmid.

[0107] Molecular construction method 2

[0108] 1) First, the D10A of the hCas9_D10A plasmid (addgene number #41816) was mutated in situ to D, and then the U6-ROSA sgRNA (SEQ ID NO: 131) element was synthesized (synthesized by GenScript) and inserted into the MfeI and MluI sites of the vector; on this basis, the linker-wt Hyperactive PBase sequence (synthesized by GenScript) was synthesized at the C-terminus of the Cas9 coding sequence to obtain the Cas9-PB-ROSA sgRNA vector (SEQ ID NO: 139). This plasmid was used as a template for subsequent mutations, and the primers AscI-up-Cas-PFN1 / PBm PR for amplifying the upstream fragment of the PB mutant (PBm), the primer AgeI-down-pA-PR N1 / PBm PF for the downstream fragment, and the primer Cas9-AscI-PF / AgeI-PB-PR for fusing the upstream and downstream fragments were designed using Snapgene3.2.1 software. The primers were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd.;

[0109] 2) Amplify the upstream and downstream fragments of PBm using a PCR instrument, 50μL reaction system: 2×Phanta Max Master Mix (Dye Plus) 25μL, ddH2O 20μL, 1.0μL 10μg / μL plasmid template, upstream primer AscI-up-Cas-PFN1 / PBm PR, downstream primer AgeI-down-pA-PR N1 / PBm PF 2μL each. PCR reaction conditions: 95℃ pre-denaturation for 2min; 95℃ denaturation for 20s, 58℃ annealing for 30s, 72℃ extension for 1min, 30 cycles; 72℃ extension for 5min;

[0110] 3) Ordinary agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.) was used to purify and recover the upstream and downstream fragments of PBm;

[0111] 4) Amplify the full-length fragment of PBm: Use the purified and recovered upstream and downstream fragments of PBm as templates, and the primers are Cas9-AscI-PF / AgeI-PB-PR. 50μL reaction system: 2×Phanta Max Master Mix (Dye Plus) 25μL, ddH2O 20μL, 10μg / μL PBm upstream and downstream fragments 0.5μL each, forward and reverse primers 2μL each. PCR reaction conditions: 95℃ pre-denaturation for 2min; 95℃ denaturation for 20s, 58℃ annealing for 30s, 72℃ extension for 2min, 30 cycles; 72℃ extension for another 5min;

[0112] 5) Purification and recovery of the full-length PBm fragment using a conventional agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.);

[0113] 6) After the PCR product of the full-length PBm fragment was recovered and purified, it was digested with restriction endonucleases AscI and AgeI-HF. The reaction system contained 0.2 μg of PBm fragment, 1 μL of restriction endonucleases AscI and AgeI-HF, and 5 μL of 10×rCutSmart buffer, and the mixture was rehydrated to 50 μL. The reaction was carried out at 37°C for 30 min. The plasmid pcDNA3.1-Cas9-PB-N347A R376E D450N was digested with the same enzymes as a vector. The reaction system contained 2 μg of plasmid, 1 μL of restriction endonucleases AscI and AgeI-HF, and 5 μL of 10×rCutSmart buffer, and the mixture was rehydrated to 50 μL.

[0114] Incubate at 37°C for 1 hour.

[0115] 7) A common agarose gel DNA recovery kit (Tiangen Biochemical Technology Co., Ltd.) was used to purify and recover the PBm full-length fragment and plasmid vector after enzyme digestion;

[0116] 8) The full-length fragment of PBm was digested and the plasmid vector was prepared using T4 DNA Ligase purchased from Novigene. The reaction system was based on the T4 DNA Ligase instruction manual and the reaction was performed at 25°C for 30 min.

[0117] 9) Take 5 μL of the ligation product and add it to 50 μL of E. coli DH5α competent cells. Take 5 μL of the ligation product and gently add it to 50 μL of DH5α cloning competent cells, flick to mix, and place on ice for 30 minutes; heat shock in a 42°C water bath for 30 seconds and immediately place on ice for 2 minutes; add 200 μL of LB medium equilibrated to room temperature. Oscillate at 37°C and 180 rpm for 1 hour; evenly spread 200 μL of bacterial solution on a solid LB plate (containing antibiotics: ampicillin working concentration 50 μg / ml); invert the LB plate and culture it in an incubator at 37°C overnight;

[0118] 10) Select positive clones and send them to Boshang Biotechnology (Shanghai) Co., Ltd. for sequencing;

[0119] 11) After the bacterial solution with correct sequencing was expanded, Cas9-PBm was extracted using the endotoxin-free plasmid mini-extraction kit from Tiangen Biochemical Technology Co., Ltd. After the extraction, the concentration and quality of the plasmid were detected by ultra-micro spectrophotometer, and the Cas9-PBm plasmid was double-digested with endonucleases AscI and AgeI-HF to verify the plasmid size.

[0120] Example 1 Detection of PBase mutant splicing transposon donor activity

[0121] The purpose of this experiment is to test the efficiency of different PBase mutants in cleaving the transposon donor. First, a tdTomato reporter gene cell line was constructed on 293F cells, and then the PBase mutant plasmid was transfected into the cell line. The efficiency of PBase mutant cleavage of the transposon donor was determined by observing the expression of the tdTomato gene.

[0122] First, a homologous recombination donor at the AAVS1 locus was synthesized ( Figure 1A , synthesized by GenScript Gene, the sequence is shown in SEQ ID NO: 15), the donor contains a homologous recombination arm integrated into the AAVS1 site, which can be site-directedly recombined into the intron of the PPP1R12C gene (located on chromosome 19). The donor contains an mRNA splicing acceptor (SA) and T2A-Puro, and T2A-Puro can be expressed by the endogenous PPP1R12C promoter. At the same time, the tdTomato expression driven by the CAG promoter on the donor is blocked by the middle transposon (expressing BSD). After the transposon is excised by PBase or its mutant, tdTomato will be expressed.

