RNA frameworks for gene editing and gene editing methods

Abstract

The present invention provides an RNA framework for gene editing and a gene editing method. The RNA framework includes a target site upstream sequence, an inserted sequence, and a target site downstream sequence in the 5'→3' direction. This gene editing method is based on the inherent mechanism of eukaryotes themselves, and transfers RNP or RNA (which can be produced by in vitro preparation) and related proteins as vectors into the cytoplasm and cell nucleus to realize gene editing of sequences or sites on the genome of a target system, such as sequence insertion, deletion, replacement, and site replacement, while at the same time combining relatively high targeting accuracy. The present invention is more suitable for clinical application than other conventional technologies because it does not introduce exogenous systems or substances such as proteins derived from prokaryotes, and does not cause double-strand breaks.

Description

[Technical field]

[0001] The present invention belongs to the field of biotechnology and relates to gene editing technology, specifically to an RNA framework for gene editing and a gene editing method. [Background technology]

[0002] Currently, gene editing technologies in the biological field mainly include TALEN, ZFN, Targetron, CRISPR / Cas9 technology, etc. Although these technologies have been relatively mature since their use, they still have obvious shortcomings. Among them, ZFN technology can only identify sequences of 9 bp length, making its targeting accuracy relatively poor; at the same time, this technology is relatively complicated, has a high detargeting rate, and has high cytotoxicity, which also makes it impossible to apply practically. On the other hand, TALEN technology is simpler than ZFN technology and has a long identification sequence, but this technology is still complicated, which hinders further application in various fields. CRISPR / Cas 9 technology is currently the mainstream gene editing technology, which is easy to operate, but has non-negligible off-targets, and the risk of DNA double-strand breaks hinders further clinical application. On the other hand, Targetron technology uses type II introns to introduce exogenous sequences into specific sites of the genome, but this technology introduces type II introns into the genome to generate a "scar", which is only excellent in the field of bacterial gene editing, but is difficult to apply to other higher organisms. All three of these technologies have various problems that hinder their application, such as off-target, technical complexity, unknown risks due to double-strand breaks, etc. In addition, these three technologies inevitably introduce genetic material or proteins that do not belong to the receptor system, which brings about unpredictable effects and seriously hinders the clinical application of the above technologies. Summary of the Invention [Problem to be solved by the invention]

[0003] In order to solve the above problems, the object of the present invention is to provide an RNA framework for gene editing that enables DNA insertion, deletion, sequence replacement, and site replacement in any region of the genome.

[0004] Another object of the present invention is to provide an RNP.

[0005] It is yet another object of the present invention to provide DNA sequences.

[0006] It is a further object of the present invention to provide a DNA vector.

[0007] It is a further object of the present invention to provide a method for gene editing.

[0008] A further object of the present invention is to provide a use of the above-mentioned RNA framework for gene editing. [Means for solving the problem]

[0009] To achieve the above objective, the present invention provides an RNA framework for gene editing, which includes, in the 5'→3' direction, a target site upstream sequence, a sequence to be inserted, and a target site downstream sequence. The upstream sequence of the target site on the RNA framework or the complementary sequence of the upstream sequence of the target site hybridizes with the upstream sequence of the target site or the complementary sequence of the upstream sequence of the target site of a target site in a eukaryotic genome or a prokaryotic genome, and the downstream sequence of the target site or the complementary sequence of the downstream sequence of the target site hybridizes with the downstream sequence of the target site or the complementary sequence of the downstream sequence of the target site of a target site in a eukaryotic genome or a prokaryotic genome, the upstream sequence of the target site and the downstream sequence of the target site are directly linked in the corresponding sequence in the genome, and the region between the upstream sequence of the target site and the downstream sequence of the target site among the sequences on the genome is the target site.

[0010] As described above, the RNA framework for gene editing further includes: downstream of the target site downstream sequence, one or more ORF2p functional start portions are directly or indirectly linked, or the target site downstream sequence is replaced or partially replaced by one or more ORF2p functional start portions, and the multiple ORF2p functional start portions are directly or indirectly linked to each other.

[0011] As described above, one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are further inserted into the ORF2p functional start portion, and when one pan-ORF1p coding sequence or one pan-ORF2p coding sequence is inserted into the ORF2p functional start portion, the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or pan-ORF2p coding sequence, and the ORF2p functional start portion includes: a) a plurality of pan-ORF1p coding sequences; or b) multiple pan-ORF2p coding sequences are inserted, or c) if the total number of pan-ORF1p coding sequences and pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequence and the pan-ORF2p coding sequence are directly or indirectly linked, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other, and the ORF2p functional initiation portion is directly or indirectly linked to the pan-ORF1p coding sequence or the pan-ORF2p coding sequence.

[0012] As described above, the gene editing RNA framework further includes: one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences linked directly or indirectly upstream of the upstream sequence of the target site, and / or within the upstream sequence of the target site, and / or within the downstream sequence of the target site, and / or downstream of the downstream sequence of the target site.

[0013] As described above, when the one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are located upstream of the target site upstream sequence, within the target site upstream sequence, within the target site downstream sequence, or downstream of the target site downstream sequence and are at the same position, a) the total number of the multiple pan-ORF1p coding sequences is two or more, or b) the total number of the multiple pan-ORF2p coding sequences is two or more, or c) the total number of the pan-ORF1p coding sequences and the pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequences and the pan-ORF2p coding sequences are directly or indirectly linked between each other, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other.

[0014] As described above, the RNA framework for gene editing further includes: downstream of the downstream sequence of the target site, one or more ORF2p functional initiation portions are further linked directly or indirectly.

[0015] As described above, when the one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are located upstream of the target site upstream sequence, within the target site upstream sequence, or within the target site downstream sequence, and are in the same position, a) the total number of the multiple pan-ORF1p coding sequences is two or more, or b) the total number of the multiple pan-ORF2p coding sequences is two or more, or c) the total number of the pan-ORF1p coding sequences and the pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequences and the pan-ORF2p coding sequences are directly or indirectly linked, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other.

