Gene editing system based on ibodomide and application thereof

Abstract

The invention relates to the field of gene editing, in particular to a gene editing system based on ibodomide and application of the gene editing system. The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (Clustered Regularly Interspaced Short Palindromic Repeats) system gene editing kit based on ibodomide termination, which is characterized in that the kit comprises coding RNA (Ribonucleic Acid), gRNA (guide Ribonucleic Acid) and ibodomide, the coding RNA comprises an RNA sequence for coding an ibodomide binding sequence and an RNA sequence for coding a CRISPR / Cas system gene editing tool enzyme, the fusion protein is used for coding an ibodomide binding sequence and a CRISPR / Cas system gene editing tool enzyme; the gRNA is used for guiding the fusion protein to edit a target gene; the ibodomide is used for terminating the gene editing effect of the fusion protein. The action period of the CRISPR / Cas system can be quantitatively and regularly controlled through the ibodomide, and off-target editing is reduced.

Description

Technical Field

[0001] The present invention relates to the field of gene editing, and in particular to a gene editing system based on ibodomide and uses thereof. Background Art

[0002] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins (Cas) editing technology can efficiently and flexibly perform precise editing of nucleic acids, and has great potential for application in research and clinical practice. However, the duration of action of this technology is difficult to accurately control, and long-term action in the body may cause off-target effects, genetic modification, genotoxicity and carcinogenic risks, and induce host immune responses and even diseases, which greatly limits its application.

[0003] The spontaneous degradation of Cas can be controlled by introducing unstable domains and stability factors into the Cas protein, but the degradation time is long; timed degradation can be achieved by connecting transcription activators to the Cas protein and introducing molecules that trigger the degradation of transcription activators, but this molecule has more than 200 amino acids, which reduces the activity of the Cas protein, and the off-target effects of the degradation molecules cause additional cytotoxicity; the proteasome can be induced to target and degrade the Cas protein through protein hydrolysis targeting chimeras, but its molecular weight is large, its absorption and action efficiency are low, and the degradation time exceeds 24 hours.

[0004] Ibodamide (C 25 H 27 N3O5, molecular weight 449.5) is a third-generation small molecule adhesive drug that has entered Phase II clinical trials. It can bind to Cereblon (CRBN) protein and substrate protein, inducing E3 ubiquitin ligase and proteasome targeted degradation of substrates. Compared with the early molecular adhesive drug pomalidomide, ibolidomide has a 20-fold higher binding affinity with CRBN protein and a 24-fold higher substrate degradation efficiency; compared with proteolysis-targeted chimeras, ibolidomide has a smaller molecular weight and better penetration and pharmacological properties.

[0005] In summary, the half-life of CRISPR / Cas technology is difficult to regulate, resulting in off-target editing and pathogenic effects; existing regulatory technologies face problems such as reduced efficiency of CRISPR / Cas technology, low regulatory efficiency and precision, and cytotoxicity. Currently, there is still a lack of a safe and reliable method to accurately control the half-life of CRISPR-Cas technology. Summary of the invention

[0006] The object of the present invention is to provide a CRISPR / Cas system gene editing kit and a corresponding gene editing method based on ibodomide termination. The kit or gene editing method of the present invention fuses a class of protein structure sequences with ibodomide binding ability on the gene editing tool enzyme. When it is necessary to terminate the action of the CRISPR / Cas system, ibodomide is bound to the tool enzyme, ibodomide binds to CRBN, induces E3 ubiquitin ligase to approach and ubiquitinate the tool enzyme, so that it is degraded, thereby terminating the action of the CRISPR / Cas gene editing system. By controlling the reaction by targeting the gene editing tool enzyme with ibodomide, the action cycle of the CRISPR / Cas system can be quantitatively and timed, reducing off-target editing, pathogenic risks and adverse immune responses, complementing the functional defects of the CRISPR / Cas technology, and assisting its research and therapeutic applications.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0008] A CRISPR / Cas system gene editing kit based on ibodomide termination, the kit comprising coding RNA, gRNA and ibodomide:

