Crimson fluorescent protein reporting system and application thereof in gene editing multi-scene evaluation

Abstract

The invention belongs to the technical field of gene editing, and particularly discloses a Crimson fluorescent protein reporting system and application thereof in gene editing efficiency multi-scene evaluation. According to the Crimson fluorescent protein reporting system, an equidirectional repetitive sequence which can be targeted by sgAlb and contains a termination codon is introduced into a Crimson gene sequence, a frame-shift mutant fluorescent reporting module is successfully constructed, and the module can recover fluorescent expression through an SSA (homologous sequence annealing repair) repair mechanism after being cut in a Cas9 / sgAlb system. The developed Crimson report cell system can be used for accurately evaluating the stability and editing efficiency of different sgRNA skeletons; the method is suitable for optimizing a virus transduction system and screening a guiding aid for efficient gene delivery; plasmid pollution in a virus vector system can be rapidly detected, and accurate distinguishing of a functional RNP compound and residual plasmid DNA is realized by comparing the proportion of Crimson positive cells before and after DNase I treatment and heat treatment; and small molecular compounds for promoting homologous directional repair can be efficiently screened.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and more specifically, to the Crimson fluorescent protein reporter system and its application in multi-scenario evaluation of gene editing. Background Technology

[0002] In recent years, the CRISPR / Cas9 system has become an important tool in the field of gene editing due to its high efficiency and ease of operation. However, there are still several technical bottlenecks that need to be addressed in practical applications: (1) The stability of the sgRNA backbone significantly affects the editing efficiency. In the existing technology, the screening of the sgRNA backbone mainly relies on sequencing analysis, which has the disadvantages of cumbersome operation, low throughput, and high cost, and lacks efficient visualization evaluation methods; (2) Low viral transduction efficiency will limit the effect of gene delivery. Screening for efficient transducers can help improve the editing efficiency of the VICEP system; (3) Potential plasmid contamination in viral vector systems may interfere with experimental results; (4) In stem cell therapy, disease model construction and gene therapy, the limited efficiency, short window and high toxicity of HDR have long restricted its practicality in sensitive cells such as iPSC (multipotent stem cells) and HSC (hematopoietic stem cells). Traditional reporter systems are mostly based on common fluorescent proteins such as GFP, which are easily affected by cell autofluorescence and cannot achieve multiple detection. In addition, existing evaluation methods are difficult to meet multiple needs such as sgRNA backbone screening, transducer screening, plasmid contamination assessment and HDR-enhanced small molecule screening at the same time. Summary of the Invention

[0003] To address the aforementioned technical challenges, this invention innovatively develops the Crimson fluorescent protein reporter system, establishing a multifunctional gene editing assessment and application platform. The Crimson fluorescent protein possesses unique advantages such as long excitation / emission wavelengths (611 / 646 nm) and good photostability, effectively avoiding interference from cellular autofluorescence. By introducing a direct repeat sequence into this gene to construct a frameshift mutant and utilizing the SSA repair mechanism to achieve fluorescence recovery, a novel technical solution is provided for gene editing research.

[0004] The development of this technology not only solves the technical shortcomings of existing assessment methods, such as low throughput and poor specificity, but more importantly, it enables integrated assessment of the entire gene editing process (including sgRNA design, guide agent screening, plasmid contamination assessment, and HDR efficiency improvement), providing important tool support for promoting the clinical application of gene editing technology.

[0005] First, this invention provides a Crimson reporter cell line for efficient integrated evaluation and multi-scenario application, the construction of which includes the following steps:

[0006] Two repetitive sequences with the same orientation were introduced into the open reading frame of the Crimson fluorescent protein gene to obtain the Crimson reporter construct of the lentiviral backbone.

[0007] The Crimson reporter construct was randomly integrated into the genome of host cells via lentiviral transduction to obtain Crimson reporter cell lines;

[0008] Furthermore, the repeating sequence has a length of 20–50 bp, and its middle region includes at least one stop codon.

[0009] Furthermore, the host cells include 293T cells, K562 cells, and HUDEP2 cells.

[0010] On the other hand, the present invention also provides an application of the Crimson fluorescent protein reporter system in multi-scenario evaluation of gene editing efficiency, and the application scenarios of the Crimson reporter cell line include the following:

[0011] 1. Assess the stability of the sgRNA backbone.

[0012] The optimized and unoptimized sgAlb proteins were assembled with Cas9 proteins to form ribonucleoprotein complexes (RNPs) and packaged into VICEP virus-like particles. These particles were then delivered into Crimson reporter cell lines via VICEP. After culturing for 24-72 hours, the proportion of Crimson-positive cells was detected by flow cytometry to evaluate the editing efficiency of different sgRNA backbones.

[0013] After delivery, the transduced Crimson reporter cell line was cultured. After culture, the proportion of Crimson positive cells was detected by flow cytometry, and the editing efficiency of the two sgRNA backbones was determined by the proportion of positive cells.

[0014] The sgRNA_v2 backbone incorporates UGCUGC stabilizing motifs on both sides of stem-loop 2, resulting in a Gibbs free energy (ΔG) that is at least 10% lower than that of the conventional sgRNA_v1 backbone; generally, a smaller ΔG value indicates greater stability.

[0015] Furthermore, the VICEP delivery system comprises: the Gag-Cas9 fusion protein; and the v1 and v2 versions of sgAlb;

[0016] Furthermore, the host cells include 293T cells.

