Precise fixed-point insertion method for super-long fragment gene and application thereof

Abstract

The invention relates to the technical field of gene editing, in particular to an ultra-long fragment gene precise fixed-point insertion method and application thereof.According to the method, virus-like particles VICEP are adopted to deliver a ribonucleoprotein complex (RNP), and an integrated defective lentivirus IDLV is adopted to deliver a donor template containing a 5 ' / 3' homologous arm; the two genes have a synergistic effect in the same time window to trigger homologous recombination repair, so that high-efficiency and low-off-target integration of a large-fragment gene at a target site is realized. By adding an inhibitor HDR promoter during infection, non-homologous end ligation (NHEJ) can be further inhibited and insertion efficiency can be improved. The method shows stable knock-in efficiency greater than or equal to 15% in various difficult-to-transfect cells such as iPSC, HUDEP2, K562 and the like, breaks through the traditional AAV capacity upper limit and electrotransfection toxicity bottleneck, and can be widely applied to gene therapy, cell engineering and synthetic biology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene editing, in particular to a method for precise site insertion of ultra-long fragment gene and application thereof. BACKGROUND

[0002] In recent years, CRISPR / Cas9 system combined with adeno-associated virus (AAV), lipid nanoparticle (LNP) or electroporation technology has been widely used in gene therapy, cell engineering and synthetic biology. However, when inserting exogenous sequences with a length greater than 4.7 kb into target loci, these mainstream platforms still face the following technical bottlenecks:

[0003] 1. Vector capacity limitation, the maximum packaging capacity of AAV vector is about 4.7 kb, and the knock-in efficiency of large fragments (≥7 kb) modules in AAV-HDR platform is usually <10%. Although LNP can theoretically encapsulate longer nucleic acids, the increased PDI and intracellular nucleases significantly reduce the effective template content.

[0004] 2. Stability and delivery efficiency, the nucleic acid degradation rate of non-viral systems (LNP, electroporation) in extracellular / intracellular environment can be as high as 70%. Although strong electric field electroporation can transiently introduce Cas9 RNP and donor, the membrane damage caused by it to primary cells such as iPSC and HSC significantly reduces the survival rate and long-term function.

[0005] 3. Limited by multi-gene collaborative insertion, the existing methods are mostly based on single-site HDR, and it is difficult to simultaneously complete the precise integration of multiple loci in the same cell, which limits the construction of complex regulatory networks, disease models and multi-target CAR-T.

[0006] It can be seen that the existing exogenous sequence insertion methods generally have the problems of capacity, activity and multi-site collaboration, which together lead to the problems of low efficiency, high off-target and high cytotoxicity of the existing technology in dealing with large-size gene fragments or multi-gene circuits.

[0007] Therefore, a method for precise site insertion of ultra-long fragment gene is needed to solve the problems of insufficient vector capacity, delivery stability and multi-gene editing ability in the prior art, to provide a universal technical platform for high-load, low-toxicity and expandable ultra-long fragment gene editing SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides a method for precise site insertion of ultra-long fragment gene and application thereof, aiming to solve the problems of insufficient vector capacity, delivery stability and multi-gene editing ability in the background art.

[0009] In one aspect, the present application provides a method for precise site insertion of ultra-long fragment gene, comprising the following steps:

[0010] a ribonucleoprotein complex is formed by combining the functional protein with the targeting single guide RNA, and the ribonucleoprotein complex is loaded into a VICEP vector;

[0011] an HDR donor template is loaded into an IDLV vector;

[0012] the loaded VICEP vector and IDLV vector are delivered to a target cell;

[0013] the target cell is cultured after adding an HDR promoter to the target cell.

[0014] Further, the HDR donor template comprises an exogenous insertion sequence with a length of 2-15 kb, and 5' homologous arms and 3' homologous arms with lengths of 500-1200 bp at both ends, respectively.

[0015] Further, the delivery is specifically: the ratio of the multiplicity of infection (MOI) of the VICEP vector and the IDLV vector is 1:(1-8).

[0016] Further, the target cell is selected from induced pluripotent stem cells, K562, HUDEP2, or Jurkat cells.

[0017] Further, the functional protein is one of SpCas9 protein, SaCas9 protein, or Cas12a protein.

[0018] Further, the exogenous insertion sequence contains a promoter-coding sequence-WPRE functional framework.

[0019] In another aspect, the present application also provides a gene editing kit for the long-fragment gene precise site insertion method, comprising:

[0020] a first viral vector VICEP, loaded with the functional protein and the targeting single guide RNA;

[0021] a second viral vector IDLV, loaded with the HDR donor template;

[0022] a liquid preparation of M3814, the concentration of the liquid preparation of M3814 being 10 mM.

[0023] wherein the first viral vector and the second viral vector are stored separately in a freeze-dried or liquid preparation, and are accompanied by instructions for use.

[0024] Further, the enzyme in the second viral vector IDLV contains a D64V inactivation mutation.

[0025] Further, the shell of the first viral vector VICEP or the second viral vector IDLV carries a CD45 antibody.

[0026] In another aspect, the application also provides an application of the long-fragment gene precise site insertion method in the construction of induced pluripotent stem cell (iPSC) disease models, CAR-T modification of T cells, monogenic genetic diseases and tumor treatment.

[0027] Compared with the prior art, the application has the beneficial effects that:

[0028] 1. The application realizes efficient site insertion of a long-fragment gene greater than 7 kb by delivering ribonucleoprotein complex (RNP) and HDR donor template to target cells respectively through double vectors.

