Crispr-mediated gene transcription activation (crispra) system and use thereof

Abstract

The present invention relates to a CRISPR-mediated gene transcription activation (CRISPRa) system and use thereof. Preferably, the present invention relates to a miniCas-based gene transcription activation system (miniCRISPRa system) and use thereof. By using a smaller Cas protein and a transcription activation domain VP64, and using a multimerization peptide segment to rapidly recruit a large amount of VP64 to a target gene, a miniCRISPRa system with "a small molecular weight, high efficiency, unbiased nature, and ease of delivery" is developed, thereby achieving efficient transcription activation of the target gene. The present invention further relates to use of the CRISPRa system in treating genetic diseases, tumors, or delaying aging.

Description

A CRISPR-mediated gene transcription activation (CRISPRa) system and its applications Technical Field

[0001] This invention relates to a CRISPR-mediated transcriptional activation (CRISPRa) system and its applications, preferably to a miniCas-based transcriptional activation system (miniCRISPRa system) and its applications. Background Technology

[0002] Gene transcription activation technology is an important tool for studying gene function and has broad application prospects in the life sciences and medicine. For example, researchers have used gene activation screening in a mouse liver injury model to identify protein-coding genes that drive hepatocyte proliferation and tumorigenesis[1], providing potential targets for drug development for liver cancer treatment. Traditional gene expression regulation usually involves introducing natural or in vitro synthesized DNA-binding proteins, such as tetracycline repressor[2], Gal4[3], TALE protein, etc.[4,5], and fusing them with gene activation or repression domains to directly regulate the expression of related genes. However, since the above DNA-binding proteins are not unique in their selection of target DNA sequences, it is difficult to achieve activation of a specific gene in this way.

[0003] CRISPR-mediated transcriptional activation (CRISPRa) works by fusing dead Cas9 (dCas9) with the VP16 transcription activation domain derived from herpesviruses, thereby activating gene transcription [6,7]. CRISPRa has broad clinical application prospects, but currently faces two major challenges: firstly, low transcriptional activation efficiency, including low gene activation efficiency or poor gene activation breadth; and secondly, difficulties in targeted delivery in vivo, such as adeno-associated virus (AAV) delivery. Effectively addressing these issues would greatly enhance the feasibility of in vivo CRISPRa applications.

[0004] Since the advent of CRISPRa, many laboratories have continuously improved it to enhance its transcriptional activation efficiency. The initial version of the CRISPRa system was called dCas9-VP64, which fused dCas9 with four VP16 transcriptional activation domains derived from herpesviruses, but the transcriptional activation efficiency was still low [6,7]. The number of VP16 fused with dCas9 protein was increased to 10 (i.e., from VP64 to VP160), which slightly improved the transcriptional activation level of the target gene [8]. Subsequently, researchers used the suntag system to recruit transcriptional activators (dCas9-24×GCN4+scFV-VP64), which combined dCas9 coupled with 24 GCN4s (dCas9-24×GCN4) with VP64 fused with the anti-GCN4 antibody scFV (scFV-VP64), which enhanced the transcriptional activation of the target gene [9]. In addition to directly fusing transcription activators with dCas9 protein, researchers have also fused transcription activators with RNA element ligands such as PCP, MCP, or N22 by coupling sgRNA with RNA elements such as PP7, MS2, Boxb, or PBS (i.e., sgRNA-PP7, sgRNA-MS2, sgRNA-Boxb, or sgRNA-PBS). Ultimately, by binding different RNA elements to their specific binding proteins (i.e., PP7-PCP, MS2-MCP, Boxb-N22, PUF-VP64, etc.), transcription activators can be recruited to target DNA sites

[0010] (Figure 1).

[0005] In addition, researchers used the phase separation principle to fuse the intrinsic disordered regions (IDR) element of the phase separation protein with the CRISPRa activation-related element VP64, thereby recruiting VP64 to the promoter site of the target gene through phase separation, thus achieving high-level activation of the gene

[0011] (Figure 2).

[0006] To further enhance the activation ability of CRISPRa, researchers fused more transactivation domains into VP64. The first method (Figure 3A) is to use the three components dCas9-VP64, sgRNA-MS2, and MCP-p65-HSF1 in combination. That is, based on the fusion of VP64 with dCas9, the transactivation domains p65 and HSF1 are recruited by sgRNA-MS2

[0012] , thereby achieving transcriptional activation. Among them, HSF1 is an activation domain derived from human heat shock factor; p65 is the NF-κB transactivation subunit, which can recruit unique transcription factors such as AP-1, ATF / CREB and SP1 and chromatin remodeling complexes

[0013] . The second method (Figure 3B) involves fusing dCas9-VP64 with the activation domains of p65 and Rta, i.e., dCas9-VP64-p65-Rta (dCas9-VPR)

[0014] , where Rta is the activation domain derived from EB virus, and p65 is the NF-κB transactivating subunit. Compared with dCas9-VP64, the dCas9-VPR system has higher transcriptional activation efficiency (Figure 3B). Similar CRISPRa systems include the VPH system and its derivative VPH-SS18, which are formed by tandem with VP48, p65, and HSF1 [15,16]. In addition, transcriptional activation can also be achieved by changing the epigenetic properties of the promoter region of the target gene to activate gene expression. For example, dCas9 is fused with p300 (dCas9-p300), and p300 can increase the level of histone acetylation in the promoter region of the target gene, thereby activating gene expression

[0017] . dCas9 can also be fused with DNA demethylase TET1 (dCas9-TET1), and TET1 can cause DNA demethylation in the promoter region of the target gene, thereby activating gene expression [18,19].

[0007] However, the existing CRISPRa system has the following key problems: (1) The gene transcription activation system based on VP64 has relatively low activation efficiency and fold; (2) Although the introduction of VPR can greatly improve the transcription activation efficiency of target genes, the use of the NF-κB subunit p65 is more inclined to activate genes downstream of NF-κB

[0014] , and many genes in the genome cannot be activated, or even inhibit gene expression, thus limiting the wide application of the system

[0020] ; (3) The gene length of dCas9-VPR is about 5.8Kb (>4.7Kb, the highest packaging load of AAV virus is 4.7kb), which cannot be delivered into the body through a single AAV package, which also poses a great challenge to the in vivo application of the CRISPRa system

[0021] .

[0008] Currently, the main delivery methods of the CRISPR system in vivo include adeno-associated virus (AAV), adenovirus (ADV), lentivirus, lipid nanoparticles (LNP), and virus-like particles (VLP). Preclinical studies mainly focus on delivery via AAV and lipid nanoparticles. AAV is a non-enveloped virus that can package a single-stranded genome of ~4.7kb

[0022] . It has good safety and has been approved by the FDA. It can be used for DNA delivery in various tissues, including eye, liver, brain tissue, cardiomyocytes, and skeletal muscle cells

[0023] . AAV requires ITR (inverted terminal repeats) sequences at both ends, so the maximum deliverable exogenous genome is ≤4.7kb

[0024] . However, the total length of the DNA elements in the SpCas9-based CRISPRa system, including the necessary promoter, transcription termination element (polyA), and corresponding sgRNA expression element, exceeds 4.7kb, thus making it impossible to package into an AAV virus. Although LNPs can deliver DNA, mRNA, and siRNA as non-viral delivery vectors, they only have a short-term effect and are not suitable for long-term activation of transcription.

[0009] To address the issue that the CRISPRa system is too large to be delivered using a single AAV, researchers have split dCas9-VPR and delivered it in vivo using two AAVs, after which it self-assembles into a complete CRISPRa (dCas9-VPR). For example, dCas9 is divided into N-terminus and C-terminus. The N-terminal protein of dCas9 is packaged with sgRNA into one AAV, and the C-terminus of dCas9 is fused with VPR and packaged into another AAV. After cells are infected with the two AAVs, both the N-terminus and C-terminus of dCas9 are expressed simultaneously, allowing for self-assembly into a complete dCas9-VPR within the cell. This is then applied to activate the expression of the Opn1mw gene in the mouse retina to alleviate vision problems caused by rhodopsin deficiency

[0025] . The prerequisite for the CRISPRa system to activate the transcription of the target gene is that both AAVs enter the same cell simultaneously and assemble correctly. To ensure that all components are packaged within one AAV, smaller Cas proteins such as SaCas9 and Cas12f can be used. For example, in the treatment of muscular dystrophy caused by LAMA2 deficiency, researchers packaged the VP64-SaCas9-VP64 fusion protein and sgRNA into an AAV to activate the LAMA1 gene in mouse muscle cells

[0026] . In addition, a single AAV of the CRISPRa system was packaged using the smallest AsCas12f protein currently available, along with a modified sgRNA replacing two MS2 loops and an MCP-p65-HSF1 (~1416bp) fusion protein, and the expression of the reporter gene luciferase was activated in mice. However, because its activation domain selects p65-HSF1, only a single sgRNA plus the CRISPRa system AAV packaging could be achieved

[0027] . Apart from the two articles mentioned above reporting the delivery of the complete CRISPRa element and sgRNA via a single AAV, there are no other reports of single AAV delivery of the CRISPRa system.