[0017] Furthermore, the editing efficiency of the sgRNA_v2 backbone is improved by at least 30% compared to the conventional sgRNA_v1 backbone, as assessed by the proportion of Crimson positive cells.

[0018] 2. Screening for more efficient delivery aids

[0019] The optimized VICEP particles were co-transduced with a specified concentration of a transducing agent into 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines;

[0020] After 24 hours of transduction, the culture medium was replaced with normal medium, and the cell lines were cultured for another 24-48 hours. After the culture was completed, the proportion of Crimson positive cells was detected by flow cytometry to evaluate the efficiency and stability of different types of transduction aids in promoting viral transduction.

[0021] The method is characterized by:

[0022] 1) Utilizing the long-wavelength emission characteristics (611 / 646nm) of Crimson fluorescent protein, interference from cell autofluorescence can be effectively reduced;

[0023] 2) The fluorescent signal activated by the SSA repair mechanism is positively correlated with viral transduction efficiency;

[0024] 3) The effectiveness of the transduction can be evaluated within 24-72 hours;

[0025] Furthermore, the method is applicable to the rapid screening of the following adjuvants:

[0026] a) Cationic polymers: including but not limited to Polybrene, DEAE-dextran, and PS (Protamine).

[0027] Sulfate);

[0028] b) Nonionic surfactants: including but not limited to Pluronic F108 and F127;

[0029] c) Lipid transduction enhancers: including but not limited to Lipofectamine and DOSPA;

[0030] Furthermore, the evaluation indicators of the method include: the Crimson positivity rate after treatment with the facilitator;

[0031] Furthermore, the advantages of the method are as follows:

[0032] 1) High detection sensitivity, capable of distinguishing transduction efficiency differences within 1%;

[0033] 2) The operation process is simplified, shortening the detection cycle by more than 50% compared with traditional methods. Crimson fluorescence detection can be performed as early as 24 hours after transduction.

[0034] 3) Compatible with 48-well or 96-well high-throughput screening modes.

[0035] 3. Verify whether VICEP contains plasmid contamination.

[0036] The VICEP involved in the evaluation method requires the following pretreatment steps before transducing Crimson reporter cell lines:

[0037] 1) DNase I enzymatic digestion (37℃ for 30 minutes) to degrade free plasmid DNA in VICEP liquid formulation;

[0038] 2) Heat treatment at 65℃ for 10 minutes to destroy the overall viral structure of VICEP;

[0039] The pretreated VICEP cells were transduced into 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines, respectively, and F108 promoter at a final concentration of 0.1% was added to the co-culture system.

[0040] 24 hours after transduction, the culture medium was replaced with normal medium, and the cell lines were cultured for another 24-48 hours. After culture, the proportion of Crimson-positive cells was detected by flow cytometry to assess whether there was residual plasmid contamination in the VICEP virus samples.

[0041] Furthermore, the plasmid contamination determination criteria include:

[0042] 1) A difference of more than 1% in the proportion of Crimson-positive cells before and after DNase I digestion indicates plasmid contamination;

[0043] 2) A Crimson-positive cell percentage >1% in the 65℃ heat-treated group indicates plasmid contamination;

[0044] Furthermore, the evaluation method is applicable to: lentiviral vector systems, adeno-associated virus vector systems, and virus-like particle delivery systems;

[0045] Furthermore, the quality control indicators of the evaluation method are as follows:

[0046] 1) It can detect plasmid contamination levels as low as 1%;

[0047] 2) The operation process is simplified, and the testing cycle can be controlled within 24-72 hours;

[0048] 3) Compatible with 48-well or 96-well plate high-throughput screening modes;

[0049] 4) The repeatability CV value of the results is <5%.

[0050] 4. Screening for effective HDR-enhancing small molecules

[0051] The Cas9 / sgRNA system was co-treated with the Crimson reporter cell line, and HDR-enhancing small molecule drugs of different concentration gradients were added and co-incubated for 24 hours.

[0052] After 24 hours of transduction, the culture medium was replaced with normal medium, and the cell line was cultured for another 24-48 hours. After incubation, the Crimson reporter cell line was analyzed by flow cytometry to obtain the proportion of Crimson-positive cells. The optimal concentration of HDR-enhancing small molecule drugs and cell adaptability were determined based on the proportion of Crimson-positive cells.

[0053] Furthermore, the HDR-enhancing small molecule drug includes: NHEJ inhibitors and HDAC inhibitors.

[0054] Furthermore, the NHEJ inhibitors include M3814 and AZD7648; the HDAC inhibitors include VPA (valproic acid), NaB (sodium butyrate), and Entinostat.

[0055] Furthermore, the concentration gradient of the added HDR-enhanced small molecule drug is 0.5–4 μM.

[0056] Furthermore, the incubation time is 48 hours.

[0057] Furthermore, when performing flow cytometry analysis on the incubated Crimson reporter cell line: fluorescence detection is performed on the Crimson reporter cell line, and cells under Crimson fluorescence are identified as Crimson positive cells.

[0058] Furthermore, when determining the optimal concentration and cell fit of HDR-enhancing small molecule drugs by the proportion of Crimson-positive cells, the proportion of Crimson-positive cells is used as a function indicating HDR efficiency, thereby obtaining the optimal concentration and cell fit of HDR-enhancing small molecule drugs.