[0123] AAVS1 sgRNA (target sequence: CACCCCACAGTGGGGCCACT, SEQ ID NO: 130) was synthesized at GenScript, and Cas9 protein (GenScript, Z03469-500) was purchased at the same time. The linear donor was amplified and purified using a high-fidelity DNA polymerase (Vazyme, P525-01). TM Electroporation was performed into 293F cells. The experimental process was to first mix 150pmol Cas9 protein and 300pmol sgRNA and incubate at room temperature for 10 minutes to obtain RNP (ribonucleoprotein complex); at the same time, 5E6 cells and 1.0μg linear donor DNA were added to the 100μL electroporation system, and then RNP was added to the electroporation system. The electroporation conditions were 1100V, 20ms / pulse*2pulse. Puromycin (1μg / mL) was used to screen positive single cell clones and Sanger sequencing was used to confirm the site-specific knock-in of the reporter gene into the AAVS1 site.

[0124] The amino acid sequences of PBase mutants PBase R315A_N347E_D450N, PBase N347E_R376E_D450N, PBaseN347A_R376E_D450N and PBase R372A_K375A_D450N are shown in SEQ ID NOs: 5, 7, 9 and 11, the amino acid sequence of wild-type PBase (wt Hyperactive PBase) is shown in SEQ ID NO: 3, and the amino acid sequence of PBaseR315A_N347E is an amino acid sequence in which R315A and N347E are replaced based on the amino acid sequence shown in SEQ ID NO: 3.

[0125] The construction of PBase mutants was carried out by introducing corresponding mutations by overlap PCR, using the synthesized pcDNA3.1-PBase as a template, and cloning into the pcDNA3.1 expression vector by HindIII and BamHI. For specific experimental details, see Molecular Construction Method 1. The coding nucleic acid sequence of wt Hyperactive-PBase is shown in SEQ ID NO:13, and the coding nucleic acid sequence of each PBase mutant is shown in SEQ ID NO:135-137, and the nucleotide sequence with ATG added at the 5' end (PBase all contains M encoded by ATG, but the ATG of PBase is removed in the coding sequence of the fusion protein). 1 μg of the pcDNA3.1-PBase mutant plasmid was transfected into the above-mentioned tdTomato reporter cells using lipofectamin 3000 reagent, and the number of tdTomato positive cells was counted 5 days after transfection, and the number of positive cells represented the corresponding PBase mutant's activity in cutting the transposon donor.

[0126] Results and conclusion: The cleavage activities of different PBase mutants decreased to varying degrees compared with the wild-type PBase. Among them, R315A_N347E, R315A_N347E_D450N, R315E_N347E_D450N and N347A_R376E_D450N all had higher cleavage activities than R372A_K375A_D450N (this mutant can be found in the articles piggyBac transposase tools for genome engineering and Find and cut-and-transfer (FiCAT) mammalian genome engineering); Y312E_N347E_D450N and N347E_R376E_D450N also showed certain cleavage activities; in addition, the triple mutation containing D450N showed higher cleavage activities than the corresponding double mutants. ( Figure 1B )

[0127] Example 2 Detection of the activity of PBase mutants in integrating transposon donors into the genome (TTAA-dependent)

[0128] The purpose of this experiment is to test the activity of different PBase mutants in cleaving the transposon donor and integrating it into the genome "TTAA" site. The experiment first constructed a transposon donor with dual promoters expressing the NeoR resistance gene and enhanced green fluorescent protein eGFP ( Figure 7 ), then 1 μg of NeoR-GFP transposon donor (sequence shown in SEQ ID NO: 21) and 0.7 μg of pcDNA3.1-PBase mutant in Example 1 were co-transfected, and the GFP positive rate, i.e., transfection efficiency and stable integration efficiency, was detected by flow cytometry 2 days and 14 days after transfection. The integration activity of different PBase mutants was calculated according to the ratio of GFP on day 14 / GFP on day 2.

[0129] Results and conclusion: The integration activities of double mutants and triple mutants were generally at a low level, and most mutants had almost no integration activity; some mutants including Y312E_N347E_D450N, R315A_N347E_D450N, R315E_N347E_D450N and N347A_R376E_D450N had integration activities close to or slightly higher than R372A_K375A_D450N. Figure 2 )

[0130] Taking into account the activity of different PBase mutants in cleaving the transposon donor (Example 1) and integrating the transposon donor into the genome (TTAA target) (Example 2), some mutants with higher cleavage activity and lower integration activity (called Exc+Int- mutants) were selected, mainly including R315A_N347E_D450N, N347A_R376E_D450N, N347E_R376E_D450N and R372A_K375A_D450N mutants in the prior art, and combined with CRISPR-Cas9 protein that can produce site-directed DNA double-strand breaks, the efficiency of site-directed integration of the Piggybac transposon donor carrying the exogenous target gene was tested.

[0131] Example 3 Construction of Cas9-PBase mutants and testing of their site-directed integration efficiency

[0132] The purpose of this experiment is to fuse the above-mentioned Exc+Int- mutants with the Cas9 gene to construct Cas9-PBase mutants, and to test the site-specific integration efficiency of different Cas9-PBase mutants through the designed and constructed Split GFP test system. The functional principle of Cas9-PBase fusion protein is as follows Fig.14 shown.

