Caffeine-regulated crisper-cas9 gene editor and preparation method and use thereof

Abstract

The present application provides a kind of caffeine regulation type CRISPR-Cas9 gene editor and its preparation method and purposes. Specifically, a kind of caffeine regulation type CRISPR-Cas9 gene editor composition of the present application, caffeine-induced dimerization system based on anti-caffeine heavy chain antibody (acVHH) is integrated with split Cas9, and constitutes CoCas9 gene editing system, can be conveniently packaged into viral vector such as AAV, so as to be efficiently delivered into cell, promote its wide application in gene editing, in vivo gene therapy and clinic.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a caffeine-regulated CRISPR-Cas9 gene editor, its preparation method, and its uses. Background Technology

[0002] CRISPR / Cas9 and its derivatives, including base editors and leader editors, can efficiently modify target sites in the eukaryotic genome under the guidance of sgRNA or pegRNA. These powerful DNA editing tools have provided a promising platform for the treatment of genetic diseases and have revolutionized the fields of biomedicine and genetic engineering. However, the persistent expression of uncontrolled Cas9 or deaminases in vivo has been shown to lead to severe genomic damage, adverse immune responses, and mutations in tumor suppressor genes or proto-oncogenes, undoubtedly raising concerns about the clinical application of gene editing. To reduce genotoxicity and cytotoxicity, various engineered inducible CRISPR / Cas9 (Cas9), base editors (CBE), and leader editors (PE) based on genetic, chemical, and physical strategies have been developed to achieve spatial and temporal control over gene editing. These systems can not only be used to mitigate or even eliminate genomic damage caused by long-term overexpression of effector proteins, but can also be embedded in genetically engineered animal models to explore the role of mutated genes in cancer or record biological events in vivo.

[0003] However, achieving efficient, low-background, and reversible spatiotemporally controlled gene editing in complex biological tissues remains a formidable challenge. Although several optogenetic techniques have been used to construct light-induced Cas9 or base editors by integrating photoinduced aggregation magnets or photosensitive cleavage groups with splitting Cas9, cleaved deaminases, or guide RNAs, this physical strategy appears unsuitable for in vivo regulation due to poor organ accessibility. Intracellular or in vivo genome editing control can also be achieved based on genetic transcriptional regulation strategies that fuse transcriptional activators or cell-specific promoters with Cas9 or sgRNAs. However, their practical application is significantly hampered by obstacles such as leaky expression and delays in transcription-to-translation.

[0004] Integrating split Cas9 or deaminase components with chemically induced dimerization (CID) systems has shown potential for developing inducible gene-editing systems. To date, several research groups have successfully used this approach to modulate Cas9 or deaminase activity and validated its ability to mitigate adverse effects by shortening the time window for gene-editing activity. However, when these systems are applied to humans or large genetically engineered animal models, the adverse cytotoxicity, poor permeability, and interference with normal metabolism associated with the chemical drugs used in these regulatory systems (such as rapamycin and doxycycline) must be considered. These drugs affect both targeted and non-targeted cells. Furthermore, limitations such as irreversibility, limited organ accessibility, high background activity, and high drug costs still need to be addressed to optimize CID-based strategies.

[0005] Therefore, there is an urgent need in this field to develop a safe, rapid, and reversible gene editing system that can efficiently avoid gene damage caused by Cas9. Summary of the Invention

[0006] The purpose of this invention is to provide a safe, rapid, and reversible gene editing system that can efficiently avoid gene damage caused by Cas9.

[0007] In a first aspect of the present invention, a caffeine-regulated CRISPR-Cas9 gene editor composition is provided, the composition comprising:

[0008] (a) The first fusion protein, comprising an N-terminal sequence element of the Cas9 nuclease and a caffeine-binding element fused together; and

[0009] (b) The second fusion protein comprises a C-terminal sequence element of the Cas9 nuclease and a caffeine-binding element fused together;

[0010] The first fusion protein and the second fusion protein bind to caffeine through the caffeine-binding element to form a caffeine-regulated CRISPR-Cas9 gene editor composition.

[0011] In another preferred embodiment, a dimer structure is formed through the interaction of the caffeine-binding element with caffeine, thereby combining the first fusion protein and the second fusion protein to form a dimer, namely a caffeine-regulated CRISPR-Cas9 gene editor composition.

[0012] In another preferred embodiment, the caffeine-regulated CRISPR-Cas9 gene editor composition is capable of entering the cell nucleus.

[0013] In another preferred embodiment, the caffeine-binding element is an anti-caffeine heavy chain antibody (acVHH).

[0014] In another preferred embodiment, the amino acid sequence of the anti-caffeine heavy chain antibody (acVHH) is shown in SEQ ID NO.3.

[0015] In another preferred embodiment, the CoCas9 gene editor composition has the function of precise genome editing.

[0016] In another preferred embodiment, the first fusion protein has a structure of formula Ia or formula Ib from the N-terminus to the C-terminus:

[0017] N1-X1-L1-Z1 (Ia)

[0018] N1-Z1-L1-X1 (Ib)

[0019] in,

[0020] N1 is a core output signal element or a core positioning signal element;

[0021] X1 is the N-terminal sequence element of the Cas9 nuclease;

[0022] L1 is either absent or linked to a peptide element;

[0023] Z1 is a caffeine-binding element;

[0024] "-" indicates a peptide bond or peptide linker.

[0025] In another preferred embodiment, the second fusion protein has a structure of formula IIa or IIb from the N-terminus to the C-terminus:

[0026] N2-X2-L2-Z2-N2 (IIa)

[0027] N2-Z2-L2-X2-N2 (IIb)

[0028] in,

[0029] N2 is a core positioning signal element or a core output signal element;

[0030] X2 is the C-terminal sequence element of the Cas9 nuclease;

[0031] L2 is either absent or linked to a peptide element;

[0032] Z2 is a caffeine-binding element;

[0033] "-" indicates a peptide bond or peptide linker.

[0034] In another preferred embodiment, the first fusion protein has a structure of formula Ia from the N-terminus to the C-terminus:

[0035] N1-X1-L1-Z1(Ia)

[0036] in,

[0037] N1 is the core output signal element;

[0038] X1 is the N-terminal sequence element of the Cas9 nuclease;

[0039] L1 is either absent or linked to a peptide element;

[0040] Z1 is a caffeine-binding element;

[0041] "-" indicates a peptide bond or peptide linker;

[0042] The second fusion protein has the structure of formula IIa from the N-terminus to the C-terminus:

[0043] N2-X2-L2-Z2-N2(IIa)

[0044] in,

[0045] N2 is the nuclear positioning signal element;

[0046] X2 is the C-terminal sequence element of the Cas9 nuclease;

[0047] L2 is either absent or linked to a peptide element;

[0048] Z2 is a caffeine-binding element;

[0049] "-" indicates a peptide bond or peptide linker.

[0050] In another preferred embodiment, the amino acid sequence of the N-terminal sequence element of the Cas9 nuclease is shown in SEQ ID NO.5.

[0051] In another preferred embodiment, the amino acid sequence of the C-terminal sequence element of the Cas9 nuclease is shown in SEQ ID NO.6.

[0052] In another preferred embodiment, the amino acid sequence of the nuclear output signal element is shown in SEQ ID NO.4.

[0053] In another preferred embodiment, the amino acid sequence of the nuclear localization signal element is shown in SEQ ID NO.7.

[0054] In another preferred embodiment, the amino acid sequence of the first fusion protein is shown in SEQ ID NO.1.

[0055] In another preferred embodiment, the amino acid sequence of the second fusion protein is shown in SEQ ID NO.2.