[0029] 2. The application ensures accurate insertion of target genes and avoids non-specific insertion and off-target effects through the homologous arms of the ribonucleoprotein complex (RNP) and the IDLV vector.

[0030] 3. The application efficiently encapsulates the two key components in viral vectors by delivering ribonucleoprotein complex (RNP) and HDR donor template separately, avoids the loading competition problem in traditional methods, and ensures a high viral titer; at the same time, through the optimization of the preparation process, the stable production of virus particles is ensured, and the potential for large-scale production is possessed.

[0031] The foregoing general description and the following detailed description are merely exemplary and explanatory, rather than limiting the present disclosure.

[0032] Other features and aspects of the present disclosure will become more apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0034] Figure 1 The long-fragment gene precise site insertion method provided by the embodiment of the present application is shown in the general schematic diagram.

[0035] Figure 2 The GAPDH site insertion experimental result graph provided by the embodiment of the present application is shown in the general schematic diagram.

[0036] Figure 3A PGK1 site insertion experiment result graph provided for the embodiment of the present application is shown in FIG. 1;

[0037] Figure 4 An AAVS1 site insertion experiment result graph provided for the embodiment of the present application is shown in FIG. 2;

[0038] Figure 5 A cell activity comparison graph after delivery of the present application and an electrotransformation system provided for the embodiment of the present application is shown in FIG. 3;

[0039] Figure 6 A specific detection result graph of the present application provided for the embodiment of the present application is shown in FIG. 4;

[0040] Figure 7 An efficiency verification result graph of the present application in iPS and other primary cells provided for the embodiment of the present application is shown in FIG. 5;

[0041] Figure 8 An effect verification graph of the present application for efficiently realizing site-specific insertion of an ultra-long fragment provided for the embodiment of the present application is shown in FIG. 6. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0043] In the description of the present application, it should be understood that the terms “center”, “upper”, “lower”, “front”, “back”, “left”, “right”, “vertical”, “horizontal”, “top”, “bottom”, “inner”, “outer” and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0044] The terms “first” and “second” are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with “first” and “second” can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of “a plurality of” is two or more.

[0045] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connecting" should be understood in a broad sense, for example, it can be fixed connection, or detachable connection, or integral connection, it can be mechanical connection, or electrical connection, it can be direct connection, or indirect connection through intermediate medium, or internal communication of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0046] Referring to Figure 1 As shown, according to the method for precise site insertion of ultralong fragment gene of the embodiment of the present application, the method comprises the following steps:

[0047] The functional protein is combined with the targeting single guide RNA to form a ribonucleoprotein complex, and the ribonucleoprotein complex is loaded into a VICEP vector;

[0048] The HDR donor template is loaded into an IDLV vector;

[0049] The loaded VICEP vector and IDLV vector are delivered to target cells;

[0050] The target cells are cultured after adding an HDR promoter to the target cells;

[0051] The HDR promoter is preferably one of M3814, AZD7648, VX-984 or RS-1;

[0052] The concentration of the HDR promoter is preferably 1-2 μM;

[0053] The culture time is preferably 72-120 h.

[0054] Specifically, the titer of the VICEP vector is greater than or equal to 1 x 10 7 TUml-1, after the target cells are cultured, it is confirmed by flow cytometry or amplification-sequencing that the exogenous sequence has been precisely integrated at the predetermined site, and the predetermined site is AAVS1, GAPDH or PGK1 locus.

[0055] It can be understood that the VICEP in the present application can precisely guide the functional protein to form a complex with the targeting single guide RNA, directionally cut the DNA sequence of the target site to produce a double-strand break (DSB), thereby starting the homologous recombination repair (HDR) or non-homologous end joining (NHEJ) pathway of the cell.

[0056] It can be understood that the competitive NHEJ pathway can be effectively inhibited by using the HDR promoter, and the activation of the HDR repair path is promoted. These small molecules enhance the HDR signal pathway, improve the gene knock-in efficiency, and reduce the occurrence of non-specific repair or insertion events, thereby improving the overall efficiency of gene editing.

[0057] In some embodiments, the HDR donor template comprises an exogenous insertion sequence with a length of 2-15 kb, and 5' and 3' homologous arms with lengths of 500-1200 bp located at both ends, respectively.

[0058] It can be understood that the IDLV serves as a donor template vector, carries a large fragment expression cassette of a target gene, and carries 5' and 3' homologous arms homologous to a target genomic site on both sides. These homologous arms are precisely inserted into the target DNA through the HDR mechanism. In the design of the IDLV, the risk of random integration is greatly reduced by the integration enzyme mutation, ensuring high specificity and high safety of gene insertion.

[0059] In some embodiments, the ratio of the multiplicity of infection (MOI) of the VICEP vector and the IDLV vector is 1:(1-8).

[0060] Specifically, the VICEP and IDLV vectors are simultaneously delivered in the target cells. The VICEP delivers a ribonucleoprotein complex (RNP) to direct the cutting of the target site, and the IDLV provides a homologous donor template for repair. The two work together in the cell to complete the precise gene insertion through the double-strand break of the RNP and the HDR repair of the IDLV donor template, respectively.

[0061] In some embodiments, the target cells are selected from induced pluripotent stem cells, K562, HUDEP2, or Jurkat cells.

[0062] Specifically, the application also performs well in different cell types. In induced pluripotent stem cells (iPSC) and erythroid progenitor cells (HUDEP2), the gene knock-in efficiency of the method of the application is 70% and 40%, respectively, which is about 2 times higher than that of the traditional method (using a single AAV vector or a lipid nanoparticle vector) of 20%-30%.