[0133] Construction of Cas9-PBase mutants: First, the D10A of the hCas9_D10A plasmid (addgene number #41816) was mutated in situ to D, and then the U6-ROSA sgRNA (SEQ ID NO: 131) element (synthesized by GenScript) was synthesized and inserted into the MfeI and MluI sites of the vector; on this basis, 3*G4S linker-wt Hyperactive PBase was further synthesized and connected to the C-terminus of Cas9 (the sequence is shown in SEQ ID NO: 1) to obtain the Cas9-PB-ROSA sgRNA vector (SEQ ID NO: 139). On this basis, the above 4 different PBase mutants replaced the wt Hyperactive PBase and were connected to the C-terminus of Cas9. For specific experimental details, please refer to Molecular Construction Method 2. The constructed Cas9-PB-ROSA sgRNA plasmid map is shown in Figure 3C(Take Cas9-wtPBase-ROSA sgRNA as an example). Considering that the first amino acid M (ATG encoding) is usually removed during protein maturation, in all fusion proteins, the PBase part lacks the first amino acid M compared to the free PBase, and retains the 2nd to 594th amino acids (GSSLDD---QSCF). The part encoding PBase in the encoding nucleic acid sequence of Cas9-PBase of each mutant (see the nucleic acid sequences shown in SEQ ID NOs: 14 and 135-137, 4183-5964, all underlined) corresponds to the 2nd to 594th amino acids of the PBase amino acid sequence. The amino acid sequences of the resulting Cas9-PBase fusion proteins Cas9-wt HyperactivePBase, Cas9-PBase R315A_N347E_D450N, Cas9-PBase N347E_R376E_D450N, Cas9-PBaseN347A_R376E_D450N and Cas9-PBase R372A_K375A_D450N are shown in SEQ ID NOs: 4, 6, 8, 10 and 12, respectively.

[0134] Split GFP system: The working principle of this system is shown in Figure 4 The basic principle is to place the N-terminal and C-terminal GFP coding sequences into the N GFP transposon donor plasmid ( Figure 3B , sequence as shown in SEQ ID NO: 17) and receiving plasmid ( Figure 3A , the sequence is shown in SEQ ID NO:16), and the intron sequence between exon 7 and exon 8 of the endogenous SMN1 gene is split into two parts (1 / 2 intron and 2 / 2 intron) and placed downstream of N GFP (1 / 2 intron) and upstream of C GFP (2 / 2 intron) respectively. If the N GFP transposon donor can be site-specifically integrated upstream of C GFP, the intron can be removed by mRNA splicing and GFP protein can be expressed. Add the target sequence (including a specific sgRNA sequence, such as the above-mentioned ROSAsgRNA) and the 2 / 2 intron sequence and the splicing acceptor sequence upstream of C GFP, and add the Bxb1 attB-BSD resistance gene to the plasmid, and the Bxb1 integrase can be used to integrate into the Bxb1 attP site. The N GFP transposon donor contains the continuously expressed BFP-2A-Puro to facilitate flow cytometry detection of transfection, integration efficiency and screening, and a splicing donor sequence and 1 / 2 intron sequence are added downstream of N GFP ( Figure 4 ). For the specific principle, please refer to doi:10.1016 / j.molcel.2018.10.037.

[0135] Construction of Split GFP cell line: First, the pPGK-Bxb1 attP sequence was site-directedly recombined into the ROSA site in HeLa cells by homologous recombination mediated by Cas9 protein (GenScript Z03469-500) and single-cell cloning was performed. The single-copy sequence integration sequence was determined by Sanger sequencing and NGS sequencing (conventional homologous recombination knock-in, which will not be described here). Then, 1 μg of the synthesized pcDNA3.1-Bxb1 integrase plasmid (Bxb1 integrase sequence reference addgene#127519) and 1.5 μg of the receptor plasmid ( Figure 3A ) were co-transfected with pPGK-Bxb1, and single-cell clones that were site-specifically integrated into the attP site of pPGK-Bxb1 were selected using blasticidin antibiotic (10 μg / mL), and the accurate sequence was determined by Sanger sequencing.

[0136] Cas9-PBase mutant site-directed integration efficiency test: The above-constructed Cas9-PBase mutant plasmid (1.3 μg, wherein the Cas9-PBase N347A_R376E_D450N-ROSA gRNA plasmid sequence is shown in SEQ ID NO: 18) and N GFP transposon donor (1.2 μg, sequence is shown in SEQ ID NO: 17) were co-transfected into Split GFP cell line using lipofectamin 3000 according to standard operating procedures. Four days after transfection, the BFP and GFP positive rates were detected by flow cytometry ( Figure 5A ) and perform data statistics ( Figure 5B ). Since the BFP positive rate represents the transfection efficiency and the GFP positive rate represents the site-specific integration ratio, the GFP / BFP ratio can reflect the site-specific integration efficiency of different Cas9-PBase mutants ( Figure 5C ).

[0137] Results and conclusion: Through the comparison of site-specific integration efficiency test, it was found that Cas9-PBase N347A_R376E_D450N was more efficient than Cas9-PBase R372A_K375A_D450N, while Cas9-PBase N347E_R376E_D450N and Cas9-PBase R315A_N347E_D450N were less efficient than Cas9-PBase R372A_K375A_D450N. Cas9-PBaseN347A_R376E_D450N was selected as the basis, new mutations were added, and mutants with improved efficiency were screened.

[0138] Example 4 Testing the on- / off-target ratio of different Cas9-PBase mutants using Split GFP cell lines

[0139] The purpose of this experiment is to test the proportion of cells with site-specific integration among all cells with stable integration. For the Cas9-PBase tool, off-targets mainly come from two aspects: on the one hand, off-target cutting guided by Cas9 through sgRNA, and on the other hand, the PBase mutant still has low "TTAA" integration activity. By detecting the positive rate of BFP stably integrated in cells and GFP integrated on-target, the proportion of on-target cells can be calculated.

[0140] The cells transfected in Example 3 were cultured in normal HeLa cell culture medium (DMEM+10% FBS+P / S) for 12 days after transfection, and then screened and enriched with 1.0 μg / mL puromycin for 10 days, and the proportion of BFP and GFP positive cells was detected by flow cytometry. The proportion of site-directed integration in the target area was calculated as 2×GFP positive percentage.