[0016] As described above, when the one or more pan-ORF1p coding sequences and / or the one or more pan-ORF2p coding sequences are located downstream of the target site downstream sequence, a) when one or more ORF2p functional start portions and one or more pan-ORF1p coding sequences are present, the one or more ORF2p functional start portions are located before or after the one or more pan-ORF1p coding sequences, or the ORF2p functional start portion is spaced apart from the pan-ORF1p coding sequence, the ORF2p functional start portion and the pan-ORF1p coding sequence are directly or indirectly linked, a plurality of the pan-ORF1p coding sequences are directly or indirectly linked to each other, a plurality of the ORF2p functional start portions are directly or indirectly linked to each other, or b) when one or more ORF2p functional start portions and one or more pan-ORF2p coding sequences are present, the one or more ORF2p functional start portions are located before or after the one or more pan-ORF2p coding sequences, or the ORF2p functional start portion is spaced apart from the pan-ORF2p coding sequence, the ORF2p functional start portion and the pan-ORF2p coding sequence are directly or indirectly linked, a plurality of the pan-ORF2p coding sequences are directly or indirectly linked to each other, a plurality of the ORF2p functional start portions are directly or indirectly linked to each other, or c) if one or more functional start portions of ORF2p, one or more pan-ORF1p coding sequences, and one or more pan-ORF2p coding sequences are present, the functional start portions of ORF2p are located before or after the one or more pan-ORF1p coding sequences, or the one or more pan-ORF2p coding sequences are located before or after the one or more pan-ORF1p coding sequences, or the ORF2p functional start portions, the pan-ORF1p coding sequences, and / or the pan-ORF2p coding sequences are present. The pan-ORF2p coding sequence is arranged with spaces between it, the ORF2p functional start portion and the pan-ORF1p coding sequence are directly or indirectly linked, the ORF2p functional start portion and the pan-ORF2p coding sequence are directly or indirectly linked, a plurality of the pan-ORF1p coding sequences are directly or indirectly linked to each other, a plurality of the pan-ORF2p coding sequences are directly or indirectly linked to each other, a plurality of the ORF2p functional start portions are directly or indirectly linked to each other, and the pan-ORF1p coding sequence and the pan-ORF2p coding sequence are directly or indirectly linked.

[0017] As described above, the inside of a single ORF2p functional start portion of one or more ORF2p functional start portions on the RNA framework is further directly or indirectly linked to one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, when one pan-ORF1p coding sequence or one pan-ORF2p coding sequence is inserted into the ORF2p functional start portion, the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or pan-ORF2p coding sequence, and ...2p coding sequence or pan-ORF2p coding sequence. If a) multiple pan-ORF1p coding sequences, or b) multiple pan-ORF2p coding sequences are inserted into the start portion, or c) the total number of pan-ORF1p coding sequences and pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequences and pan-ORF2p coding sequences are directly or indirectly linked, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other, and the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or pan-ORF2p coding sequence.

[0018] As described above, the target site downstream sequence is replaced or partially replaced by one or more ORF2p functional start portions, and if there are multiple ORF2p functional start portions, each of the ORF2p functional start portions is directly or indirectly linked to each other.

[0019] As described above, the sequence of the ORF2p functional initiation region is a short interspersed element RNA, a long interspersed element RNA, a short interspersed element derivative RNA, a long interspersed element derivative RNA, or a sequence of a functional structure that initiates ORF2p cleavage function and reverse transcription.

[0020] As stated above, the pan-ORF1p coding sequence is the ORF1p coding sequence or a modified version of the ORF1p coding sequence, and the pan-ORF2p coding sequence is the ORF2p coding sequence or a modified version of the ORF2p coding sequence.

[0021] As stated above, the RNA framework may be obtained by prokaryotic transcription, eukaryotic transcription or chemical synthesis.

[0022] As stated above, the RNA framework may be or be located in a linear RNA, or may be or be located in a circular RNA.

[0023] As described above, the linear RNA in which the RNA framework is located or the circular RNA in which the RNA framework is located can be obtained by prokaryotic transcription, eukaryotic transcription or chemical synthesis.

[0024] The transcription process can occur in vitro or in prokaryotic or eukaryotic organisms, tissues, organs or cells.

[0025] As described above, the prokaryotic transcription is transcription by a prokaryotic RNA polymerase, and the eukaryotic transcription is transcription by a eukaryotic RNA polymerase I, a eukaryotic RNA polymerase II, or a eukaryotic RNA polymerase III.

[0026] The present invention also provides an RNP obtained by binding the above-mentioned RNA framework for gene editing to ORF1p, ORF2p, an ORF1p-derived protein and / or an ORF2p-derived protein, or by binding a linear RNA in which the RNA framework is located or a circular RNA in which the RNA framework is located in the above-mentioned RNA framework for gene editing to ORF1p, ORF2p, an ORF1p-derived protein and / or an ORF2p-derived protein.

[0027] The present invention also provides DNA sequences that transcribe the above-mentioned RNA frameworks for gene editing.

[0028] The present invention also provides a DNA sequence that transcribes a linear or circular RNA in which the above-mentioned RNA framework for gene editing is located.

[0029] As mentioned above, a prokaryotic or eukaryotic promoter may further be directly or indirectly linked upstream, downstream and / or within the DNA sequence.

[0030] As stated above, the prokaryotic promoter is T7, T3, T7lac, Sp6, araBAD, trp, lac, Ptac, pL, LacUV5, Tac, pBAD or pR.

[0031] As described above, the eukaryotic promoter is CMV, pCMV, EF1a, SV40, human PGK1, mouse PGK1, Ubc, human beta actin, CAG, EFT3, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1, GAL10, GAL1, GAL10, GAL4, GAL80, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, human U6 or mouse U6.

[0032] The present invention further provides a DNA vector having the above DNA sequence.

[0033] The present invention further provides a gene editing method comprising the following steps: Step 1) of selecting a target site to be edited from a genome and determining an upstream sequence of the target site and a downstream sequence of the target site on both sides of the target site; Step 2) of producing the above-mentioned RNA framework for gene editing, or the linear RNA or circular RNA in which the above-mentioned RNA framework for gene editing is located, or the above-mentioned RNP, and / or producing the DNA vector according to claim 23; 3a) transforming or transfecting said RNA framework into a cell, tissue, organ or organism to achieve gene editing; or Step 3b) of transforming or transfecting a cell, tissue, organ or organism with the linear or circular RNA in which the RNA framework is located to achieve gene editing; or 3c) transforming or transfecting the RNP into a cell, tissue, organ or organism to achieve gene editing; or 3d) transforming or transfecting said DNA vector into a cell, tissue, organ or organism to achieve gene editing; or Step 3e) of co-transforming or co-transfecting a cell, tissue, organ or organism with the RNA framework, the linear or circular RNA in which the RNA framework is located, the RNP, and the DNA vector to achieve gene editing; or Step 3f) of co-transforming or co-transfecting one or more of the RNA framework, the linear RNA or circular RNA in which the RNA framework is located, the RNP, and the DNA vector with ORF1p, ORF2p, an ORF1p-derived protein, and / or an ORF2p-derived protein into a cell, tissue, organ, or organism to achieve gene editing.