[0063] Specifically, the gene insertion efficiency of the application in K562 cells is 90%; the gene insertion efficiency of the application in iPSC cell lines is 70%; and the gene insertion efficiency of the application in HUDEP2 cell lines is 40%.

[0064] It can be understood that the present application has wide cross-cell type adaptability and can achieve efficient gene editing in various cell types, including but not limited to iPSC (induced pluripotent stem cell), HUDEP2 (erythroid progenitor cell), K562 (leukemia cell line), etc., including difficult-to-transfect cell types, and its high efficiency and stability in various cells.

[0065] In some embodiments, the functional protein is one of SpCas9 protein, SaCas9 protein or Cas12a protein; the functional protein is preferably SpCas9 protein.

[0066] In some embodiments, the exogenous insertion sequence contains a promoter-coding sequence-WPRE functional framework.

[0067] In an embodiment, the gene editing tool kit of the long-fragment gene precise site insertion method of the present application comprises:

[0068] The first viral vector VICEP carries the functional protein and the targeting single guide RNA as a load;

[0069] The second viral vector IDLV carries the HDR donor template as a load;

[0070] A liquid preparation of M3814, the concentration of the liquid preparation of M3814 is 10mM.

[0071] The first viral vector and the second viral vector are stored separately in a freeze-dried or liquid preparation, and are accompanied by instructions for use.

[0072] Specifically, the instructions for use are used to indicate that the recommended MOI ratio of VICEP:IDLV is 1:(1-8)

[0073] In some embodiments, the enzyme in the second viral vector IDLV contains a D64V inactivation mutation.

[0074] In some embodiments, the shell of the first viral vector VICEP or the second viral vector IDLV carries a CD45 antibody.

[0075] In an embodiment, the present application also provides an application of the long-fragment gene precise site insertion method in treating β-thalassemia, sickle anemia or congenital immunodeficiency.

[0076] Embodiment 1

[0077] S1, combine SpCas9 protein with targeting single guide RNA to obtain ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into VICEP vector;

[0078] S2, load the HDR donor template with the exogenous insert sequence of 2-15 kb in length and 5' and 3' homology arms of 500-1200 bp in length on both ends into the IDLV vector;

[0079] S3, simultaneously deliver the loaded VICEP vector and IDLV vector into induced pluripotent stem cells (iPSCs) at a multiplicity of infection of VICEP:IDLV = 1:2;

[0080] S4, add 1 μM M3814 to the induced pluripotent stem cells (iPSCs) and co-culture with viruses for 24 h, and continue to culture for 120 h.

[0081] Example 2

[0082] S1, combine Cas9 protein and single guide RNA to obtain a ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into the VICEP vector;

[0083] S2, load the HDR donor template with the exogenous insert sequence of 2-15 kb in length and 5' and 3' homology arms of 500-1200 bp in length on both ends into the IDLV vector;

[0084] S3, simultaneously deliver the loaded VICEP vector and IDLV vector into induced pluripotent stem cells (iPSCs) at a multiplicity of infection of VICEP:IDLV = 1:1;

[0085] S4, add 1 μM M3814 to the induced pluripotent stem cells (iPSCs) and co-culture with viruses for 24 h, and continue to culture for 120 h.

[0086] Example 3

[0087] S1, combine Cas9 protein and single guide RNA to obtain a ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into the VICEP vector;

[0088] S2, load the HDR donor template with the exogenous insert sequence of 2-15 kb in length and 5' and 3' homology arms of 500-1200 bp in length on both ends into the IDLV vector;

[0089] S3, simultaneously deliver the loaded VICEP vector and IDLV vector into induced pluripotent stem cells (iPSCs) at a multiplicity of infection of VICEP:IDLV = 1:0.5;

[0090] S4, add 2 mM M3814 to the induced pluripotent stem cells (iPSCs) and co-culture with viruses for 24 h, replace the medium, and continue to culture for 120 h.

[0091] S5, confirm that the exogenous sequence has been precisely integrated at the predetermined site by flow cytometry, and calculate the gene insertion rate.

[0092] Example 4

[0093] S1, combine Cas9 protein and single guide RNA to obtain a ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into a VICEP vector;

[0094] S2, load a HDR donor template with an exogenous insertion sequence of 2-15 kb in length and 5' and 3' homologous arms of 500-1200 bp in length at both ends into an IDLV vector;

[0095] S3, simultaneously deliver the loaded VICEP vector and IDLV vector to erythroid progenitor cells (HUDEP2) according to an infection multiplicity of VICEP:IDLV = 1:1;

[0096] S4, add 2 mM M3814 to the erythroid progenitor cells (HUDEP2) and co-culture with viruses for 24 h, replace the medium, and continue to culture for 120 h.

[0097] S5, confirm that the exogenous sequence has been precisely integrated at the predetermined site by flow cytometry.

[0098] Example 5

[0099] S1, combine Cas9 protein and single guide RNA to obtain a ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into a VICEP vector;

[0100] S2, load a HDR donor template with an exogenous insertion sequence of 2-15 kb in length and 5' and 3' homologous arms of 500-1200 bp in length at both ends into an IDLV vector;

[0101] S3, simultaneously deliver the loaded VICEP vector and IDLV vector to K562 cells according to an infection multiplicity of VICEP:IDLV = 1:1;

[0102] S4, add 2 mM M3814 to the K562 cells and co-culture with viruses for 24 h, replace the medium, and continue to culture for 120 h.

[0103] S5, confirm the precise integration of the exogenous sequence at the predetermined site by flow cytometry.

