Gene editing system and application thereof

Abstract

The present disclosure discloses a gene editing system that specifically targets APOE4RNA, and a gene editing system that specifically targets APOE4RNA. The gene editing system can be used for treating Alzheimer's disease and other diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a gene editing system and its applications. Background Art

[0002] Human apolipoprotein E (ApoE) is a 34 kDa glycoprotein that is abundantly expressed in the central nervous system and is distributed in neurons, astrocytes, microglia, vascular wall cells, etc. After being released, ApoE binds to the ApoE receptor on the cell surface, redistributes cholesterol and other lipids into neurons and plays a key role. ApoE has three main allelic variants, ApoE2, ApoE3, and ApoE4, among which ApoE4 is associated with a reduced risk and age of onset of Alzheimer's disease (AD), while ApoE2 has a certain neuroprotective effect.

[0003] AD is a neurodegenerative disease with insidious onset and progressive development. Neurons and their connections in the patient's brain slowly degenerate, resulting in severe memory loss, intellectual disability, and decline in motor skills and communication ability, and ultimately developing into global dementia, commonly known as "senile dementia", which is the most common type of dementia (accounting for 60%-80%). The main pathological features of AD are 1) neuron loss; 2) β-amyloid plaques, extracellular aggregation of β-amyloid protein (Aβ) in neurons, which causes neuron damage and necrosis by blocking communication between synapses of neurons; 3) neurofibrillary tangles (NFTs), intracellular aggregation of highly phosphorylated Tau protein in neurons, which hinders the transport of nutrients and small molecules within neurons. Currently, there is a lack of effective drugs for significantly treating or preventing AD clinically.

[0004] ApoE4 is one of the greatest risk factors for the onset of AD. Compared with the common ApoE3 allele, carrying one ApoE4 allele increases the risk of late-onset AD by 3-4 times; carrying two alleles increases the risk by 9-15 times. In AD patients, ApoE4 is also associated with a reduced age of onset of AD. More and more evidence shows that ApoE4 promotes Aβ aggregation by inhibiting the clearance of amyloid-β (Aβ), and participates in the onset of AD by impairing microglial reactivity and lipid metabolism, affecting synaptic integrity and plasticity.

[0005] Preclinical animal experiments have shown that ApoE4 promotes Aβ deposition, blood-brain barrier leakage, etc. (Brain 145.10(2022):3582-3593.). Knockdown of ApoE4 in neurons, astrocytes, and microglia can reduce Aβ deposition, improve animal cognition and memory, and delay the progression of AD. (Frontiers in aging neuroscie nce 11(2019):14.; Journal of Neuroscience 39.37(2019):7408-7427.). ApoE3, which has only one base difference from ApoE4, plays certain functions such as promoting synaptic integrity and regulating cholesterol levels. Existing reports have shown that synaptic integrity is reduced in ApoE knockout mice. Notably, plasma ApoE is crucial for spatial learning ability and other non-neuronal degenerative diseases such as coronary heart disease. In addition, ApoE plays an anti-inflammatory role in Aβ-induced inflammation. ApoE antibodies have shown an increased risk of inflammation and a slowed motor response in mice (The Journal ofclinicalinvestigation 128.5(2018):2144-2155. Journal of Experimental Medicine 209.12(2012):2149-2156.). Therefore, in the face of ApoE heterozygous patients, silencing ApoE without selection may have potential side effects and should be treated with caution. Summary of the Invention

[0006] The first aspect of the present disclosure provides the use of a gene editing system in the preparation of a drug for diagnosing, treating, or preventing Alzheimer's disease in a subject, characterized in that:

[0007] After the gene editing system contacts a cell containing ApoE4 RNA, the level of the ApoE4 RNA or ApoE4 protein in the cell can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%,

[0008] After the gene editing system contacts cells containing ApoE3 RNA, the level of the ApoE3 RNA or ApoE3 protein in the cells may not decrease or may decrease by at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, or at most 5%.

[0009] In some embodiments of the present disclosure, the subject carries at least one ApoE4 allele.

[0010] In some embodiments of the present disclosure, the subject carries one ApoE4 allele.

[0011] In some embodiments of the present disclosure, the subject carries ApoE4 and ApoE3 alleles.

[0012] In some embodiments of the present disclosure, there is provided a gene editing system, characterized in that: the gene editing system comprises:

[0013] a guide RNA comprising a guide sequence that hybridizes to ApoE4 RNA or a polynucleotide sequence encoding the guide RNA, and

[0014] an RNA-guided nuclease or a polynucleotide sequence encoding the nuclease;

[0015] The guide RNA is capable of forming a complex with the nuclease and guiding the complex to specifically bind to the sequence of ApoE4 RNA.

[0016] In some embodiments of the present disclosure, there is provided a gene editing system, characterized in that:

[0017] the gene editing system comprises:

[0018] a guide RNA comprising a guide sequence that hybridizes to ApoE4 RNA or a polynucleotide sequence encoding the guide RNA, and

[0019] an RNA-guided nuclease or a polynucleotide sequence encoding the nuclease;

[0020] The guide RNA is capable of forming a complex with the nuclease and guiding the complex to specifically bind to the sequence of ApoE4 RNA;

[0021] After contacting the gene editing system with cells containing ApoE4 RNA, the level of the ApoE4 RNA or ApoE4 protein in the cells can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0022] After contacting the gene editing system with cells containing ApoE3 RNA, the level of the ApoE3 RNA or ApoE3 protein in the cells is not reduced or is reduced by at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10% or at most 5%.

[0023] In some embodiments of the present disclosure, the ApoE4 RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

[0024] In some embodiments of the present disclosure, the ApoE4 RNA is human ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

[0025] In some embodiments of the present disclosure, the guide RNA guides the complex to bind to and cleave the ApoE4 RNA.

[0026] In some embodiments of the present disclosure, the cells are eukaryotic cells. In some embodiments of the present disclosure, the cells are mammalian cells. In some embodiments of the present disclosure, the cells are human cells.

[0027] The reduction in the level of the said RNA can be tested using conventional methods in the art; including but not limited to qPCR methods, such as RT-qPCR methods. The level of the target RNA (ApoE4 RNA or ApoE3 RNA) in untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be tested as a negative control, and the knockdown level of the target RNA in the experimental group compared to the negative control can be calculated. The experimental group and the negative control group can use the same RNA-guided nuclease (for example, by expressing the same RNA-guided nuclease) and gRNAs with different guide sequences for editing and then compare the differences in the target RNA levels. For example, the experimental group is edited using C13-2 and the gRNAs claimed in this disclosure request, and the negative control group is edited using C13-2 and a gRNA targeting, for example, the bacterial genome. The experimental group can also be compared with known editing tools, for example, compared with the CasRx+gRNA editing tool. Methods the same as or similar to those in the examples of this disclosure can be used to test the reduction in the level of the said RNA.

[0028] In some embodiments of this disclosure, when the said complex binds to and cleaves the target RNA (ApoE4 RNA or ApoE3 RNA), the number of off-target genes is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes determined by RNA sequencing and the set of off-target genes predicted by a program. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes with downregulated expression determined by RNA sequencing and the set of off-target genes predicted by a program. Off-target gene prediction programs known in the art can be used to predict off-target genes under conventional parameter settings. Non-limiting examples are, for example, the method of prediction by the program: using the EMBOSS-water program to perform prediction in the whole genome and whole cDNA sequences of the target species (such as Homo sapiens or Mus musculus, etc.), setting the parameters as gap_extend = 0.5 & gap_extend = 10, using the forward and reverse strands of the gRNA guide sequence for alignment, and filtering the prediction results to obtain the predicted potential target genes (including on-target genes and / or off-target genes).

[0029] The reduction in the protein level encoded by the target RNA (ApoE4 RNA or ApoE3 RNA) can be tested using conventional methods in the art; including but not limited to ELISA, Western Blotting. Untreated cells or cells treated with a gene editing system targeting non-mammalian genomes can be used as negative controls, and the levels of ApoE4 or ApoE3 proteins in the experimental group compared to the negative control group are calculated. Methods the same as or similar to those in the examples of this disclosure can be used to test the reduction in the protein level encoded by the target RNA.

[0030] In some embodiments of this disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with the sequence shown in SEQ ID NO:55 (ggcccggctgggcgcggacatggaggacgtgcgcggccgcctggtgcagtaccgcggcgagg).

[0031] In some embodiments of this disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with the sequence shown in SEQ ID NO:56 (gcgcggacatggaggacgtgcgcggccgcctggtgcagtac).

[0032] In some embodiments of this disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with any one of the sequences shown in SEQ ID NOs: 1-54.

[0033] In some embodiments of this disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with any one of the sequences shown in SEQ ID NOs: 2, 31, 44 and 47.

[0034] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO: 55, with exactly 1 or 2 nucleotide mismatches (meaning a mismatch of 1 pair of complementary bases or 2 pairs of complementary bases). In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO: 55, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 32nd base C of SEQ ID NO: 55.

[0035] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO: 56, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO: 56, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 21st base of SEQ ID NO: 56.

[0036] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to any one of the sequences shown in SEQ ID NOs: 1-8, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to any one of the sequences shown in SEQ ID NOs: 1-8, with exactly 1 nucleotide mismatch.

[0037] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO: 2, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO: 2, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 13th base of SEQ ID NO: 2.

[0038] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO: 5, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO: 5, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 19th base of SEQ ID NO: 5.

[0039] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA has no mismatch at the base corresponding to the ApoE4 genotype mutation site c.334T>C.

[0040] In some embodiments of the present disclosure, the mismatch refers to other base pairing modes other than A-T, A-U, and C-G pairings.

[0041] In some embodiments of the present disclosure, the guide sequence of the guide RNA comprises the sequence shown in any one of SEQ ID NOs: 1-54.

[0042] In some embodiments of the present disclosure, the guide sequence of the guide RNA comprises the sequence shown in any one of SEQ ID NOs: 2, 31, 44, and 47.

[0043] In some embodiments of the present disclosure, the guide sequence of the guide RNA is the sequence shown in any one of SEQ ID NOs: 1-54.

[0044] In some embodiments of the present disclosure, the guide sequence of the guide RNA is the sequence shown in any one of SEQ ID NOs: 2, 31, 44, and 47.

[0045] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a scaffold sequence, and the scaffold sequence interacts with an RNA-guided nuclease. The scaffold sequence is the sequence that generally remains unchanged in the guide RNA molecule when designing the guide RNA molecule. For example, the scaffold sequence may refer to the part of the guide RNA molecule other than the guide sequence. In some embodiments of the present disclosure, the scaffold sequence is a direct repeat (DR).

[0046] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas13 protein or a fragment thereof. The Cas13 protein fragment is, for example, the nucleic acid binding domain of the Cas13 protein.

[0047] In some embodiments of the present disclosure, the Cas13 protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein. For example: LwaCas13a, LsCas13a, LbuCas13a, dLbuCas13a (R472A / H477A / R1048A / H1053A), TccCas13a, LneCas13a (LneC2c2), LbmCas13a, LbnCas13a, PpCas13a, LbfCas13a, CgCas13a, Cg2Cas13a, PspCas13b, PspCas13b H133A / H1058A, PbuCas13b, PgiCas13b, BzCas13b, RanCas13b, PguCas13b, dPguCas13b (H151A / H1121A), Cas13bt1, Cas13bt3, CcaCas13b, MisCas13b, Hgm4Cas13b, Pba4Cas13b, Bba2Cas13b, CasRx, dCasRx (R239A / H244A / R858A / H863A), CasRx_N2V8 (A134V,A140V,A141V,A143V), RspCas13d, C13-2.

[0048] In some embodiments of the present disclosure, the Cas13 protein is a Cas13d protein.

[0049] In some embodiments of the present disclosure, the Cas13 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the CasRx or C13-2 protein.

[0050] In some embodiments of the present disclosure, the Cas13 protein is a CasRx protein. In some embodiments, the Cas13 protein is dCasRx in which both HEPN domains carry mutations (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0051] In some embodiments of the present disclosure, the amino acid sequence of the Cas13 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:64.

[0052] In some embodiments of the present disclosure, the Cas13 protein is the C13-2 protein. In some embodiments, the Cas13 protein is the dead C13-2 protein.

[0053] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and cleave ApoE4 RNA.

[0054] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and inhibit the translation of ApoE4 RNA.

[0055] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a protein domain.

[0056] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a fused protein domain.

[0057] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0058] In some embodiments of the present disclosure, the subcellular localization signal is a nuclear localization signal and / or a nuclear export signal sequence.

[0059] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence.

[0060] In some embodiments of the present disclosure, the subcellular localization signal is a mitochondrial localization signal, a chloroplast localization signal sequence.

[0061] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence, and optionally a deaminase domain, a translation activation domain, or a translation inhibition domain.

[0062] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease is linked to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is linked to a regulatory sequence 2 that regulates its expression.

[0063] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and an enhancer sequence.

[0064] In some embodiments of the present disclosure, the regulatory sequence is selected from the CMV promoter, CMV enhancer, CBh promoter, U6 promoter, brain-specific promoter, and neuron-specific promoter.

[0065] In some embodiments, at least 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) guide RNAs are expressed in tandem under the regulation of the same regulatory sequence. Further, the tandem expression results in a crRNA precursor (pre-cRNA), which is then processed by an RNA-guided nuclease to obtain a mature guide RNA molecule.

[0066] In some embodiments, the polynucleotide sequence encoding the guide RNA and the polynucleotide sequence encoding the RNA-guided nuclease are located on the same vector.

[0067] In some embodiments of the present disclosure, the gene editing system is introduced into a cell or a cell-free system in any of the following ways: (i) as mRNA encoding an RNA-guided nuclease and a guide RNA, (ii) as part of a single vector or plasmid, or divided into multiple vectors or plasmids, (iii) as a separate RNA-guided nuclease and guide RNA, or (iv) as an RNP complex of an RNA-guided nuclease and a guide RNA.

[0068] The second aspect of the present disclosure provides a gene editing system, characterized in that:

[0069] After the gene editing system contacts a cell containing ApoE4 RNA, the level of the ApoE4 RNA or ApoE4 protein in the cell can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0070] After the gene editing system contacts a cell containing ApoE3 RNA, the level of the ApoE3 RNA or ApoE3 protein in the cell is not reduced or is reduced by at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, or at most 5%.

[0071] In some embodiments of the present disclosure, the subject carries at least one ApoE4 allele.

[0072] In some embodiments of the present disclosure, the subject carries one ApoE4 allele.

[0073] In some embodiments of the present disclosure, the subject carries ApoE4 and ApoE3 alleles.

[0074] In some embodiments of the present disclosure, there is provided a gene editing system, characterized in that: the gene editing system comprises:

[0075] a guide RNA comprising a guide sequence that hybridizes to ApoE4 RNA or a polynucleotide sequence encoding the guide RNA, and

[0076] an RNA-guided nuclease or a polynucleotide sequence encoding the nuclease;

[0077] The guide RNA is capable of forming a complex with the nuclease and guiding the complex to specifically bind to the sequence of ApoE4 RNA.