[0010] The CRISPRa system based on SaCas9-VP64 has low activation efficiency, while the AsCas12f1-MCP-p65-HSF1 system can achieve activation of a single sgRNA in vivo. However, MCP-p65-HSF1 is approximately 1416 bp, and the length of the dCas protein, sgRNA sequence, and necessary expression regulatory elements reaches approximately 4.5 kb, which is close to the AAV packaging limit. This means that a single AAV cannot accommodate more sgRNAs, making it difficult to achieve efficient activation of the transcriptional levels of several genes simultaneously in vivo. Studies have shown that three sgRNAs are required to achieve efficient activation of LAMA1 in a muscular dystrophy model with LAMA2 deficiency

[0026] . Furthermore, in a Duchenne muscular dystrophy (DMD) model, it is necessary to simultaneously increase the protein levels of LAMA1, LAMB1, and LAMC1 to alleviate muscular dystrophy symptoms

[0028] . This indicates that in actual disease treatment, it is usually necessary to deliver the activation system and multiple sgRNAs via a single AAV vector to achieve the best therapeutic effect.

[0011] Perez-Pinera, P et al. (RNA-guided gene activation by CRISPR-Cas9-based transcription factors, Nat Methods. 2013 Oct; 10(10): 973-6. doi: 10.1038 / nmeth.2600.) reported that by fusing four herpesvirus-derived transcription activation domains VP16 (dCas9-VP64) to the C-terminus of the dCas9 protein, the endogenous gene in human cells was effectively activated, but its activation efficiency was relatively low.

[0012] Chavez, A. et al. (Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015 Apr; 12(4):326-8. doi:10.1038 / nmeth.3312.) first reported the VPR system, which adds activation domains of p65 and Rta to VP64, thereby greatly improving the activation efficiency of CRISPRa for endogenous genes in cells. However, the VPR-based system has a bias towards gene transcriptional activation.

[0013] Zalatan, JG et al. (Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell. 2015 Jan 15; 160(1-2): 339-50. doi: 10.1016 / j.cell. 2014.11.052) mentioned that recruitment of the activation domain VP64 was achieved by adding two PP7 loops to sgRNA.

[0014] Xu, X et al. (Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell. 2021 Oct 21; 81(20):4333-4345.e4.doi:10.1016 / j.molcel.2021.08.008.) reported that a CasMini version was generated by evolving the Un1Cas12f protein and a VPR activation domain was fused to its C-terminus to create a CasMini-based CRISPRa activation system. However, the VPR-based system described above is biased towards gene transcriptional activation, and Xu, X et al. did not conduct any research on AAV-related issues.

[0015] Lyu, XY et al. (CRISPR FISHer enables high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification. Cell Res. 2022 Nov; 32(11):969-981. doi:10.1038 / s41422-022-00712-z.) describes a live-cell imaging detection method that systematically elucidates how a large number of GFP molecules are recruited to target DNA sites through phase separation to achieve imaging of single-copy sites.

[0016] Louise Bendixen et al. (CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi. Mol Ther. 2023 Jul 5;31(7):1920-1937. doi:10.1016 / j.ymthe.2023.03.024.) summarized the development and application research of existing CRISPRa systems.

[0017] Aditya Raguram et al. (Therapeutic in vivo delivery of gene editing agents. Cell. 2022 Jul 21; 185(15): 2806-2827. doi: 10.1016 / j.cell. 2022.03.045.) summarized the existing gene editing delivery methods and their advantages and disadvantages.

[0018] Michael Chavez et al. (Advances in CRISPR therapeutics. Nat Rev Nephrol. 2023 Jan; 19(1): 9-22. doi: 10.1038 / s41581-022-00636-2.) summarized the progress of CRISPRa in preclinical research.

[0019] However, these studies have not solved the problems existing in the CRISPRa system. There has been a persistent need in the field for a CRISPRa system that is "small in molecular weight, efficient, unbiased, and easy to deliver," capable of recruiting as many transcriptional regulators as possible to target genes to efficiently activate transcriptional expression.

[0020] Summary of the Invention

[0021] One object of the present invention is to develop an improved CRISPR-mediated gene transcription activation (CRISPRa) system that has a significantly enhanced target gene transcription activation effect.

[0022] Another objective of this invention is to develop a low-molecular-weight, unbiased, visualized, and efficient gene activation technology based on the miniCRISPR system. This technology can not only contain all the necessary elements in a single AAV vector, but also achieve simultaneous and efficient activation of multiple endogenous genes in vivo, providing a new treatment strategy for genetic diseases caused by congenital gene insufficiency.

[0023] The inventors have for the first time used a smaller Cas protein and the transcriptional activation domain VP64 to rapidly recruit a large amount of VP64 to the target gene using polymerized peptides, thereby developing the miniCRISPRa system, which is "small in molecular weight, efficient, unbiased, and easy to deliver," to achieve efficient transcriptional activation of the target gene.

[0024] In a first aspect, this application provides a highly efficient CRISPR-mediated gene transcription activation (CRISPRa) system, the CRISPRa system comprising:

[0025] (1) dCas protein or recombinant vector expressing dCas protein;

[0026] (2) A modified sgRNA expression vector, wherein the modified sgRNA comprises: an sgRNA backbone containing n RNA aptamers, and an sgRNA sequence specifically targeting the target gene to be activated, wherein n is an integer greater than or equal to 2;

[0027] (3) A fusion protein expression vector, wherein the fusion protein comprises: an RNA-binding motif that specifically recognizes the RNA aptamer, a polymerized peptide, and a transcription activation domain, which are operatively linked together.

[0028] Those skilled in the art will understand that fusion proteins may contain fluorescent proteins for detecting the location of a target gene in the chromosome, and, in conjunction with RNA imaging techniques, can indicate the transcriptional activation of the target gene. However, given the limitations of vectors on the size of the delivered fragment, fusion proteins typically do not contain fluorescent proteins.

[0029] In some embodiments, the transcriptional activation domain may be selected from, but is not limited to, VP64, p65-HSF1, or VPR, preferably VP64. The amino acid sequence of VP64 is SEQ ID NO:12.

[0030] In some implementations, the amino acid sequence of the dCas protein is SEQ ID NO:1.

[0031] In some implementations, the dCas protein is a miniCas protein, such as dUn1Cas12f1, whose amino acid sequence is shown in SEQ ID NO:2.

[0032] The gene transcription activation system based on miniCas in this application is called the miniCRISPRa system.

[0033] The modified sgRNA described in this invention does not alter the sequence that binds to dCas9. Instead, it modifies the stem-loop portion of the sgRNA by inserting an RNA stem-loop sequence (e.g., but not limited to PP7, MS2, or Box). For example, the RNA stem-loop sequence is located in the sgRNA backbone (i.e., the sgRNA scaffold).

[0034] In one embodiment, the modified sgRNA expression vector is driven by a U6 promoter, which may be a mouse U6 promoter (mU6) or a human U6 promoter (hU6).

[0035] The RNA stem-loop binding motif in the fusion protein specifically recognizes the RNA stem-loop sequence in the modified sgRNA expression vector; that is, the RNA stem-loop structure and the RNA binding motif are paired. In some embodiments, the RNA stem-loop structure and the RNA binding motif exist in a paired combination selected from: PP7 and PCP, MS2 and MCP, or BoxB and N22.

[0036] In a preferred embodiment, the RNA stem-loop structure and the RNA binding motif are combined as PP7 and PCP.

[0037] In some embodiments, the polymerized peptide may be selected from foldon trimer peptide, GCN4 trimer peptide, 3HB trimer peptide, or 6G6H hexamer peptide, and wherein the polymerized peptide is fused to the N-terminus or C-terminus of a transcription activation domain (e.g., VP64), or located at the N-terminus of a transcription activation domain (e.g., VP64). Preferably, the structure of the fusion protein from the N-terminus to the C-terminus is: RNA-binding motif - polymerized peptide - transcription activation domain (e.g., VP64).

[0038] In some implementations, the RNA-binding motif, the polymerized peptide, and the transcriptional activation domain (e.g., VP64) can be operably linked in any order to form a fusion protein, wherein the polymerized peptide can be located at the N-terminus, C-terminus, or middle of the fusion protein.

[0039] In a preferred embodiment, the fusion protein has a PCP-polymerized peptide-transcriptional activation domain (e.g., VP64) structure from the N-terminus to the C-terminus. In a more preferred embodiment, the fusion protein has a PCP-foldon-VP64 structure from the N-terminus to the C-terminus. In another preferred embodiment, the fusion protein has a polymerized peptide-PCP-VP64 structure from the N-terminus to the C-terminus, preferably foldon-PCP-VP64. In other embodiments, the fusion protein has a PCP-foldon-p65-HSF1 (SEQ ID NO:34) or PCP-foldon-VPR (SEQ ID NO:35) structure from the N-terminus to the C-terminus, as shown in Figure 8.

[0040] In a preferred embodiment, the polymerized peptide is a foldon trimerized peptide.

[0041] In a preferred embodiment, the RNA aptamer and the RNA binding motif are combined as PP7 and PCP, and the polymerized peptide is a foldon trimerized peptide.

[0042] n RNA stem-loop sequences represent n RNA stem-loop structures connected in tandem. These can be joined using a adapter or directly. When using an adapter, the adapter can be selected from commonly used adapters in the art. n is an integer greater than or equal to 2, for example, it can be 2, 3, 4, 5, 6, 7, or 8 or larger. There is no particular upper limit, and those skilled in the art can choose a suitable value for n according to actual needs.

[0043] In some implementations, n is any integer from 2 to 8; for example, n can be 2, 3, 4, 5, 6, 7, or 8. In a preferred implementation, n is 2.

[0044] In some implementations, the n RNA aptamers can be tandemly linked to each other in the first circular structure of the sgRNA, or directly replace the two circular structures of the sgRNA.

[0045] This is the first time that a multiplying peptide has been proposed to be used in conjunction with the CRISPRa system. By utilizing the multiplying ability of the multiplying peptide itself, a large number of transcription activation domain (e.g., VP64) proteins are recruited to the promoter region of the target gene, thereby resulting in a high level of target gene transcription effect.