[0104] Example 6

[0105] S1, combine the Cas9 protein with the targeting single guide RNA to obtain a ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into the VICEP vector;

[0106] S2, load the HDR donor template with an exogenous insertion sequence of 2-15 kb in length and 5' and 3' homologous arms of 500-1200 bp in length at both ends into the IDLV vector;

[0107] S3, simultaneously deliver the loaded VICEP vector and IDLV vector to Jurkat cells at a multiplicity of infection of VICEP:IDLV = 1:1;

[0108] S4, add 2 μM M3814 to the Jurkat cells and co-culture with the virus for 24 h, and continue to culture for 120 h.

[0109] S5, confirm the precise integration of the exogenous sequence at the predetermined site by flow cytometry.

[0110] Example 7

[0111] S1, combine the Cas9 protein with the targeting single guide RNA to obtain a ribonucleoprotein complex (CRISPR / Cas9 RNP), and load the ribonucleoprotein complex into the VICEP vector;

[0112] S2, load the HDR donor template with an exogenous insertion sequence of 2-15 kb in length and 5' and 3' homologous arms of 500-1200 bp in length at both ends into the IDLV vector;

[0113] S3, simultaneously deliver the loaded VICEP vector and IDLV vector to HUDEP2 cells at a multiplicity of infection of VICEP:IDLV = 1:4;

[0114] S4, add 2 μM M3814 to the HUDEP2 cells and co-culture with the virus for 24 h, and continue to culture for 120 h.

[0115] S5, confirm the precise integration of the exogenous sequence at the predetermined site by flow cytometry.

[0116] The effect of a super-long fragment gene precise site insertion method (hereinafter referred to as the TIVID system) of the present application is tested:

[0117] 1、The gene insertion rates of the above-mentioned embodiments 3-7 are calculated: the TIVID system of the application achieves gene insertion efficiencies of 70% and 40% in induced pluripotent stem cells (iPSC) and erythroid progenitor cells (HUDEP2), respectively, which is about 2 times higher than the traditional method (using a single AAV vector or a lipid nanoparticle vector) of 20%-30%; the TIVID system of the application has a gene insertion efficiency of 90% in K562 cells, 70% in iPSC cells, and 40% in HUDEP2 cells.

[0118] 2、GAPDH and PGK1 site insertion experiments

[0119] K562 cells were selected for the experiment, and the target genes were GAPDH and PGK1, respectively. The exogenous insertion sequence was the mNeonGreen fluorescent gene, and the homologous arms matching the exon / intron boundaries of GAPDH or PGK1 were designed.

[0120] (1) GAPDH site insertion experiment:

[0121] Experimental group:

[0122] The Cas9 protein was combined with the target single guide RNA (sgRNA) corresponding to the GAPDH site to obtain a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex was loaded into the VICEP vector;

[0123] The mNeonGreen donor template with homologous arms matching the exon / intron boundaries of GAPDH was loaded into the IDLV vector;

[0124] The above-loaded VICEP vector and IDLV vector were simultaneously delivered into K562 cells, and the MOI of the VICEP vector and the IDLV vector was 4;

[0125] After delivery, 2 μM of small molecule drug (HDR promoter M3814) was added to the K562 cells; real-time detection of exogenous gene expression mNeonGreen was performed by flow cytometry (FACS), and ICE analysis was performed at 12 days.

[0126] Control group 1 (VICEP only):

[0127] The Cas9 protein was combined with the target single guide RNA (sgRNA) corresponding to the GAPDH site to obtain a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex was loaded into the VICEP vector;

[0128] The VICEP vector containing Cas9 / sgRNA complex was delivered in K562 cells, and the MOI of the VICEP vector was 4;

[0129] The mNeonGreen expressed by the exogenous gene was detected in real time by flow cytometry (FACS), and ICE analysis was performed on day 12.

[0130] Control group 2 (IDLV only):

[0131] The mNeonGreen donor template with homologous arms matching the GAPDH exon / intron boundary was loaded into the IDLV vector;

[0132] The IDLV vector containing the mNeonGreen donor template with homologous arms matching the GAPDH exon / intron boundary was delivered in K562 cells, and the MOI of the IDLV vector was 4;

[0133] The mNeonGreen expressed by the exogenous gene was detected in real time by flow cytometry (FACS), and ICE analysis was performed on day 12.

[0134] Control group 3 (Control):

[0135] The Cas9 protein was combined with a target single guide RNA (sgRNA) corresponding to the GAPDH site to obtain a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex was loaded into the VICEP vector;

[0136] The mNeonGreen donor template with homologous arms matching the GAPDH exon / intron boundary was loaded into the IDLV vector;

[0137] The VICEP vector containing Cas9 / sgRNA complex and the IDLV vector containing the mNeonGreen donor template with homologous arms matching the GAPDH exon / intron boundary were simultaneously delivered in K562 cells, and the MOI of the VICEP vector and the IDLV vector was 4;

[0138] The mNeonGreen expressed by the exogenous gene was detected in real time by flow cytometry (FACS), and ICE analysis was performed on day 12.