[0059] On the other hand, the present invention also provides a gene editing assessment kit comprising:

[0060] a) The Crimson reporter cell line of claim 1;

[0061] b) VICEP delivery system components;

[0062] c) Instruction manual.

[0063] Compared with existing technologies, the advantages of this invention are as follows: This invention achieves real-time dynamic monitoring, visual tracking, and quantitative analysis of the gene editing process through the Crimson reporter cell line. It features simple operation, high throughput, and reliable results, providing strong technical support for gene editing tool development, delivery system optimization, and related research. Furthermore, the Crimson reporter cell line constructed in this invention allows the evaluation method to simultaneously meet multiple needs such as sgRNA backbone screening, guide agent screening, plasmid contamination assessment, and HDR-enhanced small molecule screening. (1) Significant differences in sgRNA backbone stability affect editing efficiency. In existing technologies, sgRNA backbone screening mainly relies on sequencing analysis, which is cumbersome and costly. This Crimson reporter cell line can efficiently screen sgRNA backbones with better overall performance. (2) Low viral transduction efficiency limits gene delivery. This Crimson reporter cell line can screen for highly efficient transduction agents, which can not only further improve the editing efficiency of the VICEP system, but also improve the transduction efficiency of viruses such as lentiviruses and adenoviruses. (3) Potential plasmid contamination in viral vector systems may interfere with experimental results. This Crimson reporter cell line can quickly assess the amount of plasmid residue in VICEP viral particles. (4) In stem cell therapy, low HDR efficiency or high toxicity limits the application of related technologies in sensitive cells such as pluripotent stem cells (iPSCs) and hematopoietic stem cells (HSCs). This Crimson reporter cell line can quickly screen for highly efficient small molecules that promote HDR, thereby further expanding the application of site-specific integration technologies such as the TIVID system in iPSCs and HSCs. In addition, traditional fluorescent reporter systems such as GFP are susceptible to autofluorescence interference, while Crimson fluorescent protein has unique advantages such as good photostability, which can effectively avoid cell autofluorescence interference.

[0064] The above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure.

[0065] Other features and aspects of this disclosure will become clearer from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0066] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0067] Figure 1A schematic diagram illustrating the construction of the Crimson reporter cell line is provided for embodiments of the present invention;

[0068] Figure 2 This is a graph showing the effect of the optimized sgRNA backbone on Crimson reporter cell lines according to an embodiment of the present invention.

[0069] Figure 3 The graph shows the effect of different types of promoters on Crimson reporter cell lines provided in the embodiments of the present invention.

[0070] Figure 4 The figure shows the effect of DNase I treatment and heat treatment of VICEP on Crimson reporter cell lines provided in this embodiment of the invention.

[0071] Figure 5 The figure shows the effect of HDAC inhibitor and NHEJ inhibitor on Crimson reporter cell lines provided in the embodiments of the present invention.

[0072] Figure 6 The graph shows the effect of different concentrations of AZD7648 and M3814 on the Crimson reporter cell line provided in the embodiments of the present invention.

[0073] Figure 7 The figure shows the detection results of the effects of different concentrations of AZD7648 and M3814 on the activity of HUDEP2 cells, as provided in the embodiments of the present invention.

[0074] Figure 8 The image shows a test pattern for verifying the HDR enhancement effect of the 2μM M3814 in a TIVID system, as provided in an embodiment of the present invention. Detailed Implementation

[0075] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0076] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0077] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0078] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0079] See Figure 1 As shown in the embodiments of this application, the construction of a Crimson reporter cell line for multiple application scenarios includes the following steps:

[0080] Two identically oriented repetitive sequences were introduced into the open reading frame of the Crimson fluorescent protein gene to obtain the Crimson reporter system construct.

[0081] The Crimson reporter construct was packaged into lentiviral particles and transduced into host cells to obtain a Crimson reporter cell line.

[0082] Understandably, the introduction of two oriented repetitive sequences within the open reading frame of the Crimson fluorescent protein gene causes a frameshift mutation, introducing a premature termination codon, thereby blocking Crimson fluorescent protein expression. When CRISPR / Cas9-mediated sgRNA-guided cleavage occurs between the two repetitive sequences, it induces the SSA mechanism, deleting the intermediate fragment and one repetitive sequence, thus restoring the reading frame and fluorescent expression of the Crimson gene. The recovery of the fluorescence signal represents the successful occurrence of the HDR or SSA repair event, which can be rapidly and quantitatively analyzed by flow cytometry.

[0083] Specifically, the repeating sequence is 20–50 bp in length, and its middle region includes at least one stop codon.

[0084] It is understandable that inserting two oriented repeat sequences into the middle of the original Crimson fluorescent protein gene causes a frameshift and forms an early stop codon, thus blocking Crimson expression.

[0085] In some specific embodiments, the host cells include 293T cells, K562 cells, and HUDEP2 cells.

[0086] Understandably, the Crimson reporter construct is integrated into the host cell genome to construct multiple reporter cell lines: 293T-Crimson, K562-Crimson, and HUDEP2-Crimson, to verify the HDR regulatory effects of different small molecules (M3814, AZD7648, NaB, VPA, Entinostat) in different cellular backgrounds.