[0141] Results and Conclusion: After Puromycin screening and enrichment, the BFP positive rate reached more than 90%, and the proportion of BFP&GFP double positive cells was different in different Cas9-PBase mutants ( Figure 6 ). These two ratios for each Cas9-PBase mutant were: Cas9-PBase N347A_R376E_D450N (97.5% and 20.3%), Cas9-PBase R372A_K375A_D450N (97.3% and 17.2%), Cas9-PBase N347E_R376E_D450N (94.7% and 20.2%), and Cas9-PBase R315A_N347E_D450N (96.0% and 14.8%).

[0142] From this data, it can be concluded that among the new Cas9-PBase fusion mutants, Cas9-PBase N347A_R376E_D450N and Cas9-PBase N347E_R376E_D450N have lower off-target rates than Cas9-PBase R372A_K375A_D450N, and the off-target rate of Cas9-PBase R315A_N347E_D450N is slightly higher than that of Cas9-PBase R372A_K375A_D450N.

[0143] In general, the three mutant Cas-PBs were compared with Cas9-PBase R372A_K375A_D450N, and the results were as follows: In terms of specific effects, it was found that the site-specific integration efficiency of Cas9-PBase N347A_R376E_D450N was significantly higher than that of Cas9-PBase R372A_K375A_D450N, and the off-target probability of the former was slightly lower than that of the latter. The site-specific integration efficiency of Cas9-PBase N347E_R376E_D450N was slightly lower than that of Cas9-PBase R372A_K375A_D450N, but the off-target probability of the former was significantly reduced. In most cases, the site-specific integration efficiency of Cas9-PBase R315A_N347E_D450N was comparable to that of Cas9-PBase R372A_K375A_D450N, but the off-target rate of the former was higher.

[0144] Example 5 Site-specific integration test at the HeLa cell genome safe harbor locus

[0145] The purpose of this experiment is to further test the integration efficiency of different Cas9-PBase mutants at different endogenous safe harbor sites. Three sgRNAs for safe harbor sites were designed and synthesized and constructed into different Cas9-PBase mutant vectors. The three sites and sgRNA targeting sequences are: ROSA sgRNA targeting sequence (GTCGAGTCGCTTCTCGATTA, SEQ ID NO: 132), GSH1 sgRNA targeting sequence (TTAGTCCTAGTGCCATGAAG, SEQ ID NO: 133), GSH2 sgRNA targeting sequence (CATCAGACTTGATAGCACTG, SEQ ID NO: 134).

[0146] The different Cas9-PBase mutant gRNA plasmids constructed in Example 3 (1.3 μg, the plasmid sequences of the Cas9-PBase N347A_R376E_D450N enzyme and the sgRNA expressing the three safe harbors are shown in SEQ ID NO: 18-20, and the 4501-6282 positions of the sequences 18-20 are replaced with the base sequences shown in the 4183-5964 positions of SEQ ID NO: 135-137, and the gRNA sequences of the three safe harbors of the remaining three mutants can be obtained) were co-transfected with HeLa cells by lipofectamin 3000 according to the standard operating procedures with the NeoR-GFP transposon donor (1.2 μg), and the GFP positive rate was detected by flow cytometry 3 days and 14 days after transfection. The positive rate measured 3 days after transfection is the transfection efficiency of the corresponding sample, and the positive rate measured 14 days after transfection is the stable integration efficiency of the corresponding sample. The experimental methods for HeLa cell transfection and flow cytometry are as follows:

[0147] 1) Before plasmid transfection of HeLa cells, place HeLa cells in a 6-well plate to control the cell density between 60-70%. The plasmid ratio is 1.3 μg of Cas9-PBm and 1.2 μg of NeoR-GFP transposon donor. Use Lipofectamine purchased from Thermo Scientific TM 3000 transfection reagent for cell transfection, the reaction system refers to Lipofectamine TM 3000 transfection reagent instructions, incubate overnight at 37°C in a cell culture incubator for 20 h.

[0148] 2) Remove the transfection reagent from the HeLa cells, add 250 μL of trypsin, incubate at 37°C for 3 min, add 1.5 mL of DMEM high-glucose medium containing 10% Fetal Bovine Serum to terminate the reaction, centrifuge at 200 g for 5 min, remove the supernatant, resuspend the cells and culture them in a 6 cm dish (or add antibiotics of corresponding concentrations), and continue to culture at 37°C for the described time.

[0149] 3) On the 3rd and 14th days after transfection, the supernatant of HeLa cells was removed, 600 μL of trypsin was added, and the cells were incubated at 37°C for 3 min. 5 mL of DMEM high-glucose medium was added to terminate the reaction, and the cells were centrifuged at 1300 rpm for 5 min, the supernatant was removed, and the cells were resuspended for flow cytometry detection.

[0150] 4) Flow cytometry was performed using an Attune NxT flow cytometer purchased from Thermo Company, using flow tube detection, setting the voltage parameters of transverse scattered light (FSC) and side scattered light (SSC) to FSC 60, SSC 300, and selecting the channel BL1. The number of cells tested was 50,000; the ratio of green fluorescence was detected. The ratio of the GFP ratio on the 14th day to the GFP ratio on the 3rd day represents the integration efficiency of the corresponding Cas9-PBase mutant at the corresponding site.

[0151] Results and Conclusion: The co-transfection efficiency of different Cas9-PBase mutant plasmids and NeoR-GFP transposon donors was relatively stable, about 20%-30% ( Fig. 8A ); From the perspective of stable integration efficiency of the three safe harbor sites, GSH1 has the highest efficiency, while ROSA and GSH2 sites are relatively close in efficiency, which may be due to the higher efficiency of sgRNA at the GSH1 site; by comparing different Cas9-PBase mutants at the three endogenous sites, it was found that Cas9-PBase N347A_R376E_D450N was more efficient than Cas9-PBase R372A_K375A_D450N, while Cas9-PBase N347E_R376E_D450N and Cas9-PBase R315A_N347E_D450N were less efficient than Cas9-PBase R372A_K375A_D450N, which is consistent with the results obtained in the Split GFP cell line in Example 3 ( Figure 8B and Figure 8C ).