[0009] The coding RNA includes an RNA sequence encoding an ibodomide binding sequence and an RNA sequence encoding a CRISPR / Cas system gene editing tool enzyme, and is used to encode a fusion protein of the ibodomide binding sequence and the CRISPR / Cas system gene editing tool enzyme;

[0010] The gRNA is used to guide the fusion protein to edit the target gene; and

[0011] The ibodomide is used to terminate the gene editing effect of the fusion protein.

[0012] In some specific embodiments, the ibodomide binding sequence is fused to the N-terminus, C-terminus or inside the CRISPR / Cas system gene editing tool enzyme.

[0013] In some specific embodiments, the RNA sequence encoding the ibodomide binding sequence is shown in any one of SEQ ID NOs: 1-9.

[0014] In some specific embodiments, the CRISPR / Cas system gene editing tool enzyme is selected from Cas9, Cas12, Cas13, Cas14, APOBEC1, APOBEC2, APOBEC3 and APOBEC4.

[0015] In some specific embodiments, the CRISPR / Cas system gene editing tool enzyme is Cas9, and preferably, the sequence of the encoding RNA is as shown in SEQ ID NO:10.

[0016] In some specific embodiments, the gRNA targets Lyz2, and preferably, the sequence of the gRNA is as shown in any one of SEQ ID NOs: 11-13.

[0017] In some embodiments, the kit further comprises a delivery agent for delivering the coding RNA and gRNA;

[0018] Preferably, the delivery agent is a liposome nanoparticle;

[0019] Preferably, the delivery agent is a targeted liposome nanoparticle, for example, liver targeting, kidney targeting, lung targeting, gastrointestinal tract targeting and heart targeting;

[0020] Preferably, the delivery agent comprises cationic lipid C12-200, 5A2-SC8, DLin-MC3-DMA11, DODAP, anionic lipid 14PA, didodecyldimethylammonium bromide, dioleoylphosphatidylethanolamine, cholesterol, liposomal DMG-PEG and dioleoylphosphatidic acid.

[0021] In some specific embodiments, the gene editing is selected from gene knockout, gene knock-in and single gene editing.

[0022] The present invention also provides a CRISPR / Cas system gene editing method based on ibodomide termination, the method comprising the following steps:

[0023] (1) delivering the coding RNA and gRNA to the target cell to perform gene editing on the target gene, wherein the coding RNA comprises an RNA sequence encoding an ibodomide binding sequence and an RNA sequence encoding a CRISPR / Cas system gene editing tool enzyme, and is used to encode a fusion protein of the ibodomide binding sequence and the CRISPR / Cas system gene editing tool enzyme, and the gRNA is used to guide the fusion protein to edit the target gene;

[0024] (2) adding ibodomide to terminate the gene editing of the fusion protein.

[0025] In some specific embodiments, the ibodomide binding sequence is fused to the N-terminus, C-terminus or inside the CRISPR / Cas system gene editing tool enzyme.

[0026] In some specific embodiments, the RNA sequence encoding the ibodomide binding sequence is shown in any one of SEQ ID NOs: 1-9.

[0027] In some specific embodiments, the CRISPR / Cas system gene editing tool enzyme is selected from Cas9, Cas12, Cas13, Cas14, APOBEC1, APOBEC2, APOBEC3 and APOBEC4.

[0028] In some specific embodiments, the CRISPR / Cas system gene editing tool enzyme is Cas9, and preferably, the sequence of the encoding RNA is as shown in SEQ ID NO:10.

[0029] The gRNA targets Lyz2, and preferably, the sequence of the gRNA is shown in any one of SEQ ID NOs: 11-13.