[0034] The present invention further provides a gene editing method comprising the following steps: Step 1) of selecting a target site to be edited from a genome and determining an upstream sequence of the target site and a downstream sequence of the target site on both sides of the target site; Step 2) of producing the above-mentioned RNA framework for gene editing, or a linear RNA or a circular RNA in which the above-mentioned RNA framework for gene editing is located, or the above-mentioned RNP, or the above-mentioned DNA vector; Step 3) of producing one or more helper RNAs containing an ORF2p functional initiation portion sequence, one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, and / or a helper RNP obtained by binding said helper RNA to ORF1p, ORF2p, an ORF1p-derived protein and / or an ORF2p-derived protein, and / or a helper DNA vector transcribing the ORF2p functional initiation portion, the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence; Step 4a) of co-transforming or co-transfecting the RNA framework with the helper RNA, helper RNP and / or helper DNA vector produced in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4b) of co-transforming or co-transfecting the linear RNA or circular RNA in which the RNA framework for gene editing is located and the helper RNA, helper RNP and / or helper DNA vector produced in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4c) of co-transforming or co-transfecting the RNP with the helper RNA, helper RNP and / or helper DNA vector produced in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4d) of co-transforming or co-transfecting the DNA vector with the helper RNA, helper RNP and / or helper DNA vector produced in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4e) of co-transforming or co-transfecting the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector with the helper RNA, helper RNP, and / or helper DNA vector produced in step 3) into a cell, tissue, organ, or organism to achieve gene editing; or Step 4f) of co-transforming or co-transfecting one or more of the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector, one or more of the helper RNA, helper RNP, and helper DNA vector produced in step 3), and one or more of ORF1p, ORF2p, an ORF1p-derived protein, and an ORF2p-derived protein into a cell, tissue, organ, or organism to achieve gene editing.

[0035] As described above, the RNA framework for transforming, transfecting, cotransforming or cotransfecting a cell, tissue, organ or organism, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector are one or more; In the case of a linear RNA or a circular RNA in which one of the RNA frameworks or one of the RNA frameworks for gene editing is located, or one of the RNPs or one of the DNA vectors, editing is realized at one site on the genome; When the total number of the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector is two or more, and the upstream sequence of the target site and / or the downstream sequence of the target site in the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector are different, editing or manipulation of multiple locations on the genome is achieved.

[0036] The present invention provides use of the above-mentioned RNA framework for gene editing, or a linear RNA or circular RNA in which the above-mentioned RNA framework for gene editing is located, or the above-mentioned RNP, or the above-mentioned DNA vector, as a drug for preventing and / or treating cancer, a gene-related disease or a neurodegenerative disease.

[0037] As described above, the cancer is glioma, breast cancer, cervical cancer, lung cancer, gastric cancer, colorectal cancer, duodenal cancer, leukemia, prostate cancer, endometrial cancer, thyroid cancer, lymphoma, pancreatic cancer, liver cancer, melanoma, skin cancer, pituitary tumor, germ cell tumor, meningioma, meningeal cancer, glioblastoma, various astrocytomas, various oligodendrogliomas, astrocytic oligodendrogliomas, various ependymomas, choroid plexus papilloma, choroid plexus carcinoma, chordoma, various ganglioneuromas, olfactory neuroblastoma, sympathetic nervous system neuroblastoma, pineal cell tumor, pineoblastoma, medulloblastoma, retinoblastoma, trigeminal nerve schwannoma, facial acoustic neuroma, glomus jugular tumor, hemangioreticuloma, craniopharyngioma, or granular cell tumor.

[0038] As described above, the diseases associated with the genes include Huntington's disease, fragile X syndrome, phenylketonuria, Duchenne muscular dystrophy, mitochondrial encephalomyopathy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, mucopolysaccharidosis type IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, spinal muscular atrophy, Parkinson's syndrome, albinism, red-green color blindness, achondroplasia, melauria, congenital deafness, thalassemia, sickle cell disease, and diseases associated with genetic changes. Epilepsy, myoclonus, dystonia, stroke and schizophrenia, vitamin D-resistant rickets, familial colonic polyposis syndrome, 21-hydroxylase deficiency, arginase deficiency, Alport syndrome, Angelman syndrome, Jeune syndrome, atypical hemolytic uraemic syndrome, autoimmune encephalitis, autoimmune hypophysitis, insulin autoimmune syndrome, beta-ketothiolase deficiency, biotinidase deficiency, channelopathies, primary carnitine deficiency, Castleman disease, Charcot-Marie-Tooth disease, citrullinemia, congenital adrenal hypoplasia, congenital hyperinsulinemia, congenital Myasthenic syndromes, nondystrophic myotonic syndromes, congenital scoliosis, coronary artery ectasia, congenital dyserythroplastic anemia, Erdheim-Chester disease, Fabry disease, familial Mediterranean fever, Fanconi anemia, galactosemia, Gaucher disease, generalized myasthenia gravis, Gitelman syndrome, glutaric acidemia type I, glycogen storage disease (types I and II), hemophilia, hepatolenticular degeneration, hereditary angioedema, hereditary epidermolysis bullosa, hereditary fructose intolerance, hereditary hypomagnesemia, hereditary multi-infarct dementia, hereditary spastic paraplegia, holocarboxylase synthase deficiency, hyperhomogenous leukemia, Cysteinemia, homozygous familial hypercholesterolemia, HHH syndrome, hyperphenylalaninemia, hypophosphatasia, hypophosphatemic rickets, idiopathic cardiomyopathy, idiopathic hypogonadism, idiopathic pulmonary hypertension, idiopathic pulmonary fibrosis, IgG4-related disease, congenital disorders of bile acid metabolism, isovaleric acidemia, Kallmann syndrome, Langerhans histiocytosis, Laron syndrome, Leber's hereditary optic neuropathy, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, lymphangioleiomyomatosis, lysinuric protein intolerance, lysosomal acid lipase deficiency, maple syrup urine disease, Marfan syndrome,McCune-Albright syndrome, medium-chain acyl-CoA dehydrogenase deficiency, methylmalonic acidemia, multifocal motor neuropathy, multiple acyl-CoA dehydrogenase deficiency, multiple sclerosis, myotonic dystrophy, N-acetylglutamate synthetase deficiency, neonatal diabetes mellitus, neuromyelitis optica, Niemann-Pick disease, nonsyndromic hearing loss, Noonan syndrome, ornithine carbamoyltransferase deficiency, osteogenesis imperfecta, juvenile Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Peutz-Jeghers syndrome, POEMS syndrome, porphyria, Prader-Willi syndrome, primary immunodeficiency, primary hereditary dystonia, primary light-chain amyloidosis, progressive familial intrahepatic cholestasis, progressive muscular dystrophy, propionic acidemia, pulmonary alveolar proteinosis, cystic fibrosis, retinitis pigmentosa, congenital Agranulocytosis, severe myoclonic epilepsy of infancy, Dravet syndrome, Silver-Russell syndrome, sitosterolemia, spinal-bulbar muscular atrophy, spinal muscular atrophy, spinocerebellar ataxia, systemic sclerosis, tetrahydrobiopterin deficiency, tuberous sclerosis, tyrosinemia, very long-chain acyl-CoA dehydrogenase deficiency, Williams syndrome, eczematous thrombocytopenia with immunodeficiency syndrome, X-linked agammaglobulinemia, adrenoleukodystrophy, X-linked lymphoproliferative syndrome, arteriosclerotic cerebral small vessel disease, cerebral amyloid angiopathy, autosomal recessive cerebral arteriopathy with subcortical infarction and leukoencephalopathy, autosomal dominant cerebral arteriopathy with subcortical infarction and leukoencephalopathy, cathepsin A-related arteriopathy with stroke and leukoencephalopathy, pyridoxine-dependent epilepsy, serotonin-metabolizing AADC enzyme deficiency, AADC deficiency, or hereditary nephritis.