[0056] In another preferred embodiment, the CoCas9 gene editor composition is a CRISPR-Cas9 gene editor that enters the cell nucleus under caffeine regulation.

[0057] In another preferred embodiment, the N-terminal sequence element and the C-terminal sequence element of the Cas9 nuclease are polymerized in the cell nucleus by caffeine regulation to form a complete Cas9 nuclease.

[0058] In another preferred embodiment, the Cas9 nuclease is selected from the group consisting of Cas9, SpCas9, or combinations thereof.

[0059] In another preferred embodiment, the Cas9 nuclease is derived from Streptococcus pyogenes.

[0060] In a second aspect of the invention, a gene editing system is provided, comprising a caffeine-regulated CRISPR-Cas9 gene editor composition or its encoded polynucleotide as described in the first aspect of the invention.

[0061] In another preferred embodiment, the gene editing system includes a CRISPR-Cas9 gene editing system, a lead editing system, a cytosine base editing system, and an adenine base editing system.

[0062] In another preferred embodiment, the gene editing system also includes guide RNA.

[0063] In another preferred embodiment, the sequence of the guide RNA is selected from any of the nucleotide sequences shown in SEQ ID NO. 8-14.

[0064] In a third aspect of the invention, a polynucleotide encoding the caffeine-regulated CRISPR-Cas9 gene editor composition of the first aspect of the invention is provided, the polynucleotide comprising:

[0065] (i) a first polynucleotide encoding the first fusion protein; and

[0066] (ii) A second polynucleotide, which encodes the second fusion protein.

[0067] In another preferred embodiment, the first polynucleotide and the second polynucleotide are located on the same nucleic acid chain.

[0068] In another preferred embodiment, the first polynucleotide and the second polynucleotide are located on different nucleic acid chains.

[0069] In a fourth aspect of the invention, a carrier is provided, comprising a first carrier and a second carrier, the first carrier containing the first fusion protein and the second carrier containing the second fusion protein, wherein the first fusion protein and the second fusion protein are as defined in the first aspect of the invention.

[0070] In another preferred embodiment, the vector further contains guide RNA.

[0071] In another preferred embodiment, the vector is selected from the group consisting of plasmids and viral vectors.

[0072] In another preferred embodiment, the vector is selected from adenovirus, adeno-associated virus, lentivirus, or a combination thereof.

[0073] In another preferred embodiment, the vector is adeno-associated virus.

[0074] In a fifth aspect of the invention, a genetically engineered cell is provided, the genome of which is edited using a gene editing system as described in the second aspect of the invention.

[0075] In another preferred embodiment, the cells contain the polynucleotides described in the third aspect of the invention or the carriers described in the fourth aspect of the invention.

[0076] In another preferred embodiment, the cells are selected from the group consisting of prokaryotic cells, eukaryotic cells, or combinations thereof.

[0077] In another preferred embodiment, the cells are selected from the group consisting of Escherichia coli and mammalian cells.

[0078] In another preferred embodiment, the cell is a mammalian somatic cell or stem cell.

[0079] In a sixth aspect of the invention, a composition is provided comprising the composition described in the first aspect of the invention, the gene editing system described in the second aspect of the invention, or the vector described in the fourth aspect of the invention.

[0080] In another preferred embodiment, the composition includes a pharmaceutical composition or a laboratory formulation composition.

[0081] In a seventh aspect of the invention, the use of the composition as described in the sixth aspect of the invention in the editing of a predetermined target gene is provided.

[0082] In another preferred embodiment, the gene editing includes in vivo gene editing and in vitro gene editing.

[0083] In another preferred embodiment, the gene editing includes both therapeutic and non-therapeutic gene editing.

[0084] In another preferred embodiment, the gene editing is non-diagnostic and non-therapeutic in vitro gene editing.

[0085] In another preferred embodiment, the gene editing is performed on mammalian cells (such as somatic cells of humans or non-human primates).

[0086] In another preferred embodiment, the target gene includes genes selected from the group consisting of EGFP, EMX1, GAPDH, CCR5CFTR, WAS, or Hpd genes.

[0087] In an eighth aspect of the present invention, a gene editing method is provided, comprising the steps of:

[0088] (1) Provides the target nucleic acid sequence to be edited and the caffeine-regulated CRISPR-Cas9 gene editor composition described in the first aspect of the present invention; and

[0089] (2) In the presence of the caffeine-regulated CRISPR-Cas9 gene editor composition, gene editing is performed on the provided nucleic acid target sequence to be edited.

[0090] In another preferred embodiment, the gene editing method is performed in vitro or in vivo.

[0091] In another preferred embodiment, the target sequence is selected from the group consisting of nucleotide sequences as shown in any of SEQ ID NO. 8-14.

[0092] In a ninth aspect of the present invention, a kit is provided, the kit comprising:

[0093] (a) a first container, and a first carrier located within the first container; and

[0094] (b) a second container, and a second carrier located in the second container;

[0095] The first carrier contains the first fusion protein, and the second carrier contains the second fusion protein, wherein the first fusion protein and the second fusion protein are as defined in the first aspect of the present invention.

[0096] In a tenth aspect of the invention, the use of the caffeine-regulated CRISPR-Cas9 gene editor composition of the first aspect of the invention, the gene editing system of the second aspect of the invention, or the vector of the fourth aspect of the invention in the preparation of reagents and / or pharmaceuticals for gene editing is provided.

[0097] In an eleventh aspect of the present invention, the use of the caffeine-regulated CRISPR-Cas9 gene editor composition of the first aspect of the present invention, the gene editing system of the second aspect of the present invention, or the vector of the fourth aspect of the present invention in the preparation of reagents and / or drugs for treating hereditary metabolic diseases is provided.

[0098] In another preferred embodiment, the hereditary metabolic disorder includes hereditary tyrosinemia.

[0099] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0100] Figure 1 The diagram shows a schematic of the construction of caffeine-based Cas9 (CoCas9) and the resulting image. Figures a and b show the construction strategy of CoCas9 and the structural schematics of CoCas9-N and CoCas9-C. Figure c shows that under natural conditions, the two elements CoCas9-N and CoCas9-C are isolated in the nucleus and cytoplasm, respectively, preventing their binding and function. In the presence of caffeine (Caf), CoCas9-C containing the nuclear export signal (NES, shown in red) dimers with CoCas9-N containing the NLS, recombinating into a nuclease-active dimer to form CoCas9. This shifts the balance towards nuclear transfection, allowing DNA to become the target under the guidance of sgRNA. Figure d shows a schematic diagram of CoCas9 or Cas9 transfection. By transfecting EGFP-expressing 293 cells with sgRNA targeting the EGFP gene, transgenic vectors (CoCas9-N, CoCas9-C, or Cas9), and transposases, the proportion of EGFP-negative cells was measured after 72 hours. Simultaneously, EGFP-negative cells stably expressing Cas9 and CoCas9 were sorted. Figure e shows the results of EGFP-negative cell levels detected by flow cytometry; Figure f shows the percentage of indels (insertions) resulting from controlled editing of endogenous genes (CCR5, CFTR, EMX1, GAPDH, and WAS) by CoCas9.