[0078] In some embodiments of the present disclosure, there is provided a gene editing system, characterized in that:

[0079] The gene editing system comprises:

[0080] a guide RNA comprising a guide sequence that hybridizes to ApoE4 RNA or a polynucleotide sequence encoding the guide RNA, and

[0081] an RNA-guided nuclease or a polynucleotide sequence encoding the nuclease;

[0082] The guide RNA is capable of forming a complex with the nuclease and guiding the complex to specifically bind to the sequence of ApoE4 RNA;

[0083] After contacting the gene editing system with cells containing ApoE4 RNA, the level of the ApoE4 RNA or ApoE4 protein in the cells can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0084] After contacting the gene editing system with cells containing ApoE3 RNA, the level of the ApoE3 RNA or ApoE3 protein in the cells is not reduced or is reduced by at most 95%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10% or at most 5%.

[0085] In some embodiments of the present disclosure, the ApoE4 RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

[0086] In some embodiments of the present disclosure, the ApoE4 RNA is human ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

[0087] In some embodiments of the present disclosure, the guide RNA guides the complex to bind to and cleave the ApoE4 RNA.

[0088] In some embodiments of the present disclosure, the cell is a eukaryotic cell. In some embodiments of the present disclosure, the cell is a mammalian cell. In some embodiments of the present disclosure, the cell is a human cell.

[0089] The reduction in the level of the RNA can be tested using conventional methods in the art; including but not limited to qPCR methods, such as RT-qPCR methods. The level of the target RNA (ApoE4 RNA or ApoE3 RNA) in untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be tested as a negative control, and the knockdown level of the target RNA in the experimental group compared to the negative control can be calculated. The experimental group and the negative control group can use the same RNA-guided nuclease (e.g., by expressing the same RNA-guided nuclease) and gRNAs with different guide sequences for editing, and then compare the differences in the target RNA levels. For example, the experimental group is edited using C13-2 and the gRNAs claimed in this disclosure request, and the negative control group is edited using C13-2 and a gRNA targeting, for example, the bacterial genome. The experimental group can also be compared with known editing tools, such as the CasRx+gRNA editing tool. The reduction in the level of the RNA can be tested using the same or similar methods as those in the examples of this disclosure.

[0090] In some embodiments of this disclosure, the number of off-target genes when the complex binds to and cleaves the target RNA (ApoE4 RNA or ApoE3 RNA) is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes determined by RNA sequencing and the set of off-target genes predicted by a program. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes with downregulated expression determined by RNA sequencing and the set of off-target genes predicted by a program. Off-target gene prediction programs known in the art can be used to predict off-target genes under conventional parameter settings. Non-limiting examples are as follows: the method of program prediction is to use the EMBOSS-water program to perform prediction in the whole genome and whole cDNA sequences of the target species (such as Homo sapiens or Mus musculus, etc.), set the parameters as gap_extend = 0.5 & gap_extend = 10, use the forward and reverse strands of the gRNA guide sequence for alignment, and filter the prediction results to obtain the predicted potential target genes (including on-target genes and / or off-target genes).

[0091] The reduction in the protein level encoded by the target RNA (ApoE4 RNA or ApoE3 RNA) can be tested using conventional methods in the art; including but not limited to ELISA, Western Blotting. Untreated cells or cells treated with a gene editing system targeting non-mammalian genomes can be used as negative controls, and the levels of ApoE4 or ApoE3 proteins in the experimental group compared to the negative control group can be calculated. Methods the same as or similar to those in the examples disclosed herein can be used to test the reduction in the protein level encoded by the target RNA.

[0092] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with the sequence shown in SEQ ID NO:55 (ggcccggctgggcgcggacatggaggacgtgcgcggccgcctggtgcagtaccgcggcgagg).

[0093] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with the sequence shown in SEQ ID NO:56 (gcgcggacatggaggacgtgcgcggccgcctggtgcagtac).

[0094] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with any one of the sequences shown in SEQ ID NOs: 1-54.

[0095] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with any one of the sequences shown in SEQ ID NOs: 2, 31, 44 and 47.

[0096] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO:55, with exactly 1 or 2 nucleotide mismatches (meaning 1 pair of complementary base mismatches or 2 pairs of complementary base mismatches). In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO:55, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 32nd base C of SEQ ID NO:55.

[0097] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO:56, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to SEQ ID NO:56, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 21st base of SEQ ID NO:56.

[0098] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8, with exactly 1 nucleotide mismatch.

[0099] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO:2, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO:2, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 13th base of SEQ ID NO:2.

[0100] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO:5, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guiding sequence of the guiding RNA is reverse complementary to the sequence shown in SEQ ID NO:5, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 19th base of SEQ ID NO:5.

[0101] In some embodiments of the present disclosure, the guiding sequence of the guiding RNA has no mismatch at the base corresponding to the ApoE4 genotype mutation site c.334T>C.

[0102] In some embodiments of the present disclosure, the mismatch refers to other base pairing modes other than A-T, A-U, and C-G pairings.

[0103] In some embodiments of the present disclosure, the guide sequence of the guide RNA comprises the sequence shown in any one of SEQ ID NOs: 1-54.

[0104] In some embodiments of the present disclosure, the guide sequence of the guide RNA comprises the sequence shown in any one of SEQ ID NOs: 2, 31, 44, and 47.

[0105] In some embodiments of the present disclosure, the guide sequence of the guide RNA is the sequence shown in any one of SEQ ID NOs: 1-54.

[0106] In some embodiments of the present disclosure, the guide sequence of the guide RNA is the sequence shown in any one of SEQ ID NOs: 2, 31, 44, and 47.

[0107] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a backbone sequence, and the backbone sequence interacts with the RNA-guided nuclease. The backbone sequence is the sequence that generally remains unchanged in the guide RNA molecule when designing the guide RNA molecule. For example, the backbone sequence may refer to the part of the guide RNA molecule other than the guide sequence. In some embodiments of the present disclosure, the backbone sequence is a direct repeat (DR).

[0108] In some embodiments of the present disclosure, the RNA-guided nuclease is a Cas13 protein or a fragment thereof. The Cas13 protein fragment is, for example, the nucleic acid binding domain of the Cas13 protein.

[0109] In some embodiments of the present disclosure, the Cas13 protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein. For example: LwaCas13a, LsCas13a, LbuCas13a, dLbuCas13a (R472A / H477A / R1048A / H1053A), TccCas13a, LneCas13a (LneC2c2), LbmCas13a, LbnCas13a, PpCas13a, LbfCas13a, CgCas13a, Cg2Cas13a, PspCas13b, PspCas13b H133A / H1058A, PbuCas13b, PgiCas13b, BzCas13b, RanCas13b, PguCas13b, dPguCas13b (H151A / H1121A), Cas13bt1, Cas13bt3, CcaCas13b, MisCas13b, Hgm4Cas13b, Pba4Cas13b, Bba2Cas13b, CasRx, dCasRx (R239A / H244A / R858A / H863A), CasRx_N2V8 (A134V,A140V,A141V,A143V), RspCas13d, C13-2.

[0110] In some embodiments of the present disclosure, the Cas13 protein is a Cas13d protein.

[0111] In some embodiments of the present disclosure, the Cas13 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with the CasRx or C13-2 protein.

[0112] In some embodiments of the present disclosure, the Cas13 protein is a CasRx protein. In some embodiments, the Cas13 protein is dCasRx in which both HEPN domains carry mutations (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0113] In some embodiments of the present disclosure, the Cas13 protein is a C13-2 protein. In some embodiments, the Cas13 protein is a dead C13-2 protein.

[0114] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and cleave ApoE4 RNA.

[0115] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and inhibit the translation of ApoE4 RNA.

[0116] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a protein domain.

[0117] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a fused protein domain.

[0118] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0119] In some embodiments of the present disclosure, the subcellular localization signal is a nuclear localization signal and / or a nuclear export signal sequence.

[0120] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence.

[0121] In some embodiments of the present disclosure, the subcellular localization signal is a mitochondrial localization signal, a chloroplast localization signal sequence.

[0122] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence, and optionally a deaminase domain, a translation activation domain, or a translation inhibition domain.

[0123] In some embodiments of the present disclosure, the polynucleotide sequence encoding the RNA-guided nuclease is linked to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is linked to a regulatory sequence 2 that regulates its expression.

[0124] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and an enhancer sequence.

[0125] In some embodiments of the present disclosure, the regulatory sequence is selected from a CMV promoter, a CMV enhancer, a CBh promoter, a U6 promoter, a brain-specific promoter, and a neuron-specific promoter.

[0126] In some embodiments, at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more) guide RNAs are expressed in tandem under the control of the same regulatory sequence. Further, the tandem expression results in a crRNA precursor (pre-cRNA), which is then processed by an RNA-guided nuclease to obtain a mature guide RNA molecule.

[0127] In some embodiments, the polynucleotide sequence encoding the guide RNA and the polynucleotide sequence encoding the RNA-guided nuclease are located on the same vector.

[0128] In some embodiments of the present disclosure, the gene editing system is introduced into a cell or a cell-free system in any of the following ways: (i) as an mRNA encoding an RNA-guided nuclease and a guide RNA, (ii) as part of a single vector or plasmid, or divided into multiple vectors or plasmids, (iii) as a separate RNA-guided nuclease and guide RNA, or (iv) as an RNP complex of an RNA-guided nuclease and a guide RNA.

[0129] A third aspect of the present disclosure provides a gene editing system guide RNA (gRNA), characterized in that it comprises a guide sequence that hybridizes with ApoE4 RNA.

[0130] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% sequence identity with the sequence shown in SEQ ID NO:55 (ggcccggctgggcgcggacatggaggacgtgcgcggccgcctggtgcagtaccgcggcgagg).

[0131] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% sequence identity with the sequence shown in SEQ ID NO:56 (gcgcggacatggaggacgtgcgcggccgcctggtgcagtac).

[0132] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with the sequence shown in any one of SEQ ID NOs: 1-54.

[0133] In some embodiments of the present disclosure, the guide sequence of the guide RNA has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 100% sequence identity with the sequence shown in any one of SEQ ID NOs: 2, 31, 44 and 47.

[0134] In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 55 with one or two nucleotide mismatches. In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 55 with only one nucleotide mismatch. Further, the mismatch does not occur at the 32nd base C of SEQ ID NO: 55.

[0135] In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 56 with one or two nucleotide mismatches. In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 56 with only one nucleotide mismatch. Further, the mismatch does not occur at the 21st base of SEQ ID NO: 56.

[0136] In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8 with one or two nucleotide mismatches. In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8 with only one nucleotide mismatch.

[0137] In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to the sequence shown in SEQ ID NO: 2 with one or two nucleotide mismatches. In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to the sequence shown in SEQ ID NO: 2 with only one nucleotide mismatch. Further, the mismatch does not occur at the 13th base of SEQ ID NO: 2.

[0138] In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to the sequence shown in SEQ ID NO:5, with exactly 1 or 2 nucleotide mismatches. In some embodiments of the present disclosure, the guide sequence of the guide RNA is reverse complementary to the sequence shown in SEQ ID NO:5, with exactly 1 nucleotide mismatch. Further, the mismatch does not occur at the 19th base of SEQ ID NO:5.

[0139] In some embodiments of the present disclosure, the guide sequence of the guide RNA has no mismatch at the base corresponding to the ApoE4 genotype mutation site c.334T>C.

[0140] In some embodiments of the present disclosure, the mismatch refers to a base pairing mode other than A-T, A-U, and C-G pairings.

[0141] In the present disclosure, a mismatch of X nucleotides means a mismatch of X pairs of complementary bases.

[0142] In some embodiments of the present disclosure, the guide sequence of the guide RNA comprises the sequence shown in any one of SEQ ID NOs: 1-54.

[0143] In some embodiments of the present disclosure, the guide sequence of the guide RNA comprises the sequence shown in any one of SEQ ID NOs: 2, 31, 44, and 47.

[0144] In some embodiments of the present disclosure, the guide sequence of the guide RNA is the sequence shown in any one of SEQ ID NOs: 1-54.

[0145] In some embodiments of the present disclosure, the guide sequence of the guide RNA is the sequence shown in any one of SEQ ID NOs: 2, 31, 44, and 47.

[0146] In some embodiments of the present disclosure, the guide RNA comprises a guide sequence and a backbone sequence, and the backbone sequence interacts with an RNA-guided nuclease. The backbone sequence is the sequence that generally remains unchanged in the guide RNA molecule when designing the guide RNA molecule. For example, the backbone sequence may refer to the part of the guide RNA molecule other than the guide sequence. In some embodiments of the present disclosure, the backbone sequence is a direct repeat (DR).

[0147] In some embodiments of the present disclosure, the ApoE4 RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

[0148] In some embodiments of the present disclosure, the ApoE4 RNA is human ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

[0149] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to specifically bind to the sequence of ApoE4 RNA.

[0150] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with the nuclease and guiding the complex to bind to and cleave the ApoE4 RNA.

[0151] In some embodiments of the present disclosure, the complex reduces the level of the ApoE4 RNA in a mammal (such as in a human).

[0152] In some embodiments of the present disclosure, after the complex contacts a cell containing ApoE4 RNA, the level of the ApoE4 RNA in the cell is reduced.

[0153] In some embodiments of the present disclosure, the complex reduces the level of the ApoE4 RNA in the cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the RNA level can be tested using conventional methods in the art; including but not limited to qPCR methods, such as RT-qPCR methods. The level of the target RNA (ApoE4 RNA or ApoE3 RNA) in untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be tested as a negative control, and the knockdown level of the target RNA in the experimental group compared to the negative control can be calculated. The experimental group and the negative control group can use the same RNA-guided nuclease (for example, by expressing the same RNA-guided nuclease) and gRNAs with different guide sequences for editing and then compare the differences in the target RNA levels. For example, the experimental group is edited using C13-2 and the gRNA claimed in the present disclosure, and the negative control group is edited using C13-2 and a gRNA targeting, for example, the bacterial genome. The experimental group can also be compared with known editing tools, such as the CasRx+gRNA editing tool. The reduction in the RNA level can be tested using the same or similar methods as those in the examples of the present disclosure.

[0154] In some embodiments of the present invention, the number of off-target genes when the complex binds to and cleaves the ApoE4 RNA is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes determined by RNA sequencing and the set of off-target genes predicted by a program. Non-limiting examples are as follows: the method of prediction by the program is to use the EMBOSS-water program to predict in the whole genome and whole cDNA sequences of the target species (Homo sapiens / Mus musculus), set the parameters as gap_extend = 0.5 & gap_extend = 10, use the forward and reverse strands of the gRNA guide sequence for alignment, and filter the prediction results to obtain the predicted potential target genes (including on-target genes and off-target genes).