[0046] Taking foldon trimerized peptide as an example, it is a short peptide derived from the C-terminus of T4 phage fibrin, discovered in 1999 by Av Letarov et al. (Av Letarov et al., Biochemistry (Moscow), Vol. 64, No. 7, 1999, pp. 817-823. Translated from Biokhimiya, Vol. 64, No. 7, 1999, pp. 974-981). This domain consists of three identical subunits, each including a β-hairpin structure. After foldon is fused with the target protein, the target protein can spontaneously form a trimer

[0029] . In this study, we utilize the small molecular weight and self-aggregating properties of the trimerized peptide foldon, which has not yet been reported in the field of transcriptional activation. We attempt to combine foldon, VP64, and the CRISPR system for gene transcriptional activation. Currently, there are no reports in this field regarding the application of foldon in gene transcriptional activation.

[0047] In one embodiment, a schematic diagram of the design and working principle of the CRISPRa system of the present invention is shown in Figure 4. dCas9 and sgRNA containing two PP7 molecules assemble into a Cas9 / sgRNA complex, which binds to the promoter region of the target gene. Each of the two PP7 molecules recruits two PCP-foldon-VP64 proteins. Since PCP-foldon-VP64 proteins are trimers, two PCP domains remain vacant, allowing the recruitment of additional sgRNA containing two PP7 molecules. The sgRNA, in turn, recruits even more trimer PCP-foldon-VP64 proteins. Theoretically, the trimer PCP-foldon-VP64 proteins and sgRNA can be repeatedly recruited, resulting in the recruitment of a large amount of VP64 protein in the gene promoter region, ultimately leading to high-level activation of the target gene.

[0048] In one specific implementation, the CRISPRa system includes:

[0049] (1) dCas protein or recombinant vector expressing dCas protein;

[0050] (2) A modified sgRNA expression vector, wherein the modified sgRNA comprises: an sgRNA backbone containing two RNA aptamers PP7, and an sgRNA sequence specifically targeting the target gene to be activated, i.e., the modified sgRNA is represented as sgRNA-2×PP7.

[0051] (3) A fusion protein expression vector, wherein the fusion protein comprises: an RNA-binding motif PCP that specifically recognizes the RNA aptamer PP7, a foldon trimerized peptide and VP64, which are operably linked together, for example, the fusion protein is represented as PCP-foldon-VP64.

[0052] In the specific implementation scheme described above, the CRISPRa system operates as follows: First, the dCas protein and sgRNA-2xPP7 bind to the promoter region of the target gene, at which point the two PP7 domains recruit trimerized PCP-foldon-VP64 protein. Second, the trimerized PCP-foldon-VP64 protein, having three PCP domains, can bind not only to the PP7 domains of the sgRNA at the target gene site but also to free sgRNA-2×PP7 in the cell, thereby recruiting more sgRNA to the target gene site. Third, these additionally recruited sgRNA-2×PP7 domains recruit even more trimerized PCP-foldon-VP64 protein. Fourth, the repeated recruitment by sgRNA and trimer protein results in the recruitment of a large amount of VP64 protein in the promoter region of the target gene, leading to a high level of target gene transcription efficiency.

[0053] In some implementations, the foldon trimer peptide can be replaced by any one of the GCN4 trimer peptide, 3HB trimer peptide, and 6G6H hexamer peptide.

[0054] In some implementations, the dCas protein is a miniCas protein.

[0055] In addition to dUn1Cas12f1 (SEQ ID NO:2) mentioned above, miniCas proteins can also be selected from the following, but are not limited to:

[0056] (i) The miniCas protein can be AsCas12f1

[0030] , and its corresponding sgRNA needs to be modified to have 2 PP7 loops;

[0057] (ii) The miniCas protein can be CasΦ

[0031] , and its corresponding sgRNA should also be modified to add 2 PP7 loops;

[0058] (iii) The miniCas protein can be MmCas12m

[0032] , and its corresponding sgRNA should also be modified to add 2 PP7 loops;

[0059] (iv) The miniCas protein can be AcCas12n

[0033] , and its corresponding sgRNA should also be modified to add 2 PP7 loops;

[0060] (v) The miniCas protein can be TnpB

[0034] , and its corresponding sgRNA should also be modified to add two PP7 loops;

[0061] (vi) The miniCas protein can be Fanzor

[0035] , and its corresponding sgRNA should also be modified to add 2 PP7 loops;

[0062] (vii) The miniCas protein can be IscB

[0036] , and its corresponding sgRNA should also be modified to add two PP7 loops; or

[0063] (viii) The miniCas protein can be RhCas12f1 or OsCas12f1

[0037] , and their respective sgRNAs should also be modified to add two PP7 loops.

[0064] In some implementations, the PP7 and PCP pairing combination can be replaced with the MS2 and MCP combination or the BoxB and N22 combination.

[0065] Depending on the needs of the actual application, those skilled in the art can easily select appropriate plasmids to construct the expression vectors (1) to (3) for detection. Available plasmids include, but are not limited to, PX330, pUR and AAV vectors, lentiviral vectors, retroviral vectors, or adenovirus vectors, with AAV vectors being preferred. Among these, AAV vectors are suitable for in vivo delivery.

[0066] In some implementations, all elements of the CRISPRa system (e.g., the coding sequences for the dCas protein, the modified sgRNA, and the fusion protein) can be constructed into a single vector, for example, into an AAV vector. Preferably, when intended for in vivo delivery, all elements of the CRISPRa system are constructed into a single AAV vector.

[0067] In some implementations, all components of the CRISPRa system may be packaged as lipid nanoparticles (LNPs) or virus-like particles (VLPs). However, due to the short duration of action of LNPs, CRISPRa-based therapies generally do not choose to package all components of the CRISPRa system as LNPs.

[0068] In a second aspect, this application provides a method for activating the transcription and expression of a target gene, the method comprising: constructing the CRISPRa system described in the first aspect for the target gene, and then introducing the gene activation system into cells to activate the transcription and expression of the target gene.

[0069] In some implementations, all components of the CRISPRa system (e.g., the coding sequences for the dCas protein, the modified sgRNA, and the fusion protein) are constructed into a vector (e.g., an AAV vector), and the CRISPRa system is then introduced into cells via transformation or transfection. Alternatively, theoretically, all essential components can be packaged into LNPs or VLPs before being introduced into cells via transformation or transfection. For in vivo applications, the AAV vector is particularly preferred.

[0070] In some implementations, a miniCRISPRa system is constructed using miniCas, and all elements of the miniCRISPRa system (e.g., the coding sequences for the miniCas protein, the modified sgRNA, and the fusion protein) can be constructed into an AAV vector.

[0071] In a third aspect, this application provides a kit comprising the various components of the CRISPRa system of the first aspect. These components are each contained in a separate container. Preferably, the kit further comprises an AAV vector, a lentiviral vector, a retroviral vector, or an adenovirus vector, or other clinically available vectors. Additionally, the kit may include an instruction manual containing basic information about each component and instructions for use.

[0072] In one embodiment, the kit comprises: various elements of the miniCRISPRa system of the first aspect, and an AAV vector. For example, the various elements and the AAV vector are each contained in a separate container.

[0073] In a preferred embodiment, the kit comprises an AAV vector containing the various elements of the miniCRISPRa system of the first aspect, preferably, all elements of the miniCRISPRa system of the first aspect (e.g., the coding sequence of the miniCas protein, the coding sequence of the modified sgRNA and the fusion protein) are constructed into an AAV vector.

[0074] In a fourth aspect, this application provides the use of the CRISPRa system described in the first aspect in the preparation of a kit for treating a subject with a disease, wherein the disease is a genetic disease (e.g., a disease caused by gene deletion, mutation or insufficient expression) or a tumor.

[0075] In some embodiments, the CRISPRa system of the present invention can treat diseases caused by the deletion, mutation or underexpression of pathogenic genes by activating the transcription and expression of alleles of pathogenic genes, such as sickle cell anemia due to HBB (β-globin) mutation, thalassemia due to β-globin deficiency, or muscular dystrophy due to LAMA2 deficiency.

[0076] In some implementations, the subject is a mammal, preferably a human.

[0077] In some implementations, the CRISPRa system described in the first aspect is a miniCRISPRa system, and all elements in the miniCRISPRa system (e.g., the coding sequence of the miniCas protein, the modified sgRNA, and the coding sequence of the fusion protein) can be constructed into an AAV vector.

[0078] In one exemplary embodiment, the miniCRISPRa system of this application is used to activate LAMA1 expression via AAV delivery, thereby treating or alleviating muscular dystrophy due to LAMA2 deficiency. For example, by designing sgRNA targeting the transcription start site (TSS) of the human LAMA1 gene within 500 bp, delivery of the sgRNA and activation element via a single AAV vector can activate LAMA1 expression in the muscles of MDC1A patients, thereby enhancing muscle tissue function and alleviating or treating symptoms of muscle atrophy.

[0079] In one exemplary embodiment, the miniCRISPRa system of this application is delivered via AAV to simultaneously activate the expression of LAMA1, LAMB1, and LAMC1, thereby treating or alleviating Duchenne muscular dystrophy (DMD) caused by Dystrophin gene deletion. For example, by designing sgRNAs targeting the transcription start site (TSS) of human LAMA1, LAMB1, and LAMC1 genes within 500 bp, simultaneous delivery of the three sgRNAs and activating elements via a single AAV vector can simultaneously activate the expression of LAMA1, LAMB1, and LAMC1 in the muscles of DMD patients, thereby enhancing muscle tissue function and alleviating or treating muscle atrophy symptoms caused by Dystrophin gene deletion.