[0039] As described above, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, multiple system atrophy, primary lateral sclerosis, Pick's disease, frontotemporal dementia, dementia with Lewy bodies, or progressive supranuclear palsy.

[0040] The present invention provides use of the above-mentioned RNA framework for gene editing, or a linear RNA or circular RNA in which the above-mentioned RNA framework for gene editing is located, or the above-mentioned RNP, or the above-mentioned DNA vector, as a tool for inserting a target sequence, deleting a target sequence, replacing a target sequence, deleting a target site, adding a target site, adding a target sequence, replacing a target site, inverting a target gene sequence, and / or correcting an inversion of a target gene sequence.

[0041] The present invention provides the use of the above-mentioned RNA framework for gene editing, or a linear or circular RNA in which the above-mentioned RNA framework for gene editing is located, or the above-mentioned RNP, or the above-mentioned DNA vector, in the production or amplification of a DNA template containing the above-mentioned RNA framework sequence.

[0042] The present invention provides use of the above-mentioned RNA framework for gene editing, or the linear RNA or circular RNA in which the above-mentioned RNA framework for gene editing is located, or the above-mentioned RNP, or the above-mentioned DNA vector, or the above-mentioned DNA template as a tool for improving the gene editing efficiency of TALEN, ZFN, Targetron, Prime Editor, Twin Prime Editor, CRISPR or CRISPR / Cas9 gene technology.

[0043] The present invention includes RNA, ssDNA, and / or dsDNA, including target site upstream sequences, inserted sequences, target site downstream sequences, and / or other sequences such as short interspersed elements, partial short interspersed elements, and these components can assist the homologous recombination of technologies such as TALEN, ZFN, Targetron, CRISPR, CRISPR / Cas 9, or insert sequences into target sites, facilitating RNAization of the corresponding technologies, not requiring viral transfection, and improving the efficiency of genome sequence insertion of the corresponding technologies (RNA does not need to be introduced into the nucleus after transformed cells, but can be introduced into the nucleus during the non-dividing phase under the binding and action of the corresponding proteins, such as ORF1p and / or ORF2p).

[0044] The present invention uses the downstream short interspersed elements, partial short interspersed elements, long interspersed elements, partial long interspersed elements, and the functional structure of the start ORF2p cleavage function and reverse transcription, and the long interspersed elements, especially the human LINE-1 coding ORF2p, to cut the target genome single strand, and reverse transcribe it as a primer, and finally form dsDNA, and insert the sequence to be inserted by homologous recombination into the target site on the genome. The present invention only cuts the genome single strand, and does not cause genome double strand breaks, so it has high safety.

[0045] The present invention uses RNA and RNA (also called RNP) that binds to human endogenous proteins with specific functions as the functional body to perform gene editing on the genome. Gene editing by introducing RNA into a living body is safer than DNA. In addition, in vitro synthesis and transcription of RNA, especially the production of RNA by in vitro transcription of prokaryotes, makes in vitro production of RNA easier, which is advantageous for further industrialization and commercialization.

[0046] The ORF1p and / or ORF2p in the present invention can bind to the RNA framework, and help transport RNA into the cell nucleus while protecting the RNA framework. Only when the target site upstream sequence on the vector and the corresponding complementary sequence of the target site upstream sequence on the genome are completely identical, the ORF2p expressed in the vector or cell can successfully slide from the 3' end of the ssDNA formed by reverse transcription from the vector (RNA and / or RNP) to the shearing site (target site), shear the single strand on the genome, and further mediate the formation of the vector dsDNA. The present invention has higher targeting accuracy and highly avoids dsDNA, which can have a negative effect on the genome due to its extensive non-specific production in the cell nucleus, making its safety and accuracy theoretically higher than other existing gene editing technologies. At the same time, it effectively solves the problem of the difficulty of DNA nuclear introduction using RNA or RNP as a vector, and facilitates gene editing in cells with low DNA transfection efficiency. Effect of the Invention

[0047] The beneficial effects of the present invention are as follows:

[0048] The present invention provides an RNA framework for gene editing based on a mechanism inherent to eukaryotes themselves. RNP or RNA (which can be produced by in vitro preparation) and related proteins are transferred into the cytoplasm and cell nucleus as vectors to realize gene editing of specific sequences or sites on the genome of a target system (e.g., a cell, tissue, organ, or organism), such as insertion, deletion, substitution, and site substitution of specific sequences, while at the same time combining relatively high target accuracy. The present invention does not introduce exogenous systems or substances such as proteins derived from prokaryotes, does not cause double-strand breaks, and is therefore more suitable for further practical use, such as clinical applications, than other conventional techniques. In addition, RNA can be obtained in vivo or in vitro by methods such as prokaryotic or eukaryotic promoter transcription or chemical synthesis, and in particular, the efficiency of prokaryotic promoter transcription is high, the length of the product RNA is long, and the destruction of the integrity of the target RNA due to the shearing mechanism in eukaryotic systems can be avoided. At the same time, the proteins ORF2p and / or ORF1p can also be synthesized in vitro, which is convenient for industrial mass production and commercialization. [Brief description of the drawings]

[0049]