[0101] Figure 2The results show the relationship between the duration and concentration of caffeine and CoCas9 activity. Figure a shows the deep sequencing results of the EMX1 site after transfecting EGFP-negative cells stably expressing CoCas9 with an sgRNA expression plasmid targeting the EMX1 site, followed by treatment with different concentrations of caffeine for 72 hours. Figure b shows the deep sequencing results of the EMX1 site after transfecting EGFP-negative cells stably expressing CoCas9 with an EMX1 sgRNA plasmid, followed by treatment with 100 μM caffeine for different durations. Figure c shows the deep sequencing data assessing CoCas9 reversibility. The "Caf+72h" group refers to EGFP-negative cells stably expressing CoCas9 treated with caffeine for 72 hours, followed by electrotransfection of the EMX1-targeting sgRNA into these cells, and continued culture in caffeine-containing medium. The "Caf+72h / -" group refers to... EGFP-negative cells stably expressing CoCas9 were initially treated with caffeine for 72 hours, followed by electrotransfection with sgRNA targeting the EMX1 site. The cells were then cultured in caffeine-free medium. The untreated group consisted of EGFP-negative cells stably expressing CoCas9 cultured in caffeine-free medium for 72 hours, followed by further culture in caffeine-free medium. Figure d shows the detection results of long-term Cas9 expression and DNA damage caused by CoCas9. The Cas9 group consisted of EGFP-negative cells stably expressing Cas9 cultured for 10 days followed by TUNEL staining. The CoCas9 group consisted of EGFP-negative cells stably expressing CoCas9 cultured for 3 days with caffeine, followed by 7 days in caffeine-free medium before TUNEL staining. The Control group consisted of wild-type HEK293 cells, also known as the WT group. The right figure shows the statistical proportion of TUNEL-positive cells.

[0102] Figure 3This study demonstrates that in vivo gene editing using CoCas9 can effectively treat hereditary tyrosinemia type I (HT1). Figure a shows a schematic diagram of the structure of the Hpd sgRNA expression cassette combined with the CoCas9 component driven by the CMV promoter; Figure b shows the experimental flowchart of in vivo gene therapy in an HT1 mouse model using CoCas9, where NTBC refers to Nitisinone, a drug used to treat hereditary tyrosinemia type I; rAAV refers to the recombinant adeno-associated virus vector used to deliver the CoCas9 expression plasmid; Figures c and d show the weight changes and survival rates of HT1 mouse models after NTBC withdrawal, after rAAV injection with or without caffeine feeding, and HT1 mice injected with the empty rAAV vector without NTBC feeding. In this study, the FKAC+ group refers to HT1 mice injected with rAAV and fed with caffeine; the FKAC- group refers to HT1 mice injected with rAAV but not fed with caffeine; and the FK group refers to control HT1 mice injected with empty rAAV vector. Figures e and f show the serum ALT and AST levels of FKAC+, FKAC-, and FK mice 30 days after NTBC withdrawal, respectively. Figures g and h show the expression levels of HPD protein in liver tissue collected on day 28 after NTBC treatment cessation, detected by immunohistochemistry (IHC) and Western blotting (WB), respectively. FKAC+ is... The first group refers to HT1 mice injected with rAAV and fed with caffeine; the second group refers to HT1 mice injected with rAAV but not fed with caffeine; the third group refers to control HT1 mice injected with empty rAAV vector; Figure i shows the quantitative analysis of amplicon sequencing reads from liver tissues of FKAC+, FKAC-, and FK mice, where FKAC+1#~3# refers to HT1 mice numbered 1~3 injected with rAAV and fed with caffeine; FKAC-1#~3# refers to HT1 mice numbered 1~3 injected with rAAV but not given caffeine; and HT1 mice injected with empty rAAV vector served as the control group. Detailed Implementation

[0103] Through extensive and in-depth research and screening, the inventors have developed for the first time a caffeine-regulated CRISPR-Cas9 gene editor composition (hereinafter referred to as the "CoCas9 gene editor composition"). This invention integrates a caffeine-induced dimerization system based on an anti-caffeine heavy chain antibody (acVHH) with splitting Cas9 cells to form a CoCas9 gene editing system capable of highly efficient in vivo genome editing, which can be strictly controlled by caffeine. Unexpectedly, this invention discovered that caffeine-induced acVHH homodimerization can effectively bind the N-terminus and C-terminus of splitting Cas9 cells, which respectively carry nuclear export signals (NES) and nuclear localization signals (NLS), forming a dimeric gene-editing CoCas9 with nucleus-entry capabilities. Furthermore, this invention found that caffeine in the cell nucleus can also maintain its binding with the anti-caffeine heavy chain antibody (acVHH), thereby effectively maintaining the structural integrity and stability of the CoCas9 gene editor dimer composition, and excellently restoring the genome-targeting and DNA double-strand cleavage functions of the splitting Cas9 protein through dimerization. On the other hand, by introducing acVHH, the inventors enabled the CoCas9 gene editor to be easily packaged into a recombinant adeno-associated virus vector (rAAV) with limited loading capacity, promoting its widespread application in gene editing, in vivo gene therapy, and clinical practice. This allows it to be delivered into mammals, thereby achieving efficient and controllable in vivo gene editing. Based on this, the inventors completed this invention.

[0104] the term

[0105] As used herein, the terms “caffeine-regulated CRISPR-Cas9 gene editor composition of the present invention,” “gene editor of the present invention,” “CoCas9 gene editor composition of the present invention,” “CoCas9 system of the present invention,” and “CoCas9” are used interchangeably to refer to the caffeine-regulated CRISPR-Cas9 gene editor composition described in the first aspect of the present invention.

[0106] As used herein, the term CRISPR / Cas system refers to more than one gRNA that directs a catalytically active Cas9 or a variant thereof, or an inactivated nickase Cas9 or a variant thereof, to a desired genomic site where, upon recognition of the appropriate PAM region and base sequence, a double-strand break is formed by specific Cas9 action.

[0107] As described in this article, genome editing involves any modification to genomic and / or non-genomic DNA due to the CRISPR / Cas system and its ability to induce double-strand break repair (DSB). This allows for single-base editing, knock-in, or knock-out of cells and / or organisms.

[0108] As used herein, the term "dimerization system" refers to a pair of peptides or peptide domains linked by caffeine-binding elements. As used herein, the term "dimerization" refers to a protein domain or peptide pair linked to other domains of a different type via caffeine-binding elements.

[0109] As used in this article, the term "organism" refers to any living organism.

[0110] As used herein, the term "cell" refers to a eukaryotic or prokaryotic cell, cellular organism, or multicellular organism (cell line) cultured as a single-celled entity used as a recipient of nucleic acids, and includes daughter cells of the original cell that have been genetically modified by containing nucleic acids. The term primarily refers to cells of higher-developing eukaryotic organisms, preferably vertebrates, and preferably mammals. The invention also relies on invertebrate cells, preferably plant cells. The term "cell" also refers to human cell lines and plant cells. Naturally, as a result of natural, random, or planned mutations, the offspring of a cell are not necessarily identical to the parent in morphology and its DNA complement. A "genetically modified host cell" (also called a "recombinant host cell") is a host cell in which nucleic acids have been introduced. Eukaryotic genetically modified host cells are formed by introducing suitable nucleic acids or recombinant nucleic acids into suitable eukaryotic host cells. The invention described below includes host cells and organisms containing (transiently or stably) nucleic acids according to the invention, carrying the operon record according to the invention. Suitable host cells are known in the art and include eukaryotic cells. Proteins are known to be expressed in the cells of the following organisms: humans, rodents, cattle, pigs, poultry, rabbits, etc. Host cells can include primary cell lines or cultured cell lines of immortalized origin.

[0111] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length, and is not limited to single-stranded, double-stranded, or longer-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers having a thiophosphate polymer backbone made of purine and pyrimidine bases or other naturally, chemically or biochemically modified, synthetic or derived nucleotide bases.