[0080] In another exemplary embodiment, the CRISPRa system of this application can activate the expression of HBG (γ-globin) to treat sickle cell anemia caused by HBB (β-globin) mutation or thalassemia caused by β-globin deficiency.

[0081] In theory, the CRISPRa system or miniCRISPRa system of this application can be used to treat any hereditary disease caused by gene deletion, mutation or underexpression.

[0082] In one embodiment, this application relates to a method for treating a subject with a hereditary disease caused by gene deletion, mutation, or insufficient expression, the method comprising:

[0083] (i) Design one or more sgRNAs for one or more genes or their alleles that are associated with genetic diseases and have deletions, mutations or insufficient expression, and construct one or more sgRNAs with the nucleotide sequence encoded by miniCas and the nucleotide sequence encoded by the fusion protein into an AAV vector to form the miniCRISPRa system described in the first aspect of this application;

[0084] (ii) Packaging the AAV vector described in step (i) into an AAV virus; and

[0085] (iii) The AAV virus body described in step (ii) is administered to the subject, thereby activating the transcription and expression of one or more genes or their alleles that are missing, mutated or underexpressed, thereby treating the hereditary disease.

[0086] In a fifth aspect, this application provides the use of the CRISPR system described in the first aspect in the preparation of a kit for treating tumors in a subject.

[0087] In some embodiments, the CRISPRa system is a miniCRISPRa system, and all components of the miniCRISPRa system (e.g., the coding sequence of the miniCas protein, the modified sgRNA, and the coding sequence of the fusion protein) can be constructed into an AAV vector, and the treatment of tumors includes in situ injection of the AAV vector into tumor tissue.

[0088] In some implementations, the subject is a mammal, preferably a human.

[0089] In one exemplary embodiment, the miniCRISPRa system of this application is delivered by injecting an AAV vector into tumor tissue in situ, thereby activating the expression of tumor suppressor genes in tumor cells, thereby inhibiting tumor growth and achieving the effect of curing or delaying tumor progression in cancer patients.

[0090] In one embodiment, tumors that can be treated using the miniCRISPRa system of this application include, but are not limited to, lung cancer, colorectal cancer, liver cancer, or breast cancer.

[0091] For example, but not wishing to be bound by theory, the miniCRISPRa system of this application can be used to inhibit the Hippo signaling pathway by activating the expression of VGLL4 protein in lung cancer cells, thereby inhibiting the growth of lung cancer cells

[0038] ; or by inhibiting the expression of CLMP in colorectal cancer cells to inhibit the Wnt signaling pathway, thereby inhibiting the growth of colorectal cancer cells

[0039] ; or by activating the expression of IRF2BP2 in liver cancer cells to inhibit the Hippo signaling pathway, thereby inhibiting the growth of liver cancer cells

[0040] ; or by activating the expression of leucyl tRNA synthetase (LARS) to promote the increase of tumor suppressor proteins such as EMP3 and GGT5, thereby inhibiting the occurrence and development of breast cancer.

[0092] In another exemplary embodiment, the miniCRISPRa system of this application is delivered by injecting an AAV vector into tumor tissue in situ, thereby activating the expression of tumor surface antigen-related proteins in tumor cells, thereby activating the recognition effect of the human immune system on tumor cells, enhancing the tumor immune process in tumor tissue, thereby inhibiting tumor growth, and achieving the effect of curing or delaying the progression of cancer in cancer patients.

[0093] In theory, there are no limitations on the types of tumors that can be treated using the CRISPRa system of this application. This is because each tumor has corresponding tumor surface antigens and tumor suppressor genes. Theoretically, using either the CRISPRa system or the miniCRISPRa system of this application can activate their expression, thereby inhibiting tumor cell proliferation. For example, using the CRISPRa system of this application to reactivate tumor cell surface antigens, such as IFNγ, IFNβ1, and IFNα4, can be effective against a variety of tumors. Therefore, theoretically, the strategy of using an AAV vector to deliver miniCRISPRa to activate tumor antigen and tumor suppressor gene expression for tumor treatment is applicable to all tumors.

[0094] In one embodiment, this application relates to a method for treating a tumor in a subject, the method comprising:

[0095] (i) Design one or more sgRNAs that target one or more tumor surface antigens and / or tumor suppressor genes, and construct one or more sgRNAs with a miniCas-encoded nucleotide sequence and a fusion protein-encoded nucleotide sequence into an AAV vector to form the miniCRISPRa system described in the first aspect of this application;

[0096] (ii) Packaging the AAV vector described in step (i) into an AAV virus; and

[0097] (iii) The AAV virus body described in step (ii) is administered to the subject, thereby activating the transcription and expression of one or more tumor surface antigens and / or tumor suppressor genes, thereby inhibiting tumor cell proliferation.

[0098] In a sixth aspect, this application provides the use of the CRISPR system described in the first aspect in the preparation of a kit for delaying aging.

[0099] In some implementations, the CRISPRs system or miniCRISPRs system described in this application is used to delay aging by simultaneously activating Oct4, Sox2, and Klf4.

[0100] In one embodiment, this application relates to a method for delaying aging in a subject, the method comprising:

[0101] (i) Design one or more sgRNAs targeting aging-related genes, and construct one or more sgRNAs with the nucleotide sequence encoded by miniCas and the nucleotide sequence encoded by the fusion protein into an AAV vector to form the miniCRISPRa system described in the first aspect of this application;

[0102] (ii) Packaging the AAV vector described in step (i) into an AAV virus; and

[0103] (iii) The AAV virus body described in step (ii) is administered to the subject, thereby activating the transcription and expression of one or more aging genes, thereby delaying aging.

[0104] In summary, this application has the following advantages:

[0105] (1) This application uses VP64 as the basic element to establish the system, which greatly expands the range of target genes that can be activated;

[0106] (2) This application recruits a large number of transcription activators to the target gene site by means of mutual recruitment of sgRNA and VP64, which increases the activation fold of the target gene by thousands of times, for example, more than two thousand times, compared to the traditional dCas9-VP64.

[0107] (3) By introducing the miniCas protein and VP64 fused with polymerized peptides, this application reduces the size of the gene activation system to less than 4kb, which can not only realize the construction into a single AAV vector for in vivo delivery, but also provide a feasibility for activating multiple genes simultaneously in vivo.

[0108] (4) The above-mentioned “small molecular weight, high efficiency and no bias” miniCas-based CRISPRa system developed in this application is applicable to various Cas and sgRNA substitutions, providing a potential treatment strategy for genetic diseases caused by insufficient congenital gene expression due to gene deletion or mutation.

[0109] The above is an overview and therefore includes simplifications, generalizations, and omissions of details where necessary. Therefore, those skilled in the art will recognize that this overview is merely illustrative and not intended to be limiting in any way. Other aspects, features, and advantages of the methods described herein, the CRISPRa system, and / or other subjects will become apparent from the teachings presented herein. This overview is provided to simply introduce some alternative concepts, which will be further described in the detailed description below. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid to determining the scope of the claimed subject matter. Furthermore, the contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety. Attached Figure Description

[0110] By referring to the following accompanying drawings, those skilled in the art will more readily understand the technical solution of the present invention. These drawings form part of the present invention.

[0111] Figure 1 shows a schematic diagram of how sgRNA stem-loop structure modification recruits transcription activators.

[0112] Figure 2 shows a schematic diagram of recruiting VP64 or VPR to the promoter of the target gene to activate gene expression.

[0113] Figure 3 shows schematic diagrams of the dCas9-p65-HSF1 activation system (A) and the dCas9-VPR activation system (B).

[0114] Figure 4 shows the principle of the novel high-efficiency gene activation system of this application recruiting VP64 through polypeptide fragments.

[0115] Figure 5 shows the multi-gene activation test of VP64 and VPR performed in Example 1 of this application.

[0116] Figure 6 shows the experimental results of Example 2 of this application: (A) the effect of different polymerized peptides on the activation efficiency of the CRISPRa system, and (B) the effect of different combinations of RNA loops and RNA binding domains on the activation of CRISPRa.

[0117] Figure 7 shows the effect of different combinations of PCP, polymerized peptide, and VP64 position on the gene transcription activation effect of the CRISPRa system as detected in Example 2 of this application.

[0118] Figure 8 shows the experimental results of Example 2 of this application: (A) The improved scheme of foldon peptide based on the VPR system (shown as PCP-foldon-VPR) has a significantly better effect on the transcriptional activation of ASCL1, HBG and IL1B genes than the VPR system; (B) The improved scheme of foldon peptide based on the p65-HSF1 system (shown as PCP-foldon-p65-HSF1) has a significantly better effect on the transcriptional activation of ASCL1 gene than the p65-HSF1 system.

[0119] Figure 9 shows the activation effect of the miniCRISPRa system of this application on VPR repressor genes (i.e., GAB1 and PDGFRB) verified in Example 2 of this application.

[0120] Figure 10 shows a comparison of the results of activation of endogenous gene transcription in cells by the miniCRISPRa system of this application (multiplying peptides induce the recruitment of multiple VP64, denoted as PCP-foldon-VP64) and conventional dCas9-VP64 in Example 3 of this application.

[0121] Figure 11 shows that the CRISPRa system of this application was verified in Example 3 of this application to activate the expression of the myc gene in mice, thereby inducing tumorigenesis. T in the H&E staining image represents the tumor region.