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[0050] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to enable those skilled in the art to more easily understand the advantages and features of the present invention and to more clearly define the protection scope of the present invention, the embodiments of the present invention are described in detail and completely below. Conventional techniques such as CRISPR can cause double-stranded breaks and easily introduce random sequences or mutations, reducing the efficiency of target sequence introduction. The RNA framework for gene editing according to the present invention is based on the transposon machinery, which is widespread in eukaryotes, and the remodeling mechanism mediated by it, which modifies genomic components, such as repeat sequences and individual gene copies, which may result in the reduction or addition of pathogenic triple nucleotide repeats in degenerative central nervous system pathologies such as Huntington's disease and fragile X syndrome, the promotion of the amplification of the HIV genome in proliferating immune cells into the human genome, and the gain or loss of specific gene copies during embryonic development and tumorigenesis. The RNA framework and corresponding RNP according to the present invention do not cause double-strand breaks and carry out genome integration via homologous recombination, which is safer and more advantageous for practical application. The RNA framework for gene editing provided by the present invention uses RNA or RNP as a vector, and the inserted sequence, which is ready to be inserted into a selected gene site (target site) on the genome, is pre-determined to be located on the genome through the target site upstream sequence (target site upstream sequence) and target site downstream sequence (target site downstream sequence) on both sides of the inserted sequence on the RNA or RNP (the upstream of the target site refers to the 5'-direction sequence of the target site on any single strand of the genome, and the downstream of the target site refers to the 3'-direction sequence of the target site on the corresponding single strand of the genome), and at the same time, the inserted sequence is pre-determined to be located on the genome through the target site upstream sequence (target site upstream sequence) and target site downstream sequence (target site downstream sequence) on both sides of the inserted sequence on the RNA or RNP, and at the same time, the inserted sequence is pre-determined to be located on the genome through the target site upstream sequence (target site upstream sequence) and target site downstream sequence (target site downstream sequence) on the genome (the upstream of the target site refers to the 5'-direction sequence of the target site on any single strand of the genome, and the downstream of the target site refers to the 3'-direction sequence of the target site on the corresponding single strand of the genome). The LINE RNA, short interspersed element derivative RNA, long interspersed element derivative RNA, and / or ORF2p shearing function and functional structure to initiate reverse transcription and the proteins ORF2p (open reading frame 2 protein, L1 endonuclease) and / or ORF1p (open reading frame 1 protein) expressed in long interspersed elements are used to precisely insert the LINE RNA into the target site in the genome.ORF1p and / or ORF2p can bind to the RNA vector and help transport the RNA into the cell nucleus while protecting the RNA vector, and ORF2p expressed in the vector or cell can successfully slide from the 3' end of the ssDNA formed by vector (RNA or / and RNP) reverse transcription to the shearing site (target site) only when the target site upstream sequence on the vector is completely consistent with the corresponding complementary sequence of the target site upstream sequence on the genome, shearing the single strand on the genome and further mediating the formation of vector dsDNA, so that the present invention has higher targeting accuracy and highly avoids dsDNA, the widespread and non-specific generation of which can adversely affect the genome in the cell nucleus, and thus the safety and accuracy of the present invention are theoretically higher than other existing gene editing technologies. At the same time, the problem of DNA being difficult to enter the cell nucleus using RNA or RNP as a vector is effectively solved, facilitating gene editing in cells with low DNA transfection efficiency. Short interspersed element RNA, long interspersed element RNA, short interspersed element derivative RNA, long interspersed element derivative RNA and / or ORF2p shearing function and functional structure for initiating reverse transcription are connected downstream of the above RNA framework, long interspersed element (LINE) code (encoding), particularly ORF2p by human LINE-1 expression, is bound, the target genome single strand is incised, and reverse transcribed using this as a primer, finally forming dsDNA, and the sequence to be inserted (sequence to be inserted) is inserted into the target site on the genome by homologous recombination. The present invention only incises the genome single strand and does not cause genome double strand breaks, so it is highly safe. The present invention produces RNA by in vivo or in vitro expression in eukaryotic or prokaryotic systems and cells, tissues, organs, or organisms, and produces the required proteins ORF1p and / or ORF2p in the target system or outside the target system (ex vivo), and introduces the RNA or RNP format vector into the target system, e.g., cells, tissues, organs, or organisms, to achieve the goal of gene editing, facilitating industrial mass production and commercialization. In addition, since there is no splicing mechanism of precursor mRNA in eukaryotic systems in prokaryotic systems or in vitro expression, the RNA framework and downstream connectable short interspersed element RNA, short interspersed element derivative RNA, long interspersed element, long interspersed element derivative RNA, and / or functional structures of the initiating ORF2p shearing function and reverse transcription can be expressed without any hindrance without potential splicing risks, improving the production efficiency and gene editing effect of the present invention. Therefore, the present invention can perform precise sequence deletion, sequence replacement, and individual site replacement through homologous recombination or genome repair of the recipient system, such as prokaryotes or eukaryotes, after targeting the insertion of a desired sequence into the genome. At the same time, based on the technical principle of the present invention, the present invention can continue to design a vector through the new site formed after the sequence has been inserted by the present invention, and the inserted sequence has no theoretical upper limit in length, and can achieve gene editing purposes such as sequence insertion, deletion, replacement, and site replacement in various and various forms, and the method of use is flexible. In addition, the present invention can also perform gene editing on CNV and its ends to stably change, extend, shorten, or change its expression sequence, etc., to achieve the purpose of changing or stabilizing the gene expression and state of cells, tissues, organs, or organisms. The sequence to be inserted in the RNA framework provided by the present invention may be an exogenous sequence or an endogenous sequence, and the length of the sequence inserted at one time is 1 bp to 20000 bp. By inserting multiple times, it is possible to realize genome insertion of DNA sequences of any length. The nucleotide sequence length of the upstream sequence of the target site can be 1 bp-20000 bp, and the nucleotide sequence length of the downstream sequence of the target site can be 1 bp-20000 bp. The present invention relates to short interspersed elements, long interspersed elements and the proteins involved in their production, such as ORF1p, ORF2p and other types of open reading frame proteins (ORFp). Short interspersed elements (SINEs) are mainly