[0155] In some embodiments of the present disclosure, the complex reduces the level of the protein encoded by the ApoE4 RNA in a mammal (such as in a human).

[0156] In some embodiments of the present disclosure, after the complex contacts a cell containing the target RNA, the level of the protein encoded by the ApoE4 RNA in the cell is reduced. In some embodiments of the present disclosure, the protein encoded by the ApoE4 RNA is the ApoE4 protein. In some embodiments of the present disclosure, the complex reduces the level of the ApoE4 protein in the cell.

[0157] In some embodiments of the present disclosure, the complex reduces the level of the ApoE4 protein in the cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. Conventional methods in the art can be used to test the reduction in the level of the protein encoded by the ApoE4 RNA; including but not limited to ELISA and Western Blotting. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls, and the knockdown level of the ApoE4 protein in the experimental group compared to the negative control group can be calculated.

[0158] A fourth aspect of the present disclosure provides a isolated nucleic acid, characterized in that it encodes a guide RNA according to any one of the present disclosure.

[0159] A fifth aspect of the present disclosure provides a vector, the vector comprising a polynucleotide sequence encoding a guide RNA according to any one of the present disclosure, and a regulatory sequence for regulating the expression of the guide RNA.

[0160] In some embodiments of the present disclosure, the vector is a viral vector. In some embodiments of the present disclosure, the vector is an adeno-associated virus vector, an adenovirus vector or a lentivirus vector.

[0161] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and an enhancer sequence.

[0162] In some embodiments of the present disclosure, the regulatory sequence is selected from CMV promoter, CMV enhancer, CBh promoter, U6 promoter, brain-specific promoter and eye-specific promoter.

[0163] In some embodiments of the present disclosure, the regulatory sequence is selected from pol III promoters (e.g., U6 and H1 promoters), pol II promoters (e.g., retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter (optionally with CMV enhancer), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter or EF1α promoter.

[0164] In some embodiments, the promoter is a constitutive promoter, which is continuously active and not regulated by external signals or molecules. Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV40, EF1α, CAG and β-actin promoters. In some embodiments, the promoter is an inducible promoter regulated by external signals or molecules (e.g., transcription factors).

[0165] In some embodiments, the promoter is a tissue-specific promoter that can be used to drive tissue-specific expression of an RNA-guided nuclease or guide RNA. Suitable muscle-specific promoters include, but are not limited to, CK8, MH CK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and the synthetic promoter SPc5-12. Suitable immune cell-specific promoters include, but are not limited to, the B29 promoter (B cells), CD14 promoter (monocytes), CD43 promoter (leukocytes and platelets), CD68 (macrophages), and SV40 / CD43 promoter (leukocytes and platelets). Suitable blood cell-specific promoters include, but are not limited to, the CD43 promoter (leukocytes and platelets), CD45 promoter (hematopoietic cells), INF-β (hematopoietic cells), WASP promoter (hematopoietic cells), SV40 / CD43 promoter (leukocytes and platelets), and SV40 / CD45 promoter (hematopoietic cells). Suitable pancreas-specific promoters include, but are not limited to, the elastase-1 promoter. Suitable endothelial cell-specific promoters include, but are not limited to, the Fit-1 promoter and the ICAM-2 promoter. Suitable neuron tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), SYN1 promoter (neurons), and NSE / RU5' (mature neurons). Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocytes). Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts). Suitable lung-specific promoters include, but are not limited to, the SP-B promoter (lung). Suitable liver-specific promoters include, but are not limited to, the SV40 / Alb promoter. Suitable heart-specific promoters include, but are not limited to, α-MHC.

[0166] In some embodiments of the present disclosure, the promoter is the chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., the chicken β-actin promoter) contains an enhancer sequence, such as the cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter contains a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter contains a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art know that a short CMV enhancer can be used with a long CB promoter, and a long CMV enhancer can be used with a short CB promoter. In some embodiments of the present disclosure, the promoter is the CBh promoter. In some embodiments of the present disclosure, the regulatory sequence contains an HRE enhancer element. In some embodiments of the present disclosure, the regulatory sequence contains a tandem NRS element and an HRE enhancer element.

[0167] The sixth aspect of the present disclosure provides a vector system, wherein the vector system comprises a polynucleotide sequence encoding the guide RNA of the present disclosure and a second regulatory sequence regulating the expression of the guide RNA; and a polynucleotide sequence encoding the RNA-guided nuclease and a first regulatory sequence regulating the expression of the RNA-guided nuclease.

[0168] In some embodiments of the present disclosure, the vector system comprises one or more vectors.

[0169] In some embodiments of the present disclosure, the vector system comprises multiple vectors. The polynucleotide sequence encoding the guide RNA and the second regulatory sequence regulating the expression of the guide RNA are located on the second vector, and the polynucleotide sequence encoding the RNA-guided nuclease and the first regulatory sequence regulating the expression of the RNA-guided nuclease are located on the first vector.

[0170] In some embodiments of the present disclosure, the regulatory sequence is a promoter sequence. In some embodiments of the present disclosure, the regulatory sequence is an enhancer sequence. In some embodiments of the present disclosure, the regulatory sequence is a promoter and enhancer sequence.

[0171] The seventh aspect of the present disclosure provides an adeno-associated virus vector, wherein the adeno-associated virus vector contains DNA encoding an RNA-guided nuclease and the guide RNA of the present disclosure.

[0172] In some embodiments of the present disclosure, the AAV vector comprises a ssDNA genome that contains the coding sequences of an RNA-guided nuclease and a guide RNA flanked by ITRs.

[0173] The eighth aspect of the present disclosure provides a lipid nanoparticle, wherein the lipid nanoparticle comprises the guide RNA described in the present disclosure and an mRNA encoding the RNA-guided nuclease.

[0174] The ninth aspect of the present disclosure provides a lentiviral vector, wherein the lentiviral vector comprises the guide RNA described in the present disclosure and an mRNA encoding an RNA-guided nuclease; optionally, the lentiviral vector is pseudotyped with an envelope protein; optionally, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0175] The tenth aspect of the present disclosure provides a ribonucleoprotein complex, wherein the ribonucleoprotein complex is formed by the guide RNA described in the present disclosure and an RNA-guided nuclease.

[0176] The eleventh aspect of the present disclosure provides a virus-like particle, wherein the virus-like particle comprises a ribonucleoprotein complex formed by the guide RNA described in the present disclosure and an RNA-guided nuclease; optionally, the RNA-guided nuclease is fused to a gag protein.

[0177] The twelfth aspect of the present disclosure provides a eukaryotic cell, which eukaryotic cell comprises the gene editing system, guide RNA, nucleic acid, vector, and / or vector system described in the present disclosure; optionally, the eukaryotic cell is a mammalian cell; further optionally, the eukaryotic cell is a human cell.

[0178] In some embodiments of the present disclosure, the eukaryotic cell comprises the gene editing system described in the present disclosure.

[0179] The thirteenth aspect of the present disclosure provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the gene editing system, guide RNA, nucleic acid, vector, and / or vector system described in any one of the present disclosure.

[0180] In some embodiments of the present disclosure, the pharmaceutical composition comprises the gene editing system described in any one of the present disclosure.

[0181] In some embodiments of the present disclosure, the pharmaceutical composition comprises the guide RNA, nucleic acid, vector, and / or vector system described in any one of the present disclosure.

[0182] In some embodiments of the present disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0183] The fourteenth aspect of the present disclosure provides the use of a gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated virus vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in any one of the following or in the preparation of a reagent for achieving any one of the following scenarios:

[0184] Cleaving one or more ApoE4 RNA molecules or nicking one or more ApoE4 RNA molecules, activating or upregulating one or more ApoE4 RNAs, activating or inhibiting the translation of one or more ApoE4 RNA molecules, inactivating one or more ApoE4 RNA molecules, visualizing, labeling or detecting one or more ApoE4 RNA molecules, binding to one or more ApoE4 RNA molecules, transporting one or more ApoE4 RNA molecules, and masking one or more ApoE4 RNA molecules.

[0185] In some embodiments of the present disclosure, there is provided the use of a gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated virus vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in any one of the following or in the preparation of a reagent for achieving any one of the following scenarios:

[0186] Cleaving ApoE4 RNA molecules, inhibiting the translation of ApoE4 RNA molecules, and binding to ApoE4 RNA molecules.

[0187] In some embodiments of the present disclosure, there is provided the use of a gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated virus vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in cleaving ApoE4 RNA molecules or in the preparation of a reagent for cleaving ApoE4 RNA molecules.

[0188] In some embodiments of the present disclosure, there is provided the use of a gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated virus vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell or pharmaceutical composition according to any one of the present disclosure in binding to ApoE4 RNA molecules or in the preparation of a reagent for binding to ApoE4 RNA molecules.

[0189] In some embodiments of the present disclosure, the ApoE4 RNA is pre-mRNA or mature mRNA. In some embodiments of the present disclosure, the ApoE4 RNA is mature mRNA. In some embodiments of the present disclosure, the ApoE4 RNA is human ApoE4 RNA. In some embodiments of the present disclosure, the ApoE4 RNA is human Apo E4 mRNA.

[0190] The fifteenth aspect of the present disclosure provides a method for diagnosing, treating or preventing a disease or disorder, characterized by administering an effective amount of the gene editing system, guide RNA, nucleic acid, vector, vector system, adeno-associated virus vector, lipid nanoparticle, lentiviral vector, ribonucleoprotein complex, virus-like particle, eukaryotic cell and / or pharmaceutical composition according to the present disclosure to a sample of a subject in need or to a subject in need.

[0191] In some embodiments of the present disclosure, there is provided a method for diagnosing, treating or preventing a disease or disorder, characterized by administering an effective amount of the gene editing system, eukaryotic cell and / or pharmaceutical composition according to the present disclosure to a sample of a subject in need or to a subject in need.

[0192] In some embodiments of the present disclosure, there is provided a method for diagnosing, treating or preventing a disease or disorder, characterized by administering an effective amount of the gene editing system according to the present disclosure to a sample of a subject in need or to a subject in need.

[0193] In some embodiments of the present disclosure, the disease or disorder is a disease or disorder related to ApoE4 RNA; optionally, the disease or disorder is a disease or disorder caused by the expression of ApoE4 RNA.

[0194] In some embodiments of the present disclosure, the ApoE4 RNA is pre-mRNA or mature mRNA. In some embodiments of the present disclosure, the ApoE4 RNA is mature mRNA. In some embodiments of the present disclosure, the ApoE4 RNA is human ApoE4 RNA. In some embodiments of the present disclosure, the ApoE4 RNA is human Apo E4 mRNA.

[0195] In some embodiments of the present disclosure, the disease or disorder is Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figure 1 . Shown is a schematic diagram of the corresponding positions of the screened sg1-sg8 gRNAs on the target nucleic acid. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0197] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0198] Definition section:

[0199] As used herein, the term "gene editing system" refers to a protein, nucleic acid, or combination thereof that, when introduced into a cell, is capable of modifying an endogenous target nucleic acid sequence. The gene editing system includes, but is not limited to, the CRISPR-Cas system, the TALEN system, and the ZFN system. For example, the RNA-guided nucleases described in this disclosure are each independently selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Ca s8, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12f / CasZ, Cas12g, Cas12h, Cas12i, Csy

[0200] 1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas13a, Cas13b, Cas13c, Cas13d, Cas13e, Cas13f, TnpB, IscB, IsrB, Fancor; or a system of fragments thereof (non-limiting examples such as nucleic acid binding domain fragments).

[0201] In some embodiments of the present invention, the RNA-guided nuclease is independently selected from Cas9, Cas12, Cas13, TnpB, IscB, IsrB, Fancor nucleases; or fragments thereof, including but not limited to nucleic acid binding domain fragments.

[0202] In some embodiments of the present invention, the Cas9 is optionally selected from SpCas9, SaCas9, Nme2Cas9, Nme3Cas9, CjCas9, NmCas9, FnCas9, PpnCas9, FrCas9, SauCas9, SauriCas9, ScaCas9, St1Cas9, BlatCas9, CdiCas9, GeoCas9, fragments thereof, and mutants or fragments of mutants thereof. In some embodiments of the present invention, the RNA-guided nuclease is optionally selected from AsCpf1, enAsCas12a (addgene plasmid #196724), dFnCas12a (addgene plasmid #136379), ErCas12a, LbCas12a D832A, LbCas12aH759A, LbCas12a E795L, FnCas12a3, FnCas12a D917A, AsCas12a R1226A, AsCas12a D908A, AsCas12a E174R / S542R, AsCas12a (S542R / K548V / N552R), PrCas12a, PxCas12a, PcCas12a, PdCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12a, CMaCas12a, CMtCas12a, HkCas12a, Lb5Cas12a, ErCas12a, TsCas12a, FnCpf1, LbCas12a, ttHsCas12a, AaCas12b, AaCas12b D570A, AaCas12bQ119F / E475R / E758R, BhCas12b, BvCas12b, BrCas12b, AkCas12b, AmCas12b, BsCas12b, OspCas12c, Cas12c2 (addgene plasmid #183072), Cas12c_4 (addgene plasmid #183071), Cas12c1 (addgene plasmid #120872), CasY.1 (from Katanobacteria), CasY.2 (from Vogelbacteria), CasY.3 (from Vogelbacteria), CasY.4 (from Parcubacteri a), CasY.5 (from Komeilibacteria), CasY.6 (from Kerfeldbacteria), PlmCasX, Dpb CasX, Un1Cas12f, CnCas12f1, enRhCas12f1, AsCas12f1, SpaCas12f1, Cas12g1 (add gene plasmid #120879), Cas12h (SEQ ID NO:1) of WO2021113522A1, Cas12i1 (addgene plasmid #171670), Cas12i2 (addgene plasmid #188275), Cas12i1 (addge ne plasmid #120882), Cas12i2 (addgeneplasmid #120883), Cas12i proteins named Cas12f.4 / Cas12f.5 / Cas12f.6 in CN111757889B, dSiCas12i (D1049A), SiCas12i, Si2Cas12i, WiCas12i, Wi2Cas12i, Wi3Cas12i, SaCas12i, Sa2Cas12i, Sa3Cas12i, WaCas12i, Wa2Cas12i, xCas12i, hfCas12Max, Cas12i-Max (addgeneplasmid #188276), Cas12i1D647A (addgene plasmid #171671), Cas12i-HiFi (addgeneplasmid #188269), Ca s12i1 D647A, Cas12j3 (addgene plasmid #188497), Cas12j2 (addgene plasmid #188498), AsCas12j-2 (addgene plasmid #191655), Cas12j-8 (addgeneplasmid #19496.