[0112] As used herein, the term “recombination” refers to a specific nucleic acid (DNA or RNA) as the product of various combinations of cloning, restriction, and / or ligation that produce constructs having structural coding or non-coding sequences different from those of endogenous nucleic acids in the natural host system.

[0113] CoCas9 gene editing system

[0114] In the pursuit of highly safe and practical chemically induced spatiotemporally controlled gene editing strategies, this invention has noticed a caffeine-induced dimerization system based on an anti-caffeine heavy chain antibody (acVHH), named COSMO. Considering caffeine's minimal cytotoxicity (associated with reduced risk of various chronic diseases), broad organ accessibility (capable of crossing the blood-brain barrier), and cost-effectiveness, this CID system is considered an ideal solution for developing regulated gene editing systems. In this invention, by integrating COSMO with a splitting Cas9, a novel chemically induced genome editing system for rapid, controlled genome editing is established, named CoCas9.

[0115] Based on experiments at the mammalian cell level, this invention demonstrates that caffeine can effectively modulate CoCas9 activity, with minimal background activity in the absence of caffeine. The rapid response and reversibility provided by COSMO enable the induction system developed in this invention to efficiently circumvent Cas9-induced genomic damage. Furthermore, the successful treatment of inherited metabolic diseases via rAAV-mediated CoCas9 demonstrates the potential of the caffeine-induced gene editing system developed in this invention for in vivo gene therapy. Therefore, the CoCas9 gene editing system provided by this invention is a safe, rapid, and reversible chemically induced genome engineering gene editing system suitable for mammalian cells and in vivo.

[0116] The present invention provides a caffeine-regulated CRISPR-Cas9 gene editor composition comprising a first fusion protein and a second fusion protein (as described in the first aspect of the present invention).

[0117] Preferably, the gene editor of this invention is based on the integration of the caffeine-induced dimerization system (COSMO) of anti-caffeine heavy chain antibody (acVHH) with splitting Cas9, establishing a chemically induced genome editing system for rapid and controlled genome editing, named CoCas9, which exhibits minimal background activity in the absence of caffeine. The rapid response and reversibility provided by COSMO enable the developed induction system to efficiently circumvent genome damage caused by Cas9 and deaminases. Furthermore, CoCas9 mediated by rAAV has successfully treated inherited metabolic diseases, promoting its widespread application in gene editing, gene therapy, and clinical settings. This provides a safe, rapid, and reversible chemically induced genome engineering gene editing platform for mammalian cells and in vivo.

[0118] There are no particular limitations on the various elements used in the caffeine-regulated CRISPR-Cas9 gene editor of this invention; elements known in the art or derived similar elements can be used. Those skilled in the art can obtain the corresponding elements using conventional methods, such as PCR, fully artificial chemical synthesis, and enzyme digestion, and then link them together using well-known DNA ligation techniques to form the construct assembly of this invention. Inserting the caffeine-regulated CRISPR-Cas9 gene editor (first nucleic acid element and second nucleic acid element) of this invention into a foreign vector constitutes the vector assembly of this invention (first vector and second vector).

[0119] Hereditary tyrosinemia Type I (HT1)

[0120] Hereditary tyrosinemia type I (HT1) is a rare genetic disorder caused by a deficiency of an enzyme called fumarate acetoacetate hydrolase (FAH) in the liver and kidney tissues, leading to impaired tyrosine metabolism.

[0121] Hereditary tyrosinemia type I presents with diverse clinical manifestations, and can be classified as neonatal or acute, or chronic. Early symptoms of the acute type primarily include irritability, vomiting, diarrhea, fever, hypoglycemia, and liver failure symptoms such as hepatomegaly, jaundice, and bleeding tendency; the condition deteriorates rapidly. The chronic type mainly manifests as progressive cirrhosis and renal tubular dysfunction. Without treatment, hereditary tyrosinemia type I can lead to liver and kidney complications, and even be life-threatening.

[0122] NTBC

[0123] NTBC stands for Nitisinone, a drug used to treat hereditary tyrosinemia type I (HT1). Its full name is "2-(2-nitro-4-trifluoromethyl-benzoyl)-cyclohexane-1,3-dione". NTBC is one of the main treatment methods for HT1 patients, significantly improving their quality of life and prognosis. However, it requires lifelong use and strict dosage control and monitoring under the guidance of a physician. Furthermore, NTBC treatment does not completely cure HT1, but rather controls disease progression; therefore, patients also need regular liver function tests and long-term follow-up.

[0124] The main advantages of this invention include:

[0125] (1) This invention is the first to utilize existing protein structure information of Cas9 to develop a safe, rapid and reversible caffeine-regulated CRISPR-Cas9 gene editor.

[0126] (2) This invention is the first to use caffeine-induced acVHH to effectively combine the N-terminus and C-terminus of split Cas9 cells, which are respectively carrying nuclear output signal (NES) and nuclear localization signal (NLS), to form a dimer with the ability to enter the nucleus.

[0127] (3) The caffeine used in this invention is a small molecule with better safety, broad biocompatibility and low cost, making it an ideal inducing molecule for gene editing tools.

[0128] (4) This invention can effectively restore the function of genome targeting and DNA double-strand cutting by dimerizing the split Cas9 protein. By giving or removing caffeine, precise control of Cas9 gene editing activity is achieved, reducing the many risks caused by long-term or overexpression of Cas9 protein and reducing the possibility of genome damage.

[0129] (5) The present invention can be conveniently packaged into a recombinant adeno-associated virus vector (rAAV) with limited loading capacity, and can be delivered into mammals, thereby achieving efficient and controllable in vivo gene editing at the in vivo level, and is expected to be widely used in the field of in vivo gene therapy.

[0130] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0131] The experimental methods involved in this application are as follows:

[0132] Laboratory animals:

[0133] All mouse experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of the Guangzhou Institutes of Biomedicine and Health (GIBH). WT and HT1 mice were housed in an environment with a 12-hour light-dark cycle, provided with adequate standard rodent feed and water. When necessary, HT1 homozygous mice were given drinking water (Sigma, PHR1731) containing 10 mg / L NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexenedione).

[0134] Cell culture and transfection:

[0135] HEK293 and HEK293T cells were cultured and passaged in DMEM medium (Dulbecco's modified Eagle's medium; HyClone, SH30243.01) supplemented with 10% (v / v) fetal bovine serum (FBS; Gibco, 10270-106). Cells were then cultured in a cell culture incubator at 37°C and 5% CO2.

[0136] For electroporation transfection: via Neon TM The plasmid was transfected into HEK293 cells using a Life Technology transfection system at 1150V, a pulse duration of 20ms, and a pulse count of 2. For Cas9 editing, 2 μg sgRNA, 4 μg CoCas9-N, 4 μg CoCas9-C, and 4 μg PBase, or 2 μg sgRNA, 8 μg Cas9, and 4 μg PBase were transfected into the cells. In the experimental group, electroporated cells were evenly seeded in two wells, one treated with caffeine and the other untreated. 72 hours after transfection, transfected cells were collected for flow cytometry analysis to determine the proportion of EGFP-negative cells. EGFP-negative cells were then sorted and cultured to obtain transgenic cells. 2 μg of sgRNA targeting the endogenous gene was electroporated into the sorted cells to assess the endogenous gene editing efficiency.

[0137] For polyethyleneimine (PEI)-based transfection: Cells were digested and seeded into 24-well plates. After 12–24 hours, when the cell density was 60–80%, 3 μg of PEI (Sigma-Aldrich, 408727) and 1 μg of total plasmid were transfected into the cells according to the transfection reagent instructions. In the experimental group, the plasmid quantity was doubled as described above, and cells in two wells were transfected: one well was treated with caffeine, and the other well was not. Transfected cells were treated with puromycin at a concentration of 1 μg / ml for 24 hours. 72 hours after transfection, cells were collected for flow cytometry analysis to determine the proportion of EGFP-positive cells or to detect endogenous gene editing efficiency.