classified into Alu elements (e.g., Alu Jo, Alu Jb, Alu Sq, Alu Sx, Alu Sp, Alu Sc, Alu Sg, Alu S, Alu Y, Alu Yb8, Alu Ya5, Alu Ya8, Alu J, etc.) in primates (including humans), SVA elements, various mammalian-wide interspersed repeat elements (MIRs) families common in mammals, such as MIR and MIR 3, Mon-1 in monotremes, B1 and B2 elements in rodents, C-element in rabbits, HE1 family in zebrafish, SINE SmaI in salmon, Anolis SINE2 and Sauria SINE3 in reptiles, and others. SINEs, such as IdioSINE1, IdioSINE2, SepiaSINE, Sepioth-SINE1, Sepioth-SINE2A, Sepioth-SINE2B and OegopSINE in invertebrates such as squid, and p-SINE1 in plants such as rice. Long interspersed elements mainly include six types of LINEs, namely L1 (L1RE1 (L1.2, LRE1) and LRE2 in humans, various LINE2 (L2) and various LINE3 (L3), Ta elements and R2, RandI, L1, RTE, I and Jockey, as well as other types of LINEs, such as mouse LINE 1, eel LINE UnaL2, insect LINE R2, zebrafish LINE ZfL2-1 and ZfL2-2, algae L1, silkworm LINE SART1, monocotyledonous plant L1, fungus Tad1, fish L2 and some mammalian RTE. LINEs also include L1s, L1spa, L1Orl, L1.2, F subfamily of L1, TF subfamily and so on.SINEs and LINEsThese structures are widely present in various animals and plants and are scattered throughout the genome, and each organism has its own unique SINEs and their functionally corresponding LINEs. The DNA sequence corresponding to an Alu element is referred to as the Alu sequence. The main characteristics of SINEs are that they are relatively short retrotransposons distributed throughout the genome, containing an internal RNA polymerase III promoter, ending with an A- or T-rich tail or a short simple repeat sequence, and realize reverse transcription using LINEs, the right half of whose transcripts contain a reverse transcription functional structure. The characteristics of LINEs are that they are transposons distributed widely throughout the genome, containing reverse transcriptase coding sequences. SINEs and their corresponding LINEs in their corresponding species all reconstruct the genome through a similar mechanism. The basic principle of this mechanism is to connect the lariat structure generated by processing pre-mRNA with the SINE right half, which has a reverse transcription functional structure generated by shearing the SINE transcript. These are called partial short interspersed element RNAs, and their corresponding coding DNA sequences are called partial short interspersed element sequences, for distinction. They are in different short interspersed element RNAs in different species, and their shearing sites are different. The natural shearing site of short interspersed element RNA is generally located at the middle front position of the full length, and for short interspersed element RNA with a total length of about 100 to 400 nt, the natural shearing site is generally located at the 100th to 250th nt. For example, for an Alu element with a total length of about 300 bp, the shearing site of the transcript RNA (i.e., the scAlu shearing site or natural shearing site below is generally located before the middle polyA sequence of the Alu transcript, and the actual situation may vary) is located at the 118th nt. The sheared product contains the Alu right monomer, the middle polyA repeat sequence of the Alu element transcript other than the Alu right monomer, or 2 to 3 bases upstream thereof, and the 3' polyA repeat sequence after the right monomer, and can be called a partial Alu. For distinction, the corresponding coding DNA sequence is called a partial Alu sequence. On the other hand, for the transcript RNA of various MIRs with a total length of about 260 nt, the shearing site can be observed in the range of 100 to 150 nt. In fact, regardless of location, after shearing, the remaining right part of the transcript can function as long as it contains an intact reverse transcription functional structure.The secondary structure of the reverse transcription functional structure forms a special structure, usually Ω-shaped, and its primary structure is characterized by containing two sequences separated by an intermediate spacing sequence (intermediate spacer sequence). These two sequences do not contain a corresponding intermediate spacing sequence on the genome, and can bind to the complementary sequence of the sequence that directly connects the two sequences, and the LINE-encoded ORF2p can bind to the sequence located 3' of the two sequences in the ORF2p functional initiation structure in the above-mentioned transcript, and can incise the genome single strand at the genomic site corresponding to the gap between the two sequences and start reverse transcription. Furthermore, the corresponding transcription product (RNA sequence) of the Alu element is referred to as a complete Alu. A DNA sequence that contains a functional structure for reverse transcription and is capable of initiating reverse transcription, but differs in sequence from conventional short interspersed elements (e.g., short interspersed elements with some mutations but with a special structure and function) is called a short interspersed element-like (SINE-like) sequence, and the corresponding RNA sequence is called a short interspersed element-like RNA. In addition, the RNA sequence including the reverse transcription functional structure and the ORF2p binding sequence and the structure of the initiating ORF2p shear genome and reverse transcription is called "ORF2p binding (e.g., having a polyA sequence, usually located on the right foot of the reverse transcription functional structure "Ω" structure) to initiate ORF2p shear function and reverse transcription functional structure", which can be abbreviated to "initial ORF2p shear function and reverse transcription functional structure", and can also be called "ORF2p function initiating structure", "ORF2p binding to initiate ORF2p shear function and reverse transcription functional structure" The transcript can form a 0 secondary structure due to its own or external causes, and ORF2p is formed by the initiating ORF2p shear function and reverse transcription functional structure. The Alu element can bind to the "Ω" notch and be located in the 3' direction of the "Ω" structure. Through the proteins ORF1p and ORF2p expressed in the corresponding types of LINEs, such as the functionally corresponding LINE-1 and the corresponding LINE-2 of each MIR element, "convert RNA into double-stranded DNA" is realized, and it binds to complementary, identical or similar sequences on the genome, where the RNA (transcript) transcribed is a single-stranded DNA generated by reverse transcription, and a double-stranded DNA generated by using the single-stranded DNA as a primer using the genome sequence, that is, a conversion product of the transcript, and after forming a specific 0 structure, it completes the insertion into the genome through the homologous recombination mechanism. In addition, LINE can also achieve the above-mentioned RNA-to-double-stranded DNA conversion and genome insertion by transcribing its downstream sequence (i.e., 3' transduction) and binding to a complementary sequence on the genome to form a 0 structure. Taking the Alu element and its corresponding supporting function LINE-1 as an example, the pre-mRNA generated after gene expression can generate overlapping lassos on the sequence by shearing. This can occur in any region of the pre-mRNA, except that the shear strengths that generate these lariat sequences are different. Because lariat sequences are generated upstream and downstream of exons (lasso sequences that do not contain exons), the shear strength based on sequence differences will be higher than other surrounding lariat structures (such as lariat sequences that contain exons in their sequences). This makes it easier for exons to be completely excised during pre-mRNA processing, inhibiting the generation of other lariat sequences.At the same time, ORF1p generated by LINE-1 can protect the nucleic acid bound to it, and both ORF1p and ORF2p generated by LINE can localize and transport the bound nucleic acid to the nucleus. In addition, ORF2p can bind to the special Ω