[0152] Example 6 Detection of the targeted integration ratio of Cas9-PBase mutants at the GSH1 site of HeLa cells and sequence characteristics of the integration junction

[0153] The purpose of this experiment is to further test the targeted integration ratio of different Cas9-PBase mutants at the GSH1 safe harbor site and to detect the sequence characteristics of the integration junction region.

[0154] The cells with stable integration of different Cas9-PBase mutants at the GSH1 site in Example 5 were GFP-positive sorted into single cells, and about 80-90 single cell clones were cultured separately. Then, these clones were PCR-tested for site-specific integration, and PCR primers (SEQ ID NO: 126, 127 were combined with SEQ ID NO: 128, 129 in pairs) were respectively bound to the donor and the upstream and downstream of the sgRNA target. A portion of the amplified bands were sequenced, and the sequence of the donor-genome junction region was compared and observed.

[0155] Experimental results and conclusions: According to the site-specific integration detection of all clones, the site-specific integration ratios of different Cas9-PBase mutants were 38 / 86 for Cas9-PBase N347A_R376E_D450N, 29 / 80 for Cas9-PBase R315E_N347E_D450N, 40 / 96 for Cas9-PBase R372A_K375A_D450N, and 56 / 96 for Cas9-PBase N347E_R376E_D450N ( Fig.10 ). According to the results of identification on single cells, it can be concluded that in the new Cas9-PBase fusion mutants, Cas9-PBase N347A_R376E_D450N and Cas9-PBase N347E_R376E_D450N have lower off-target rates than Cas9-PBase R372A_K375A_D450N, and Cas9-PBase R315A_N347E_D450N has a slightly higher off-target rate than Cas9-PBase R372A_K375A_D450N. This conclusion is consistent with the off-target ratio conclusion tested in the Split GFP cell line in Example 4.

[0156] Sequencing of PCR bands of some site-specific integrated single-cell clones revealed that compared with the theoretical integration sequence, most clones had small base deletions or base insertions (Indel) in the junction region, and the base deletions or insertions did not have an obvious quantitative or sequential regularity, which was consistent with the gene insertion characteristics of non-homologous recombination ( Fig. 9 ).

[0157] Example 7 Testing the site-specific integration efficiency of other mutants at the N347 site

[0158] The N347 site is a key site for the binding of PBase and the upstream and downstream "TTAA" of the targeted DNA. The purpose of this experiment is to test other mutations in this site and test the corresponding site-specific integration efficiency.

[0159] Using Cas9-PBase R376E_D450N as a template, the PBase N347 position was mutated into 19 other amino acid forms, and different Cas9-PBase N347x_R376E_D450N (x represents various amino acid mutations) mutants were tested for site-directed integration on SplitGFP cells. The experimental method is the same as described in Example 3 and will not be repeated here.

[0160] Experimental results and conclusions: The co-transfection efficiency of different Cas9-PBase N347x_R376E_D450N mutants and N GFP transposon donors was similar ( Fig.11A), but the site-specific integration ratios were quite different. Among them, the mutation of N347 to S, T, Y, Q, C, G, A, E and other amino acids all had higher site-specific integration efficiency ( Fig. 11B and Fig. 11C ), since both E and D amino acid residues are negatively charged, based on the fact that E can reduce the off-target rate, it is speculated that mutation to D can also achieve a similar effect.

[0161] Example 8 Testing the site-specific integration efficiency of other mutants at the D450 site

[0162] The D450 site is a key site that affects the efficiency of PBase splicing transposon donors. The purpose of this experiment is to test other mutations in this site and test the corresponding site-specific integration efficiency.

[0163] Using Cas9-PBase N347A_R376E as a template, the PBase D450 position was mutated into other amino acid forms, and different Cas9-PBase N347A_R376E_D450x (x represents different amino acid mutations) mutants were tested for site-directed integration on SplitGFP cells. The experimental method is the same as described in Example 3 and will not be repeated here.

[0164] Experimental results and conclusions: The co-transfection efficiency of different Cas9-PBase N347A_R376E_D450x mutants and N GFP transposon donors was similar ( Fig. 12A ), but the site-specific integration ratio is quite different. Among them, D450N has the highest efficiency, while D450 has a lower efficiency when it is not mutated. Mutations to A, S, T, C, Y and other amino acids have a significant improvement over non-mutated ones ( Fig. 12B and Fig. 12C ).

[0165] Example 9 Adding other PBase site mutations to Cas9-PBase N347A_R376E_D450N to improve site-directed integration efficiency

[0166] The site where PBase and ITR sequences bind may affect the efficiency of PBase cleaving the transposon donor, and thus affect the efficiency of Cas9-PBase mutant cleaving and site-directed integration of the transposon donor. Therefore, mutating the binding site of the ITR sequence may improve the site-directed integration efficiency of the Cas9-PBase mutant. The purpose of this experiment is to further explore the effect of the binding site mutation of the ITR sequence on the site-directed integration efficiency in the Cas9-PBaseN347A_R376E_D450N mutant.

[0167] According to the resolved structure of PBase protein and ITR (PDB number 6x67), the key sites of PBase and ITR binding were analyzed, and these sites were mutated into positively charged or other polar amino acids. The mutant plasmid construction process is the same as described in Example 1, and will not be repeated here.

[0168] The transfection and site-directed integration efficiencies of the constructed different Cas9-PBase N347A_R376E_D450N MutX mutants were tested in Split cells, using the same methods as in Examples 3, 7, and 8. The site-directed integration efficiency of Cas9-PBase N347A_R376E_D450N was defined as 100%, and Cas9-PBase N347A_R376E_D450NMutX mutants with higher efficiency were screened.