[0139] The results are shown in Figure 2

[0140] Figure 2 ​In the middle, part A depicts the IDLV donor construction map for GAPDH site mNeonGreen knock-in, which details the positions of 5' homologous arm, 3' homologous arm, E2A self-splicing sequence and mNeonGreen insertion fragment, and labels the target region of sgGAPDH and its corresponding mutation site;

[0141] Part B shows the flow cytometry detection results of exogenous mNeonGreen expression at GAPDH site at 0, 3, 6, 9 and 12 days in a line chart, in which "VICEP only" and "IDLV only" are used as negative controls, and the knock-in efficiency of the combined delivery group is compared under the conditions of adding 2 μM M3814 and not adding 2 μM M3814, and the MOI of VICEP and IDLV in each group is 4, and the data in the figure show that 2 μM M3814 has good KI% improvement effect, and the KI reaches a relatively stable level at day 3;

[0142] Part C shows the Indel editing results of GAPDH site at day 12 in a column chart, which compares the Indel rates under different delivery methods (IDLV only, VICEP only and combined delivery) and different drug treatments (control, 2 μM M3814), and the data in the figure show that under the condition of MOI of VICEP being 4, the editing efficiency of each group reaches a near-saturated state, and can reach an editing efficiency of about 90%.

[0143] (2) PGK1 site insertion experiment:

[0144] Experimental group:

[0145] The Cas9 protein is combined with the target single guide RNA (sgRNA) corresponding to the mNeonGreen gene to obtain a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex is loaded into a VICEP vector;

[0146] An mNeonGreen donor template with homologous arms matching the PGK1 exon / intron boundary is loaded into an IDLV vector;

[0147] The above loaded VICEP vector and IDLV vector are simultaneously delivered in K562 cells, and the MOI of the VICEP vector and the IDLV vector is 4;

[0148] After delivery, 2 μM small molecule drug (HDR promoter M3814) is added to the K562 cells; real-time detection of exogenous gene expression mNeonGreen is performed by flow cytometry (FACS), and at day 12, ICE analysis is performed.

[0149] Control group 1 (VICEP only):

[0150] The Cas9 protein was combined with a target single guide RNA (sgRNA) corresponding to the mNeonGreen gene to obtain a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex was loaded into a VICEP vector

[0151] The VICEP vector containing the Cas9 / sgRNA complex was delivered in K562 cells, and the MOI of the VICEP vector was 4;

[0152] The mNeonGreen expressed by the exogenous gene was detected in real time by flow cytometry (FACS), and at the same time on day 12, ICE analysis was performed.

[0153] Control group 2 (IDLV only):

[0154] The mNeonGreen donor template with homologous arms matching the PGK1 exon / intron boundary was loaded into an IDLV vector;

[0155] The IDLV vector containing the mNeonGreen donor template with homologous arms matching the PGK1 exon / intron boundary was delivered in K562 cells, and the MOI of the IDLV vector was 4;

[0156] The mNeonGreen expressed by the exogenous gene was detected in real time by flow cytometry (FACS), and at the same time on day 12, ICE analysis was performed.

[0157] Control group 3 (Control):

[0158] The Cas9 protein was combined with a target single guide RNA (sgRNA) corresponding to the PGK1 site to obtain a Cas9 / sgRNA complex, and the Cas9 / sgRNA complex was loaded into a VICEP vector;

[0159] The mNeonGreen donor template with homologous arms matching the PGK1 exon / intron boundary was loaded into an IDLV vector;

[0160] The VICEP vector containing the Cas9 / sgRNA complex and the IDLV vector containing the mNeonGreen donor template with homologous arms matching the PGK1 exon / intron boundary were simultaneously delivered in K562 cells, and the MOI of the VICEP vector and the IDLV vector was 4;

[0161] Real-time detection of mNeonGreen expression of exogenous gene was performed by flow cytometry (FACS), and ICE analysis was performed at day 12.

[0162] Results are shown in Figure 3

[0163] Figure 3 In the above, part A depicts the IDLV donor construction map for PGK1 site mNeonGreen knock-in, and the positions of 5' homologous arm, 3' homologous arm, E2A self-splicing sequence and mNeonGreen insertion fragment are marked in detail, and the targeting region of sgPGK1 and its corresponding mutation site are also labeled; part B shows the flow cytometry detection results of exogenous mNeonGreen expression of PGK1 site at 0, 3, 6, 9, 12 days in a line chart, and "VICEP only" and "IDLV only" are used as negative controls, and the knock-in efficiency of the combined delivery group is compared under the condition of whether adding 2 μM M3814; part C shows the Indel editing results of PGK1 site at day 12 in a column chart, and the Indel rates under different delivery modes (IDLV only, VICEP only and combined delivery) and different drug treatments (control, 2 μM M3814) are compared, and the data in the figure show that under the condition of MOI of VICEP being 4, the editing efficiency of each group can reach about 65%-90% editing efficiency.

[0164] (3) In summary

[0165] As shown in Figure 2 B and Figure 3 B, GAPDH and PGK1 both showed obvious green fluorescence signals at around day 3. The insertion rate of GAPDH reached a relatively stable platform at day 3, while PGK1 had nearly doubled during the period from day 3 to day 6. This difference suggests that there is a difference in the time window for HDR integration at different gene sites. On the other hand, without the addition of small molecules, the KI% is relatively low, but after adding 2 μM M3814, the KI efficiency can be increased to 10%-20%, indicating that M3814 can effectively promote HDR.

[0166] As shown in Figure 2 C and Figure 3 C, ICE analysis showed that the MOI=4 VICEP we gave was sufficient, and all could reach an editing efficiency of 65-90%.

[0167] ​The above results show that the application can obtain high-efficiency and stable site insertion at both GAPDH and PGK1 sites in K562 cells, different small molecule drugs have a certain universal promoting effect on the HDR process, and there are significant differences in time course and integration efficiency between different gene sites. In summary, these findings lay a methodological foundation for the practical application of the TIVID system in gene therapy and large-scale engineering cell modification.