[0138] Flow cytometry detection:

[0139] To determine the proportion of EGFP fluorescent cells, transfected cell samples were digested with 0.25% trypsin, washed with 1× phosphate-buffered saline (PBS), and resuspended. High-quality analysis was then performed using a FACS AriaIIU flow cytometer. EGFP-negative cells were sorted and retrieved for further culture to test endogenous gene editing efficiency. Data were subsequently analyzed using FlowJo V10.

[0140] Genomic DNA extraction and genotyping:

[0141] Two methods were used to extract whole-genome DNA: (i) For HEK293 or HEK293T cells, a small number of cells were collected and added to 10 μl of lysis buffer (0.45% NP-40 plus 0.6% proteinase K), incubated at 56°C for 60 min, and then incubated at 96°C for 10 min; (ii) For neonates or virally infected mice, genomic DNA was extracted from tail or liver tissue using the TIANamp Genomic DNA Purification Kit (TIANGEN, DP304-03) following the manufacturer's instructions. Cell lysates or extracted DNA were then used as templates for PCR amplification. PCR products were directly sent to Sanger sequencing.

[0142] Sanger sequencing detection and analysis editing efficiency:

[0143] To assess the gene editing efficiency at the target site, specific primers were used to amplify the target region, and 2 μL of cell lysis buffer or 100 ng of extracted DNA was used to amplify the DNA fragment containing the editing site. PCR conditions were: 95°C for 5 minutes; 35 cycles, each consisting of 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 8 seconds; a final extension step of 72°C for 5 minutes; and maintenance at 12°C. Editing efficiency was analyzed using EditR based on Sanger sequencing results.

[0144] Amplicon sequencing and data analysis:

[0145] First, PCR was performed on cell lysates or extracted liver genomic DNA using site-specific primers. The amplified PCR products were then purified using an agarose gel extraction kit. Equal volumes of the purified products were mixed to prepare a DNA library, which was then sent to Annoroad Genetics Co., Ltd. for amplicon deep sequencing using the Nova Seq platform. The expected genotype and insertions / deletions were identified by analyzing the protospacer sequences read from the sequences.

[0146] TUNEL staining:

[0147] Cells containing transgenic CoCas9-N / C and Cas9 components were cultured in vitro for 10 days (Cells containing transgenic CoCas9-N / C: 3 days of caffeine treatment, 7 days of no caffeine treatment), and then seeded onto lysine-containing cell slides before TUNEL staining. 24 hours later, the cells were stained according to the instructions of the TUNEL FITC apoptosis detection kit (Vazyme). The cell slides were then observed using a Carl Zeiss LSM800 confocal microscope.

[0148] Production and purification of rAAV:

[0149] This was accomplished by Guangzhou Paizhen Biotechnology Co., Ltd. The optimal HPD-sgRNA, along with the U6 promoter and either the CoCas9-N or CoCas9-C fragment, was packaged into AAV8, designated AAV8-CoCas9-N and AAV8-CoCas9-C, respectively. The purified AAV8 was stored at -80°C.

[0150] rAAV and caffeine delivery:

[0151] rAAV8-CoCas9-N and rAAV8-CoCas9-C (50 μL of each vector injected into each mouse, at a concentration of 3 x 10^13 gene copies / mL) were thawed and mixed. This viral mixture was introduced into six 15-day-old HT1 mice via tail vein injection. Seven days after injection, NTBC administration was discontinued in Fah-KO (FK) mice, FK mice receiving rAAV delivery but not caffeine (FKA-), and Fah-KO mice receiving AAV delivery and caffeine (FKA+). For FK mice receiving AAV delivery, caffeine was administered intraperitoneally (ip) at a concentration of 0.005 mg / 1 g / day for the first week after AAV injection, and caffeine-infused water at a concentration of 1 mg / ml was provided for drinking for one week. One month after AAV delivery, FKA+ mice were euthanized, and their livers were collected to analyze HPD gene editing efficiency.

[0152] Hematological analysis:

[0153] Blood samples were collected from the ophthalmic vein. Serum was separated by centrifugation at 9000g for 20 minutes and stored at -80℃ until biochemical analysis. Blood biochemical indicators were tested by the Guangdong Provincial Animal Quality Monitoring Center.

[0154] Western blotting detection:

[0155] Liver tissues from WT, FK, FKA-, and FKA+ mice were collected and dissolved using RIPA buffer containing a proteinase K inhibitor. After homogenization, the samples were placed on ice and agitated for 30 minutes, then centrifuged at 14,000 rpm for 30 minutes at 4°C to obtain the supernatant. The total protein concentration of the samples was determined using a BCA protein quantification kit, followed by addition of SDS buffer and boiling at 100°C for 15 minutes before loading. Equal volumes of protein were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with TBST containing 5% skim milk for 2 hours, then incubated overnight at 4°C with mHPD antibody (proteintech, 10442-1-AP; 1:2000 dilution). The next day, the membrane was washed three times with TBST, then incubated with the corresponding horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. The membrane was washed three times with TBST for 8 minutes each time. The protein signal was then detected using a high-sensitivity luminescent solution and finally visualized using a MiniChemi™ 830.

[0156] Immunohistochemical detection:

[0157] Liver tissues obtained from WT, FK, FKA-, and FKA+ mice were fixed in 4% paraformaldehyde for 2 days and then embedded in paraffin for sectioning. The paraffin-embedded sections were dewaxed with xylene, then rehydrated sequentially in ethanol solutions of decreasing concentrations (100%, 90%, 80%, 70%, and 50%), and finally washed with distilled water. The sections were placed at 95°C for antigen retrieval and then cooled to room temperature. Immunohistochemical staining was then performed on the sections using mHPD antibody.

[0158] Example 1: Caffeine-controlled Cas9 (CoCas9) exhibits strictly controllable nuclease activity.

[0159] (1) To construct a caffeine-inducible Cas9 system, this application first requires the Cas9 protein to be cleaved to insert an anti-caffeine heavy chain antibody (acVHH). Combining COSMO technology with the CRISPR / Cas9 system, Cas9 is cleaved into two inactive parts at Glu573, and the caffeine-binding element acVHH (SEQ ID NO.3) is linked to the N-terminus of Cas9 (as shown in SEQ ID NO.5) and the C-terminus of Cas9 (as shown in SEQ ID NO.6), respectively named CoCas9-N (as shown in SEQ ID NO.1) and CoCas9-C (as shown in SEQ ID NO.2). Figure 1 (Figure a)

[0160] (2) To prevent the spontaneous recombination of the N-terminus and C-terminus of Cas9 into a functional protein, the nuclear output signal (NES, shown in red, as shown in SEQ ID NO.4) is attached to the N-terminus of CoCas9, and the nuclear localization signal (NLS, shown in green, as shown in SEQ ID NO.7) is attached to the C-terminus of CoCas9, thus spatially separating these two fragments into different cellular regions. Figure 1 (See Figure b). Under natural conditions, these two elements are isolated in the nucleus and cytoplasm, respectively, preventing their binding and function. In the presence of caffeine, CoCas9-N containing NES dimers with CoCas9-C containing NLS, reforming into a dimer with nuclease activity. This shifts the balance towards intranuclear importation, guiding DNA to become the target under the guidance of sgRNA. Figure 1 (Figure C in the middle)