[0030] In some embodiments, the encoding RNA and gRNA are delivered to target cells via liposome nanoparticles;

[0031] Preferably, the liposome nanoparticles are targeted liposome nanoparticles, for example, liver-targeted, kidney-targeted, lung-targeted, gastrointestinal-tract-targeted and heart-targeted;

[0032] Preferably, the agents used for the LNP delivery include cationic lipid C12-200, 5A2-SC8, DLin-MC3-DMA11, DODAP, anionic lipid 14PA, didodecyldimethylammonium bromide, dioleoylphosphatidylethanolamine, cholesterol, liposomal DMG-PEG and dioleoylphosphatidic acid.

[0033] In some specific embodiments, the methods for introducing the gene editing system include: (1) introduction at the cellular level through liposome nanoparticle transfection; (2) introduction at the animal level through intravenous injection of liposome nanoparticles.

[0034] In some specific embodiments, the gene editing is selected from gene knockout, gene knock-in and single gene editing. Technical Effects

[0035] The novel gene editing system proposed in the present invention uses the small molecule drug ibodomide to target and degrade the modified proteins in the CRISPR / Cas gene editing system, thereby achieving control over the action time of the CRISPR / Cas system. Compared with the prior art, it has the following significant advantages:

[0036] 1) The action time of the CRISPR / Cas system can be controlled and regulated to reduce the risk of off-target editing and pathogenicity; it has a wide range of applications and can modify various gene editing tool enzymes in the CRISPR / Cas gene editing system to function for different gene editing purposes.

[0037] 2) The time required to stop the action of the CRISPR / Cas system is significantly reduced. At the cellular level, compared with other current technologies that take more than 24 hours to control, this technology can achieve rapid degradation within 1 hour.

[0038] 3) The drug ibolidomide used has a smaller molecular weight, stronger permeability, and better pharmacokinetics than protein-targeted hydrolysis chimeras; it has fewer toxic side effects and higher safety than stabilizing factors in degradation inhibition schemes; and the third-generation molecular adhesive drug ibolidomide has a stronger binding affinity to CRBN protein and higher efficiency than previous-generation molecular adhesive drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 This is a flow chart of the working method of the gene editing system described in the present invention.

[0041] Figure 2 Schematic diagram of the modified RNA sequence of the present invention.

[0042] Figure 3 Schematic diagram of the binding sequence protein structure used in the present invention.

[0043] Figure 4 The figure is a flow chart of a method according to an embodiment of the present invention. DETAILED DESCRIPTION

[0044] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. In addition, the present invention can also be implemented in other ways different from those described herein, and therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below. The materials, reagents, detection methods, etc. used in the following implementation methods, if not otherwise specified, can all be obtained from commercial sources. Example 1

[0045] This embodiment discloses a method for inducing the expression of B6..Fas lpr Lyz2 Akaluc .Crbn I391V / I391VThe method for knocking out the Lyz2 gene in mice and terminating the effect of the gene editing system by ibodomide specifically comprises the following steps:

[0046] Step S1. Design of RNA sequence encoding ibodomide binding sequence

[0047] The RNA sequence of the ibodomide binding sequence was obtained through public database and molecular docking verification, and the RNA sequence encoding the ibodomide binding sequence was obtained through codon optimization: Based on the public database, the amino acid sequence of the zinc finger binding domain of the CRBN protein binding to the molecular glue protein substrate was obtained. Codon optimization was performed using public tools such as IDT-Codon Optimization Tool for parameters such as the target species for gene editing, the restriction endonuclease sites to be excluded, the secondary structure of the nucleic acid, and the GC content, and RNA sequences were designed to obtain multiple RNA sequences encoding the ibodomide binding sequence (SEQ ID NO: 1-9).