[0203] 6), ShCas12k, N7Cas12k, AcCas12k, Cas12k-TniQ (addgene plasmid #181787), Cas12k-TnsC (addgene plasmid #181789), Cas12l, MmCas12m, MmCas12mΔZF (H549A, C552A), dCas12m-ΔZF (D485A, H549A, C552A), AcCas12n, dAcCas12n (D240), TnpB Actinomadura_cellulosilytica_strain_DSM_45823, TnpB Actinomadura_namibien sis_strain_DSM_44197, TnpB Actinomadura_umbrina_strain_DSM_43927_$, TnpB Actinoplanes_lobatus_strain_DSM_43150 (TnpB-1 and TnpB-2), TnpB Alicyclobacillus_macr osporagiidus_strain_DSM_17980, TnpB Haloactinospora_alba_Strain_DSM_45015, TnpB Lipingzhangella_halophila_strain_DSM_102030, TnpB Meiothermus_Silvanus_DSM_9946, TnpB QNFX01000004, ISDra2 TnpB (PDB: 8H1J), KraIscB-1, AwaIscB, OgeuIscB, GtFz1 (from Guillardia theta), SpuFz1 (from Spizellomyces punctatus), NlovFz2 (from Percolozoa Naegleria lovaniensis), MmeFz2 (from Mercenariamercenaria), their fragments, and fragments of their mutants or mutants.

[0204] The term RNA-guided nuclease refers to a polypeptide that binds to a specific target RNA sequence in a sequence-specific manner, and the polypeptide is guided to the target RNA by a guide RNA that complexes with the polypeptide and hybridizes to the target sequence on the target RNA. Cleavage of the target sequence by the RNA-guided nuclease can result in strand breakage. Although an RNA-guided nuclease can cleave the target sequence upon binding, the term RNA-guided nuclease also includes nuclease-inactivated RNA-guided nucleases that are capable of binding but not cleaving the target sequence. The RNA-guided nucleases described in this disclosure include, but are not limited to, wild-type RNA-guided nucleases (e.g., C13-2, CasRx, etc.), variants thereof (e.g., mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off-target effects, mutants with reduced collateral effects), or functional fragments or fusion proteins thereof.

[0205] As used herein, the term Cas protein is a CRISPR-associated (Cas) polypeptide or protein that, when complexed or functionally combined with one or more guide RNAs, can be directed to a target sequence in a target RNA and can sometimes subsequently bind to or cleave the target RNA.

[0206] In some embodiments, the gene editing system used in the methods described herein is a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) nuclease system, which is an engineered nuclease system based on a bacterial system that can be used for mammalian genome engineering.

[0207] As used herein, the term knockdown refers to a measurable decrease in the level of a target RNA in a genetically modified cell compared to the level of the target RNA in a corresponding control cell that does not contain a genetic modification that reduces expression. For example, the level of the target RNA in the genetically modified cell is decreased by >0%, ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% compared to the level of the target RNA in the control cell. Those skilled in the art will readily understand how to use gene editing-mediated inhibition techniques to knockdown a target RNA or a portion thereof based on the details described herein.

[0208] As used herein, the terms gene editing system guide RNA, guide RNA, guide polynucleotide, guide RNA, and gRNA are used interchangeably. The term guide RNA is used to refer to a molecule in a gene editing system that forms a complex with an RNA-guided nuclease and guides the complex to specifically bind to a target sequence in a sequence-specific manner. The guide RNA comprises a guide sequence and a backbone sequence, and the guide sequence can hybridize with the target sequence. When the RNA-guided nuclease is a Cas protein, especially a Cas13 protein, the backbone sequence is usually a direct repeat sequence.

[0209] As used herein, the terms "guide sequence" and "targeting domain" are used interchangeably and refer to a continuous nucleotide sequence in a gRNA that has partial or complete complementarity with a target sequence in a target RNA and can hybridize with the target sequence in the target RNA through base pairing facilitated by an RNA-guided nuclease. Complete complementarity between the guide sequence of the present invention and the target sequence is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a gene editing complex.

[0210] Suitable direct repeat (DR) sequences can exist in the CRISPR locus structures of prokaryotes (such as bacteria and archaea) and can be obtained through experimental screening; they can also be obtained by sequence modification or optimization on this basis. Non-limiting examples include the deletion, substitution, or addition of 1, 2, 3, 4, or more complementary base pairs in the double-stranded complementary region of the secondary structure of the DR sequence, and the deletion, substitution, or addition of nucleotides in the loop of the stem-loop structure of the secondary structure of the DR sequence (for example, an aptamer sequence can be inserted into the loop). The size of the direct repeat sequence is usually dozens of nucleotides, and partial fragments thereof are reverse complementary to each other, which means that a secondary structure is formed inside the RNA molecule, such as a stem-loop structure (often called a hairpin structure), and other fragments are unstructured. When the RNA-guided nuclease is a Cas protein, the direct repeat sequence is a constant part of the gRNA molecule, which contains a strong secondary structure, which is beneficial to the interaction between the Cas protein and the gRNA molecule.

[0211] The term "hybridization" or "hybridizing" refers to the process by which fully or partially complementary polynucleotide strands come together under suitable hybridization conditions to form a double-stranded structure or region, including the association between nucleic acids caused by hydrogen bonds. As used herein, the term hybridization includes cases in which the double-stranded structure or region contains one or more bulges or mismatches. The strength of hybridization and hybridization (i.e., the strength of the association between nucleic acids) is affected by factors such as the degree of complementarity between nucleic acids, the stringency of the conditions involved, and the Tm of the hybrid formed. Although hydrogen bonds generally form between adenine and thymine, adenine and uracil, or cytosine and guanine, other non-classical base pairs can also form hydrogen bonds. It is expected that modified nucleotides can form hydrogen bonds through non-classical pathways that allow or promote hybridization.

[0212] As used herein, the term target RNA refers to a polynucleotide containing a target sequence, representing a specific sequence or its reverse complementary sequence that one wishes to bind to, target, or modify using a gene editing system. For example, it can be a complete mature mRNA molecule or a pre-mRNA molecule.

[0213] As used herein, the term target sequence refers to a short segment of a target RNA molecule that can be complementary (fully or partially) to the guide sequence of a gRNA molecule. The gene editing complex or CRISPR complex is specifically localized to the target sequence by the guide sequence and performs corresponding functions at or near this position. The length of the target sequence is often dozens of nt (nucleotides). For example, it can be about 10 nt, about 20 nt, about 30 nt, about 40 nt, about 50 nt, about 60 nt.

[0214] As used herein, the term cleavage / cleaving refers to the breaking of a covalent bond (such as a covalent phosphodiester bond) in the ribose-phosphate backbone of a polynucleotide.

[0215] The ability of a guide RNA-guided complex to bind sequence specifically to a target RNA can be evaluated by any suitable assay. For example, the components of a gene editing system sufficient to form a CRISPR complex, including the guide RNA to be tested, can be provided to a host cell having the corresponding target RNA molecule, e.g., by transfection of a vector encoding the components of the CRISPR complex, and then preferential cleavage within the target sequence can be evaluated. Similarly, cleavage of a target RNA sequence can be evaluated in vitro by providing the target RNA, the components of the CRISPR complex, including the guide RNA to be tested and a control guide RNA different from the test guide RNA, and comparing the ability to bind the target RNA or the rate of cleavage of the target RNA between the test and control guide RNAs. The ability of a guide RNA-guided complex to cleave a target RNA can also be evaluated by the assays described above.

[0216] As used herein, the term "sequence identity" (identity or percent identity) is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both of the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared × 100%. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% sequence identity. Generally, comparisons are made when the two sequences are aligned to yield maximum sequence identity. Such alignments can be performed using publicly available and commercially available alignment algorithms and programs, such as but not limited to ClustalΩ, MAFFT, Probcons, T-Coffee, Probalign, BLAST, which can be reasonably selected for use by one of ordinary skill in the art. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, e.g., including any algorithms required to achieve a better or optimal alignment over the full length of the sequences being compared, and any algorithms required to achieve a better or optimal alignment of local regions of the sequences being compared.

[0217] As used herein, the term regulatory sequence 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). Regulatory sequences include those elements that direct continuous expression of a nucleotide sequence in many types of host cells and those elements that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., neuronal cells, lymphocytes). Regulatory sequences can also direct expression in a time-dependent manner such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue-specific or cell type-specific. The term regulatory sequence also encompasses enhancer elements such as WPRE, CMV enhancer, SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit β-globin. Those skilled in the art will appreciate that the design of an expression vector can depend on factors such as the choice of host cell to be transformed, the desired level of expression, etc. The vector can be introduced into a host cell to produce the RNA-guided nuclease and / or guide RNA described herein.

[0218] As used herein, the term promoter has the meaning commonly ascribed to it in the art.

[0219] As used herein, the term enhancer has the meaning commonly ascribed to it in the art.

[0220] As used herein, when referring to a nucleotide sequence / DNA / RNA encoding a protein, RNA, CRISPR complex, the coding sequence can be codon-optimized. For example, the coding sequence is codon-optimized for expression in a eukaryotic cell environment, codon-optimized for expression in a mammalian cell environment, or codon-optimized for expression in a human cell environment.

[0221] As used herein, the term "codon optimization" refers to the process of altering the codons of a given gene in such a way that the polypeptide sequence encoded by the gene remains the same while the altered codons improve the expression process of the polypeptide sequence. For example, if a polypeptide belongs to a human protein sequence and is expressed in Escherichia coli, expression is generally improved if the DNA sequence is codon-optimized to change human codons to codons that are more effective for expression in Escherichia coli.

[0222] As used herein, the term pharmaceutically acceptable excipient refers to a diluent, adjuvant, pharmaceutical carrier, or other excipient administered together with an active ingredient. Its selection depends on the use and the intended method of administration. The excipient should not be incompatible with the active ingredient, for example, producing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition. The pharmaceutical composition can be prepared by known methods in the field of pharmaceutical preparation.

[0223] When referring to an RNA sequence, "t" in the sequence can be used interchangeably with "u". When referring to a "guide sequence", "t" in the sequence can be used interchangeably with "u". When referring to an "inverted repeat sequence", "t" in the sequence can be used interchangeably with "u".

[0224] Guide RNA

[0225] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease (also referred to as a gene editing complex) and guiding the specific binding of the complex to the sequence of the target RNA.

[0226] In some embodiments of the present disclosure, the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and cleave the target RNA (such as ApoE4 RNA).

[0227] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell.

[0228] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. The reduction in the level of the target RNA can be tested by conventional methods in the art; including but not limited to the qPCR method as described in the examples. Untreated cells or cells treated with a gene editing system targeting a non-mammalian genome can be used as negative controls, and the knockdown level of the target RNA in the experimental group compared to the negative control can be calculated.

[0229] In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 5%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 40%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 80%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 85%. In some embodiments of the present disclosure, the complex reduces the level of the target RNA in the cell by at least 90%.

[0230] In some embodiments of the present invention, the number of off-target genes when the complex binds and cleaves the target RNA is less than 40, less than 35, less than 30, less than 25, less than 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. The number of off-target genes can be determined by conventional methods in the art. In some embodiments, the number of off-target genes is determined by taking the intersection of the set of differentially expressed genes determined by RNA sequencing and the set of off-target genes predicted by a program. Non-limiting examples are as follows: for the method of program prediction, use the EMBOSS-water program to predict in the whole genome and whole cDNA sequences of the target species (Homo sapiens / Mus musculus), set the parameters as gap_extend = 0.5 & gap_extend = 10, use the forward and reverse strands of the gRNA guide sequence for alignment, and filter the prediction results to obtain the predicted potential target genes (including on-target genes and off-target genes).

[0231] In some embodiments of the present disclosure, the guide sequence comprises 20 - 40, 20 - 35, 20 - 30, or 25 - 30 nucleotides.

[0232] In some embodiments of the present disclosure, the guide sequence hybridizes to the target RNA with no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide mismatch.

[0233] In some embodiments of the present disclosure, the guide sequence has 100% sequence identity with the target RNA sequence, i.e., it is completely complementary.

[0234] In some embodiments of the present disclosure, the guide sequence is located at the 3'-end or 5'-end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 3'-end of the direct repeat sequence. In some embodiments of the present disclosure, the guide sequence is located at the 5'-end of the direct repeat sequence.

[0235] In some embodiments of the present disclosure, the direct repeat sequence comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the sequence shown in SEQ ID NO: 65 or 66.

[0236] In some embodiments of the present disclosure, the direct repeat sequence comprises the sequence shown in SEQ ID NO: 65 or 66. In some embodiments of the present disclosure, the direct repeat sequence consists of the sequence shown in SEQ ID NO: 65 or 66.

[0237] In some embodiments of the present disclosure, the guide RNA comprises an aptamer sequence.

[0238] In some embodiments of the present disclosure, the aptamer sequence is inserted into the loop of the stem-loop structure of the secondary structure of the direct repeat sequence of the guide RNA.

[0239] In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides. The modifications include but are not limited to 2'-O-methyl, 2'-O-methyl-3'-thiolate or 2'-O-methyl-3'-thiol-PACE modifications. In some embodiments of the present disclosure, the guide RNA comprises modified nucleotides selected from deoxyribonucleotides, locked nucleic acids (LNA). In some embodiments, the guide RNA comprises at least one chemically modified nucleotide. In some embodiments, the guide RNA is a hybrid RNA-DNA guide, i.e., some of the RNA nucleotides in the guide RNA are replaced by DNA nucleotides. In some embodiments, the guide RNA is a hybrid RNA-LNA (locked nucleic acid) guide, i.e., some of the RNA nucleotides in the guide RNA are replaced by LNA nucleotides.

[0240] In some embodiments, multiple guide RNAs are part of an array (which can be part of a vector, such as a viral vector or plasmid). For example, a guide array including the sequence DR - spacer - DR - spacer - DR - spacer can include 3 unique unprocessed guide RNAs (one for each DR - spacer sequence). Once introduced into a cell or cell - free system, the array is processed by an RNA - guided nuclease such as a Cas protein into multiple individual mature guide RNAs. This allows for multiplexing, such as delivering multiple guide RNAs to a cell or system to target multiple target RNAs or multiple regions within a single target RNA.

[0241] In some embodiments of the present disclosure, the target RNA is located in the nucleus and / or cytoplasm of a eukaryotic cell.

[0242] Target RNA

[0243] The gene - editing systems and compositions described in the present disclosure can be used to target one or more target RNA molecules, such as target RNA molecules present in a biological sample. In some embodiments, the target RNA is pre - mRNA or mRNA (mature mRNA).

[0244] In some embodiments of the present disclosure, the target RNA is ApoE4 RNA. In some embodiments of the present disclosure, the target RNA is human ApoE4 RNA. In some embodiments of the present disclosure, the target RNA is human ApoE4 mRNA.

[0245] In some embodiments of the present disclosure, the target RNA is ApoE4 RNA or a fragment thereof.

[0246] In some embodiments of the present disclosure, the target RNA is ApoE4 mRNA or a fragment thereof.

[0247] In some embodiments of the present disclosure, the target RNA is ApoE4 pre - mRNA or a fragment thereof.

[0248] RNA - guided nuclease

[0249] In some embodiments of the present disclosure, the RNA - guided nuclease is a Cas protein.