[0168] 3. AAVS1 safe site insertion experiment

[0169] To further verify the universality of the application, especially in the application of safe sites, the application selects the classic gene editing target AAVS1 for insertion experiment. The AAVS1 site is located in the open chromatin region of human chromosome 19, and is a well-verified safe site suitable for long-term gene expression and does not interfere with the function of endogenous genes. Compared with the mNeonGreen insertion of GAPDH or PGK1, the insertion fragment is larger, about 2.5 kb.

[0170] K562 cells were selected for the experiment, the target gene was AAVS1 safe site, and the exogenous insertion sequence was GFP or BFP fluorescent gene. The 5' and 3' homologous arms homologous to the AAVS1 genomic region were designed.

[0171] (1) When the exogenous insertion sequence is GFP, the specific experimental steps are as follows:

[0172] Experiment group: combine the Cas9 protein and the target single guide RNA (sgRNA) corresponding to the AAVS1 site to obtain a Cas9 / sgRNA complex, and load the Cas9 / sgRNA complex into the VICEP vector;

[0173] Load the 5' and 3' homologous arms homologous to the AAVS1 genomic region and the GFP fluorescent gene into the IDLV vector;

[0174] Simultaneously deliver the above loaded VICEP vector and IDLV vector in K562 cells, the MOI of the VICEP vector is 4, and the MOI of the IDLV vector is designed in a gradient dose of 0-16;

[0175] After delivery, 2 μM of small molecule drug (HDR promoter M3814) is added to the K562 cells;

[0176] After 12 days of culture, the gene insertion efficiency and stability of the AAVS1 site are verified by flow cytometry and Sanger sequencing.

[0177] Control group: combine Cas9 protein with the target single guide RNA (sgRNA) corresponding to the AAVS1 site to obtain a Cas9 / sgRNA complex, and load the Cas9 / sgRNA complex into the VICEP vector;

[0178] Load the 5' and 3' homologous arms homologous to the AAVS1 genomic region and the GFP fluorescent gene into the IDLV vector;

[0179] Simultaneously deliver the above-mentioned loaded VICEP vector and IDLV vector in K562 cells, the MOI of the VICEP vector is 4, and the MOI of the IDLV vector is gradient-dosed from 0 to 16;

[0180] After 12 days of culture, the gene insertion efficiency and stability of the AAVS1 site are verified by flow cytometry and Sanger sequencing.

[0181] (2) When the exogenous insertion sequence is BFP, the GFP fluorescent gene in the above-mentioned specific experimental steps is replaced by a BFP fluorescent gene.

[0182] (3) The results are shown in Figure 4 .

[0183] In the figure: part A shows the IDLV donor design schematic for inserting GFP or BFP at the AAVS1 site. The donor contains the EFla promoter-driven GFP / BFP gene, the Wpre element, and the 5' and 3' homologous arms (HA) at both ends of the insertion fragment, which realizes HDR-mediated GFP gene knock-in through cooperation with sgAAVS1-guided VICEP.

[0184] Parts B and C show the results of columnar graph analysis of the Indel ratio of the AAVS1 site and the GFP gene knock-in efficiency at day 12 in K562 cells by TIVID delivery of the GFP Figure 4 B) or BFP Figure 4 C) donor under different drug treatments (control, 2 μM M3814) and different MOIs of the DLV vector. The overall KI% is increased by nearly 1-2 times after the addition of small molecules.

[0185] It can be seen that with the increase of MOI, the fluorescence signal positive rate (KI%) of GFP or BFP integration at the AAVS1 site shows an increasing trend: when MOI = 1, the KI rate may be only 5%, but when MOI is increased to 8, the knock-in efficiency of the 2 μM M3814 group can soar to the level of 80%, and further increasing MOI can even reach the near-saturated editing state of 90% ( Figure 4 B, C).

[0186] The above results collectively indicate that the AAVS1 site can successfully integrate about 2.5 kb sequences under the system of the present application, and the editing efficiency is significantly positively correlated with the MOI of IDLV. In general, the AAVS1 large capacity insertion test provides strong experimental evidence and optimization ideas for subsequent higher level gene therapy applications.

[0187] 4. Cell activity detection

[0188] Control experiment design:

[0189] Control group: combine Cas9 protein and sgRNA corresponding to the AAVS1 site to obtain Cas9 / sgRNA complex, load the Cas9 / sgRNA complex into the VICEP vector; load the 5' and 3' homologous arms homologous to the AAVS1 genomic region and the GFP fluorescent gene into the IDLV vector; simultaneously deliver the above loaded VICEP vector and IDLV vector in iPS cells, the MOI of the VICEP vector and IDLV vector is 2 and 4 respectively, and after the delivery is completed, 2 μM small molecule drug (HDR promoter M3814) is not added in the iPS cells; real-time detection of cell activity by flow cytometry.

[0190] Experimental group 1: combine Cas9 protein and sgRNA corresponding to the AAVS1 site to obtain Cas9 / sgRNA complex, load the Cas9 / sgRNA complex into the VICEP vector; load the 5' and 3' homologous arms homologous to the AAVS1 genomic region and the GFP fluorescent gene into the IDLV vector; simultaneously deliver the above loaded VICEP vector and IDLV vector in iPS cells, the MOI of the VICEP vector and IDLV vector is 2 and 4 respectively, and after the delivery is completed, 2 μM small molecule drug (HDR promoter M3814) is not added in the iPS cells; real-time detection of cell activity by flow cytometry.

[0191] Experimental group 2: under the condition of ensuring consistency with experimental group one, use electroporation + plasmid system to insert GFP or BFP into AAVS1 site and deliver to iPS cells, and do not add M3814 subsequently;

[0192] Experimental group 3: based on the experimental method of experimental group 2, add the step of adding 2 μM small molecule drug (HDR promoter M3814).