secondary structure of Alu element transcripts and mediate subsequent single-stranded genome cleavage, reverse transcription, and auxiliary genome integration. As mentioned above, the transcription product of an Alu element can be excised at a specific site to generate a partial Alu, and the socket structure generated by the excision of the pre-mRNA can connect from its 3' end to the remaining part of the Alu element transcript that contains the excised reverse transcription functional structure (i.e., a partial Alu). ORF2p can be recruited via ORF2p binding sequences, such as A-rich sequences, and is located on the 3' leg of the Ω structure formed by binding to partial Alu secondary structures. It identifies sequences in the genome that match the sequences on both legs of Ω (mainly UU / AAAA, with no continuity between U and A, i.e., there is a notch), cuts a single strand at the genomic site opposite the Ω notch, and reverse transcribes the unwound strand using the complementary sequence on the genome as a primer. This process is called target-primed reverse transcription. This is called reverse transcription (TPRT), and ORF2p moves to the 3' end of the single-stranded DNA formed as reverse transcription proceeds, and the generated single-stranded DNA sequence can bind to the complementary sequence on the genome and form an Ω structure at the corresponding insertion site on the genome. Since the sequence to be inserted does not exist at the corresponding insertion site on the genome, both sides of the sequence to be inserted on the single-stranded DNA exist on both sides of the insertion site on the genome, and ORF2p slides into the Ω structure in the 3' to 5' direction along the matching sequence, identifying a 6-bit nucleotide sequence on the genome that is complementary to the sequence on both sides of the Ω bottom notch (gap), and the nucleotides, which are mainly 4 nucleotides at 3' and 2 nucleotides at 5', form a double-stranded DNA through the process as described above. Note that only a perfectly matching sequence can slide ORF2p to the shearing site, which ensures the accuracy of its target.The double-stranded DNA finally produced is again bound in an "Ω" shape to both sides of the corresponding insertion point on the genome that matches the "Ω" end sequence, and when ORF2p identifies a middle discontinuous portion ("Ω" notch) of six nucleotides (mainly four nucleotides at 3' and two nucleotides at 5'), it creates two single-stranded notches on the genome corresponding to the notch (gap) portion and on the other strand to which it is bound by the endonucleotide cleavage function of ORF2p, and can insert the middle circular portion into the genome by the homologous recombination mechanism. In this process, ORF1p can promote the formation of functional secondary structures and further structures of the RNA used, and can promote the mutual binding and action of functional RNA and the genome that interacts with it. Meanwhile, by changing the inserted sequence, other effects such as deletion and substitution can be achieved through homologous recombination. In the above process, the annealing and dissociation functions of ORF1p, which are also encoded by LINE, can play an auxiliary role, stabilizing the binding of the nucleic acid to the secondary structure and genome in the above genome reconstruction process, and promoting the separation of the nucleic acid from the genome after binding and action. In addition, ORF1p has high RNA affinity and the ability to localize in the cell nucleus. ORF2p can only cleave one strand of the genome double strand and cannot generate double-strand breaks, so it has high safety. A similar mechanism applies to other SINE and LINE combinations. Changes in local copy number mutations in pathological processes such as embryonic development and tumor development, and the preference for short interspersed elements due to the insertion of missing HIV-1 genomes into the human genome may be one embodiment of this mechanism in nature. It has been reported that the transcribed mRNA sequence can be integrated into the genome under the cooperation of ORF1p and ORF2p, but since the transcription template is a pure exogenous non-homologous sequence, it cannot target specific sites in the genome and does not connect fragments with reverse transcription functional structures, which reduces efficiency, is random, and difficult to control.The present invention redesigns the transcription sequence to connect with sequences having reverse transcription functional structures, such as various short interspersed element RNAs or partial short interspersed element RNAs, through various active or passive means, to achieve a relatively accurate and efficient gene editing effect. In addition, ORF2p and ORF1p can also bind with long interspersed element RNAs to mediate translocation activity. The part of the long interspersed element RNA 3' part that can form a special first or second structure (for example, the corresponding transcription product (RNA sequence) of the 3'UTR part) can be cut out and named as partial long interspersed element RNA. The partial long interspersed element RNA can be connected to a corresponding position, such as downstream of the RNA frame that functions according to the above-mentioned principle. A schematic diagram of the above basic principle is shown in Figure 1. According to the above principle, other sequences (e.g., sequences not homologous to the upstream sequence of the target site) should be avoided as much as possible upstream of the upstream sequence of the target site in the RNA framework. At the same time, the upstream sequence of the target site in the RNA framework should be as close as possible to or the same as the corresponding upstream sequence of the target site on the genome, so as to improve gene editing efficiency. In the present invention, the RNA sequence of a short interspersed element transcript in nature (including mutations such as natural mutations) is called short interspersed element RNA, and the RNA sequence of a long interspersed element transcript in nature (including mutations such as natural mutations) is called long interspersed element RNA. In the present invention, short interspersed element derivative RNA or long interspersed element derivative RNA means adding other sequences to short interspersed element RNA or long interspersed element RNA, cutting out a part of the sequence, adding, reducing or rearranging a functional structure sequence, generating a similar sequence that exerts a similar function, mixing two element sequences, especially functional parts, etc., or a combination of the above changes, and short interspersed element derivative RNA or long interspersed element derivative RNA and short interspersed element RNA or long interspersed element RNA have 50% or more similarity in any continuous 10 bp sequence and above. Short interspersed element derivative RNA is a sequence that changes based on a part of short interspersed element RNA and other natural short interspersed element RNA sequences, and long interspersed element derivative RNA is a sequence that changes based on a part of long interspersed element RNA and other natural long interspersed element RNA sequences. In addition, 7SLRNA, which has relatively high similarity to short interspersed element RNA, also belongs to the partial short interspersed element derivative RNA. The functional structures for initiating ORF2p shearing function and reverse transcription include one or more of short interspersed element RNA, long interspersed element RNA, short interspersed element derivative RNA, long interspersed element derivative RNA, and analogous short interspersed element RNA. The short interspersed element RNA, the long interspersed element RNA, the short interspersed element derivative RNA, the long interspersed element derivative RNA, and / or the functional structures initiating ORF2p shearing function and reverse transcription are collectively referred to as the ORF2p functional start portion. The ORF1p coding sequence in the present invention refers to the RNA sequence of the natural coding sequence of ORF1p