[0250] In some embodiments of the present disclosure, the RNA - guided nuclease is selected from wild - type RNA - guided nucleases (including but not limited to CasRx, C13 - 2, etc.), their variants (including but not limited to mutants with complete loss of cleavage activity, mutants with partial loss of cleavage activity, mutants with increased cleavage activity, mutants with reduced off - target / by - stander effects), or their functional fragments or fusion proteins.

[0251] In some embodiments of the present disclosure, the RNA-guided nuclease comprises any one or more of the following fusion domains (which may be referred to as fusion proteins at this time): subcellular localization signals, deaminase domains, translation activation domains, translation inhibition domains, RNA methylation domains, RNA demethylation domains, nuclease domains, splicing factor domains, reporter tags, and affinity tags.

[0252] Exemplary fusion domains (such as fused heterologous protein domains) include domains that can cleave RNA (e.g., PIN endonuclease domain, NYN domain, SMR domain from SOT1, or RNase domain from staphylococcal nuclease), domains that can affect RNA stability (e.g., tristetraprolin (TTP) or domains from UPF1, EXOSC5, and STAU1), domains that can edit nucleotides or ribonucleotides (e.g., cytidine deaminase, PPR protein, adenosine deaminase, ADAR family proteins, or APOBEC family proteins), domains that can activate translation (e.g., eIF4E and other translation initiation factors, yeast poly(A)-binding protein, or domains of GLD2), domains that can inhibit translation (e.g., Pumilio or FBF PUF proteins, deadenylase, CAF1, Argonaute proteins), domains that can methylate RNA (e.g., domains from m6A methyltransferase factors such as METTL14, METTL3, or WTAP), domains that can demethylate RNA (e.g., human alkylation repair homolog 5), domains that can affect splicing (e.g., RS-rich domain of SRSF1, Gly-rich domain of hnRNP A1, alanine-rich motif of RBM4, or proline-rich motif of DAZAP1), domains that can achieve affinity purification or immunoprecipitation (e.g., FLAG, HA, biotin, or HALO tag), and domains that can achieve proximity-based protein labeling and recognition (e.g., biotin ligase such as BirA or peroxidase such as APEX2 to biotinylate target DNA-interacting proteins).

[0253] In some embodiments, the fusion domain comprises an adenosine deaminase domain. In some embodiments, an RNA-guided nuclease having a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to an adenosine deaminase domain to direct A-to-I deaminase activity of an RNA transcript in mammalian cells. For example, an adenosine deaminase domain engineered based on ADAR2 for targeted A-to-I RNA editing is fused. In other embodiments, the adenosine deaminase domain is covalently linked or fused to an adaptor protein that is capable of binding an aptamer sequence inserted or appended to the guide RNA, thereby allowing the adenosine deaminase domain to non-covalently link to the RNA-guided nuclease complexed with the guide RNA.

[0254] In some embodiments, the fusion domain comprises a cytosine deaminase domain. In some embodiments, an RNA-guided nuclease having a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to a cytosine deaminase domain to direct C-to-U deaminase activity of an RNA transcript in mammalian cells. For example, a cytosine deaminase domain evolved from ADAR2 for targeted C-to-U RNA editing is used. In other embodiments, the cytosine deaminase domain is covalently linked or fused to an adaptor protein that is capable of binding an aptamer sequence inserted or appended to the guide RNA, thereby allowing the cytosine deaminase domain to non-covalently link to the RNA-guided nuclease complexed with the guide RNA.

[0255] In some embodiments, the fusion domain comprises a splicing factor domain. In some embodiments, an RNA-guided nuclease having a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to a splicing factor domain to direct alternative splicing of a target RNA in mammalian cells. Non-limiting examples of splicing factor domains include the RS-rich domain of SRSF1, the Gly-rich domain of hnRNPA1, the alanine-rich motif of RBM4, or the proline-rich motif of DAZAP1. In other embodiments, the splicing factor domain is covalently linked or fused to an adaptor protein that is capable of binding an aptamer sequence inserted or appended to the guide RNA, thereby allowing the splicing factor domain to non-covalently link to the RNA-guided nuclease complexed with the guide RNA.

[0256] In some embodiments, the fusion domain comprises a translation activation domain. In some embodiments, an RNA-guided nuclease with a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to a translation activation domain to activate or increase the expression of a target RNA. Non-limiting examples of translation activation domains include eIF4E and other translation initiation factors, the yeast poly(A)-binding protein, or domains of GLD2. In other embodiments, the translation activation domain is covalently linked or fused to an adaptor protein that can bind an aptamer sequence inserted or appended to the guide RNA, thereby allowing the translation activation domain to non-covalently associate with the RNA-guided nuclease complexed with the guide RNA.

[0257] In some embodiments, the fusion domain comprises a translation repression domain. In some embodiments, an RNA-guided nuclease with a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to a translation repression domain to repress or decrease the expression of a target RNA. Non-limiting examples of translation repression domains include Pumilio or FBF PUF proteins, deadenylases, CAF1, Argonaute proteins. In other embodiments, the translation repression domain is covalently linked or fused to an adaptor protein that can bind an aptamer sequence inserted or appended within the guide RNA, thereby allowing the translation repression domain to non-covalently associate with the RNA-guided nuclease complexed with the guide RNA.

[0258] In some embodiments, the fusion domain comprises an RNA methylation domain. In some embodiments, an RNA-guided nuclease with a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to an RNA methylation domain for methylation of a target RNA. Non-limiting examples of RNA methylation domains include m6A domains such as METTL14, METTL3, or WTAP. In other embodiments, the RNA methylation domain is covalently linked or fused to an adaptor protein that can bind an aptamer sequence inserted or appended to the guide RNA, thereby allowing the RNA methylation domain to non-covalently associate with the RNA-guided nuclease complexed with the guide RNA.

[0259] In some embodiments, the fusion domain comprises an RNA demethylase domain. In some embodiments, an RNA-guided nuclease having a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to an RNA demethylase domain for demethylation of a target RNA. Non-limiting examples of RNA demethylase domains include human alkylation repair homolog 5 or ALKBH5. In other embodiments, the RNA demethylase domain is covalently linked or fused to an adaptor protein that is capable of binding an aptamer sequence inserted or appended to the guide RNA, thereby allowing the RNA demethylase domain to non-covalently associate with the RNA-guided nuclease complexed with the guide RNA.

[0260] In some embodiments, the fusion domain comprises a ribonuclease domain. In some embodiments, an RNA-guided nuclease having a mutant HEPN domain or a catalytically inactivated RNA-guided nuclease is covalently linked or fused to a ribonuclease domain to cleave a target RNA. Non-limiting examples of ribonuclease domains include the PIN endonuclease domain, the NYN domain, the SMR domain from SOT1, or the RNase domain from staphylococcal nuclease.

[0261] In some embodiments, the fusion domain comprises an affinity tag or a reporter domain. In some embodiments, an RNA-guided nuclease is covalently linked or fused to a reporter domain, such as a fluorescent protein. Non-limiting examples of reporter domains include GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP. In some embodiments, an RNA-guided nuclease is covalently linked or fused to an affinity tag, such as a purification tag. Non-limiting examples of affinity tags include HA-tag, His-tag (e.g., 6-His), Myc-tag, E-tag, S-tag, calmodulin tag, FLAG-tag, GST-tag, MBP-tag, Halo tag, or biotin.

[0262] In some embodiments of the present disclosure, the fusion domain is located at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the RNA-guided nuclease. In some embodiments of the present disclosure, 0, 1, 2, 3, 4, or more of the fusion domains are fused to the N-terminus, C-terminus, or both the N-terminus and C-terminus of the RNA-guided nuclease, respectively.

[0263] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked to the fusion domain with or without a linker sequence; that is, the RNA-guided nuclease is directly covalently linked to the fusion domain (without a linker sequence), or can be covalently linked through a linker sequence. Typically, the linker sequence consists of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0264] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a subcellular localization signal.

[0265] In some embodiments of the present disclosure, the RNA-guided nuclease comprises a subcellular localization signal and a deaminase domain.

[0266] In some embodiments of the present disclosure, the subcellular localization signal is optionally selected from a nuclear localization signal and a nuclear export signal.

[0267] In some embodiments, the RNA-guided nuclease is fused to at least one subcellular localization signal. Exemplary subcellular localization signals include organelle localization signals such as a nuclear localization signal (NLS), a nuclear export signal (NES), or a mitochondrial localization signal.

[0268] In some embodiments, the RNA-guided nuclease is fused to at least 1 heterologous NLS. In some embodiments, the RNA-guided nuclease is fused to at least 2 NLSs. In some embodiments, the RNA-guided nuclease is fused to at least 3 NLSs. In some embodiments, the RNA-guided nuclease is fused to at least 1 N-terminal NLS and at least 1 C-terminal NLS. In some embodiments, the RNA-guided nuclease is fused to at least 2 C-terminal NLSs. In some embodiments, the RNA-guided nuclease is fused to at least 2 N-terminal NLSs.

[0269] In some embodiments, the NLSs are independently selected from SPKKKRKVEAS, GPKKKRKVAAA, PKKKRKV, KRPAATKKAGQAKKKK, PAAKRVKLD, RQRRNELKRSP, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, VSRKRPRP, PPKKARED, POPKKKPL, SALIKKKKKMAP, DRLRR, PKQKKRK, RKLKKKIKKL, REKKKFLKRR, KRKGDEVDGVDEVAKKKSKK, or RKCLQAGMNLEARKTKK.

[0270] In some embodiments, the RNA-guided nuclease is fused to a heterologous NES. In some embodiments, the RNA-guided nuclease is fused to at least two NESs. In some embodiments, the RNA-guided nuclease is fused to at least three NESs. In some embodiments, the RNA-guided nuclease is fused to at least one N-terminal NES and at least one C-terminal NES. In some embodiments, the RNA-guided nuclease is fused to at least two C-terminal NESs. In some embodiments, the RNA-guided nuclease is fused to at least two N-terminal NESs.

[0271] In some embodiments, the NES is independently selected from adenovirus type 5 E1B NES, HIV Rev NES, MAPK NES, or PTK2 NES.

[0272] In some embodiments, the RNA-guided nuclease is fused to an NLS and an NES, and there is a cleavable linker between the NLS and the NES. In some embodiments, the NES promotes the production of delivery particles (e.g., virus-like particles) containing the RNA-guided nuclease in the production cell line. In some embodiments, cleavage of the linker in the target cell can expose the NLS and promote nuclear localization of the RNA-guided nuclease in the target cell.

[0273] In some embodiments of the present disclosure, the RNA-guided nuclease is covalently linked to the fusion domain with or without a linker sequence; that is, the RNA-guided nuclease is directly covalently linked to the fusion domain (without a linker sequence), or can be covalently linked through a linker sequence. Typically, the linker sequence consists of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0274] Cas protein

[0275] In some embodiments of the present disclosure, the Cas protein is Cas9 protein, Cas12 protein, or Cas13 protein. In some embodiments, the Cas protein is Cas12a protein, Cas12b protein, Cas12c protein, Cas12d protein, Cas12e protein, Cas12f protein, Cas12g protein, Cas12h protein, Cas12i protein, Cas12j protein, Cas12k protein.

[0276] In some embodiments of the present disclosure, the Cas protein comprises any one or more of the following fusion domains: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag.

[0277] In some embodiments of the present disclosure, the Cas protein comprises a subcellular localization signal.

[0278] In some embodiments of the present disclosure, the Cas protein comprises a subcellular localization signal and a deaminase domain.

[0279] In some embodiments of the present disclosure, the subcellular localization signal is optionally selected from a nuclear localization signal and a nuclear export signal.

[0280] In some embodiments of the present disclosure, the Cas protein is covalently linked to the fusion domain with or without a linker sequence; that is, the Cas protein is directly covalently linked to the fusion domain (without a linker sequence), or can also be covalently linked through a linker sequence. Typically, the linker sequence consists of 1-100, 1-50, 1-30, 1-20, 1-10, or 1-5 amino acids.

[0281] Cas13 protein

[0282] In some embodiments of the present disclosure, the Cas13 protein is a Cas13a protein, a Cas13b protein, a Cas13c protein, or a Cas13d protein. For example: LwaCas13a, LsCas13a, LbuCas13a, dLbuCas13a (R472A / H477A / R1048A / H1053A), TccCas13a, LneCas13a (LneC2c2), LbmCas13a, LbnCas13a, PpCas13a, LbfCas13a, CgCas13a, Cg2Cas13a, PspCas13b, PspCas13b H133A / H1058A, PbuCas13b, PgiCas13b, BzCas13b, RanCas13b, PguCas13b, dPguCas13b (H151A / H1121A), Cas13bt1, Cas13bt3, CcaCas13b, MisCas13b, Hgm4Cas13b, Pba4Cas13b, Bba2Cas13b, CasRx, dCasRx (R239A / H244A / R858A / H863A), CasRx_N2V8 (A134V,A140V,A141V,A143V), RspCas13d, C13-2.

[0283] In some embodiments of the present disclosure, the Cas13 protein is a Cas13d protein.

[0284] In some embodiments of the present disclosure, the Cas13 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the CasRx or C13-2 protein.

[0285] In some embodiments of the present disclosure, the Cas13 protein is a CasRx protein. In some embodiments, the Cas13 protein is dCasRx in which both HEPN domains carry mutations (R239A, H244A in HEPN-1 and R858A, H863A in HEPN-2).

[0286] In some embodiments of the present disclosure, the amino acid sequence of the Cas13 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:64.

[0287] In some embodiments of the present disclosure, the Cas13 protein is the C13-2 protein. In some embodiments, the Cas13 protein is the dead C13-2 protein.

[0288] In some embodiments, the Cas13 protein contains 1, 2, 3, 4, 5, or 6 mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein shown in SEQ ID NO:64. In some embodiments, the amino acid residues at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and / or H790 of the reference protein shown in SEQ ID NO:64 of the Cas13 protein are mutated to A (alanine).

[0289] In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R210 and H215 of the reference protein shown in SEQ ID NO:64. In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R750 and H755 of the reference protein shown in SEQ ID NO:1. In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R785 and H790 of the reference protein shown in SEQ ID NO:64.

[0290] In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R210, H215, R750, and H755 of the reference protein shown in SEQ ID NO:64.

[0291] In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R750, H755, R785, and H790 of the reference protein shown in SEQ ID NO:64.

[0292] In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R210, H215, R785, and / or H790 of the reference protein shown in SEQ ID NO:64.

[0293] In some embodiments, the Cas13 protein contains mutations at positions corresponding to amino acid residues R210, H215, R750, H755, R785, and H790 of the reference protein shown in SEQ ID NO:64.

[0294] In some embodiments, the corresponding positions of R210, R750, or R785 are mutated to A. In some embodiments, the corresponding positions of H215, H755, or H790 are mutated to A. In some embodiments, the corresponding positions of R210, H215, R750, H755, R785, and H790 are all mutated to A.