[0169] Experimental results and conclusions: After screening, it was found that on the basis of the Cas9-PBase N347A_R376E_D450N triple mutation, the fourth mutation was added. The proportion of the site-specific integration efficiency improved by the newly added fourth site mutation MutX is shown in the numbers in brackets after the mutation: A166K (49.30%), N286R (138.52%), S289R (25.74%), G302R (15.16%), Q441R (36.87%), S498N (7.89%), E520R (27.01%), A521R (78.63%), T523R (32.89%), T557R (153.26%).

[0170] ( Fig.13 )

[0171] Cas9 amino acid sequence (including NLS) (SEQ ID NO: 1)

[0172]

[0173] Linker 3*G4S amino acid sequence (SEQ ID NO:2)

[0174] GGGGSGGGGSGGGGS wt Hyperactive PBase amino acid sequence (SEQ ID NO:3)

[0175] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0176] Cas9-wt Hyperactive PBase amino acid sequence (SEQ ID NO:4)

[0177] GSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSE ILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDR FDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGI KILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGT VRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVD TLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0178] Note: The underlined part is the PBase part.

[0179] Amino acid sequence of PBase R315A_N347E_D450N (SEQ ID NO:5)

[0180] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGAGTQTNGVPLGEYYVKELSKPVHGSCRNITCDEWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0181] Amino acid sequence of Cas9-PBase R315A_N347E_D450N (SEQ ID NO:6)

[0182] GSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSE ILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDR FDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGI KILMMCDSGTKYMINGMPYLGAGTQTNGVPLGEYYVKELSKPVHGSCRNITCDEWFTSIPLAKNLLQEPYKLTIVGT VRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVD TLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0183] Note: The underlined part is the PBase part.

[0184] Amino acid sequence of PBase N347E_R376E_D450N (SEQ ID NO:7)

[0185] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDEWFTSIPLAKNLLQEPYKLTIVGTVRSNKEEIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0186] Amino acid sequence of Cas9-PBase N347E_R376E_D450N (SEQ ID NO:8)

[0187] GSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSE ILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDR FDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGI KILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDEWFTSIPLAKNLLQEPYKLTIVGT VRSNKEEIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVD TLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0188] Note: The underlined part is the PBase part.

[0189] Amino acid sequence of PBase N347A_R376E_D450N (SEQ ID NO:9)

[0190] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDAWFTSIPLAKNLLQEPYKLTIVGTVRSNKEEIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0191] Amino acid sequence of Cas9-PBase N347A_R376E_D450N (SEQ ID NO:10)

[0192] GSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSE ILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDR FDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGI KILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDAWFTSIPLAKNLLQEPYKLTIVGT VRSNKEEIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVD TLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0193] Note: The underlined part is the PBase part.

[0194] Amino acid sequence of PBase R372A_K375A_D450N (SEQ ID NO:11)

[0195] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0196] Amino acid sequence of Cas9-PBase R372A_K375A_D450N (SEQ ID NO:12)

[0197] GSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSE ILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFF TDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDR FDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGI KILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGT VASNAREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVD TLNQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF

[0198] Note: The underlined part is the PBase part.

[0199] wt Hyperactive PBase encoding DNA sequence (SEQ ID NO: 13)

[0200]

[0201] Cas9-wt Hyperactive PBase encoding DNA sequence (SEQ ID NO: 14)

[0202] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCA GAGCGACGACGAACTGGTGGGCGAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCG ACACCGAGGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAG AACGTGATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAA GAACAAGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGA GGGGCCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATC AGCGAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGA CACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCA CCGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATC AGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGAT CTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGG GCTTCAGAGGCAGATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATG TGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCT GGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACAACTGGTTCA CCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAG CGGGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCT GACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCA ACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGGACCAGATG TGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTG CATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGC GGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGG GACAACATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAA GAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGA TCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0203] Note: The underlined part is the part encoding PBase.

[0204] Cas9-PBase N347A_R376E_D450N encoding DNA sequence (SEQ ID NO: 135)

[0205] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGC GAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCG ACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGG CAGCTCCCTGGCCAGCAACAGAATCCTGACCCT CGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAATGGACGACAAGAGCATCAGACCC ACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTCATCCACCAGTGCATCCAGAACT ACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCAGATGCCCCTTCAGAGTGTACAT CCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGGCACCAAGTACATGATCAACGGC ATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGC CTGTGCATGGCAGCTGCAGGAACATCACCTGCGACgcCTGGTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCA GGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGgagGAGATCCCAGAGGTGCTGAAGAACAGC AGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCG CCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGT GATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGaACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAG ACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCAACAGCTTCATCATCTACAGCCA CAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCA GCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAACATCAGCAACATCCTGCCCAAG GAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAG CAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGT GCCAGAGCTGTTTCTGA

[0206] Note: The underlined part is the part encoding PBase.

[0207] Cas9-PBase N347E_R376E_D450N encoding DNA sequence (SEQ ID NO: 136)

[0208] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGC GAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCG ACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGG CAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACAAGCACTGCTGGTCC ACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGGCCCCACCAGAATGT GCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTG GACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACACCAACGAGGACGAG ATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCACCGACGACCTGTTCG ACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAAT GGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTC ATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCA GATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGG CACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTAC TACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACgagTGGTTCACCAGCATCC CCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGgaGGAGAT CCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTG GTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGA GCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGaACCAGATGTGCAG CGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCA ACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAAC CTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAA CATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGA CCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGC CGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0209] Note: The underlined part is the part encoding PBase.