[0161] (3) Evaluation of editing efficiency: The CoCas9 system was integrated into the piggyBac transposon vector to simulate the possibility of long-term effector protein persistence. sgRNAs targeting the EGFP gene sequence (as shown in SEQ ID NO. 8), expression plasmids expressing CoCas9-N and CoCas9-C, or the complete Cas9 protein, were designed and co-transfected with the sgRNA expression plasmids into HEK293 reporter cell lines with the fluorescent protein EGFP gene integrated at the hROSA26 site. After 24 hours, one group of cells transfected with CoCas9 was induced with 0.1 mM caffeine, while the other group was not induced. After 72 hours, the percentage of EGFP-negative cells generated by gene editing was quantified by flow cytometry to evaluate the editing performance of CoCas9 or Cas9, and whether CoCas9 would edit the target site under caffeine-free conditions. Figure 1 (Figure d in the middle)

[0162] The results showed that in the presence of CoCas9 and the sgRNA targeting EGFP, approximately 36% of the fluorescence intensity was inactivated in the presence of caffeine, reaching about 80% of the intact Cas9 level (approximately 46%). In the absence of caffeine, only about 3% of EGFP-negative cells were detected, which is similar to the level in the untreated control group (2%). Figure 1 (Image from the Chinese e-graph).

[0163] Building upon CoCas9's ability to induce editing of heterologous sequences, this study aimed to investigate whether CoCas9 could also perform controlled editing of genomic sites with different sequence backgrounds and characteristics. Therefore, EGFP-negative cells generated by Cas9 or caffeine-induced CoCas9 (i.e., CoCas9Caf+) editing of the EGFP gene were sorted to serve as tool cell lines for evaluating the performance of Cas9 or CoCas9 in targeting endogenous gene editing. Genomic sites targeting five endogenous genes (CCR5, CFTR, EMX1, GAPDH, and WAS) were then selected as target sites for sgRNA design (sgRNA sequences are shown in SEQ ID NO. 9-13, respectively). The constructed sgRNA expression plasmids were then electroporated into EGFP-negative cell lines. After 72 hours of caffeine or decaffeine-free treatment, cells were collected for deep sequencing to calculate the percentage of insertions and deletions (indels).

[0164] The results are as follows Figure 1 As shown in f, for caffeine-induced CoCas9 (CoCas9 Caf+), its editing efficiency reached an average of about 91% of that of the complete Cas9. Specifically, for sgRNA sites targeting CCR5, the average proportion of indels after complete Cas9 editing was 55.30%, while the average proportion after CoCas9 Caf+ editing was 47.63%; for sgRNA sites targeting CFTR, the average proportion of indels after complete Cas9 editing was 54.08%, while the average proportion after CoCas9 Caf+ editing was 40.82%; for sgRNA sites targeting EMX1, the average proportion of indels after complete Cas9 editing was 60.10%, while the average proportion after CoCas9 Caf+ editing was 51.64%; for sgRNA sites targeting GAPDH, the average proportion of indels after complete Cas9 editing was 62.67%, while the average proportion after CoCas9 Caf+ editing was 59.21%; and for sgRNA sites targeting WAS, the average proportion of indels after complete Cas9 editing was 63.87%, while the average proportion after CoCas9 Caf+ editing was 73.60%.

[0165] In the absence of caffeine (CoCas9 Caf-), the frequency of CoCas9 indels dropped to extremely low levels, with no significant difference from the untreated control group. For sgRNA sites targeting CCR5, the average indel proportion in the Control group was approximately 0.24%, while the average indel proportion after CoCas9 Caf- editing was approximately 0.27%. For sgRNA sites targeting CFTR, the average indel proportion after full Cas9 editing was approximately 2.18%, while the average indel proportion after CoCas9 Caf- editing was approximately 2.29%. For sgRNA sites targeting EMX1, the average indel proportion in the Control group was approximately 0.06%, while the average indel proportion after CoCas9 Caf- editing was approximately 0.40%. For sgRNA sites targeting GAPDH, the average indel proportion in the Control group was approximately 0.27%, while the average indel proportion after CoCas9 Caf- editing was approximately 0.59%. For sgRNA sites targeting WAS, the average indel proportion in the Control group was approximately 1.55%, while the average indel proportion after CoCas9 Caf- editing was approximately 2.09%. Figure 1 (Figure f in the middle)

[0166] The above data confirms that CoCas9 can achieve efficient and strictly controllable gene editing at mammalian genomic sites under the regulation of caffeine.

[0167] Example 2: Gene Editing Performance Testing of CoCas9

[0168] This study tested the editing properties of CoCas9 against genomic DNA under different caffeine concentrations to further evaluate its editing performance. Quantifying the relationship between the duration and concentration of caffeine and CoCas9 activity is crucial for achieving precise spatiotemporal control and minimizing potential side effects. Therefore, time and concentration gradient experiments were performed by co-transfecting CoCas9 and intact Cas9 transgenic EGFP-negative cells with EMX1 sgRNA, followed by treatment with different concentrations of caffeine (0 μM (untreated control), 0.1 μM, 10 μM, and 100 μM) and treatment durations of 100 μM caffeine (0 hours (untreated control), 0.25 hours, 0.5 hours, 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours). The performance of CoCas9 at different caffeine concentrations and treatment times was evaluated based on deep sequencing data targeting the EMX1 site. Compared with the untreated control, treatment with 0.1 μM caffeine for 72 hours or 100 μM caffeine for 15 minutes significantly induced gene editing. Figure 2The diagram (ab) shows that caffeine can rapidly and effectively activate CoCas9, enabling gene editing in mammalian cells.

[0169] Ideally, induced gene editing tools would be able to turn activation on or off as needed. To assess the reversibility of CoCas9, EGFP-negative cells stably expressing CoCas9 were first cultured in caffeine-containing medium for 72 hours, followed by electroporation of the cells with sgRNA targeting the EMX1 site (sgRNA sequence shown in SEQ ID NO. 9). After electroporation, the cells were divided into two groups: one group was replaced with caffeine-free medium and cultured (i.e., the Caf+72h / - group), while the other group continued to be cultured in caffeine-containing medium (i.e., the Caf+72h group). An untreated group was also included, which was decaffeinated throughout the entire treatment. Figure 2 The deep sequencing data of c showed that CoCas9 cleavage activity was only observed in cells treated with continuous caffeine (Caf+72h group), while the Caf+72h / - group showed extremely low levels of indels that were not different from the untreated group, indicating that CoCas9 is a reversible genome editing system that can achieve rapid functional shutdown after caffeine withdrawal.

[0170] Uncontrolled and persistent expression of Cas9 in mammalian cells has been shown to induce DNA damage. To further illustrate how the reversibility of CoCas9 can avoid these adverse side effects, this invention evaluated the advantages of CoCas9 in avoiding DNA damage using TUNEL fluorescence staining assays:

[0171] First, an sgRNA expression plasmid targeting the EMX1 site (sgRNA sequence shown in SEQ ID NO. 9) was transfected into EGFP-negative cells expressing CoCas9 or the complete Cas9 transgene to mimic targeted genome editing. Then, CoCas9-expressing cells were exposed to caffeine for 72 hours and cultured in decaffeinated medium for one week (CoCas9 group). Simultaneously, cells containing the complete Cas9 transgene were cultured as a control for ten consecutive days (Cas9 group). A control group (control group) was also established, containing cells without the editing system. Afterward, TUNEL staining was performed on all three groups of cells. The TdT-catalyzed FITC labeling after staining indicated apoptotic cells with DNA breaks.