[0122] Figure 12 shows the Un1Cas12f1 system-based sgRNA loop modification design schemes (A and B) applicable to multiple VP64 recruitment systems induced by polymerized peptides in Example 4 of this application, and the activation effects (C, D and E) on multiple endogenous genes (i.e., HBB, INFγ and ILIRN) in different cell lines (i.e., 293T, U2OS and HeLa cells).

[0123] Figure 13 shows the effect of miniCRISPRa, as verified in Example 4 of this application, on the simultaneous activation of the expression of three genes, ASCL1, HBG, and HBB, in 293T cells.

[0124] Figure 14 shows the design pattern of the sgRNA targeting sequence in the application of the CRISPRa system based on miniCas protein in the treatment of β-thalassemia in Example 5 of this application.

[0125] Figure 15 shows the transcriptional activation effect of the miniCRISPRa system verified in Example 5 of this application on the human HBG gene.

[0126] Figure 16 shows a schematic diagram of the miniCRISPRa system constructed in Example 7, which can simultaneously activate the INFγ, INFα4, and INFβ1 genes, and the treatment regimen for tumor-bearing mice.

[0127] Figure 17 shows that in Example 7, in situ administration of the AAV-miniCRISPRa of this application to tumor-bearing mice slowed tumor growth. The blue arrows indicate the time points when the recombinant AAV virus (containing the miniCRISPRa system) was injected.

[0128] Figure 18 shows that in Example 7, in situ application of the AAV-miniCRISPRa of this application to tumor-bearing mice reduced tumor size.

[0129] Figure 19 shows that in Example 7, the in situ injection of AAV-miniCRISPRa of this application into tumor-bearing mice simultaneously activated the expression of IFNγ, IFNα4 and IFNβ1, resulting in increased expression levels of the three genes.

[0130] Figure 20 shows the CD8+ expression in tumor tissue of tumor-bearing mice after in situ injection of AAV-miniCRISPRa of this application in Example 7. + The cells have increased infiltration. The black arrow indicates CD8. + . cells.

[0131] Figure 21 shows a schematic diagram of the miniCRISPRa system constructed in Example 8, which can simultaneously activate the Oct4, Sox2 and Klf4 genes, and the treatment regimen for aging mice.

[0132] Figure 22 shows that in Example 8, when the AAV-miniCRISPRa system of this application was applied to aging mice, the expression levels of Oct4, Sox2 and Klf4 genes were upregulated to varying degrees in the colon and liver of the mice.

[0133] Figure 23 shows the X-gal staining results of the spleen, colon and liver of mice in Example 8. It shows that the X-gal staining of aged mice (22 months old) treated with the AAV-miniCRISPRa system was lighter than that of the control group and similar to that of young mice (3 months old).

[0134] Figure 24 shows the H&E staining results of the spleen and colon of mice in Example 8. It shows that the colon wall (thickness) of aged mice (22 months old) in the AAV-miniCRISPRa treatment group is better than that of the control group and similar to that of young mice (3 months old); the germinal centers of the spleen of aged mice in the miniCRISPRa treatment group are better than those of the control group and similar to those of young mice.

[0135] Figure 25 shows the design principle diagram of the high-efficiency activation system miniCRISPRa in Example 4.

[0136] Sequence Description

[0137] The sequences used in this application are summarized as follows:

[0138] The amino acid sequence of the dCas protein (SEQ ID NO:1)

[0139] The amino acid sequence of the miniCas protein: Detailed Implementation

[0140] While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

[0141] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Additionally, the scope provided in the specification and appended claims includes all values ​​between endpoints and breakpoints.

[0142] definition

[0143] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0144] The term CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) refers to a repetitive sequence within the genome of a prokaryotic organism. It represents an immune weapon developed during the evolutionary struggle between bacteria and viruses. In short, when infected by a virus, the virus can integrate its genes into the bacterial genome and utilize the bacteria's cellular tools for gene replication. However, to eliminate the invading viral genes, bacteria evolved the CRISPR-Cas9 system. Using this system, bacteria can silently remove the integrated viral genes from their own chromosomes—a unique bacterial immune system. CRISPR technology was discovered in the early 1990s and, with subsequent research, has rapidly become the most popular gene-editing tool in fields such as human biology, agriculture, and microbiology.

[0145] Generally, the term "CRISPR system" refers to transcripts and other elements involved in the expression of CRISPR-associated (Cas) genes or guiding their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr pairing sequences (covering "direct repeats" and partial direct repeats processed by tracrRNA in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or other sequences and transcripts derived from CRISPR loci. In some embodiments, one or more elements of the CRISPR system are derived from a type I, II, or III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a specific organism containing an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex (also referred to as the pre-interstitial region in the context of an endogenous CRISPR system) at the site of the target sequence. In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not required, provided that sufficient complementarity exists to induce hybridization and promote the formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondrion or chloroplast. A sequence or template that can be used for recombination into a target locus including the target sequence is referred to as an "edit template," "edit polynucleotide," or "edit sequence." In this invention, the exogenous template polynucleotide may be referred to as an edit template. In one aspect of this invention, the recombination is homologous recombination.

[0146] Cas refers to CRISPR-associated genes, and can also refer to the expression products of these genes (called CRISPR enzymes or Cas9 enzymes). Currently discovered Cas types include Cas1 to Cas10. Cas genes and CRISPR have co-evolved and together constitute a highly conserved system.

[0147] dCas9 refers to "dead Cas9," a Cas9 protein without DNA cleavage catalytic activity (e.g., through mutations in D10A and H840A). It is typically a Cas protein or a fusion protein containing one or more NLS nuclei.

[0148] miniCas refers to a miniature Cas protein, that is, a Cas protein with approximately 500 amino acids. MiniCas proteins that can be used in this application include, but are not limited to: dUn1Cas12f1 (SEQ ID NO:2), AsCas12f1 (SEQ ID NO:3), CasΦ (SEQ ID NO:4), OsCas12f1 (SEQ ID NO:5), RhCas12f1 (SEQ ID NO:6), IscB (SEQ ID NO:7), Fanzor (SEQ ID NO:8), TnpB (SEQ ID NO:9), AcCas12n (SEQ ID NO:10), or MmdCas12m (SEQ ID NO:11).

[0149] “sgRNA”: guide RNA that binds to Cas9 (or dCas9). The sgRNA used in this system also carries an RNA aptamer that binds to the RNA-binding motif, such as PP7, MS2, or BoxB.

[0150] PP7: The binding region of RNA binding motifs other than Cas9 (or dCas9) that are fused with guide RNA (sgRNA), and generally binds to PCP.

[0151] PCP: Recognizes the phage capsid binding motif of PP7.

[0152] Foldon is a short peptide derived from the C-terminus of T4 phage fibrin. This domain consists of three identical subunits, each of which includes a β-hairpin structure. When foldon is fused with the target protein, the target protein can spontaneously form a trimer (AV Letarov et al., Biochemistry (Moscow), Vol. 64, No. 7, 1999, pp. 817-823. Translated from Biokhimiya, Vol. 64, No. 7, 1999, pp. 974-981).

[0153] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) as defined by ligation analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeled components.

[0154] "Complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence via conventional Watson-Crick or other non-conventional types. The complementarity percentage indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Complete complementarity" means that all consecutive residues in a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. As used herein, “substantially complementary” refers to a complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% in a region having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids hybridizing under stringent conditions.

[0155] "Gene transcription" refers to the process by which an organism synthesizes RNA in the cell nucleus or cytoplasm using one strand of DNA as a template, under the action of RNA polymerase.

[0156] "Gene expression" refers to the process of transcription from a DNA template into polynucleotides (such as mRNA or other RNA transcripts) and / or the subsequent translation of transcribed mRNA into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotides originate from genomic DNA, expression may also involve the splicing of mRNA in eukaryotic cells.

[0157] Generally, and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid molecule linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or without free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and a wide variety of other polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which another DNA fragment can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virus-derived DNA or RNA sequence is present in a vector used to package a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also contain polynucleotides carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) are capable of autonomous replication in the host cells in which they are introduced. Other vectors (e.g., non-episodic mammalian vectors) integrate into the genome of the host cell after introduction and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of genes they are operatively linked to. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA technology are typically in plasmid form.

[0158] Recombinant expression vectors may contain the nucleic acids of the present invention in a form suitable for nucleic acid expression in host cells. This means that these recombinant expression vectors contain one or more regulatory elements selected based on the host cell to be used for expression, said regulatory elements being operatively linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operatively linked" is intended to indicate that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows the expression of that nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell). In the present invention, the term "recombinant vector expressing dCas protein" refers to a recombinant expression vector containing a nucleotide sequence encoding the dCas protein, which, upon introduction into a host cell, is capable of expressing the dCas protein. The term "sgRNA expression vector" refers to a vector containing an sgRNA sequence. Similarly, "modified sgRNA expression vector" refers to a vector containing the modified sgRNA described in the present invention. The term "fusion protein expression vector" refers to a vector containing a nucleotide sequence encoding a fusion protein.

[0159] The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory sequences are described, for example, in Goeddel, *GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY*, 185, Academic Press, San Diego, California, 1990. Regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct expression of that nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type specific.

[0160] Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of host cells to be transformed and the desired expression level. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein (e.g., regularly spaced clustered short palindromic repeats (CRISPR) transcripts, proteins, enzymes, their mutant forms, their fusion proteins, etc.).