[0295] In some embodiments, the Cas13 protein is obtained by introducing mutations in the RxxxxH motifs at positions 210-215, 750-755, and / or 785-790 of the sequence shown in SEQ ID NO:64.

[0296] In some embodiments, the Cas13 protein is obtained by introducing 1, 2, 3, 4, 5, or 6 mutations at positions R210, H215, R750, H755, R785, and / or H790 of the sequence shown in SEQ ID NO:64. In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R750, H755, R785, and / or H790 of the sequence shown in SEQ ID NO:64 to A (alanine).

[0297] In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R785, and H790 of the sequence shown in SEQ ID NO:64 to A. In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R750, and H755 of the sequence shown in SEQ ID NO:64 to A. In some embodiments, the Cas13 protein is obtained by mutating positions R750, H755, R785, and H790 of the sequence shown in SEQ ID NO:64 to A. In some embodiments, the Cas13 protein is obtained by mutating positions R210, H215, R750, H755, R785, and H790 of the sequence shown in SEQ ID NO:64 to A.

[0298] In some embodiments, compared to the reference protein shown in SEQ ID NO:64, the Cas13 protein comprises any one or more mutations at the corresponding positions of the following amino acid residues of the reference protein shown in SEQ ID NO:64: R11, N34, R35, R47, R58, R63, R64, N68, N87, N265, N274, R276, R290, R294, N299, N303, R308, R314, R320, R328, N332, R341, N346, R358, N372, N383, N390, N394, R47+R290, R47+R314, R290+R314, R47+R290+R314, R308+N68, N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N87+N68+N265, T7, A16, S260, A263, M266, N274, F288, M302, N303, L304, V305, I311, D313, H324, P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, S418.

[0299] In some embodiments, the Cas13 protein is obtained by introducing any one or more mutations at the following positions in the sequence shown in SEQ ID NO:64: R11, N34, R35, R47, R58, R63, R64, N68, N87, N265, N274, R276, R290, R294, N299, N303, R308, R314, R320, R328, N332, R341, N346, R358, N372, N383, N390, N394, R47+R290, R47+R314, R290+R314, R47+R290+R314, R308+N68, N394+N68, N87+N68, R308+N265, N394+N265, N87+N265, R308+N68+N265, N87+N68+N265, T7, A16, S260, A263, M266, N274, F288, M302, N303, L304, V305, I311, D313, H324, P326, H327, N332, N346, T353, T360, E365, A373, M380, S382, K395, Y396, D402, D411, S418.

[0300] In some embodiments, the C13-2 protein, a protein similar to the C13-2 protein, a mutant of the C13-2 protein, and functional fragments or fusion proteins thereof can form a complex with the direct repeat sequence containing the sequence shown in SEQ ID NO: 65 or 66 as described in this disclosure, and then specifically target the target RNA (such as APOE4 RNA) described in this disclosure under the guidance of the guide sequence.

[0301] Nucleotide sequence encoding a guide RNA or an RNA-guided nuclease

[0302] In some embodiments, the nucleotide sequence encoding the RNA-guided nuclease is a plasmid. In some embodiments, the nucleotide sequence encoding the RNA-guided nuclease is part of a viral vector genome, such as the DNA genome of an AAV vector flanked by ITRs. In some embodiments, the nucleotide sequence encoding the RNA-guided nuclease is an mRNA.

[0303] In some embodiments of this disclosure, the nucleotide sequence encoding a guide RNA or an RNA-guided nuclease is codon-optimized.

[0304] In some embodiments of this disclosure, the nucleotide sequence encoding the guide RNA is DNA. In some embodiments, the sequence of the DNA is codon-optimized.

[0305] In some embodiments of this disclosure, the nucleotide sequence encoding the RNA-guided nuclease is DNA or mRNA. In some embodiments, the sequence of the DNA is codon-optimized. In some embodiments, the sequence of the mRNA is codon-optimized.

[0306] In some embodiments of this disclosure, codon optimization is for expression in the desired cell type. In some embodiments, codon optimization is performed for expression in a eukaryotic cell environment. In some embodiments, codon optimization is performed for expression in a mammalian cell environment. In some embodiments, codon optimization is performed for expression in a human cell environment.

[0307] Typically, codon optimization refers to modifying a nucleic acid sequence to enhance its expression in a host cell of interest by replacing at least one codon of the original sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more frequently used in the genes of that host cell while maintaining the original amino acid sequence. Computational programs or algorithms for performing codon optimization are also available, and for example, the following tools or algorithms can be used for codon optimization: ExpOptimizer, Codon OptimWiz, NGTM Codon, Codon optimization, Synthetic GeneDesigner, and DNAWorks, etc.

[0308] vector

[0309] Vectors can contain any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, can be partially obtained from natural sources, and can contain natural, non-natural, or altered nucleotides. Suitable vectors include those designed for expression, such as plasmids and viruses.

[0310] In some embodiments, the recombinant vector contains regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate.

[0311] In some embodiments, the recombinant vector optionally includes gene vector elements (nucleic acids), such as selectable marker regions, lac operons, CMV promoters, CAG promoters, tac promoters, T7 RNA polymerase promoters, SP6 RNA polymerase promoters, SV40 promoters, IRES sequences, WPRE elements, ITR sequences, FLAG tag coding regions, c-myc tag coding regions, polyHis tag coding regions, HA tag coding regions, MBP tag coding regions, GST tag coding regions, polyA coding regions, SV40 polyadenylation signals, SV40 origins of replication, Col E1 origins of replication, lox P sites, or Cre recombinase coding regions.

[0312] regulatory sequence

[0313] In some embodiments of the present disclosure, the regulatory sequence comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof.

[0314] Promoter

[0315] In some embodiments of the present disclosure, the vector comprises a pol III promoter (e.g., U6 and H1 promoters), a pol II promoter (e.g., the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, or the EF1α promoter), or a pol III promoter and a pol II promoter.

[0316] In some embodiments of the present disclosure, the promoter is a constitutive promoter that is continuously active and not regulated by external signals or molecules. Suitable constitutive promoters include, but are not limited to, CMV, RSV, SV40, EF1α, CAG, and β-actin promoters. In some embodiments, the promoter is an inducible promoter that is regulated by external signals or molecules (e.g., transcription factors).

[0317] In some embodiments of the present disclosure, the promoter is a tissue-specific promoter, which can be used to drive tissue-specific expression of the Cas13 protein. Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and its variants, and SP c5-12 synthetic promoter. Suitable immune cell-specific promoters include, but are not limited to, B29 promoter (B cells), CD14 promoter (monocytes), CD43 promoter (leukocytes and platelets), CD68 (macrophages), and SV40 / CD43 promoter (leukocytes and platelets). Suitable blood cell-specific promoters include, but are not limited to, CD43 promoter (leukocytes and platelets), CD45 promoter (hematopoietic cells), INF-β (hematopoietic cells), WASP promoter (hematopoietic cells), SV40 / CD43 promoter (leukocytes and platelets), and SV40 / CD45 promoter (hematopoietic cells). Suitable pancreas-specific promoters include, but are not limited to, elastase-1 promoter. Suitable endothelial cell-specific promoters include, but are not limited to, Fit-1 promoter and ICAM-2 promoter. Suitable neuron tissue / cell-specific promoters include, but are not limited to, GFAP promoter (astrocytes), SYN1 promoter (neurons), and NSE / RU5' promoter (mature neurons). The neuron tissue / cell-specific promoter can be GFAP promoter, SYN1 promoter. Suitable kidney-specific promoters include, but are not limited to, NphsI promoter (podocytes). Suitable bone-specific promoters include, but are not limited to, OG-2 promoter (osteoblasts, odontoblasts). Suitable lung-specific promoters include, but are not limited to, SP-B promoter (lungs). Suitable liver-specific promoters include, but are not limited to, SV40 / Alb promoter. Suitable heart-specific promoters include, but are not limited to, α-MHC.

[0318] In some embodiments of the present disclosure, the promoter is the chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., the chicken β-actin promoter) contains an enhancer sequence, such as the cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter contains a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter contains a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art know that a short CMV enhancer can be used with a long CB promoter, and a long CMV enhancer can be used with a short CB promoter. In some embodiments of the present disclosure, the promoter is the CBh promoter.

[0319] Enhancer

[0320] In some embodiments of the present disclosure, the enhancer is selected from WPRE, CMV enhancer, SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit β-globin.

[0321] In some embodiments of the present disclosure, the enhancer is located upstream of the promoter element; however, it can also be located downstream or within the coding sequence regulated by the promoter and still maintain its function. Therefore, the enhancer or a part thereof can be present in the RNA sequence transcribed from the coding sequence.

[0322] In some embodiments of the present disclosure, the enhancer can be located within 100, 200, 300, 400, 500 or more base pairs upstream or downstream of the coding sequence regulated by the promoter.

[0323] In some embodiments of the present disclosure, the enhancer increases the expression of the coding sequence to a level higher than the increased expression provided by the promoter.

[0324] Adeno-associated virus vector (AAV vector)

[0325] In some embodiments of the present disclosure, the guide RNA or gene editing system described herein is packaged in an AAV vector, such as packaged into an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, AAV PHP.B2, AAV PHP.B3, AAV PHP.A, AAV PHP.eB, AAV PHP.eS, AAV2.7m8, AAV8.7m8, AAV ShH10, AAV rh10 or AAVrh74 capsid.

[0326] In some embodiments of the present disclosure, the guide RNA or gene editing system described herein is packaged into an AAV2, AAV5, AAV6, AAV8, AAV9 or AAV PHP.eB capsid.

[0327] In some embodiments of the present disclosure, the AAV vectors described herein are optionally selected from: AAV2 / 2, AAV2 / 3, AAV 2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV2 / 12, AAV2 / 13, AAV2 / PHP.B, AAV2 / PHP.B2, AAV2 / PHP.B3, AAV2 / PHP.A, AAV2 / PHP.eB, AAV2 / PHP.eS, AAV2 / 2.7m8, AAV2 / 8.7m8, AAV2 / ShH10, AAV2 / rh10 and AAV2 / rh74.

[0328] In some embodiments of the present disclosure, the AAV vectors described herein are optionally selected from: AAV2 / 2, AAV2 / 5, AAV 2 / 6, AAV2 / 8, AAV2 / 9, AAV2 / PHP.eB.

[0329] In some embodiments, the gene editing system described herein is packaged in an AAV vector that comprises an engineered capsid having tissue tropism, such as an engineered eye tissue tropism capsid.

[0330] Lipid nanoparticles

[0331] In some embodiments of the present disclosure, in addition to the RNA payload (mRNA encoding an RNA-guided nuclease and guide RNA), the lipid nanoparticle (LNP) further comprises four components: a cationic or ionizable lipid, cholesterol, a helper lipid, and a PEG-lipid. In some embodiments, the cationic or ionizable lipid includes cKK-E12, C12-200, ALC-0315, DLin-MC3-DMA, DLin-KC2-DMA, FTT5, Moderna SM-102, and Intellia LP01. In some embodiments, the PEG-lipid comprises PEG-2000-C-DMG, PEG-2000-DMG, or ALC-0159. In some embodiments, the helper lipid includes DSPC.

[0332] Lentiviral vector

[0333] In some embodiments of the present disclosure, the lentiviral vector is pseudotyped with a homologous or heterologous envelope protein such as VSV-G. In some embodiments, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

[0334] Aptamer / aptamer sequence

[0335] In some embodiments, the guide polynucleotide further comprises an aptamer sequence. In some embodiments, the aptamer sequence is inserted into a loop of the guide polynucleotide. In some embodiments, the aptamer sequence is appended to the end of the guide polynucleotide.

[0336] In some embodiments, the aptamer sequence includes an MS2 aptamer sequence, a PP7 aptamer sequence, or a Qβ aptamer sequence.

[0337] Adaptor protein

[0338] In some embodiments, the gene editing system further comprises a fusion protein comprising an adaptor protein and a fusion domain, or a nucleic acid encoding the fusion protein, wherein the adaptor protein is capable of binding to the aptamer sequence.

[0339] In some embodiments, the adaptor protein includes an MS2 bacteriophage coat protein (MCP), a PP7 bacteriophage coat protein (PCP), or a Qβ bacteriophage coat protein (QCP). In some embodiments, the fusion domain comprises a cytosine deaminase domain, an adenosine deaminase domain, a translation activation domain, a translation inhibition domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, or an affinity or reporter tag or domain.

[0340] RNP complex (ribonucleoprotein complex)

[0341] In some embodiments of the present disclosure, the RNP complex (ribonucleoprotein complex) can be delivered to eukaryotic cells, mammalian cells or human cells by microinjection or electroporation. In some embodiments, the ribonucleoprotein complex can be packaged in virus-like particles and delivered in vivo to a mammalian or human subject.

[0342] Virus-like particles

[0343] In some embodiments of the present disclosure, engineered virus-like particles (VLPs) are pseudotyped with homologous or heterologous envelope proteins such as VSV-G. In some embodiments, the RNA-guided nuclease is fused to the gag protein (such as MLV gag) via a cleavable linker, where cleavage of the linker in the target cell exposes the NLS located between the linker and the RNA-guided nuclease. In some embodiments, the fusion protein comprises (e.g., from 5' to 3') a gag protein (e.g., MLV gag), one or more NESs, a cleavable linker, one or more NLSs, and an RNA-guided nuclease. In some embodiments, the RNA-guided nuclease is fused to a first dimerization domain that is capable of dimerizing or heterodimerizing with a second dimerization domain fused to a membrane protein, where the presence of a ligand promotes the dimerization and enriches the RNA-guided nuclease or the fusion protein into the VLPs.

[0344] Eukaryotic cells

[0345] In some embodiments of the present disclosure, the eukaryotic cells are mammalian cells or human cells. In some embodiments, the eukaryotic cells are primary eukaryotic cells, stem cells, tumor / cancer cells, circulating tumor cells (CTCs), blood cells (e.g., T cells, B cells, NK cells, Tregs, etc.), hematopoietic stem cells, specialized immune cells (such as tumor-infiltrating lymphocytes or tumor-suppressive lymphocytes), stromal cells in the tumor microenvironment (such as cancer-associated fibroblasts, etc.). In some embodiments, the cells are brain or neuronal cells of the central or peripheral nervous system (e.g., neurons, astrocytes, microglia, retinal ganglion cells, rod / cone cells, etc.).

[0346] Disease or disorder

[0347] In some embodiments of the present disclosure, the disease or disorder refers to a disease or disorder caused by the expression of a target RNA (e.g., ApoE4 RNA).

[0348] In some embodiments of the present disclosure, the disease or disorder refers to a disease or disorder caused by the abnormally high expression of a target RNA.