[0087] In some specific embodiments, the Crimson fluorescent protein reporter system described above is applied in multiple scenarios for evaluating gene editing efficiency, and its application scenarios include the following four areas:

[0088] 1) Accurately assess the stability and editing efficiency of different sgRNA backbones.

[0089] Different versions of sgRNA were assembled with Cas9 protein into RNP complexes, which were then introduced into Crimson reporter cell lines via the VICEP delivery system. After culture, the proportion of Crimson positive cells was detected by flow cytometry to evaluate the editing efficiency of different sgRNA backbones.

[0090] Specifically, firstly, UGCUGC stabilizing motifs were introduced on both sides of stem-loop 2 of the sgRNA_v2 backbone. Then, sgAlb_v1 and sgAlb_v2 were packaged with Cas9 using a virus-like packaging system to form VICEPs.

[0091] The VICEP and 293T-Crimson reporter cell line were co-cultured for 24 hours. After incubation, the medium was replaced with normal medium and cultured for another 24-48 hours. The Crimson reporter cell line was then analyzed by flow cytometry to obtain the proportion of Crimson-positive cells. The proportion of Crimson-positive cells was used to determine whether sgRNA_v2 could significantly improve Cas9 / sgRNA stability and VICEP yield.

[0092] Understandably, the introduction of two aligned repetitive sequences into the open reading frame of the Crimson fluorescent protein gene causes a frameshift mutation and introduces an early stop codon, thereby blocking the expression of Crimson fluorescent protein. When CRISPR / Cas9-mediated sgRNA-guided cleavage occurs between the two repetitive sequences, it induces the SSA mechanism, deleting the intermediate fragment and one repetitive sequence, thus restoring the reading frame and fluorescent expression of the Crimson gene. The recovery of the fluorescence signal indicates the successful occurrence of the CRISPR / Cas9-mediated editing event, which can be rapidly and quantitatively analyzed by flow cytometry.

[0093] 2) Suitable for optimizing viral transduction systems to screen for highly efficient gene delivery aids.

[0094] VICEP particles were co-transduced with different transduction aids into the Crimson reporter cell line. The proportion of Crimson positive cells was detected after 48-72 hours to evaluate the transduction enhancement effect of the transduction aids.

[0095] Specifically, VICEP-sgAlb and the Crimson reporter cell line are co-treated, with a specified concentration of a transduction aid added simultaneously for 24 hours of co-incubation. After incubation, the virus solution and transduction aid are removed, and the cells are cultured in fresh medium for another 1-2 days. Flow cytometry is then performed on the Crimson reporter cell line to obtain the proportion of Crimson-positive cells. The proportion of Crimson-positive cells is used to determine the transduction aid that significantly promotes viral transduction.

[0096] Specifically, the conductive agent includes:

[0097] Cationic polymers: Polybrene, DEAE-dextran, PS (Protamine Sulfate);

[0098] Nonionic surfactants: Pluronic F108, F127;

[0099] Lipid transduction enhancers: Lipofectamine, DOSPA.

[0100] 3) Rapid detection of plasmid contamination in viral vector systems

[0101] VICEP samples were treated with DNase I and heat-treated at 65°C before being transduced into Crimson reporter cell lines. The presence of plasmid contamination was determined by the difference in the proportion of Crimson positive cells.

[0102] Specifically, VICEP-sgAlb needs to be subjected to DNase I digestion (37℃ for 30 minutes) or heat treatment at 65℃ for 10 minutes before transducing Crimson reporter cell lines. The pretreated VICEP is then transduced into 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines, and co-cultured with 0.1% F108 promoter for 24 hours.

[0103] After incubation, the virus solution and incubator were removed, and fresh culture medium was used to continue culturing for 1-2 days. The Crimson reporter cell line was then subjected to flow cytometry to obtain the proportion of Crimson positive cells and to assess whether there was residual plasmid contamination in the VICEP virus sample.

[0104] Specifically, the criteria for determining plasmid contamination are as follows:

[0105] The difference in the proportion of Crimson-positive cells before and after DNase I treatment was >1%;

[0106] The proportion of Crimson-positive cells in the 65℃ heat treatment group was >1%.

[0107] 4) Capable of efficiently screening small molecule compounds that promote homology-directed repair (HDR).

[0108] The Cas9 / sgRNA system was co-treated with the Crimson reporter cell line, and after incubation with gradient concentrations of HDR-enhanced small molecule drugs for 48 hours, the optimal concentration of the small molecule and cell adaptability were determined by flow cytometry detection of the proportion of Crimson-positive cells.

[0109] Specifically, the Cas9 / sgRNA system and the Crimson reporter cell line can be co-treated by adding different gradient concentrations of HDR-enhancing small molecule drugs and co-incubating for 24 hours.

[0110] After incubation, the incubated Crimson reporter cell line was analyzed by flow cytometry to obtain the proportion of Crimson-positive cells. The optimal concentration and cell fit of the HDR-enhancing small molecule drug were determined based on the proportion of Crimson-positive cells. Specifically, the VICEP carrier titer was ≥1×10⁻⁶. 7 After culturing the target cells, TUml-1 was used to confirm by flow cytometry that the exogenous sequence had been precisely integrated at the predetermined site.