[0172] The results showed that, compared with the untreated control group (WT), the number of TUNEL-positive cells in the group containing intact Cas9 was significantly increased, indicating that sustained expression of intact Cas9 leads to severe DNA damage. In contrast, CoCas9-expressing cells in a caffeine-free environment did not show significant TUNEL positivity due to the dissociation of various components, demonstrating that genome editing using CoCas9 can effectively avoid unnecessary DNA damage and apoptosis. Figure 2 d).

[0173] The data above demonstrate that CoCas9 represents an innovative platform for regulating Cas9, offering improved genetic editing efficiency, minimal background activity, and a range of safety advantages.

[0174] Example 3: In vivo gene editing using CoCas9 to treat hereditary tyrosinemia

[0175] This invention utilizes a mouse model of hereditary tyrosinemia type 1 (HT1) with a neomycin expression cassette inserted in exon 5 of the Fah gene to investigate the controllability and therapeutic potential of CoCas9 in in vivo gene therapy for hereditary diseases.

[0176] Previous studies have shown that inhibiting upstream HPD enzymes in the tyrosine metabolism pathway with drugs, or through Cas9-mediated Hpd gene knockout, can prevent the accumulation of toxic metabolites and rescue HT1-induced fatal liver failure. Therefore, the inventors designed an sgRNA (wherein the mouse mHpd sgRNA sequence is shown in SEQ ID NO. 14) that has been shown to introduce indel mutations into the Hpd gene, and integrated an Hpd sgRNA expression cassette driven by the pU6 promoter with CMV-driven CoCas9 components (CoCas9-N containing NES and CoCas9-C containing NLS) into a recombinant adeno-associated virus vector (rAAV). Figure 3 a. ITR refers to the inverted terminal repeat sequence in the rAAV vector that is crucial for genome replication, progeny genome generation and packaging, and the persistence of exogenous genes; EFs-NS refers to enhancers; WPREs refers to posttranscriptional regulatory elements.

[0177] To achieve efficient liver editing, this invention further packages the aforementioned rAAV vector plasmid containing two parts of the CoCas9 system into individual rAAV8, a serotype with high affinity for hepatocytes. Each rAAV vector, at a dose of 5E11 genome copies (5E11GC), is then administered via tail vein injection to 14-day-old HT1 mice. Figure 3(See Figure b). Simultaneously, some HT1 mice injected with the empty rAAV vector were designated as a control group (FahKO group, or FK group or FKUntreated group). Three days later (Day 0), all HT1 mice injected with rAAV were further divided into two groups. One group received 50 mg / kg caffeine via intraperitoneal injection (ip) for 7 days, followed by one week of caffeine-containing drinking water (1 mg / ml) (FK AAV+ / Caf+ group, or FKAC+ group). The other group received no caffeine treatment (FK AAV+ / Caf- group, or FKAC- group). Mice in the FKAC+, FKAC-, and FK groups continued NTBC treatment for seven days after Day 0 to ensure the survival of all hepatocytes and prevent the proliferation of corrective cells. NTBC refers to Nitisinone, an HPD enzyme inhibitor used to treat hereditary tyrosinemia type I.

[0178] After NTBC treatment was discontinued, the body weight and survival of mice in the FKAC+ and FKAC- groups, which continued to receive rAAV injections, and the FK Untreated control group, which received no treatment, were monitored regularly. The results showed that HT1 mice treated with rAAV and induced by caffeine (FKAAV+ / Caf+ group) survived to the end of the experiment, while HT1 mice treated with rAAV and given caffeine-free water (FKAAV+ / Caf- group) experienced significant weight loss after NTBC cessation and died successively within 20 days of NTBC withdrawal, similar to the untreated control group (FK Untreated). Figure 3 cd).

[0179] In the analysis of blood biochemical parameters, HT1 mice receiving caffeine-induced alanine aminotransferase (ALT) and AST (aspartate aminotransferase) levels were normal, showing no significant difference compared to age-matched wild-type mice (WT). In contrast, HT1 mice injected with rAAV but not receiving caffeine-induced alanine aminotransferase (FKAC-) showed severely abnormal ALT and AST levels, consistent with the FK group. Figure 3 ef).

[0180] To further investigate whether the restoration of the HT1 phenotype was due to HPD-deficient hepatocytes generated by caffeine-induced CoCas9 editing, FKAC+ mice were sacrificed on day 28 after NTBC treatment was stopped, and HPD levels in the collected liver tissues were detected by Western blotting (WB) and immunohistochemistry (IHC).

[0181] IHC staining results showed that a large number of HPD-negative hepatocytes were detected in the livers of caffeine-induced FKAC+ mice, while no HPD-negative hepatocytes were detected in the livers of uncaffeine-induced FKAC- mice, indicating that the rescue of the HT1 phenotype was associated with a significant reduction in HPD-positive cells. Figure 3 g). Correspondingly, Western blot analysis showed that the expression level of murine HPD protein (mHPD) in the liver of caffeine-induced FKAC+ mice was significantly reduced, while the mHPD level in the non-caffeine-induced (FKAC-) group was consistent with that in the WT or uninjected rAAV control group (FK). Figure 3 h).

[0182] Subsequently, genomic DNA was extracted from the liver tissues of all mice injected with rAAV and control HT1 mice, and the Hpd genomic region was amplified by PCR to quantify the in vivo gene editing efficiency of caffeine-induced CoCas9.

[0183] Quantitative analysis of amplicon depth sequencing reads revealed that the in vivo gene editing efficiency of CoCas9 induced by caffeine ranged from 39.99% to 50.57% (49.82% for FKAC+1#, 39.99% for FKAC+2#, and 50.57% for FKAC+3#, respectively). In contrast, the other three HT1 mice injected with rAAV treated with decaffeinated water showed low efficiencies, ranging from 0.54% to 2.70% (0.69% for FKAC-1#, 0.54% for FKAC-2#, and 2.7% for FKAC-3#, respectively), close to the background level of untreated HT1 mice (0.12%). Figure 3 i).

[0184] The above results demonstrate that the CoCas9 system can achieve efficient in vivo genome editing, and its potential in in vivo gene therapy can be demonstrated through strict control of caffeine.

[0185] discuss

[0186] Uncontrolled adverse consequences associated with effector proteins can significantly limit the application of gene editing systems in complex biological environments, necessitating substantial efforts to develop editing tools capable of rapid and reversible editing to precisely target desired sites at specific time points. Existing engineered chemically induced Cas9 has been validated for providing robust, efficient, and controllable genome editing. However, the clinical application of regulatory molecules remains a significant challenge due to their narrow safety profiles and even substantial risks.

[0187] For example, the rapamycin-induced FKBP-FRB dimerization system has been used to generate controlled Cas9, CBE, and ABE. Although rapamycin can treat various diseases and even delay aging, it has been reported to severely interfere with the endogenous mTOR signaling pathway, limiting its application in certain biological settings. While engineering optimization techniques such as Raplog (AP2-1967), iRap, and cRap can partially alleviate rapamycin-induced interference, they may lead to higher dosages and increased usage costs. Of particular note is the fact that the presence of rapamycin has been shown to inhibit double-strand break repair and high levels of DNA damage response, which, combined with Cas9-induced genetic damage, is undoubtedly extremely dangerous for in vivo gene editing therapy. Furthermore, the clinical implementation of chemically induced gene editing tools is also limited by delayed onset of action and insufficient reversibility, urgently requiring the exploration of more advanced regulatory systems.