[0349] In some embodiments, the pharmaceutical composition is delivered in vivo to a human subject. The pharmaceutical composition can be delivered by any effective route, and a therapeutically effective amount of the pharmaceutical composition can be delivered to a subject in need thereof. Exemplary routes of administration include, but are not limited to, topical administration (including, but not limited to, topical patches, ointments, gels, topical liquid preparations), intravenous infusion, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, subcutaneous injection, intradermal injection, intraventricular injection, intravascular injection, intracerebellar injection, intraocular injection, subretinal injection, intravitreal injection, intracameral injection, intratympanic injection, intranasal administration, and inhalation.

[0350] In some embodiments of the present disclosure, a therapeutically effective amount of the gene editing system or pharmaceutical composition described in the present disclosure is delivered to a subject in need thereof using a suitable delivery method, which can knockdown the expression of ApoE4 without reducing or hardly reducing the expression of ApoE3, thereby treating diseases such as Alzheimer's disease. For example, the gRNAs with guide sequences sg2, sg2-23, sg5-13, and sg5-16 disclosed in the present disclosure have a high knockdown efficiency for ApoE4 and little non-specific knockdown effect on ApoE3, and can be used to treat diseases such as Alzheimer's disease.

[0351] Based on the common general knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain the preferred embodiments of the present invention.

[0352] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or according to the product specifications.

[0353] Examples

[0354] Example 1. Test of Editing Efficiency

[0355] This example relates to a newly discovered highly active Cas13 protein, namely C13-2 protein (also known as CasRfg.4), whose amino acid sequence is shown in SEQ ID NO:64.

[0356] Amino acid sequence of C13-2 (SEQ ID NO:64):

[0357] MSKDKKTKAKRMGVKALLAHGEDKLTMTTFGKGNRSKIEFTEGYHGRALETPKHFGIRGFEVRRIDENVDLCGDLEEGKTIEALLVNPSEKVGEDYLKLKGTLEKRFFGREFPHDNIRIQLIYNILDIYKILGMNVADILYALGNMQDTELDIDMFGQSLNNEDNLKECLKRMRPYMGYFGDIFKISPKGENIADREHNKKVLRCISVLRNATAHDKQDEYPWFKSSDIYETKIFKADMWKIIKDQYREKIKKVNKDFLSKNAVNMAILFDLLNARDVEQKKQITDEFYRFTIRKDGKNLGMNLVKIREIIIDRYASGLRDKKHDPHRQKINVIADFLIFRALSQNQGIIDKTVSSLRLTKDEEEKDHVYQNAAELVWGMVSNCLTPYFNDPKNKYILKYKDAKTPGDFEDWITSKISEDDGEPFVKVLSFLCNFLEGKEINELLTAYIHKFECIQDFLNVISSLGENVQFQPRFALFNNASFAQNVAVQLRILASIGKMKPDLTEAKRPLYKAAIRMLCPPEKWEKYTSDEWLEKNMLLNSEDRKNDKKKKQVNPFRNFIAGNVIESRRFMYLVRYSKPKAVRAIMQNRSIVNYVLHRLPSEQVHRYASVFPENFADLEQEIDFLTKKLFEFSFEELLHEKDVILNNSRSHKPSLEIERLKAITGLYLSVAYIAIKNIVKANARYYIAFAVFERDKELVKAKDARIQTKIPETDFPDYFCLTQYYLDRDEEKKFPGDPRDKEAFFEHLRKTKRHFSKQWREWLNEKIADAKSSQATGLLLREARNDVEHLNVLRAIPDYIQDFRHGEKGETAMNSYFELYHYLMQRLMLKNTELDLSHWSGWIMRSGRPDRDLIQIAFVSLAYNLPRYRNLTKEHHFDDTVLQKIREKESLD

[0358] The natural direct repeat corresponding to C13-2 is:

[0359] 5’-GGAAGATAACTCTACAAACCTGTAGGGTTCTGAGAC-3’ (SEQ ID NO:65).

[0360] The C13-2-BsaI plasmid (the sequence is shown in SEQ ID NO:57) was prepared by an experimental outsourcing service company. The plasmid contains the coding sequence of the codon-optimized C13-2 protein (driven by CMV for expression) and the coding sequence of the direct repeat sequence (driven by U6 for expression).

[0361] The following method was used to construct each C13-2-gRNA plasmid:

[0362] First, gRNA was designed for the site where ApoE3 and ApoE4 differ, namely the 334T→C mutation site. After annealing the forward and reverse primers corresponding to the gRNA guide sequence, it was ligated to the digested product of the C13-2-BsaⅠ plasmid by T4 ligase to obtain the plasmid.

[0363] Specifically, the sense strand and antisense strand of the DNA sequence were synthesized by conventional methods. Corresponding to the C13-2-BsaⅠ plasmid, the sense strand was added with agac at the 5'-end of the guide sequence, and the antisense strand was added with aaaa at the 5'-end of the reverse complementary sequence of the guide sequence. The above-mentioned sense strand and antisense strand of the DNA sequence were mixed and annealed to form a double-stranded DNA with sticky ends.

[0364] The C13-2-BsaⅠ plasmid was digested with BsaⅠ to be linearized and reacted at 37°C for 2 hours. The digested product was subjected to 1% agarose gel electrophoresis, and the digested product was recovered by cutting the gel.

[0365] The digested plasmid, annealed primers and DNA Ligation Kit Ver.2.1 were mixed and incubated at 16°C in a PCR instrument for 1 hour to complete the ligation of the annealed product and the linearized backbone. After the reaction product was transformed into Escherichia coli Stbl3 competent cells, it was cultured in a medium containing the corresponding antibiotic, and colony PCR reaction was carried out. Positive clones were screened out and plasmids were extracted, and verified by sanger sequencing to obtain each C13-2-gRNA plasmid, which can express C13-2 and gRNA.

[0366] Table 1. gRNA guide sequence

[0367] Number Guide sequence (5’-3’) SEQ ID NO sg1 caccaggcggccgcGcacgtcctccatg 1 sg2 ccaggcggccgcGcacgtcctcca 2 sg3 ggccgcGcacgtcctccatgtccg 3 sg4 tgcaccaggcggccgcGcacgtcc 4 sg5 actgcaccaggcggccgcGcacgt 5 sg6 gtactgcaccaggcggccgcGcac 6 sg7 ccgcGcacgtcctccatgtccg 7 sg8 cgcGcacgtcctccatgtccgcgc 8

[0368] Construct a HEK293T stable cell line overexpressing ApoE4

[0369] Construct a vector Lv-ApoE4-T2a-GFP (SEQ ID NO: 58) for overexpressing the ApoE4 gene and the EGFP gene. Among them, ApoE4 and EGFP are separated by the 2A peptide. Package the Lv-ApoE4-T2a-GFP plasmid into lentivirus and transduce 293T cells to form a cell line stably overexpressing the ApoE4 gene.

[0370] Detection of editing efficiency

[0371] Strictly follow the usage steps of Lipofectamine 2000 (Thermo Fisher) reagent from Life Tech company to transfect the C13-2-gRNA plasmid into the HEK293T stable transfected cell line overexpressing ApoE4 (the negative control group transfects the C13-2-BsaI plasmid). One day before transfection, seed the HEK293T stable transfected cells overexpressing ApoE4 in good condition at 1×10 5 / well into a 24-well plate. On the day of transfection, add 500 ng of the corresponding volume of the recombinant gRNA plasmid to 50 μl of OPTI-MEM (Thermo) respectively, and add 1 μl of LipofacTamine 2000 to 50 μl of OPTI-MEM medium. After mixing, let it stand at room temperature for 5 mins, then mix the diluted plasmid DNA and Lipofactamine2000, and let it stand at room temperature for 15 mins. Then add 100 μl of the plasmid to each well of the 24-well plate cells, and gently shake the culture plate to mix. Incubate at 37 °C for 72 h.

[0372] Use the Aike Rui universal RNA extraction kit to extract the total RNA of 293T cells, and then use the Evo M-MLV reverse transcription kit to perform gDNA digestion and reverse transcription reaction to obtain the corresponding cDNA. The Q-PCR reaction is detected using the SYSB Green ProTaq HS premixed qPCR kit, with GAPDH as the internal reference, and the ApoE4 RNA level after cleavage of ApoE4 RNA in each group is obtained. Each pair of primers for Q-PCR is parallelly replicated 3 times for each sample. The Q-PCR primers are shown in Table 2 below.

[0373] The relative expression level of the knocked-down ApoE4 RNA is calculated by the 2-ΔΔCT calculation method, and the editing efficiency value (i.e., the knockdown efficiency / knockdown efficiency) is obtained by subtracting this relative value from 1. The average value is taken for each group. The results are shown in Table 3 below.

[0374] Table 2. Primers used for Q-PCR

[0375] Primer name Sequence (5’→3’) Sequence number ApoE4-F CGGACATGGAGGACGTGC SEQ ID NO:59 ApoE4-R CTGGTACACTGCCAGGCG SEQ ID NO:60 GAPDH-F CCATGGGGAAGGTGAAGGTC SEQ ID NO:61 GAPDH-R GAAGGGGTCATTGATGGCAAC SEQ ID NO:62

[0376] Table 3. Knockdown efficiency of ApoE4 RNA

[0377]

[0378]

[0379] As can be seen from the data in the table, the editing activities of the gRNAs with guide sequences sg2 and sg5 are significantly higher.

[0380] Example 2. Mismatch affects non-specific targeting of ApoE3

[0381] Select sg2 and sg5, and introduce a single mutation at each base of the gRNA guide sequence to obtain a new guide sequence, as shown in Table 4.

[0382] Construct each C13-2-gRNA plasmid expressing the gRNA containing the new guide sequence by a method similar to that in Example 1:

[0383] It is obtained by annealing the forward and reverse primers corresponding to the gRNA guide sequence and then ligating them with the digested product of the C13-2-BsaⅠ plasmid by T4 ligase.

[0384] Specifically, the sense strand and antisense strand of the DNA sequence are synthesized by conventional methods. Corresponding to the C13-2-BsaⅠ plasmid, the sense strand has agac added at the 5'-end of the guide sequence, and the antisense strand has aaaa added at the 5'-end of the reverse complementary sequence of the guide sequence. The sense strand and antisense strand of the above DNA sequence are mixed and annealed to form a double-stranded DNA with sticky ends.

[0385] The C13-2-BsaⅠ plasmid is digested with BsaⅠ to be linearized and reacted at 37°C for 2 hours. The digested product is subjected to 1% agarose gel electrophoresis, and the digested product is recovered by cutting the gel.

[0386] Mix the digested plasmid, annealed primers and DNA Ligation Kit Ver.2.1, incubate at 16°C in a PCR instrument for 1 hour to complete the ligation of the annealed product and the linearized backbone. After the reaction product is transformed into Escherichia coli Stbl3 competent cells, it is cultured in a medium containing the corresponding antibiotic, and colony PCR reaction is carried out to screen out positive clones and extract plasmids, and sanger sequencing verification is carried out to obtain each C13-2-gRNA plasmid.

[0387] Table 4. gRNA-related sequences

[0388]

[0389]

[0390]

[0391] Construct a vector Lv-ApoE3-T2a-GFP (SEQ ID NO: 63) that overexpresses the ApoE3 gene and the EGFP gene. Among them, ApoE3 and EGFP are separated by a 2A peptide. Package the Lv-ApoE3-T2a-GFP plasmid into lentivirus and transduce 293T cells to form a cell line that stably overexpresses the ApoE3 gene.

[0392] Detection of editing efficiency

[0393] Strictly follow the steps of using Lipofectamine 2000 (Thermo Fisher) reagent from Life Tech company to transfect the C13-2-gRNA plasmid into HEK293T cells overexpressing ApoE4 and HEK293T cells overexpressing ApoE3 respectively (the negative control group transfects the C13-2-BsaI plasmid). One day before transfection, seed the well-conditioned HEK293T cells overexpressing ApoE4 and HEK293T cells overexpressing ApoE3 into 24-well plates at 1×10 5 / well. On the day of transfection, add 500 ng of the corresponding volume of recombinant gRNA plasmid to 50 μl of OPTI-MEM (Thermo) respectively, add 1 μl of LipofacTamine 2000 to 50 μl of OPTI-MEM medium respectively, mix well and let stand at room temperature for 5 mins, then mix the diluted plasmid DNA with Lipofactamine 2000, let stand at room temperature for 15 mins, and then add 100 μl of the plasmid to each well of the 24-well plate cells, gently shake the culture plate to mix well. Incubate at 37°C for 72 h.

[0394] After extracting the total RNA of 293T cells using the Aikerui universal RNA extraction kit, use the Evo M-MLV reverse transcription kit to perform gDNA digestion and reverse transcription reaction to obtain the corresponding cDNA. The Q-PCR reaction is detected using the SYSB Green ProTaq HS premixed qPCR kit, with GAPDH as the internal reference, to obtain the levels of ApoE4 and ApoE3 RNA after cleavage of ApoE4 and ApoE3 RNA in each group. Each pair of primers for each sample in Q-PCR is done in 3 replicates in parallel. The Q-PCR primers are shown in Table 5 below.

[0395] Calculate the relative expression levels of ApoE4 or ApoE3 RNA after knockdown by the 2-ΔΔCT calculation method, and subtract this relative value from 1 to obtain the editing efficiency value (i.e., knockdown efficiency). Take the average value for each group, as shown in Table 6 below.

[0396] Table 5. Primers used for Q-PCR of ApoE 293T cells

[0397]

[0398]

[0399] Table 6. Knockdown efficiency of ApoE3 / ApoE4 RNA

[0400]

[0401]

[0402] As can be seen from the data in the table, the gRNAs with guide sequences sg2, sg2-23, sg5-13, and sg5-16 have a small non-specific knockdown effect on ApoE3 while maintaining a high knockdown efficiency for ApoE4.

[0403] Although the specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are only examples, and various changes or modifications can be made to these embodiments without departing from the principle and essence of the present disclosure.

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

Claims

1. Use of a gene editing system in the preparation of a drug for diagnosing, treating or preventing Alzheimer's disease in a subject, characterized in that: After the gene editing system contacts a cell containing ApoE4 RNA, the level of the ApoE4 RNA or ApoE4 protein in the cell can be reduced by at least 5%. After the gene editing system contacts a cell containing ApoE3 RNA, the level of the ApoE3 RNA or ApoE3 protein in the cell is not reduced or is reduced by up to 90%; Optionally, the subject carries at least one ApoE4 allele; Optionally, the subject carries ApoE4 and ApoE3 alleles.

2. The use according to claim 1, wherein the gene editing system comprises: a guide RNA comprising a guide sequence that hybridizes to ApoE4 RNA, or a polynucleotide sequence encoding the guide RNA, and RNA-guided nuclease or a polynucleotide sequence encoding the same; The guide RNA is capable of forming a complex with the nuclease and guiding the complex to bind to the sequence-specific ApoE4 RNA; Optionally, the ApoE4 RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

3. The use according to claim 2, wherein the guide RNA guides the complex to bind to and cleave the ApoE4 RNA.

4. The use according to claim 2, wherein the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in SEQ ID NO: 55; Further, the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in SEQ ID NO:56; Still further, the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 1-54.