[0111] Understandably, the introduction of two identically oriented repetitive sequences into the open reading frame of the Crimson fluorescent protein gene causes a frameshift mutation and introduces a premature stop codon, thereby blocking the expression of Crimson fluorescent protein. When CRISPR / Cas9-mediated sgRNA-guided cleavage occurs between the two repetitive sequences, it induces the SSA (homological sequence annealing repair) mechanism, deleting the intermediate fragment and one repetitive sequence, thus restoring the reading frame and fluorescent expression of the Crimson gene. The recovery of the fluorescence signal represents the successful occurrence of the HDR or SSA repair event, which can be rapidly and quantitatively analyzed by flow cytometry.

[0112] Understandably, the Crimson reporter construct is integrated into the host cell genome to construct multiple reporter cell lines: 293T-Crimson, K562-Crimson, and HUDEP2-Crimson, to verify the HDR regulatory effects of different small molecules (M3814, AZD7648, NaB, VPA, Entinostat) in different cellular backgrounds.

[0113] Specifically, the HDR-enhancing small molecule drugs include: NHEJ inhibitors and HDAC inhibitors.

[0114] Specifically, the NHEJ inhibitors include M3814 and AZD7648; the HDAC inhibitors include VPA (valproic acid), NaB (sodium butyrate), and Entinostat.

[0115] Specifically, M3814 and AZD7648 were used at concentrations of 0.5-4 μM, while VPA, NaB, and Entinostat were used at concentrations of 1 mM, 2 mM, and 5 μM, respectively.

[0116] Specifically, the incubation time is 24 hours.

[0117] Specifically, when performing flow cytometry on the incubated Crimson reporter cell line: fluorescence detection is performed on the Crimson reporter cell line, and Crimson cells under Crimson fluorescence are identified as Crimson positive cells.

[0118] Specifically, when determining the optimal concentration and cell fit of HDR-enhancing small molecule drugs by the proportion of Crimson-positive cells, the proportion of Crimson-positive cells is used as a function indicating HDR efficiency, thereby obtaining the optimal concentration and cell fit of HDR-enhancing small molecule drugs.

[0119] The application of the efficient screening of HDR-enhancing small molecules can screen for matching HDR-enhancing small molecules for the TIVID system or other site-specific integration techniques that rely on HDR repair.

[0120] In some specific embodiments of validating the application of HDR-enhancing small molecules screened from Crimson reporter cell lines to TIVID technology, the HDR donor template contains a 5.9kb exogenous insertion sequence, and 5′ and 3′ homologous arms of 500-1200bp at both ends, respectively.

[0121] Understandably, IDLV, as a donor template vector, carries a large expression cassette of the target gene and is flanked by 5' and 3' homologous arms that are homologous to the target genomic site. These homologous arms are precisely inserted into the target DNA via the HDR mechanism. In the design of IDLV, the risk of random integration is significantly reduced through integrase mutations, ensuring high specificity and high safety of gene insertion.

[0122] Example 1: Rapid evaluation of the effects of the sgAlb_v1 and v2 backbones on Cas9 / sgRNA stability and editing efficiency using Crimson reporter cell lines

[0123] S1. Introduce UGCUGC base pairs at Stem loop 2 of the existing sgAlb scaffold and name them sgAlb_v2. The unoptimized version is sgAlb_v1. Select mFold to predict the Gibson free energy of sgAlb_v1 and v2.

[0124] S2. Pack the sgAlb_v1 and v2 with Gag-SpCas9 respectively to form virus-like particles, to obtain VICEP-sgAlb_v1 and VICEP-sgAlb_v2 particles.

[0125] S3. The above-mentioned VICEP was co-cultured with the 293T-Crimson reporter cell line at different volume gradients, and F108 was added at a final concentration of 0.1% and incubated for 24 hours.

[0126] S4. After incubation, replace the co-culture system containing VICEP with normal fresh culture medium. Continue culturing for 24-48 hours, then perform flow cytometry to detect the percentage of Crimson-positive cells.

[0127] The results are as follows Figure 2As shown in the figure: AB represents the schematic diagram of the sgRNA backbone of sgAlb_v1 and sgAlb_v2, C represents the schematic diagram of the core vector required for VICEP packaging, and D represents the flow cytometry detection results of 293T-Crimson transduction after sgAlb_v1 and sgAlb_v2 are packaged into VICEP particles. The results show that the introduction of UGCUGC base pairs into sgAlb_v2 can significantly improve the stability between Ca9 / sgAlb and the yield of VICEP.

[0128] Example 2: Rapid screening of more effective promoters using Crimson reporter cell lines

[0129] S1. Package the sgAlb_v2 with Gag-SpCas9 into virus-like particles to obtain VICEP-sgAlb_v2 particles;

[0130] S2. Concentrate the VICEP-sgAlb_v2 particles by 100 times;

[0131] S3. Take 1 μL of the 100-fold concentrated VICEP and co-culture it with 50,000 293T-Crimson reporter cells, while adding different types of transduction aids (Protamine Sulfate, PS, Polybrene, and Synperonic F108, F108) and incubating for 24 hours; the VICEP transduction group without the added transduction aids serves as the control.

[0132] S4. After incubation, replace the co-culture system containing VICEP with normal fresh culture medium. Continue culturing for 24-48 hours, then perform flow cytometry to detect the percentage of Crimson-positive cells.

[0133] The results are as follows Figure 3 As shown in the figure: A, C, and D represent the bar charts of the proportion of Crimson-positive cells transduced into 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines by VICEP-sgAlb_v2 and different types of promoters, respectively. The results indicate that F108 showed more stable performance overall compared to the other two promoters.