[0188] In this invention, a caffeine-induced CoCas9 system was generated using the COSMO system comprising an optimized anti-caffeine heavy chain single-domain antibody (acVHH). The targeted editing activity of this engineered chemically controlled editing tool is strictly regulated by caffeine in mammalian cells. Under caffeine induction, CoCas9 exhibits targeted gene editing activity comparable to Cas9 and significantly reduces off-target effects, ensuring its effectiveness in in vivo therapeutic gene editing.

[0189] Furthermore, this invention demonstrates that CoCas9 can effectively edit target sites within a very short time window and exhibits good reversibility after metabolism or ligand withdrawal treatment. This will provide further safety assurance and more precise control for the clinical application of CoCas9.

[0190] Compared to other small molecule or inducer-based chemical induction systems, caffeine exhibits minimal cytotoxicity or significant side effects and has even been shown to be associated with a reduced risk of various chronic diseases or all-cause mortality. This undoubtedly lays the foundation for the safety of using caffeine for in vivo gene editing regulation. With its higher bioavailability and better compatibility with multiple routes of administration, using caffeine for in vivo induced gene editing will have significant advantages. In practical applications, this invention successfully remodeled the metabolic pathways of a Fah gene knockout HT1 mouse model using in vivo inducible editable CoCas9.

[0191] Due to the compact size of COSMO (only 118 residues) and the minimal chemical complexity of acVHH (194 Da), the sgRNA cassette can be packaged in two components of CoCas9 (CoCas9-N and CoCas9-C), allowing for more efficient targeted editing. Based on high-throughput sequencing of liver tissue from induced and uninduced samples, this invention further confirms that in vivo expression of CoCas9 can be strictly controlled by caffeine and effectively achieves efficient genome-targeted editing. More importantly, this invention restored multiple lethal phenotypes in HT1 mice through this regulatory editing, thereby achieving effective treatment for the HT1 mouse model. This is the first successful attempt at in vivo gene therapy for genetic diseases using regulatory gene editing tools, and a powerful demonstration of the significant potential of CoCas9 in in vivo gene therapy, further proving the advantages of CoCas9 in applications such as in vivo gene therapy for genetic diseases.

[0192] The sequence information involved in this invention is as follows:

[0193] SEQ ID NO.1: Amino acid sequence of CoCas9-N (underlined text represents NES sequence, italic text represents Cas9-N end sequence, bold text represents linker, and bold text represents anti-caffeine heavy chain antibody (acVHH) sequence)

[0194]

[0195]

[0196] SEQ ID NO.2: Amino acid sequence of CoCas9-C (underlined text represents NLS sequence, bold text represents anti-caffeine heavy chain antibody (acVHH) sequence, bold text represents linker, italic text represents Cas9-C terminal sequence)

[0197]

[0198] SEQ ID NO.3: Amino acid sequence of acVHH (N-terminus to C-terminus)

[0199] APEVQLQASGGGLVQAGGSLRLSCTASGRTGTIYSMAWFRQAPGKEREFLATVGWSSGITYYMDSVKGRFTISRDNAKNSAYLQMNSLKPEDTAVYYCTATRAWSVGYDYWGQGTQVTVSH

[0200] SEQ ID NO.4: Amino acid sequence of the nuclear output signal NES (N-terminus to C-terminus)

[0201] MLDLASLIL

[0202] SEQ ID NO.5: Amino acid sequence of the N-terminus of Cas9 (N-terminus - C-terminus)

[0203] DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE

[0204] SEQ ID NO.6: Amino acid sequence of the C-terminus of Cas9 (N-terminus - C-terminus)

[0205] CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0206] SEQ ID NO.7: Amino acid sequence of the nuclear localization signal (N - terminus - C - terminus)

[0207] PKKKRKV

[0208] SEQ ID NO.8: sgRNA sequence targeting the EGFP gene (5'-terminus - 3'-terminus)

[0209] TACCAGCAGAACACCCCCA

[0210] SEQ ID NO.9: sgRNA sequence targeting the CCR5 gene (5' end to 3' end)

[0211] TATTCAGGCCAAAGAATTCC

[0212] SEQ ID NO.10: sgRNA sequence targeting the CFTR gene (5' end to 3' end)

[0213] TGGAACAGAGTTTCAAAGTA

[0214] SEQ ID NO.11: sgRNA sequence targeting the EMX1 gene (5' end to 3' end)

[0215] GAGTCCGAGCAGAAGAAGAA

[0216] SEQ ID NO.12: sgRNA sequence targeting the GAPDH gene (5' end to 3' end)

[0217] AGCCCCAGCAAGAGCACAAG

[0218] SEQ ID NO.13: sgRNA sequence targeting the WAS gene (5' end to 3' end)

[0219] TGGATGGAGGAATGAGGAGT

[0220] SEQ ID NO.14: sgRNA sequence targeting the mHpd gene (5' end to 3' end)

[0221] CAACCCAGAAGGTCACCGAG

[0222] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A caffeine-regulated CRISPR-Cas9 gene editor composition, characterized in that, The composition comprises: (a) The first fusion protein, comprising an N-terminal sequence element of the Cas9 nuclease and a caffeine-binding element fused together; and (b) The second fusion protein comprises a C-terminal sequence element of the Cas9 nuclease and a caffeine-binding element fused together; The first fusion protein and the second fusion protein bind to caffeine through the caffeine-binding element to form a caffeine-regulated CRISPR-Cas9 gene editor composition.

2. The composition according to claim 1, characterized in that, The caffeine-binding element is an anti-caffeine heavy chain antibody (acVHH).

3. The composition according to claim 1, characterized in that, The first fusion protein has the structure of formula Ia from the N-terminus to the C-terminus: N1-X1-L1-Z1(Ia) in, N1 is the core output signal element; X1 is the N-terminal sequence element of the Cas9 nuclease; L1 is either absent or linked to a peptide element; Z1 is a caffeine-binding element; "-" indicates a peptide bond or peptide linker; The second fusion protein has the structure of formula IIa from the N-terminus to the C-terminus: N2-X2-L2-Z2-N2(IIa) in, N2 is the nuclear positioning signal element; X2 is the C-terminal sequence element of the Cas9 nuclease; L2 is either absent or linked to a peptide element; Z2 is a caffeine-binding element; "-" indicates a peptide bond or peptide linker.

4. The composition according to claim 1, characterized in that, The N-terminal and C-terminal sequence elements of the Cas9 nuclease are polymerized in the cell nucleus under caffeine regulation to form the complete Cas9 nuclease.

5. A gene editing system, characterized in that, It contains the caffeine-regulated CRISPR-Cas9 gene editor composition as described in claim 1, or the polynucleotide encoded thereon.

6. A polynucleotide encoding the caffeine-regulated CRISPR-Cas9 gene editor composition of claim 1, characterized in that, The polynucleotides include: (i) a first polynucleotide encoding the first fusion protein; and (ii) A second polynucleotide, which encodes the second fusion protein.

7. A carrier, characterized in that, It includes a first carrier and a second carrier, the first carrier containing the first fusion protein and the second carrier containing the second fusion protein, wherein the first fusion protein and the second fusion protein are as defined in claim 1.

8. A genetically engineered cell, characterized in that, The genome of the cell was edited using the gene editing system as described in claim 5.

9. A composition, characterized in that, It comprises the composition of claim 1, the gene editing system of claim 5, or the vector of claim 7.

10. A gene editing method, characterized in that, Including the following steps: (1) Provide the target nucleic acid sequence to be edited and the caffeine-regulated CRISPR-Cas9 gene editor composition of claim 1; and (2) In the presence of the caffeine-regulated CRISPR-Cas9 gene editor composition, gene editing is performed on the provided nucleic acid target sequence to be edited.

Images