[0161] Example

[0162] By referring to the following embodiments, those skilled in the art will more clearly understand the technical solution and its effects of the present invention. Those skilled in the art should understand that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention in any way. The scope of protection of the present invention is defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art can make corresponding modifications to the embodiments of the present invention, and these modifications are also included within the scope of the present invention.

[0163] Tables 1 and 2 below list the main experimental instruments and reagents used in the following examples. Unless otherwise specified, all reagents and chemicals used in the examples are commercially available.

[0164] Table 1. Main Experimental Instruments

[0165] Table 2. Reagents and Chemicals

[0166] Example 1. Unbiased validation of the VP64-based gene transcription activation (CRISPRa) system

[0167] Literature reports the use of the dCas9-VP4 system for CRISPRa screening. We selected 20 genes and activated them in the 293T cell line using dCas9-VP64 and dCas9-VPR, respectively.

[0168] The inventors found that VP64 has a certain degree of activation effect on all genes, but VPR does not have a significant activation effect on a few genes, and VPR also has an inhibitory effect on GAB1 and PDGFRB. This indicates that VPR has a bias in gene activation effect and its applicability is not as good as the VP64 system (Figure 5).

[0169] Example 2. Development of a high-efficiency CRISPRa system based on VP64

[0170] 2.1 Screening for peptides that promote polymerization

[0171] 1) Cell line selection: 293T cells (purchased from the American Type Culture Collection (ATCC));

[0172] 2) Selection of polymerized peptides: foldon, 3HB, 6G6H and GCN4;

[0173] 3) Control group: dCas9-VP64;

[0174] 4) Target gene: ASCL1;

[0175] 5) Specific experimental methods: In 293T cells, wild-type 293T cells were used as a blank control, and dCas9-VP64 was used as a control group. The transcriptional activation efficiency of PCP-foldon-VP64, PCP-3HB-VP64, PCP-6G6H-VP64, and PCP-GCN4-VP64 on the ASCL1 gene was compared. The polymerized peptides with the highest activation fold for the target gene were finally screened using qPCR. The qPCR results (Figure 6A) showed that the polymerized peptides foldon, 3HB, 6G6H, and GCN4 significantly enhanced the gene transcriptional activation of the CRISPRa system, with the foldon trimer showing the best effect. As shown in Figure 6A, compared with the relative mRNA levels of the blank control, foldon, 3HB, 6G6H, and GCN4 increased by 1053-fold, 689-fold, 512-fold, and 681-fold, respectively, while dCas9-VP64 only increased by 2.5-fold.

[0176] 2.2 Screening for suitable sgRNA stem-loops and their corresponding RNA-binding motifs

[0177] 1) Cell line selection: 293T cells;

[0178] 2) Selection of sgRNA stem-loop and its corresponding RNA binding motif: PCP-PP7, MCP-MS2, N22-Boxb;

[0179] 3) Control group: dCas9-VP64;

[0180] 4) Target gene: ASCL1;

[0181] 5) Specific experimental methods: In 293T cells, wild-type 293T cells were used as a blank control, and dCas9-VP64 was used as a control group. The activation efficiency of PCP-VP64 / PCP-foldon-VP64, MCP-VP64 / MCP-foldon-VP64, and N22-VP64 / N22-foldon-VP64 on the ASCL1 gene was compared. Using qPCR detection, the stem-loop sgRNA with the highest activation fold towards the target gene and its corresponding ligand were finally screened.

[0182] Based on the q-PCR results (Figure 6B), we found that the PCP-PP7 combination had the best enhancement effect on the CRISPRa system. As shown in Figure 7, compared with the blank control (i.e., wild-type 293T cells, WT) and the dCas9-VP64 control, PCP-PP7, MCP-MS2, and N22-BoxB all significantly increased the relative mRNA level. Among them, the PCP-PP7 combination increased the relative mRNA level by about 350-fold, the MCP-MS2 combination by about 40-fold, and the N22-BoxB combination by about 15-fold.

[0183] 2.3 Determine the linking sequence of the RNA-binding motif, polymerized peptide, and VP64 in the fusion protein.

[0184] 1) Cell line selection: 293T cells;

[0185] 2) The linking sequence of RNA binding motif, polymerized peptide, and VP64 (three elements, with a total of 6 possible combinations (from N-terminus to C-terminus), as shown in Figure 8: foldon-VP64-PCP, PCP-foldon-VP64, PCP-VP64-foldon, foldon-PCP-VP64, VP64-PCP-foldon, or VP64-foldon-PCP);

[0186] 3) Control group: dCas9-VP64;

[0187] 4) Target gene: ASCL1;

[0188] 5) Specific experimental methods: In 293T cells, wild-type 293T cells were used as a blank control, and dCas9-VP64 was used as a control group. The transcriptional activation efficiency of the ASCL1 gene by the above-mentioned combinations was compared. The fusion protein with the highest fold increase in transcriptional activation of the target gene was finally screened by qPCR detection.

[0189] The results are shown in Figure 7. Compared with the blank control, the fusion protein with PCP-foldon-VP64 from the N-terminus to the C-terminus had the highest CRISPRa system transcriptional activation effect (3000-fold). foldon-PCP-VP64 increased the relative mRNA level by 2563-fold, VP64-foldon-PCP increased the relative mRNA level by 1136-fold, PCP-VP64-foldon increased the relative mRNA level by 409-fold, VP64-PCP-foldon increased the relative mRNA level by 183-fold, foldon-VP64-PCP increased the relative mRNA level by 62-fold, while dCas9-VP64 only increased the relative mRNA level by 6-fold.

[0190] 2.4 Constructing a high-efficiency activation system based on p65-HSF1 and VPR architecture

[0191] The above text indicates that foldon peptide can enhance the transcriptional activation of VP64. Here, we aim to investigate whether foldon peptide can enhance the transcriptional activation of p65-HSF1 and VPR.

[0192] 1) Cell line selection: 293T cells;

[0193] 2) The linking sequence of RNA binding motif, polymerized peptide foldon and VPR, and p65-HSF1 (three elements, PCP-foldon-HSF1, PCP-foldon-VPR);

[0194] 3) Control group: dCas9-VPR or dCas9-VP64+PCP-p65-HSF1;

[0195] 4) Target genes: ASCL1, HBG, IL1B;

[0196] 5) Specific experimental methods: In 293T cells, wild-type 293T cells were used as a blank control, and dCas9-VPR or dCas9-VP64+PCP-foldon-p65-HSF1 were used as control groups. The transcriptional activation efficiency of the above combinations on ASCL1, HBG, and IL1B genes was compared. The fold change in transcriptional activation of the target genes was detected by qPCR.

[0197] As shown in Figure 8A, PCP-foldon-VPR significantly activated the expression of ASCL1, HBG, and IL1B genes compared to dCas9-VPR (ASCL1: 1481-fold vs. 89-fold; HBG: 18988-fold vs. 811-fold; IL1B: 53119-fold vs. 2041-fold). As shown in Figure 8B, PCP-foldon-p65-HSF1 significantly activated the expression of ASCL1 gene compared to dCas9-VP64+PCP-p65-HSF1 (1304-fold vs. 483-fold). These results indicate that the introduction of foldon peptides not only significantly enhanced transcriptional activation in the VP64 system but also significantly enhanced the transcriptional activation capacity of the p65-HSF1 and VPR systems.

[0198] 2.5 Regarding the VPR mentioned above, due to its activation bias, it not only has no transcriptional activation effect on certain genes (e.g., GAB1 and PDGFRB, see Figure 5) but also has a transcriptional repression effect. This application's CRISPRa system is needed to verify whether it has a transcriptional activation effect on these genes.

[0199] 1) Cell line selection: 293T cells;

[0200] 2) The linking sequence of RNA-binding motif, polymerized peptide, and VP64 (PCP-foldon-VP64)

[0201] 3) Control group: dCas9-VP64;

[0202] 4) Target genes: GAB1 and PDGFRB;

[0203] The results, as shown in Figure 9, indicate that the CRISPRa system of this application also has a significant transcriptional activation effect on the genes GAB1 and PDGFRB, which cannot be activated by VPR, and the transcriptional activation level is significantly higher than that of dCas9-VP64.

[0204] Example 3. Functional validation of the CRISPRa system of this application in vitro and in vivo in mice.

[0205] 3.1 In vitro validation of transcriptional activation using the CRISPRa system, which recruits multiple VP64s based on polymerized peptides.

[0206] 1) Cell line selection: 293T cells, HeLa cells, and U2OS cells were all purchased from ATCC;

[0207] 2) Genes to be activated: ASCL1, HBG, IL1B;

[0208] 3) Control group: dCas9-VP64;

[0209] 4) Experimental methods: In the above three cell lines, wild-type cells were used as blank control and dCas9-VP64 was used as control group. The CRISPRa system with the highest gene activation efficiency obtained in Example 2 (shown as PCP-foldon-VP64 in Figure 10) was used to perform activation experiments on the three genes ASCL1, HBG and IL1B respectively, and the activation efficiency was detected by qPCR.

[0210] The results are shown in Figure 10. These results indicate that, compared with the control group dCas9-VP64, the CRISPRa system of this application (shown as PCP-foldon-VP64) significantly improves the transcriptional activation efficiency of multiple endogenous target genes in different cell lines.

[0211] 3.2 Validation of gene transcription activation of the CRISPRa system in mice.