[0210] Cas9-PBase R315A_N347E_D450N encoding DNA sequence (SEQ ID NO: 137)

[0211] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGC GAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCG ACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGG CAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACAAGCACTGCTGGTCC ACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGGCCCCACCAGAATGT GCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTG GACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACACCAACGAGGACGAG ATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCACCGACGACCTGTTCG ACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAAT GGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTC ATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCA GATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGG CACCAAGTACATGATCAACGGCATGCCCTACCTGGGCgccGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTAC TACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACgagTGGTTCACCAGCATCC CCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGCGGGAGAT CCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTG GTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGA GCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGaACCAGATGTGCAG CGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCA ACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAAC CTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAA CATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGA CCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGC CGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0212] Note: The underlined part is the part encoding PBase.

[0213] AAVS1-PB reporter-BSD-tdTomato plasmid sequence (SEQ ID NO: 15)

[0214]

[0215] Receiving plasmid sequence (SEQ ID NO: 16)

[0216]

[0217] N GFP transposon donor plasmid sequence (SEQ ID NO:17)

[0218] Cas9-PBase N347A_R376E_D450N-ROSA gRNA plasmid sequence (SEQ ID NO: 18)

[0219] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACG ACGAACTGGTGGGCGAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGA GGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTG ATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACA AGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGG CCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGC GAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACA CCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCAC CGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATC AGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGA TCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCT GGGCTTCAGAGGCAGATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATG ATGTGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGC CCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACgcCTG GTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGC AACAAGgagGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACG GCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGC CAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTG aACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCA ATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAA GAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAG AGATACCTGCGGGACAACATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAAC CCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAA ATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0220] Note: The underlined part is the part encoding PBase.

[0221] Cas9-PBase N347A_R376E_D450N-GSH1 gRNA plasmid sequence (SEQ ID NO: 19)

[0222] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACG ACGAACTGGTGGGCGAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGA GGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTG ATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACA AGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGG CCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGC GAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACA CCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCAC CGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATC AGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGA TCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCT GGGCTTCAGAGGCAGATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATG ATGTGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGC CCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACgcCTG GTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGC AACAAGgagGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACG GCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGC CAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTG aACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCA ATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAA GAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAG AGATACCTGCGGGACAACATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAAC CCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAA ATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0223] Note: The underlined part is the part encoding PBase.

[0224] Cas9-PBase N347A_R376E_D450N-GSH2 gRNA plasmid sequence (SEQ ID NO: 20)

[0225] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACG ACGAACTGGTGGGCGAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGA GGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTG ATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACA AGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGG CCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGC GAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACA CCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCAC CGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATC AGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGA TCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCT GGGCTTCAGAGGCAGATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATG ATGTGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGC CCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACgcCTG GTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGC AACAAGgagGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACG GCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGC CAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTG aACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCA ATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAA GAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAG AGATACCTGCGGGACAACATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAAC CCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAA ATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0226] Note: The underlined part is the part encoding PBase.

[0227] NeoR-GFP transposon donor plasmid sequence (SEQ ID NO:21)

[0228]

[0229] U6-ROSA sgRNA (SEQ ID NO:131)

[0230] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTCGAGTCGCTTCTCGATTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT

[0231] wt Hyperactive PBase-encoding DNA sequence (SEQ ID NO:138)

[0232]

[0233] Cas9-PB-ROSA sgRNA vector (SEQ ID NO: 139):

[0234] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACG ACGAACTGGTGGGCGAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGA GGAAGCCTTCATCGACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTG ATCGAGCAGCCTGGCAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACA AGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGG CCCCACCAGAATGTGCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGC GAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACA CCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCAC CGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATC AGATGCCTGAGAATGGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGA TCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCT GGGCTTCAGAGGCAGATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATG ATGTGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGC CCCTGGGCGAGTACTACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACAACTG GTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGC AACAAGCGGGAGATCCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACG GCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGC CAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTG GACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCA ATATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAA GAAATTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAG AGATACCTGCGGGACAACATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAAC CCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAA ATGCAAAAAAGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0235] Note: The underlined part is the part encoding PBase.

[0236] Cas9-PBase R372A_K375A_D450N encoding DNA sequence (SEQ ID NO: 140)

[0237] GGCTCTAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAACTGGTGGGC GAGGACAGCGACAGCGAGGTCAGCGACCACGTGTCCGAGGACGACGTGCAGTCCGACACCGAGGAAGCCTTCATCG ACGAGGTGCACGAAGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCTGG CAGCTCCCTGGCCAGCAACAGAATCCTGACCCTGCCCCAGAGAACCATCAGAGGCAAGAACAAGCACTGCTGGTCC ACCTCCAAGAGCACCAGGCGGAGCAGAGTGTCCGCCCTGAACATCGTGCGGAGCCAGAGGGGCCCCACCAGAATGT GCAGAAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTG GACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCAGCGCCACCTTCAGAGACACCAACGAGGACGAG ATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGAAAGGACAACCACATGAGCACCGACGACCTGTTCG ACAGATCCCTGAGCATGGTGTACGTGTCCGTGATGAGCAGAGACAGATTCGACTTCCTGATCAGATGCCTGAGAAT GGACGACAAGAGCATCAGACCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTC ATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGATGAGCAGCTGCTGGGCTTCAGAGGCA GATGCCCCTTCAGAGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGG CACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGAGGCACCCAGACAAACGGCGTGCCCCTGGGCGAGTAC TACGTGAAAGAACTGAGCAAGCCTGTGCATGGCAGCTGCAGGAACATCACCTGCGACAACTGGTTCACCAGCATCC CCCTGGCCAAGAACCTGCTGCAGGAACCCTACAAGCTGACCATCGTGGGCACCGTGgccAGCAACgccCGGGAGAT CCCAGAGGTGCTGAAGAACAGCAGATCCAGACCTGTGGGAACAAGCATGTTCTGCTTCGACGGCCCCCTGACCCTG GTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGA GCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGaACCAGATGTGCAG CGTGATGACCTGCAGCAGAAAGACCAACAGATGGCCCATGGCCCTGCTGTACGGCATGATCAATATCGCCTGCATCA ACAGCTTCATCATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAATTCATGCGGAAC CTGTACATGAGCCTGACCTCCAGCTTCATGAGAAAGAGACTGGAAGCCCCCACCCTGAAGAGATACCTGCGGGACAA CATCAGCAACATCCTGCCCAAGGAAGTGCCAGGAACAAGCGACGACAGCACCGAGGAACCCGTGATGAAGAAGAGGA CCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAATGCAAAAAAGTGATCTGC CGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCTGA

[0238] Note: The underlined part is the coding sequence of the PBase mutant.