[0048] Step S2. Construction of RNA sequence encoding fusion protein (fusion protein of ibodomide binding sequence and SpCas9)

[0049] like Figure 2 As shown, the RNA encoding the ibodomide binding sequence is inserted into the mRNA encoding the protease SpCas9 to construct an RNA sequence encoding the ibodomide binding sequence and the SpCas9 fusion protein. In the constructed fusion protein, the ibodomide binding sequence can be inserted into the corresponding position of the N-terminus of the protease, the corresponding position of the C-terminus, or inside thereof. As an example, in this embodiment, an RNA sequence IKZF3ZF1 (SEQ ID NO: 6) encoding the ibodomide binding sequence designed in step S1 is inserted into the 5 end of the mRNA of the protease SpCas9, and the RNA sequence after the insertion of IKZF3ZF1 is shown in SEQ ID NO: 10.

[0050] Step S3. gRNA design

[0051] Design gRNA for the gene to be knocked out, Lyz2. Specifically, query the target gene sequence of the target species through public databases such as NCBI, design parameters such as the target gene region, the type of Cas protein provenance used, and the adjacent pattern of the PAM original, and design gRNA through gRNA design tools such as Crispor. In this example, gene knockout was performed on the B6 mouse target gene Lyz2, and SpCas9 protease and 20bp-NGG-Sp Cas9, SpCas9-HF1, eSpCas911 PAM region were used for design, and SEQ ID NO: 11-13 were obtained as exemplary gRNAs.

[0052] Step S4. Preparation of liposome nanoparticles targeting spleen immune cells.

[0053] Cationic lipid C12-200, dioleoylphosphatidylethanolamine, cholesterol, liposome DMG-PEG, and dioleoylphosphatidic acid were mixed at a molar fraction of 24.5:11.2:32.6:1.8:30, and then ethanol was added at a volume fraction of 17.5% to prepare liposome nanoparticles. RNA was dissolved in a 10 millimolar citrate buffer (pH = 4.0) at a concentration of 6.66 grams per liter at room temperature, and the liposome solution and RNA solution were quickly mixed at a volume ratio of 3 / 1 to obtain an RNA-LNP mixed solution with a total lipid / RNA weight ratio of 40 / 1. The RNA is a mixture of the RNA sequence encoding the fusion protein described in step S2 and the gRNA described in step S3, and the molar ratio of the two is 1:3.

[0054] Step S5. In vivo gene editing.

[0055] Ten B6..Fas of the same age and sex were collected. lpr Lyz2 Akaluc .Crbn I391V / I391V The mice were randomly divided into two groups, AB, with five mice in each group. Group A was treated with ibodomide to terminate gene editing, while group B was not treated with termination editing. All mice were injected with 50 μL of 2.5 mg / kg RNA-LNP mixed solution through the ophthalmic vein on days 0, 2, and 4. For mice in group A, ibodomide was orally administered at a dose of 30 mg / kg once a day starting on the seventh day after the injection of the RNA-LNP mixed solution for two weeks.

[0056] Step S6. Verification of gene editing effect.

[0057] The mice were imaged using a small animal in vivo imager on days 0, 7, 14, and 21 after gene editing to obtain the intensity and distribution of the mouse autofluorescence. lpr Lyz2 Akaluc .Crbn I391V / I391V Under normal physiological conditions, the Lyz2 gene in mice expresses luciferase, which binds to the substrate expressed by the Fas gene to emit spontaneous fluorescence. Since the targeted knockout of the Lyz2 gene in the spleen of mice resulted in the non-expression of luciferase, the spontaneous fluorescence of the spleen of mice in group B weakened over time; the fluorescence intensity of the spleen of mice in group A decreased before the administration of ibodomide, indicating the effect of gene knockout; after the use of ibodomide to terminate the gene knockout, the fluorescence intensity remained stable and then rebounded, indicating that the gene editing system terminated. The mice were killed on the 21st day, and the spleen was taken to extract DNA, and the knockout of the Lyz2 gene was verified by sequencing (see Figure 4 ).