[0212] 1) Selection of mouse strain: Fah expression-deficient mice (from Dr. Markus Grompe's research group (Oregon Stem Cell Center, Oregon Health & Science University, Portland, 97239, Oregon, USA)). These mice have a point mutation in the fumarylacetoacetate hydrolase (Fah) gene, resulting in the loss of Fah protein function, which leads to the accumulation of toxic metabolites of tyrosine in hepatocytes, thereby causing extensive and persistent liver damage. Fah-deficient mice can maintain normal survival by routine administration of Nitisinone (NTBC), or by introducing the Fah gene with normal function into mouse hepatocytes, or by directly transplanting normal hepatocytes to restore liver function

[0041] . 8-10 week old mice were used in the experiment, each weighing 20-25g, with 3 mice in each group (the experiment was conducted in duplicate).

[0213] 2) Gene to be activated: Myc gene;

[0214] 3) The injection groups are shown in Table 3:

[0215] Table 3. Injection regimens for mice

[0216] In Table 3, IR / DR is the recognition sequence of transposase SB11 (K33A), which transposses into the genome under the action of SB11; hU6 is the promoter of human small RNA; CAG and hPGK are promoters that can initiate mRNA transcription; Fah represents the fumarate diacetate hydrolase gene; PolyA is the transcription termination element; P2A and T2A are self-cleaving peptides that can cause a single transcription product to produce multiple proteins.

[0217] 4) Injection method and plasmid injection dosage: Tail vein high-pressure injection, 30 μg plasmid per mouse, injection volume of 2.7 ml, injection completed within 7 seconds;

[0218] 5) Specific experimental method: Using tail vein hypertension, Fah - / - Two plasmids listed in Table 3 were injected into mice, with three mice injected with each plasmid, for a total of six mice (three mice per group). Six weeks later, liver tissue was collected from the mice, the number of liver tumors was counted, and H&E staining, immunohistochemistry (IHC) detection, and qPCR detection were performed.

[0219] The results, shown in Figure 11, demonstrate that the CRISPRa system of this application can efficiently activate Myc gene transcription in mice and ultimately lead to tumorigenesis. This result proves that the CRISPRa system of this application can activate gene transcription and expression in vivo.

[0220] Example 4. Establishment of a transcriptional activation system based on miniCas protein (miniCas) (miniCRISPRa)

[0221] Millions worldwide suffer from genetic diseases, and therapeutic gene editing technology, which can fundamentally cure these diseases by correcting defective genes, is considered the most promising treatment option for conquering them. However, the prospect of achieving in vivo gene editing therapy requires efficient and safe gene delivery systems to deliver therapeutic genes to relevant organs and tissues in the human body. To date, the most widely used delivery method in clinical practice is adeno-associated virus (AAV) vectors, which have good safety, high biocompatibility, and low immune response. However, in practical applications, the packaging capacity of AAV vectors is limited (maximum 4.7kb), making it difficult to deliver the aforementioned gene transcription activation systems into the body with a single AAV vector, which greatly limits the potential of these systems in practical gene therapy applications. Although we have replaced the larger VPR gene with the VP64 small peptide and achieved the same or even better activation effect as VPR, the presence of the Cas9 protein (molecular weight 4.5kb) remains the biggest obstacle to the current construction and packaging of AAV vectors. Therefore, establishing a miniature Cas protein activation system is crucial.

[0222] To verify whether the above principle of recruiting transcription factors based on polymerized peptides can work in the miniCas system, we need to explain several key techniques and experimental protocols:

[0223] (1) Analyze the ribonucleoprotein structure of miniCas protein and its corresponding sgRNA, and determine the possible positions in sgRNA where PP7 can be replaced or added based on the structure.

[0224] (2) Verify that the binding of sgRNA with added PP7 to miniCas protein is unaffected, and verify the transcriptional activation ability of the miniCas-based CRISPRa system by verifying the activation of transcription of multiple genes in cells.

[0225] 1) Control group: minidCas-VP64;

[0226] 2) Cell line selection: 293T cells;

[0227] 3) Gene selection: IFNγ, HBB, IL1RN;

[0228] 4) Experimental group: minidCas-P2A-PCP-foldon-VP64;

[0229] The results are shown in Figure 12. These results indicate that by replacing Stem1 and AR:R2 in the sgRNA backbone with PP7 (Figure 12, A and B), the modified sgRNA and minidCas protein significantly enhance activation by recruiting multiple VP64s, and exhibit excellent activation effects on multiple genes (e.g., HBB, INFγ, and ILIRN) in different cells (i.e., 293T, U2OS, and HeLa cells) (Figure 12, CE). We have named this activation system miniCRISPRa. Figure 25 illustrates the design principle of this system: the trimeric PCP-foldon-VP64 and the sgRNA with two PP7s interact, causing the transcriptional activator VP64 to be recruited in large quantities to the promoter region of the target gene, ultimately leading to high-level activation of the target gene.

[0230] In addition, the miniCRISPRa system, which is based on the application, was constructed by using three sgRNAs targeting the ASCL1, HBG, and HBB genes respectively. Transfection of 293T cells with the miniCRISPRa system can simultaneously activate the expression of these three genes in the cells (Figure 13).

[0231] Example 5. Application of the miniCas protein transcriptional activation system (miniCRISPRa system) in disease treatment

[0232] 5.1 Treatment of β-hemoglobinopathies using the miniCas transcriptional activation system (miniCRISPRa system)

[0233] Beta-hemoglobinopathies are serious autosomal single-gene inherited disorders in humans, primarily including beta-thalassemia and sickle cell disease. Normal adult hemoglobin HbA (α2β2) is a tetramer composed of two α-globin chains and two β-globin chains. Beta-hemoglobinopathies are caused by a genetic mutation in the HBB gene, leading to abnormal β-globin production. Patients lack normal hemoglobin and require lifelong blood transfusions to alleviate symptoms. Globally, over 3% of the population are carriers of beta-hemoglobinopathies, and hundreds of thousands of newborns are diagnosed with the condition each year.

[0234] Human hemoglobin tetramers undergo changes during development, a process known as "globin switching." During embryonic development, the fetal hemoglobin HbF tetramer (α2γ2), primarily composed of α-globin and γ-globin, transports oxygen. After birth, the γ-globin gene (HBG1 / 2) is silenced and replaced by β-globin. The silencing of HBG1 / 2 expression is regulated by various transcription factors, with BCL11A and ZBTB7A being two major transcriptional repressors in this regulatory network.

[0235] Previous studies have shown that reactivating fetal hemoglobin expression can alleviate or even cure β-hemoglobinopathies. The current main treatment strategy involves collecting hematopoietic stem cells / progenitor cells (HSPCs) from the patient, targeting and destroying BCL11A in vitro using the Cas9 nuclease, and then re-infusing the edited HSPCs into the patient. This results in the production of normal red blood cells expressing HbF, thus achieving the therapeutic goal. However, this strategy still has limitations: the Cas9 nuclease can cause DNA double-strand breaks, posing a safety risk of chromosomal deletions, translocations, cell cycle arrest, or apoptosis in HSPCs. Furthermore, targeting and destroying the enhancer sequence of BCL11A is an indirect way to activate HbF expression, and in some patients, high levels of HbF expression cannot be activated; however, higher HbF expression levels correlate with better clinical remission in β-hemoglobinopathies. Therefore, further exploration of safer and more efficient strategies for reactivating γ-globin is needed.

[0236] We attempted to use the CasMini gene activation system to target the BCL11A and ZBTB7A binding motifs located on the HBG1 / 2 promoter. On the one hand, it competitively binds to the binding motifs of BCL11A and ZBTB7A, thereby relieving the inhibitory effect of BCL11A and ZBTB7A on HBG (Figure 14). On the other hand, it efficiently reactivates γ-globin, thereby achieving the goal of treating β-hemoglobinopathies.

[0237] We attempted to activate HBG in 293T cells using the miniCas gene activation system. The results are shown in Figure 15, indicating that HBG expression can be activated by targeting these two sites (i.e., the binding motifs of BCL11A and ZBTB7A on the HBG1 / 2 promoter).

[0238] Example 6. Multigene transcriptional activation effects of the CRISPRa system of this application in different cells.

[0239] The CRISPRa system reported by Xu, X. et al. (Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol Cell. 2021 Oct 21;81(20):4333-4345.e4.doi:10.1016 / j.molcel.2021.08.008.) uses a VPR fusion at the C-terminus of dCasMini to activate multiple genes in cells. However, this VPR-related system is biased towards gene transcription activation and has no obvious activation effect on a few genes. Moreover, VPR has an inhibitory effect on some genes (such as GAB1 and PDGFRB). The CRISPRa system proposed in this application is based on the VP64 system, which has a wider range of gene transcription activation and can activate target genes efficiently without bias. Its future applications will be more extensive.

[0240] The CRISPRa system disclosed by Tomohiro Hino et al. (AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Cell, 2023, 186(22): p.4920-4935.e23) uses AsCas12f in combination with modified sgRNA and MCP-p65-HSF1 protein to package a single AAV and activate gene expression in vivo. However, the CRISPRa system of this application is based on Un1Cas12f protein and trimerized PCP-foldon-VP64 fusion protein to achieve high-level activation in a small volume, and can simultaneously activate the transcription and expression of multiple genes in vivo through the delivery of a single AAV.

[0241] This invention is based on the VP64 activation system, which can significantly improve its activation effect. Its excellent performance can be seen from the activation effect on multiple endogenous genes at the cellular level.

[0242] 1) Cell line selection: 293T cells, U2OS cells, and HeLa;

[0243] 2) Endogenous gene selection: ASCL1, HBG, and IL1B;

[0244] 3) Control group: dCas9-VP64+sgRNA, total plasmid amount 1000ng;

[0245] 4) Experimental group: dCas9+PCP-foldon-VP64+sgRNA, total plasmid amount 1000ng;

[0246] 5) Blank control group: dCas9-VP64, no sgRNA, total plasmid amount 1000ng;

[0247] 6) For cell culture, use 24-well plates, one well for one experiment, in duplicate, and conduct the experiment when the cell density reaches 70%–80%.