[0134] Example 3: Rapid assessment of plasmid residue status in the VICEP system using Crimson reporter cell lines

[0135] S1. Package the sgAlb_v2 with Gag-SpCas9 into virus-like particles to obtain VICEP-sgAlb_v2 particles;

[0136] S2. Concentrate the VICEP-sgAlb_v2 particles by 100 times;

[0137] S3. Before transducing the target cells, perform the following pretreatment steps on the above-mentioned 100-fold concentrated VICEP:

[0138] 1) DNase I enzymatic digestion (37℃ for 30 minutes) to degrade free plasmid DNA in VICEP liquid formulation;

[0139] 2) Heat treatment at 65℃ for 10 minutes to destroy the overall viral structure of VICEP;

[0140] Theoretically, if the transduction effect mainly originates from residual DNA, DNase I enzymatic digestion will lead to the degradation of residual plasmid DNA, thereby reducing the proportion of Crimson positivity in this group. Similarly, heat treatment will destroy the overall viral structure of VICEP, and if residual plasmid DNA remains, it will result in residual Crimson positivity signals.

[0141] The VICEP transduction group that did not undergo the above treatment served as a control.

[0142] S4. Take 1 μL of the treated VICEP and co-culture it with 50,000 of the 293T-Crimson, K562-Crimson and HUDEP2-Crimson reporter cell lines respectively, and add 0.1% F108 to incubate for 24 hours.

[0143] S5. After incubation, replace the co-culture system containing VICEP with normal fresh culture medium. Continue culturing for 24-48 hours, then perform flow cytometry to detect the percentage of Crimson-positive cells.

[0144] The results are as follows Figure 4 As shown in the figure: Figure A represents the bar chart of the proportion of Crimson-positive cells transduced into the 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines by VICEP-sgAlb_v2 after DNase I digestion (37℃ for 30 minutes) and the control group VICEP-sgAlb_v2, respectively; Figure B represents the bar chart of the proportion of Crimson-positive cells transduced into the 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines by VICEP-sgAlb_v2 after heat treatment at 65℃ for 10 minutes and the control group VICEP-sgAlb_v2, respectively. These results indicate that the function delivered by VICEP mainly comes from its encapsulated Cas9 / sgRNA protein-ribonucleic acid complex (RNP), rather than plasmid DNA contamination.

[0145] Example 4: Effects of HDAC inhibitors and NHEJ inhibitors on Crimson reporter cell lines:

[0146] S1. Two repetitive sequences with the same orientation are introduced into the open reading frame of the Crimson fluorescent protein gene to obtain the Crimson reporter construct.

[0147] S2. The Crimson reporter construct is integrated into the cellular genomes of 293T cells, K562 cells, and HUDEP2 cells, respectively, to obtain 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines.

[0148] S3. The 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines were transduced into the VICEP system carrying SpCas9 and sgAlbVICEP for co-culture. At the same time, 0.5 μM of M3814 and AZD7648, 1 mM VPA, 2 mM NaB, 5 μM Entinostat, and 0.1% F108 were added and incubated for 24 hours.

[0149] S4. After incubation, the incubated 293T-Crimson, K562-Crimson, and HUDEP2-Crimson reporter cell lines were analyzed by flow cytometry.

[0150] The results are as follows Figure 5 As shown in the figure: A represents the detection results of the 293T-Crimson reporter cell line, B represents the detection results of the K562-Crimson reporter cell line, and C represents the detection results of the HUDEP2-Crimson reporter cell line. The results show that HDCA inhibitors have a significant inhibitory effect on HDR in K562 and HUDEP2 blood cells, but 0.5 μM M3814 and AZD7648 can effectively promote HDR repair in all three reporter cell lines.

[0151] Example 5: Effects of different concentrations of AZD7648 and M3814 on Crimson reporter cell lines:

[0152] S1. Two repetitive sequences with the same orientation are introduced into the open reading frame of the Crimson fluorescent protein gene to obtain the Crimson reporter construct.

[0153] S2. The Crimson reporter construct is integrated into the cellular genomes of K562 cells and HUDEP2 cells, respectively, to obtain K562-Crimson and HUDEP2-Crimson reporter cell lines;

[0154] S3. The Cas9 / sgRNA system was co-cultured with the K562-Crimson and HUDEP2-Crimson reporter cell lines, respectively, and 0.1% F108 and AZD7648 or M3814 at gradient concentrations of 0.5, 1, 2, 3, and 4 μM were added and incubated for 24 hours.

[0155] S4. After incubation, the K562-Crimson and HUDEP2-Crimson reporter cell lines with different concentrations of AZD7648 and M3814 were subjected to flow cytometry analysis.

[0156] The results are as follows Figure 6 As shown: AB represents the detection results of the K562-Crimson reporter cell line with different concentrations (0.5, 1, 2, 3, 4 μM) of AZD7648 (A) or M3814 (B), respectively; CD represents the detection results of the HUDEP2-Crimson reporter cell line with different concentrations (0.5, 1, 2, 3, 4 μM) of AZD7648 (C) or M3814 (D), respectively. The results show that 0.5 μM AZD7648 or M3814 both have good HDR promotion effects, and the editing efficiency is dose-dependent.