[0193] The results are shown in Figure 5 Figure 5 ​Part A, B: The effect of different delivery methods (electroporation + plasmid, TIVID system) on iPS cell proliferation after 72h when GFP (A) or BFP (B) was inserted into AAVS1 site; Part C: The effect of electroporation + plasmid and TIVID system on iPS cell cycle after 6h treatment, the flow cytometry results show that there is no significant difference between the Cycling proportion of the electroporation group and the TIVID group, indicating that the cell cycle disturbance is small; Part D: The proportion of Annexin V labeled apoptotic cells after 6h treatment under different delivery methods, it can be seen that the apoptosis rate of the electroporation + plasmid group is significantly increased, and the TIVID system shows lower cell stress; Part E, F: Comparison of GFP (E) or BFP (F) insertion efficiency at AAVS1 site after 6 days when different delivery methods (electroporation + plasmid, TIVID) are used, the insertion efficiency of the TIVID technology is significantly higher than that of the electroporation + plasmid system in iPS cells.

[0194] It can be seen that the TIVID system of the present application significantly improves the HDR efficiency while maintaining a higher cell activity: compared with electroporation of plasmid, the number of cells after TIVID treatment is more Figure 5 A, B) ; cell cycle and apoptosis detection was performed 6h after electroporation or transduction, the results support that the cell cycle distribution is normal ( Figure 5 C) and the apoptosis level is lower ( Figure 5 D) after TIVID treatment; at the same time, the GFP and BFP knock-in efficiency at the AAVS1 site is also significantly higher (Fig. E-F), especially after combining with the HDR enhancer M3814, the KI rate can reach up to 80%. This shows that TIVID not only has superior editing performance, but also has lower cell toxicity, and is suitable for precise editing scenarios with higher requirements for cell activity.

[0195] 5. Specific detection

[0196] Design control experiments:

[0197] VICEP-sgPGK1 and two kinds of IDLV delivery experiments were performed on PGK1 site, which are: IDLV-GFP for PGK1 containing GFP fluorescent gene and 5' and 3' homologous arms homologous to PGK1 site, IDLV-BFP for AAVS1 containing BFP fluorescent gene and 5' and 3' homologous arms homologous to AAVS1 site

[0198] Experimental method:

[0199] Experimental group: VICEP-sgPGK1 and IDLV-GFP for PGK1 were delivered into K562 cells, the MOI of VICEP-sgPGK1 was 4, and the MOI of IDLV-GFP for PGK1 was 12; 2 μM of small molecule drug (HDR promoter M3814) was added to K562 cells, and after 6 days of culture, specific detection was performed by flow cytometry.

[0200] Control group 1: under the condition of ensuring that other condition steps are the same as those of the experimental group, IDLV-GFP for PGK1 is not delivered and only VICEP-sgPGK1 is delivered;

[0201] Control group 2: under the condition of ensuring that other condition steps are the same as those of the experimental group, VICEP-sgPGK1 is not delivered and only IDLV-GFP for PGK1 is delivered;

[0202] Control group 3: under the condition of ensuring that other condition steps are the same as those of the experimental group, IDLV-GFP for PGK1 delivered in the experimental group is replaced by IDLV-BFP for AAVS1.

[0203] The results are shown in Figure 6 The A part of the figure is a flow cytometry showing the expression distribution of the exogenous fluorescent gene BFP or GFP under the VICEP-sgPGK1 and two IDLV delivery experiments on the PGK1 site, and the experimental results show that only the matching IDLV-GFP for PGK1 can achieve efficient site-specific knock-in of the fluorescent gene; the B part of the figure is a column chart showing the KI% statistics of the exogenous fluorescent gene BFP or GFP under the VICEP-sgAAVS1 and two IDLV delivery experiments on the PGK1 site.

[0204] It can be seen that under the condition of using a single VICEP and IDLV delivery, only under the condition that the sgRNA and the homologous arm of the donor are completely matched, significant fluorescent gene knock-in can be observed; in the non-matching group, the fluorescent signal of non-specific knock-in is extremely low, and the knock-in efficiency is usually less than 1% (B), which indicates that the TIVID system has good site dependence and editing specificity. This modular, programmable and high-specificity feature not only far exceeds the traditional single-carrier editing strategy, but also highlights the broad application prospects of the TIVID system in constructing multi-gene regulation networks, precise cell function modification and high-order gene therapy programs. Figure 6

[0205] 6. Efficiency verification of the TIVID system of the present application on iPS and other primary cells

[0206] The results are shown in Figure 7 Figure 7 ​​Part A shows the column chart analysis of the knock-in efficiency of the fluorescent gene at the AAVS1 site in HUDEP2 cells on day 6 under different drug treatment conditions by TIVID delivering an equal amount of GFP and BFP donors; Part B shows the analysis of the gene editing effect of VICEP combined with IDLV delivery in iPS cells, and the right side line chart shows the GFP knock-in efficiency (KI%) of the combined delivery of VICEP (sgAAVS1, MOI = 2) at different IDLV MOI gradients (0-4) with the addition of 2 μM M3814 drug treatment and untreated control groups.

[0207] It can be seen that the TIVID system has good applicability in various cell types, and shows better knock-in sensitivity than K562 cells in iPS cells Figure 7 B); iPS cells have been widely used in disease model construction, drug screening, and cell therapy research related to regenerative medicine due to their differentiation potential and unlimited proliferation ability; the research results indicate that the TIVID system can achieve efficient exogenous gene site-specific integration in iPS cells, providing a powerful tool for constructing more complex and functional pluripotent stem cell platforms, and expanding the technical boundaries for further application in precision treatment and personalized medicine.