5. The use according to claim 2, wherein the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 55, with only one nucleotide mismatch; Further, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 56, and has only 1 nucleotide mismatch; Further, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8, with only one nucleotide mismatch; Furthermore, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 2 and 5, with only one nucleotide mismatch.

6. The use according to claim 5, wherein the guide sequence of the guide RNA has no mismatch at the base corresponding to the ApoE4 genotype mutation site c.334T>C, Optionally, the mismatch is not located at base C at position 32 of SEQ ID NO:55; Further, the mismatch is not located at base 21 of SEQ ID NO:56; Further, the mismatch is not located at the 13th base of SEQ ID NO:2 and / or the mismatch is not located at the 19th base of SEQ ID NO:5; Furthermore, the guide sequence of the guide RNA is optionally selected from the sequence shown in any one of SEQ ID NOs: 1-54.

7. The use according to claim 2, wherein the guide RNA comprises a guide sequence and a backbone sequence, and the backbone sequence interacts with the RNA-guided nuclease.

8. The use according to claim 2, wherein the RNA-guided nuclease is a Cas13 protein or a fragment thereof, for example including but not limited to a nucleic acid binding domain fragment. Optionally, the Cas13 protein is Cas13a protein, Cas13b protein, Cas13c protein or Cas13d protein; Optionally, the Cas13 protein has at least 50% sequence identity with CasRx or the sequence shown in SEQ ID NO:

64.

9. The use according to claim 2, wherein the guide RNA is capable of forming a complex with an RNA-guided nuclease and guiding the complex to bind to and cleave ApoE4 RNA; or The guide RNA is capable of forming a complex with the RNA-guided nuclease and directing the complex to bind and inhibit translation of ApoE4 RNA.

10. The use according to claim 2, wherein the RNA-guided nuclease comprises a protein domain; Optionally, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, Deaminase domain, translation activation domain, translation repression domain, RNA methylation domain, RNA demethylation domain, nuclease domain, splicing factor domain, reporter tag and affinity tag; Further optionally, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence; Still further optionally, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence, and optionally a deaminase domain, a translation activation domain or a translation repression domain.

11. The use according to claim 2, wherein the polynucleotide sequence encoding the RNA-guided nuclease is connected to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is connected to a regulatory sequence 2 that regulates its expression.

12. A gene editing system, characterized in that: After the gene editing system contacts a cell containing ApoE4 RNA, the level of the ApoE4 RNA or ApoE4 protein in the cell can be reduced by at least 5%. After the gene editing system contacts a cell containing ApoE3 RNA, the level of the ApoE3 RNA or ApoE3 protein in the cell is not reduced or is reduced by up to 90%; Optionally, the subject carries at least one ApoE4 allele; Optionally, the subject carries ApoE4 and ApoE3 alleles.

13. The gene editing system of claim 1, comprising: a guide RNA comprising a guide sequence that hybridizes to ApoE4 RNA, or a polynucleotide sequence encoding the guide RNA, and RNA-guided nuclease or a polynucleotide sequence encoding the same; The guide RNA is capable of forming a complex with the nuclease and guiding the complex to bind to the sequence-specific ApoE4 RNA; Optionally, the ApoE4 RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA.

14. The gene editing system of claim 13, wherein the guide RNA guides the complex to bind to and cleave the ApoE4 RNA.

15. The gene editing system of claim 13, wherein the guide sequence of the guide RNA is the same as SEQ ID NO: O:55 The sequences have at least 80% sequence identity; Further, the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in SEQ ID NO:56; Still further, the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 1-54.

16. The gene editing system of claim 13, wherein the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 55, with only one nucleotide mismatch; Further, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO:56, and has only one nucleotide mismatch; Further, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8, with only one nucleotide mismatch; Furthermore, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 2 and 5, with only one nucleotide mismatch.

17. The gene editing system of claim 13, wherein the guide sequence of the guide RNA has no mismatch at the base corresponding to the ApoE4 genotype mutation site c.334T>C, Optionally, the mismatch is not located at base C at position 32 of SEQ ID NO:55; Further, the mismatch is not located at base 21 of SEQ ID NO:56; Still further, the mismatch is not located at the 13th base of SEQ ID NO:2 and / or the mismatch is not located at the 19th base of SEQ ID NO:

5.

18. The gene editing system of claim 13, wherein the guide sequence of the guide RNA is selected from any one of SEQ ID NOs: 1-54.

19. The gene editing system of claim 13, wherein the guide RNA comprises a guide sequence and a backbone sequence, wherein the backbone sequence interacts with the RNA-guided nuclease; Optionally, the backbone sequence is a direct repeat sequence; further optionally, the direct repeat sequence comprises a sequence having at least 50% sequence identity with the sequence shown in SEQ ID NO: 65 or 66.

20. The gene editing system according to claim 13, wherein the RNA-guided nuclease is Cas13 protein or Its fragments, Examples include, but are not limited to, nucleic acid binding domain fragments; Optionally, the Cas13 protein is Cas13a protein, Cas13b protein, Cas13c protein or Cas13d protein; Optionally, the Cas13 protein has at least 50% sequence identity with CasRx or the sequence shown in SEQ ID NO:

64.

21. The gene editing system of claim 13, wherein the guide RNA is capable of forming a complex with the RNA-guided nuclease and directing the complex to bind to and cleave ApoE4 RNA; or The guide RNA is capable of forming a complex with the RNA-guided nuclease and directing the complex to bind and inhibit translation of ApoE4 RNA.

22. The gene editing system of claim 13, wherein the RNA-guided nuclease comprises a protein domain; Optionally, the RNA-guided nuclease comprises any one or more of the following: a subcellular localization signal, a deaminase domain, a translation activation domain, a translation repression domain, an RNA methylation domain, an RNA demethylation domain, a nuclease domain, a splicing factor domain, a reporter tag, and an affinity tag; Further optionally, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence; Still further optionally, the RNA-guided nuclease comprises a nuclear localization signal and / or a nuclear export signal sequence, and optionally a deaminase domain, a translation activation domain or a translation repression domain.

23. The gene editing system of claim 13, wherein the polynucleotide sequence encoding the RNA-guided nuclease is connected to a regulatory sequence 1 that regulates its expression, and the polynucleotide sequence encoding the guide RNA is connected to a regulatory sequence 2 that regulates its expression; Optionally, the regulatory sequence is a promoter sequence and / or an enhancer sequence; Further optionally, the regulatory sequence is selected from CMV promoter, CMV enhancer, CBh promoter, U6 promoter, brain-specific promoter and neural-specific promoter.

24. The gene editing system of claim 13, wherein the gene editing system can be introduced into a cell or a cell-free system in any of the following ways: (i) as mRNA encoding an RNA-guided nuclease and a guide RNA, (ii) as part of a single vector or plasmid, or divided into multiple vectors or plasmids, (iii) as separate RNA-guided nucleases and guide RNAs, or (iv) as an RNP complex of an RNA-guided nuclease and a guide RNA.

25. A gene editing system guide RNA, characterized in that It contains a guide sequence that hybridizes to ApoE4 RNA.

26. The guide RNA of claim 25, wherein the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in SEQ ID NO: 55; Further, the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in SEQ ID NO:56; Still further, the guide sequence of the guide RNA has at least 80% sequence identity with the sequence shown in any one of SEQ ID NOs: 1-54.

27. The guide RNA of claim 25, wherein the guide sequence of the guide RNA is reverse complementary to SEQ ID NO: 55, with only one nucleotide mismatch; Further, the guide sequence of the guide RNA is reverse complementary to SEQ ID NO:56, and has only one nucleotide mismatch; Further, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 1-8, with only one nucleotide mismatch; Furthermore, the guide sequence of the guide RNA is reverse complementary to the sequence shown in any one of SEQ ID NOs: 2 and 5, with only one nucleotide mismatch.

28. The guide RNA of claim 25, wherein the guide sequence of the guide RNA has no mismatch at the base corresponding to the ApoE4 genotype mutation site c.334T>C, Optionally, the mismatch is not located at base C at position 32 of SEQ ID NO:55; Further, the mismatch is not located at base 21 of SEQ ID NO:56; Further, the mismatch is not located at the 13th base of SEQ ID NO:2 and / or the mismatch is not located at the 19th base of SEQ ID NO:5; Furthermore, the guide sequence of the guide RNA is optionally selected from the sequence shown in any one of SEQ ID NOs: 1-54.

29. The guide RNA of claim 25, comprising a guide sequence and a backbone sequence, wherein the backbone sequence interacts with the RNA-guided nuclease; Optionally, the backbone sequence is a direct repeat sequence; further optionally, the direct repeat sequence comprises a sequence having at least 50% sequence identity with the sequence shown in SEQ ID NO: 65 or 66.

30. The guide RNA of claim 25, wherein the nuclease guided by the RNA is a Cas13 protein or a fragment thereof, for example including but not limited to a nucleic acid binding domain fragment. Optionally, the Cas13 protein is Cas13a protein, Cas13b protein, Cas13c protein or Cas13d protein; Optionally, the Cas13 protein has at least 50% sequence identity with CasRx or the sequence shown in SEQ ID NO:

64.

31. The guide RNA of claim 25, wherein the guide RNA is capable of forming a complex with an RNA-guided nuclease and directing the complex to bind to and cleave ApoE4 RNA; or The guide RNA is capable of forming a complex with the RNA-guided nuclease and directing the complex to bind and inhibit translation of ApoE4 RNA.

32. An isolated nucleic acid, characterized in that It encodes the guide RNA according to any one of claims 25-31.

33. A vector comprising a polynucleotide sequence encoding the guide RNA according to any one of claims 25 to 31, and a regulatory sequence for regulating the expression of the guide RNA.

34. A vector system, wherein: The vector system comprises a polynucleotide sequence encoding the guide RNA according to any one of claims 25 to 31 and a second regulatory sequence regulating the expression of the guide RNA; and a polynucleotide sequence encoding an RNA-guided nuclease and a first regulatory sequence regulating the expression of the RNA-guided nuclease; Optionally, the vector system comprises one or more vectors; Optionally, the regulatory sequence is a promoter sequence and / or an enhancer sequence.

35. An adeno-associated virus vector, characterized in that The adeno-associated viral vector comprises DNA encoding an RNA-guided nuclease and a guide RNA according to any one of claims 25-31.

36. A lipid nanoparticle, characterized in that The lipid nanoparticle comprises the guide RNA according to any one of claims 25-31 and an mRNA encoding the nuclease guided by the RNA.

37. A lentiviral vector, characterized in that The lentiviral vector comprises the guide RNA according to any one of claims 25-31 and an mRNA encoding an RNA-guided nuclease.

38. The lentiviral vector of claim 37, wherein the lentiviral vector is pseudotyped with an envelope protein; optionally, the mRNA encoding the RNA-guided nuclease is linked to an aptamer sequence.

39. A ribonucleoprotein complex, characterized in that The ribonucleoprotein complex is formed by the guide RNA according to any one of claims 25-31 and the RNA-guided nuclease.

40. A virus-like particle, characterized in that The virus-like particle comprises a ribonucleoprotein complex formed by the guide RNA according to any one of claims 25 to 31 and an RNA-guided nuclease.

41. The virus-like particle of claim 40, wherein the RNA-guided nuclease is fused to the gag protein.

42. A eukaryotic cell, characterized in that The eukaryotic cell comprises the gene editing system according to any one of claims 12-24, the guide RNA according to any one of claims 25-31, the nucleic acid according to claim 32, the vector according to claim 33, or the vector system according to any one of claims 34; Optionally, the eukaryotic cell is a mammalian cell.

43. A pharmaceutical composition, characterized in that It comprises the gene editing system according to any one of claims 12-24, the guide RNA according to any one of claims 25-31, the nucleic acid according to claim 32, the vector according to claim 33 or the vector system according to any one of claims 34.

44. The pharmaceutical composition of claim 43, comprising a pharmaceutically acceptable excipient.

45. The gene editing system according to any one of claims 12-24, the guide RNA according to any one of claims 25-31, the nucleic acid according to claim 32, the vector according to claim 33, the vector system according to any one of claims 34, the adeno-associated virus vector according to claim 35, the lipid nanoparticle according to claim 36, the lentiviral vector according to claim 37 or 38, the ribonucleoprotein complex according to claim 39, the virus-like particle according to claim 40 or 41, the eukaryotic cell according to claim 42, or the pharmaceutical composition according to claim 43 or 44 in any of the following or in the preparation of an agent for implementing any of the following schemes: Cleave or nick one or more ApoE4 RNA molecules, activate or upregulate one or more ApoE4 RNA, activate or inhibit translation of one or more ApoE4 RNA molecules, inactivate one or more ApoE4 RNA molecules, visualize, label or detect one or more ApoE4 RNA molecules, bind one or more ApoE4 RNA molecules, transport one or more ApoE4 RNA molecules, and mask one or more ApoE4 RNA molecules.

46. ​​The gene editing system according to any one of claims 12-24, the guide RNA according to any one of claims 25-31, the nucleic acid according to claim 32, the vector according to claim 33, the vector system according to any one of claims 34, the adeno-associated virus vector according to claim 35, the lipid nanoparticle according to claim 36, the lentiviral vector according to claim 37 or 38, the ribonucleoprotein complex according to claim 39, the virus-like particle according to claim 40 or 41, the eukaryotic cell according to claim 42, or the pharmaceutical composition according to claim 43 or 44, in any of the following or in the preparation of an agent for implementing any of the following schemes: Cleaves ApoE4 RNA molecules, inhibits translation of ApoE4 RNA molecules, and binds to ApoE4 RNA molecules.

47. The use according to claim 45 or 46, wherein the target RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA; Optionally, the ApoE4 RNA is human ApoE4 RNA.

48. A method for diagnosing, treating or preventing a disease or condition, characterized in that: Administering an effective amount of the gene editing system according to any one of claims 12-24, the guide RNA according to any one of claims 25-31, the nucleic acid according to claim 32, the vector according to claim 33, the vector system according to any one of claims 34, the adeno-associated virus vector according to claim 35, the lipid nanoparticle according to claim 36, the lentiviral vector according to claim 37 or 38, the ribonucleoprotein complex according to claim 39, the virus-like particle according to claim 40 or 41, the eukaryotic cell according to claim 42, or the pharmaceutical composition according to claim 43 or 44 to a sample of a subject in need or to a subject in need; Optionally, the disease or condition is a disease or condition associated with ApoE4 RNA; Optionally, the disease or condition is a disease or condition caused by the expression of ApoE4 RNA; Optionally, the ApoE4 RNA is mammalian ApoE4 pre-mRNA and / or ApoE4 mature mRNA; Further optionally, the ApoE4 RNA is human ApoE4 RNA; Optionally, the disease or condition comprises: Alzheimer's disease.

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