[0157] Example 6: Effects of different concentrations of AZD7648 and M3814 on the proliferation of HUDEP2 cells:

[0158] S1. Two repetitive sequences with the same orientation are introduced into the open reading frame of the Crimson fluorescent protein gene to obtain the Crimson reporter construct.

[0159] S2. Integrate the Crimson reporter construct into the genome of HUDEP2 cells to obtain the HUDEP2-Crimson reporter cell line;

[0160] S3. The Cas9 / sgRNA system was co-cultured with the HUDEP2-Crimson reporter cell line, and 0.1% F108 and AZD7648 or M3814 at gradient concentrations of 0.5, 1, 2, 3, and 4 μM were added and incubated for 24 hours.

[0161] S4. After incubation, the activity of the HUDEP2-Crimson reporter cell lines supplemented with different concentrations (0.5, 1, 2, 3, 4 μM) of AZD7648 and M3814 was tested.

[0162] The results are as follows Figure 7 As shown: A represents the activity test results of the HUDEP2-Crimson reporter cell lines with different concentrations of AZD7648, and B represents the activity test results of the HUDEP2-Crimson reporter cell lines with different concentrations of M3814. The results show that 4 μM AZD7648 exhibits certain cytotoxicity, but M3814 has significant cytotoxicity at 2 μM.

[0163] Example 7: Validating the screening results of Crimson reporter cell lines using the TIVID system:

[0164] To further validate the universality of the HDR-enhancing small molecules screened using the Crimson reporter cell line in the TIVID system and other HDR-dependent site-specific integration technologies, this application selected the classic gene-editing target AAVS1 to test the effects of AZD7648 and M3814 in the TIVID system. The AAVS1 site is located in an open chromatin region of human chromosome 19, a well-validated safe site suitable for long-term gene expression without interfering with endogenous gene function. We chose this site for HDR-dependent site-specific integration of ultra-long fragments.

[0165] S1. The IDLV double-cut donor vector required for constructing the TIVID system: The exogenous insert sequence is an HBB-GFP fusion gene driven by the HBG promoter. The insert sequence contains approximately 600bp 5' and 3' homologous arms and sgDocut sequences on both sides, which are homologous to the target editing region of the AAVS1 gene. The total length of the sequence between sgDocuts is approximately 7.1Kb;

[0166] S2. Package the above-mentioned double-cut HBB-GFP donor into the IDLV system, with an effective biological titer ≥1x10⁻⁶. 7 TU / ml;

[0167] S3. Package Cas9 / sgAAVS1 targeting the AAVS1 site into the VICEP system, achieving an effective biological titer ≥1x10⁻¹. 7 TU / ml;

[0168] S4. K562, HUDEP2 and human hematopoietic stem and progenitor cells (HSPC) were selected for experiments, with the target gene being the AAVS1 safe harbor site.

[0169] S5. VICEP and IDLV targeting the AAVS1 site were co-cultured with target cells, where the multiple of infection (MOI) of VICEP and IDLV was... Figure 8 Clearly labeled. Add F108 to the above system at a final concentration of 0.1% and AZD7648 or M3814 at 1-2 μM and incubate for 24 hours.

[0170] S6. After incubation, the target cells with different concentrations of AZD7648 and M3814 were subjected to flow cytometry to evaluate the feasibility of using the HDR enhancer obtained by Crimson screening in the previous stage in the TIVID system.

[0171] The results are as follows Figure 8 As shown:

[0172] In the figure: Part A shows a schematic diagram of the IDLV donor design for inserting HBB-GFP at the AAVS1 site. The donor contains an HBB-GFP fusion gene driven by an HBG mutant promoter and 5' and 3' homologous arms (HA) located at both ends of the insert fragment. Through synergy with sgAAVS1-guided VICEP, HDR-mediated HBB-GFP gene knock-in is achieved.

[0173] Part B is shown in K562 cells, delivered via TIVID (HBB-GFP). Figure 8 A) Bar chart analysis of the HBB-GFP gene knock-in efficiency at the AAVS1 site on day 6 under different drug treatments (control, 2 μM M3814) and different MOIs of the DLV vector. The overall KI% increased nearly 5-fold after adding 2 μM M3814.

[0174] Part C is shown in HUDEP2 cells, delivered via TIVID (HBB-GFP). Figure 8 A) Bar chart analysis of the HBB-GFP gene knock-in efficiency at the AAVS1 site on day 6 under different drug treatments (control, 1-2 μM AZD7648 or M3814). The data show that the overall KI% increased in a dose-dependent manner after adding AZD7648 or M3814.

[0175] Part D shows Figure 8 In experiment C, HUDEP2 cells were analyzed by RT-qPCR, and the relative expression of T87Q-HBB mRNA conjugated with GFP was analyzed by bar chart. The data showed that the overall relative expression of T87Q-HBB mRNA increased in a dose-dependent manner after the addition of AZD7648 or M3814.

[0176] Part E is shown in HSPC cells, delivered via TIVID (HBB-GFP). Figure 8 A) Bar chart analysis of the HBB-GFP gene knock-in efficiency at the AAVS1 site on day 12 under different drug treatments (control, 1 μM M3814). Data shows that the overall KI% increased 2-3 times after adding 1 μM M3814.