[0248] 7) PEI was used as the transfection reagent, 3 μl per well, and the transfection time was 72 h;

[0249] For each of the selected endogenous target genes ASCL1, HBG, and IL1B, the blank control group (dCas9-VP64, no sgRNA), the control group (dCas9-VP64+sgRNA), and the experimental group (dCas9+PCP-foldon-VP64+sgRNA) were transfected into three cell types (i.e., 293T cells, U2OS cells, and HeLa cells), respectively. RNA was extracted from the cells using Trizol 72 h after transfection. Gene expression levels were then detected by Q-PCR.

[0250] As shown in Figure 10, the CRISPRa system of this application significantly enhances the transcriptional activation of the target gene by 587–3000 times compared to the basic dCas9-VP64 system. This indicates that the CRISPRa system of this application can greatly improve the transcriptional activation effect of VP64.

[0251] Example 7. Application of the CRISPRa system of this application in inhibiting tumor growth by activating tumor immune-related genes.

[0252] Wang, G. et al. (Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity. Nature Immunology 20(11):1494-1505.) reported that activating mouse melanoma surface antigen genes via AAV delivery of the CRISPRa activation system can increase the immunogenicity of mouse tumor cells, thereby activating the mouse immune system and enhancing the killing effect of mouse immune cells on tumor cells, thus achieving the effect of treating tumors

[0042] . This indicates that the mouse immune system can be activated by AAV to achieve the effect of treating tumors. IFNγ, IFNα4, and IFNβ1 are important tumor immune regulatory factors. Increasing the levels of IFNγ, IFNα4, and IFNβ1 in tumor tissue can enhance the function of mouse immune cells, thereby achieving the purpose of treating tumors.

[0253] We screened for sgRNAs that could activate the transcription of IFNγ, IFNα4, and IFNβ1 genes in mice and constructed an AAV vector that could simultaneously activate these three genes (Figure 16). By delivering the AAV vector into tumor tissue via orthotopic injection, we found that the tumor volume growth in the AAV-miniCRISPRa injection group was slower (Figure 17), and the tumor mass was smaller (Figure 18). Simultaneously, we detected activation of the three genes IFNγ, IFNα4, and IFNβ1 in the tumor tissue (Figure 19). IHC analysis revealed CD8+ in the tumor tissue of the experimental group. + The cells showed increased infiltration (Figure 20). These results indicate that our CRISPRa system can simultaneously activate the expression of multiple (e.g., three) tumor immune factors in tumor tissue, thereby achieving a therapeutic effect on tumors.

[0254] Example 8. Application of the CRISPRa system of this application in reversing aging in mice by simultaneously activating three genes, Oct4, Sox2, and Klf4, with a single AAV.

[0255] Ocampo, A. et al. (In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming. Cell 167(7):1719-1733.e1712) reported that simultaneous overexpression of the three proteins Oct4, Sox2, and Klf4 (abbreviated as OSK) in premature aging mice could reverse the phenotype of premature aging mice. However, this study was achieved by constructing transgenic mice that overexpress OSK, so there is no possibility of clinical application

[0043] . In addition, some research groups have delivered the expression regulatory elements of Oct4, Sox2, and Klf4 to aged mice through dual AAV, thereby achieving the reversal of aging in aged mice

[0044] . However, this study used dual AAV. Traditional AAV-based activation requires two AAVs simultaneously infecting a single cell to be effective, thus necessitating a relatively large AAV dosage. Our CRISPRa system, however, is small enough to integrate three sgRNAs capable of activating the Oct4, Sox2, and Klf4 genes (OSK genes) along with the miniCRISPRa system into a single AAV vector. This allows for OSK gene activation in multiple tissues with a smaller dosage. We screened three sgRNAs capable of activating Oct4, Sox2, and Klf4, and then constructed... A single AAV plasmid integrating three sgRNAs and miniCRISPRa was synthesized (Figure 21). The AAV plasmid was packaged into an AAV-9 virus and then injected into 22-month-old aged mice via tail vein. AAV-9-dUn1Cas12f without sgRNA served as the control group, AAV9-miniCRISPRa-OSK as the experimental group, and 3-month-old young mice as the control. One month later, the spleen, liver, and colon tissues of the mice were removed. Q-PCR detection revealed that OSK expression levels were upregulated in the liver and colon tissues of the experimental group mice. Figure 22). X-gal staining revealed that the spleen, liver, and colon tissues of aged mice treated with AAV-OSK showed lighter X-gal staining, similar to those of young mice (Figure 23). H&E staining showed that the colon wall thickness in the miniCRISPRa treatment group was higher than that in the control group, similar to that in young mice. The spleen morphology of the aged mice in the experimental group was significantly better than that in the control group, and similar to that in young mice. Specifically, the germinal centers of the spleen in the aged mice in the miniCRISPRa treatment group were significantly better than those in the control group, and similar to those in young mice (Figure 24).The above results indicate that the aging phenotype of mouse tissues was reversed after treatment with AAV9-miniCRISPRa-OSK (i.e., the miniCRISPRa system of this application targeting Oct4, Sox2 and Klf4 genes, sgRNA and miniCas targeting the three genes and PCP-foldon-VP64 constructed in an AAV vector). This means that the miniCRISPRa system of this application can be used to delay aging.

[0256] Those skilled in the art will further recognize that the invention can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention discloses only exemplary embodiments thereunder, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the invention.

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Claims

1. A CRISPR-mediated gene transcription activation (CRISPRa) system, said CRISPRa system comprising: (1) dCas protein or recombinant vector expressing dCas protein; (2) A modified sgRNA expression vector, wherein the modified sgRNA comprises: an sgRNA backbone containing n RNA aptamers, and an sgRNA sequence specifically targeting the target gene to be activated, wherein n is an integer greater than or equal to 2; (3) A fusion protein expression vector, wherein the fusion protein comprises: an RNA-binding motif that specifically recognizes the RNA aptamer, a polymerized peptide, and VP64, which are operatively linked together.

2. The CRISPRa system according to claim 1, wherein the dCas protein is a miniature Cas protein, preferably: dUn1Cas12f1 (SEQ ID NO:2), AsCas12f1 (SEQ ID NO:3), CasΦ (SEQ ID NO:4), OsCas12f1 (SEQ ID NO:5), RhCas12f1 (SEQ ID NO:6), IscB (SEQ ID NO:7), Fanzor (SEQ ID NO:8), TnpB (SEQ ID NO:9), AcCas12n (SEQ ID NO:10), or MmdCas12m (SEQ ID NO:11).

3. The CRISPRa system according to claim 1, wherein the modified sgRNA expression vector is driven by a U6 promoter, preferably, the U6 promoter is a mouse U6 promoter (mU6) or a human U6 promoter (hU6).

4. The CRISPRa system according to claim 1, wherein the RNA aptamer and the RNA binding motif are present in a paired combination, the combination being selected from: PP7 and PCP, MS2 and MCP, or BoxB and N22, preferably PP7 and PCP.

5. The CRISPRa system according to claim 1, wherein n is any integer from 2 to 8, preferably 2.

6. The CRISPRa system according to claim 1, wherein the polymerized peptide is located at the N-terminus, C-terminus, or middle of the fusion protein, and wherein the polymerized peptide is selected from foldon trimer peptide, GCN4 trimer peptide, 3HB trimer peptide, or 6G6H hexamer peptide, preferably foldon trimer peptide.

7. The CRISPRa system according to claim 1, wherein the transcriptional activation domain is selected from VP64, p65-HSF1 or VPR, preferably VP64.

8. The CRISPRa system according to claim 1, wherein each element of the CRISPRa system is constructed into a vector, preferably, the coding sequence of the dCas protein, the modified sgRNA and the coding sequence of the fusion protein are constructed into a vector, or all elements of the CRISPRa system are packaged into lipid nanoparticles (LNPs) or virus-like particles (VLPs), wherein the vector is a viral vector, such as an AAV vector, a lentiviral vector, a retroviral vector or an adenovirus vector, preferably an AAV vector.

9. A method of activating transcription and expression of a gene of interest, the method comprising: The CRISPRa system of any one of claims 1-8 is constructed for the target gene, and then the gene activation system is introduced into the cell to transcribe and activate the expression of the target gene.

10. A kit comprising the CRISPRa system of any one of claims 1-8, preferably further comprising a viral vector, such as an AAV vector, a lentiviral vector, a retroviral vector, or an adenovirus vector, preferably an AAV vector.

11. Use of the CRISPRa system of any one of claims 1-8 in the preparation of a kit for treating a subject with a hereditary disease, wherein the hereditary disease is a disease caused by gene deletion, mutation or insufficient expression.

12. Use of the CRISPRa system according to any one of claims 1-8 in the preparation of a kit for treating a subject's tumor or delaying aging.

13. The use according to claim 11 or 12, wherein the subject is a mammal, preferably a human.

14. The use according to claim 11 or 12, wherein the dCas protein in the CRISPRa system is a miniature Cas protein and comprises: one or more sgRNAs targeting one or more genes or alleles associated with a hereditary disease having deletions, mutations, or insufficient expression, or one or more sgRNAs targeting one or more genes associated with tumors or aging; and wherein the coding sequence of the dCas protein of the CRISPRa system, the modified sgRNA, and the coding sequence of the fusion protein are constructed into an AAV vector.

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