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CRISPR / Cas system based on ibodomide termination and a gene editing kit based on the system, characterized in that: The kit includes coding RNA, gRNA, liposome nanoparticle material and ibodomide: The coding RNA includes an RNA sequence encoding an ibodomide binding sequence and an RNA sequence encoding a CRISPR / Cas system gene editing tool enzyme, and is used to encode a fusion protein of the ibodomide binding sequence and the CRISPR / Cas system gene editing tool enzyme; The gRNA is used to guide the fusion protein to edit the target gene; and, The ibodomide is used to terminate the gene editing effect of the fusion protein.

2. The kit according to claim 1, characterized in that The RNA sequence encoding the ibodomide binding sequence is shown in any one of SEQ ID NOs: 1-9.

3. The kit according to claim 1, characterized in that The gene editing tool enzyme is selected from Cas9, Cas12, Cas13, Cas14, APOBEC1, APOBEC2, APOBEC3 and APOBEC4; preferably, the gene editing tool enzyme protein is Cas9; more preferably, the sequence of the encoding RNA is as shown in SEQ ID NO:

10.

4. The kit according to claim 1, characterized in that The gRNA targets Lyz2, and preferably, the sequence of the gRNA is shown in any one of SEQ ID NOs: 11-13.

5. The kit according to any one of claims 1 to 4, characterized in that The kit also includes a delivery agent for delivering the coding RNA and gRNA; Preferably, the delivery agent is a liposome nanoparticle; Preferably, the delivery agent is a targeted liposome nanoparticle, for example, liver targeting, kidney targeting, lung targeting, gastrointestinal tract targeting and heart targeting; Preferably, the delivery agent comprises cationic lipid C12-200, 5A2-SC8, DLin-MC3-DMA11, DODAP, anionic lipid 14PA, didodecyldimethylammonium bromide, dioleoylphosphatidylethanolamine, cholesterol, liposomal DMG-PEG and dioleoylphosphatidic acid.

6. A CRISPR / Cas system gene editing method based on ibodomide termination, characterized in that: The method comprises the following steps: (1) delivering the coding RNA and gRNA to the target cell to perform gene editing on the target gene, wherein the coding RNA comprises an RNA sequence encoding an ibodomide binding sequence and an RNA sequence encoding a CRISPR / Cas system gene editing tool enzyme, and is used to encode a fusion protein of the ibodomide binding sequence and the CRISPR / Cas system gene editing tool enzyme, and the gRNA is used to guide the fusion protein to edit the target gene; (2) adding ibodomide to terminate the gene editing of the fusion protein.

7. The method according to claim 6, characterized in that The RNA sequence encoding the ibodomide binding sequence is shown in any one of SEQ ID NOs: 1-9.

8. The method according to claim 5, characterized in that The CRISPR / Cas system gene editing tool enzyme is selected from Cas9, Cas12, Cas13, Cas14, APOBEC1, APOBEC2, APOBEC3 and APOBEC4; preferably, the CRISPR / Cas system gene editing tool enzyme is Cas9, and preferably, the sequence of the encoding RNA is as shown in SEQ ID NO:

10.

9. The method according to claim 6, characterized in that The gRNA targets Lyz2, and preferably, the sequence of the gRNA is shown in any one of SEQ ID NOs: 11-13.

10. The method according to any one of claims 1 to 4, characterized in that: Delivering the coding RNA and gRNA to target cells via liposome nanoparticles; Preferably, the liposome nanoparticles are targeted liposome nanoparticles, for example, liver-targeted, kidney-targeted, lung-targeted, gastrointestinal-tract-targeted and heart-targeted; Preferably, the liposome nanoparticles include cationic lipids C12-200, 5A2-SC8, DLin-MC3-DMA11, DODAP, anionic lipids 14PA, didodecyldimethylammonium bromide, dioleoylphosphatidylethanolamine, cholesterol, liposomal DMG-PEG and dioleoylphosphatidic acid.

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