Claims

1. A PBase protein, characterized in that The PBase protein, based on the amino acid sequence shown in SEQ ID NO: 3, has only one of the following amino acid mutations: (1) N347S+R376E+D450N; (2) N347T+R376E+D450N; (3) N347Y+R376E+D450N; (4) N347Q+R376E+D450N; (5) N347C+R376E+D450N; (6) N347G+R376E+D450N; (7) N347A+R376E+D450N; (8) N347E+R376E+D450N; (9) N347A+R376E+D450N+A166K; (10) N347A+R376E+D450N+N286R; (11) N347A+R376E+D450N+S289R; (12) N347A+R376E+D450N+G302R; (13) N347A+R376E+D450N+Q441R; (14) N347A+R376E+D450N+S498N; (15) N347A+R376E+D450N+E520R; (16) N347A+R376E+D450N+A521R; (17) N347A+R376E+D450N+T523R; (18) N347A+R376E+D450N+T557R.

2. A fusion protein, comprising the PBase protein according to claim 1 fused to a site-specific DNA nuclease, and the original functions of the PBase protein and the specific DNA nuclease are not changed after fusion; the site-specific DNA nuclease and the PBase protein are connected by a linker; the linker is 3*G4S; the specific DNA nuclease includes an NLS sequence, and the structure of the fusion protein from the N-terminus to the C-terminus is: specific DNA nuclease-NLS-linker-PBase protein; The PBase protein in the fusion protein lacks the methionine M at position 1 in the amino acid sequence; The site-specific DNA nuclease is Cas9.

3. The fusion protein according to claim 2, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO: 8 or 10.

4. An isolated nucleic acid encoding the PBase protein according to claim 1 and / or the fusion protein according to claim 2 or 3.

5. The isolated nucleic acid according to claim 4, wherein The isolated nucleic acid encoding the PBase protein is a nucleotide sequence such as the nucleotide sequence shown at positions 4183-5964 of SEQ ID NO: 135 or 136, or the isolated nucleic acid encoding the fusion protein is a nucleotide sequence such as the nucleotide sequence shown in SEQ ID NO: 135 or 136.

6. A gene integration system, comprising the PBase protein according to claim 1 or the fusion protein according to claim 2 or 3, or the isolated nucleic acid according to claim 4 or 5; and piggyBac Transposon donor nucleic acid.

7. The gene integration system according to claim 6, characterized in that The fusion protein includes the PBase protein and the Cas system, and the Cas system includes a Cas protease and a guide RNA (sgRNA).

8. The gene integration system according to claim 7, characterized in that The Cas protease is Cas9 or Cas12 protease.

9. A gene delivery system, comprising one or more vectors, wherein the vectors comprise one or more of the PBase protein according to claim 1, the fusion protein according to claim 2 or 3, the isolated nucleic acid according to claim 4 or 5, and the gene integration system according to any one of claims 6 to 8, and the vectors further comprise a vector carrying an exogenous target gene. piggyBac Transposon donor nucleic acid.

10. The gene delivery system according to claim 9, wherein The vector is an adenovirus vector, a lipid nanoparticle, a lentivirus vector, a ribonucleoprotein complex or a virus-like particle.

11. A gene integration method, comprising the following steps: Using the gene integration system as described in any one of claims 6 to 8, integrating the exogenous target gene into the genome of the host cell; The method is not for diagnostic and / or therapeutic purposes.

12. The integration method according to claim 11, characterized in that: The gene integration system enters the host cell via the gene delivery system according to claim 9 or 10.

13. A pharmaceutical composition comprising one or more of the PBase protein according to claim 1, the fusion protein according to claim 2 or 3, the isolated nucleic acid according to claim 4 or 5, the gene integration system according to any one of claims 6 to 8, and the gene delivery system according to claim 9 or 10.

14. A kit comprising one or more of the PBase protein according to claim 1, the fusion protein according to claim 2 or 3, the isolated nucleic acid according to claim 4 or 5, the gene integration system according to any one of claims 6 to 8, the gene delivery system according to claim 9 or 10, and the pharmaceutical composition according to claim 13.

15. A kit of parts, comprising kit A, wherein kit A comprises one or more of the PBase protein according to claim 1, the fusion protein according to claim 2 or 3, the isolated nucleic acid according to claim 4 or 5, the gene integration system according to any one of claims 6 to 8, the gene delivery system according to claim 9 or 10, the pharmaceutical composition according to claim 13 and the test kit according to claim 14.

16. The kit of claim 15, wherein: The kit also includes kit B, which includes reagents for detecting or screening whether gene integration is successful.

17. The kit of claim 16, wherein: The reagent is a detection reagent that specifically binds to the integrated gene.

18. The kit of claim 17, wherein: The detection reagent is a probe or an antibody.

19. Use of one or more of the PBase protein according to claim 1, the fusion protein according to claim 2 or 3, the isolated nucleic acid according to claim 4 or 5, the gene integration system according to any one of claims 6 to 8, the gene delivery system according to claim 9 or 10, the pharmaceutical composition according to claim 13 and the kit according to claim 14 in the preparation of an agent for integrating a gene, wherein the use is for non-diagnostic or therapeutic purposes.

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