[0208] 7. Site-specific insertion of ultra-long fragments

[0209] Experimental methods:

[0210] As shown in Figure 8 A, the constructed HDR template contains a ZIM3-dCas9-Crimson expression framework with EF1α promoter, P2A element, WPRE and other regulatory modules, with a total length of 8.3 kb;

[0211] IDLV dose was increased at different MOI gradients (1 to 32), and VICEP was delivered at a fixed MOI = 4 in K562 cells, and low-dose DNA-PK inhibitor M3814 (2 μM) was set at the same time to evaluate its effect on HDR efficiency.

[0212] The results are shown in Figure 8 Figure 8 ​In the middle, part A is the schematic diagram of IDLV donor design for inserting Zim3-dCas9 (CRISPRi system) expressing Crimson fluorescence at AAVS1 site. The donor contains Zim3-dCas9 gene driven by EF1α promoter and Crimson gene, Wpre element and 5' and 3' homologous arms (HA) at both ends of the insertion fragment, which realizes HDR-mediated GFP gene knock-in by synergizing with VICEP guided by sgAAVS1; the column chart in part B shows that the Indel efficiency of AAVS1 site has reached a saturated editing state under the condition of MOI = 4 of VICEP; the line chart in part C shows the efficiency of inserting Zim3-dCas9 CRISPRi system at AAVS1 site in K562 cells by TIVID technology. The right line chart shows the Zim3-dCas9 knock-in efficiency (KI%) under different IDLV MOI gradients (0-32) combined with the delivery of VICEP (sgAAVS1), the addition of 2 μM M3814 drug treatment and the untreated control group, and the current data shows that KI% is significantly positively correlated with MOI, indicating that the current dCas9 insertion has not yet reached the platform period.

[0213] The results show that the knock-in positive rate (KI%) of the fluorescent reporter protein Crimson is significantly positively correlated with IDLV MOI, and there is no platform period trend when IDLV MOI is within 32. When MOI = 32, the KI rate of the M3814 group has reached about 20%. Combined with the editing efficiency of AAVS1 site shown in B, the cells are in a saturated editing state at this time, suggesting that there is still room for improvement for the HDR system. The results prove that the TIVID platform has strong fragment length adaptability and good HDR performance, and under appropriate condition optimization (such as MOI control, small molecule enhancer synergism), it can be applied to the stable integration of ultra-long fragments. Figure 8 B shows the editing efficiency of AAVS1 site, at this time the cells are in a saturated editing state, suggesting that there is still room for improvement for the HDR system. The results prove that the TIVID platform has strong fragment length adaptability and good HDR performance, and under appropriate condition optimization (such as MOI control, small molecule enhancer synergism), it can be applied to the stable integration of ultra-long fragments.

[0214] It can be seen that the TIVID system has feasibility and efficiency in realizing the site-specific insertion of ultra-long fragments, further expanding its application potential in complex gene editing scenarios.

[0215] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. A method for precise site-specific insertion of ultra-long gene fragments, characterized in that, Includes the following steps: A ribonucleoprotein complex is obtained by combining a functional protein with a targeting single RNA, and the ribonucleoprotein complex is loaded into the VICEP vector; Load the HDR donor template into the IDLV carrier; The loaded VICEP vector and IDLV vector are simultaneously delivered to the target cells; The target cells were cultured after the HDR promoter was added to them.

2. The method for precise site-specific insertion of ultra-long gene fragments according to claim 1, characterized in that, The HDR donor template contains an exogenous insertion sequence of 2-15kb in length, and 5′ homologous arms and 3′ homologous arms of 500-1200bp in length located at both ends.

3. The method for precise site-specific insertion of ultra-long gene fragments according to claim 1, characterized in that, The ratio of the multiples of infection of the VICEP vector and the IDLV vector is 1:(1-8).

4. The method for precise site-specific insertion of ultra-long gene fragments according to claim 2, characterized in that, The target cells are selected from induced pluripotent stem cells, K562, HUDEP2, or Jurkat cells; 5. The method for precise site-specific insertion of ultra-long gene fragments according to claim 1, characterized in that, The functional protein is one of SpCas9, SaCas9, or Cas12a proteins.

6. The method for precise site-specific insertion of ultra-long gene fragments according to claim 1, characterized in that, The exogenous insertion sequence contains a promoter-encoding sequence-WPRE functional framework.

7. A gene editing toolkit for use in a precise site-directed insertion method for ultra-long gene fragments as described in any one of claims 1-6, characterized in that, include: The first viral vector, VICEP, carries the aforementioned functional protein and the aforementioned target single-channel RNA. The second viral vector IDLV has the HDR donor template as its payload. A liquid formulation of M3814, wherein the concentration of the liquid formulation of M3814 is 10 mM. The first and second viral vectors are stored separately in lyophilized or liquid formulations and are accompanied by instructions for use.

8. The gene editing toolkit according to claim 7, characterized in that, The enzyme in the second viral vector IDLV contains the D64V inactivation mutation.

9. The gene editing toolkit according to claim 7, characterized in that, The outer shell of the first viral vector VICEP or the second viral vector IDLV carries CD45 antibodies.

10. The application of the precise site-directed insertion method for ultra-long gene fragments as described in any one of claims 1-6 in the construction of induced pluripotent stem cell (iPSC) disease models, CAR-T modification of T cells, and treatment of single-gene genetic diseases and tumors.

Images