[0177] Based on the results of Examples 4-7, it can be concluded that the small molecule compounds that promote HDR obtained by screening using the Crimson reporter cell lines also perform excellently in the TIVID system.

[0178] The efficient screening method for HDR-enhancing small molecules employed in this application allows for the rapid evaluation of the regulatory effects of various small molecule inhibitors on HDR repair efficiency. Previous literature reports that NHEJ inhibitors such as M3814 and AZD7648 can significantly enhance CRISPR / Cas9 system-induced HDR efficiency, while HDAC inhibitors (NaB, VPA, Entinostat) may indirectly regulate the repair pathway by affecting chromatin structure. Our experimental results show that HDAC inhibitors have almost no significant effect in 293T cells, but exhibit a trend of significantly inhibiting HDR repair in K562 and HUDEP2 cells. Figure 5 In contrast, the promoting effects of NHEJ inhibitors were more consistent and varied.

[0179] Further gradient experiments showed that M3814 and AZD7648 at concentrations of 0.5–1 μM significantly increased the proportion of K562-Crimson and HUDEP-Crimson positive cells, with minimal impact on cell proliferation and viability. Figure 6 ).

[0180] However, in HUDEP2 cells, significant cytotoxicity began to appear when the concentration of M3814 exceeded 2 μM, while AZD7648 showed lower cytotoxicity at the same concentration. Figure 7 It shows better biocompatibility.

[0181] Furthermore, we tested the effects of 1-2 μM AZD7648 or M3814 in the TIVID system and found that they worked well in the TIVID cells.

[0182] In summary, the experiments demonstrated that the Crimson reporter system's advantages as a high-throughput, small-molecule screening platform are evident in this study, providing an efficient and feasible technical path for the systematic screening and optimization of HDR enhancers under different cell types and complex editing conditions.

[0183] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Crimson fluorescent protein reporter system, characterized in that, Includes the following steps: Constructing the Crimson reporter cell line: Two 20-50 bp homologous repeat sequences were introduced into the open reading frame of the Crimson fluorescent protein gene, with a stop codon in the middle region, to obtain the Crimson reporter construct with a lentiviral backbone, and then integrated into the host cell genome.

2. An application of the Crimson fluorescent protein reporter system as described in claim 1 in multi-scenario evaluation of gene editing efficiency, characterized in that, The Crimson reporter cell line is used for at least one of the following applications: (1) sgRNA backbone stability assessment: sgRNAs with different backbones were assembled with Cas9 protein into RNP complexes and packaged into VICEP viral particles. These complexes were then delivered into Crimson reporter cell lines via VICEP. After culturing, the proportion of Crimson-positive cells was detected by flow cytometry to evaluate the editing efficiency of different sgRNA backbones. (2) Screening of viral transduction aids: VICEP particles were co-transduced with different transduction aids into the Crimson reporter cell line. The proportion of Crimson positive cells was detected after 48-72 hours to evaluate the transduction enhancement effect of different transduction aids. (3) Plasmid contamination detection: VICEP samples were treated with DNase I (37℃ for 30 minutes) and heat-treated (65℃ for 10 minutes) before being transduced into Crimson reporter cell lines. Flow cytometry was performed 48-72 hours later, and the presence of plasmid contamination was determined by the difference in the proportion of Crimson positive cells. (4) HDR-enhanced small molecule screening: The Cas9 / sgRNA system was co-treated with the Crimson reporter cell line, and after incubation with gradient concentrations of HDR-enhanced small molecule drugs for 48 hours, the optimal concentration of the small molecule and cell adaptability were determined by flow cytometry detection of the proportion of Crimson-positive cells.

3. The application according to claim 2, characterized in that, The VICEP delivery system comprises a Gag-Cas9 fusion protein and an optimized sgRNA_v2 backbone, wherein the sgRNA_v2 introduces UGCUGC stabilizing motifs on both sides of stem-loop 2.

4. The Crimson fluorescent protein reporter system according to claim 1, characterized in that, The host cells were selected from 293T cells, K562 cells, and HUDEP2 cells.

5. The application according to claim 2, characterized in that, The conductive agent includes: a) Cationic polymers: Polybrene, DEAE-dextran, PS (Protamine Sulfate); b) Nonionic surfactants: Pluronic F108, F127; c) Lipid transduction enhancers: Lipofectamine, DOSPA.

6. The application according to claim 2, characterized in that, The criteria for determining plasmid contamination are as follows: a) The difference in the proportion of Crimson-positive cells before and after DNase I treatment was >1%; b) The proportion of Crimson-positive cells in the 65℃ heat treatment group was >1%.

7. The application according to claim 2, characterized in that, The HDR-enhancing small molecule drug includes: a) NHEJ inhibitors: M3814, AZD7648; b) HDAC inhibitors: VPA (valproic acid), NaB (Sodium butyrate), Entinostat.

8. The application according to claim 2, characterized in that, The detection sensitivity of the Crimson reporting system is: a) Can distinguish transduction efficiency differences within 1%; b) It can detect plasmid contamination levels as low as 1%; c) The repeatability CV value of the results is <5%.

9. The application according to claim 2, characterized in that, The application uses a 48-well or 96-well plate high-throughput screening mode, and the detection cycle is controlled within 24-72 hours.

10. A gene editing assessment kit, characterized in that, Include: a) The Crimson reporter cell line of claim 1; b) VICEP delivery system components; c) Instruction manual.

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