in the long interspersed element on the genome, and the ORF2p coding sequence refers to the RNA sequence of the natural coding sequence of ORF2p in the long interspersed element on the genome. The modified sequence of the ORF1p coding sequence in the present invention can be modified by the ORF1p coding sequence, i.e., the natural ORF1p sequence, or the natural ORF1p sequence containing various mutations or mutations. The modified sequence of the ORF2p coding sequence can be modified from the ORF2p coding sequence, i.e., the natural ORF2p sequence, or the natural ORF2p sequence containing various mutations and mutations. Related modifications include, based on the native ORF1p coding sequence or the native ORF2p coding sequence, adding other sequences, truncating a part of the sequence, adding, reducing or rearranging functional structural sequences, generating similar sequences that perform similar functions, mixing (fusing) the whole or part of the ORF1p coding sequence and / or the ORF2p coding sequence, particularly the functional sequences therein, with one or more other proteins (including ORF1p and ORF2p) to form corresponding fusion protein coding sequences, etc., or a combination of the above modifications. The protein resulting from the ORF1p coding sequence is called ORF1p, the protein resulting from the ORF2p coding sequence is called ORF2p, and the protein generated from the modified sequence of the ORF1p coding sequence or the modified sequence of the ORF2p coding sequence is called ORF1p-derived protein or ORF2p-derived protein, respectively. The modified sequence of the ORF1p coding sequence or the modified sequence of the ORF2p coding sequence and its expression ORF1p-derived protein or ORF2p-derived protein should still have the above-mentioned functions and characteristics. The ORF1p coding sequence and modified sequences of the ORF1p coding sequence are collectively referred to as pan-ORF1p coding sequences, and the ORF2p coding sequence and modified sequences of the ORF2p coding sequence are collectively referred to as pan-ORF2p coding sequences. ORF2p contains several functional domains that have been identified so far, mainly the endonuclease region (aa:1-239), the cryptic region (aa:240-347), the Z region (aa:380-480), the reverse transcriptase region (aa:498-773), and the cysteine-rich domain (aa:1130-1147). Because the endonuclease machinery is required to practice the present invention, adding an endonuclease domain, a partial endonuclease domain, or a modified structure having 50% or more amino acid identity to the endonuclease domain sequence in native ORF2p to the ORF2p-derived protein can enhance the action of the ORF2p-derived protein in the present invention. Meanwhile, it has been found that the function of the significance reserved region can reduce the cytotoxicity of the endonuclease region and increase the nuclear localization of the protein or polypeptide fragment in which it exists.In the present invention, more nuclear localization can increase the gene editing effect, and lower cytotoxicity is also advantageous for the practical application of the present invention, so adding or adding a longer significance reserved region to the ORF2p derivative protein can promote the gene editing efficiency of the present invention to a certain extent. It has now been discovered that the function of the cysteine-rich region can promote the binding of ORF2p-derived proteins with nucleic acids. Since the endonuclease action of ORF2p requires the assistance of special nucleic acids and their secondary structures for initiation, in the present invention, the gene editing efficiency of ORF2p can be improved or is required by adding a cysteine-rich region, a partial cysteine-rich region, or a modified structure having 50% or more amino acid homology with the cysteine-rich region sequence in the native ORF2p to the ORF2p-derived protein. The Z region can be a PCNA-bound form, which can promote the role of ORF2p in the present invention. Therefore, in the present invention, the gene editing efficiency of ORF2p-derived proteins can be improved or is necessary by adding a Z region, a part of a Z region, or a modified structure having 50% or more amino acid homology with the Z region sequence in the native ORF2p to the ORF2p-derived protein. In addition to the above-mentioned regions in native ORF2p, other regions are added or partially added to the ORF2p-derived protein, or are modified into the ORF2p-derived protein, and the gene editing efficiency of the present invention can be similarly improved to some extent by adding or partially adding other regions of native ORF2p that maintain 50% or more homology with the original sequence. The distribution of each region in the ORF2p-derived protein can be arranged, rearranged, randomly ordered, or shuffled according to the natural ORF2p. Other regions can be added between or within each region. The amino acids in ORF2p and the ORF2p-derived protein can be replaced with corresponding conservative substitutions (e.g., mutual substitutions between Phe, Trp, and Tyr, mutual substitutions between Leu, Ile, and Val, mutual substitutions between Gln and Asn, mutual substitutions between basic amino acids Lys, Arg, and His, mutual substitutions between acidic amino acids Asp and Glu, and mutual substitutions between hydroxyl amino acids Ser and Thr). In addition, the ORF2p-derived protein contains more homologous conserved amino acid sequences between human-derived ORF2p and other genera such as mouse ORF2p, or the gene editing efficiency of the ORF2p-derived protein can be increased. The base sequence in the modified sequence of the ORF2p coding sequence encoding the ORF2p-derived protein can be replaced with a different codon sequence of the same amino acid. We now find that ORF1p contains several functional domains, mainly the N-terminal domain, the coiled coil domain, the RNA recognition motif, and the C-terminal domain. Higher nucleic acid binding affinity and nucleic acid molecule companion activity can enhance the effects of the present invention, while the RNA recognition module and C-terminal region in ORF1p have the above-mentioned functions in the derived protein, so that the ORF1p-derived protein contains an RNA recognition module and / or C-terminal region, a partial RNA recognition module and / or C-terminal region, or a modified structure having 30% or more amino acid homology with the RNA recognition module or C-terminal region sequence in natural ORF1p, thereby enhancing the action of the ORF1p-derived protein in the present invention. On the other hand, the curled helical region in ORF1p acts to improve the nucleic acid binding affinity and promote the translocation activity by forming a trimer of ORF1p, so the ORF1p-derived protein contains a curled helical region, a partial curled helical region, or a modified structure having 30% or more amino acid homology with the curled helical region in the natural ORF1p, which can enhance the function of the ORF1p-derived protein in the present invention. In addition, since the N-terminal region also acts on the normal function of ORF1p, the ORF1p-derived protein contains an N-terminal region, a part of the N-terminal region, or a modified structure having 30% or more amino acid homology with the N-terminal region in the natural ORF1p, and can enhance the action of the ORF1p-derived protein in the present invention. Except for the above-mentioned regions in native ORF1p, other regions are added or partially added to the ORF1p-derived protein, or are modified into the ORF1p-derived protein, and the gene editing efficiency of the present invention can be improved to some extent by adding or partially adding other regions of native ORF1p that maintain 50% or more homology with the original sequence. Since protein phosphorylation affects the normal function of ORF1p, adding a conserved proline-induced protein kinase (PDPK) site in ORF1p to an ORF1p-derived protein can enhance the function of the ORF1p-derived protein in the present invention. The distribution of each region in the ORF1p-derived protein can be arranged according to the natural ORF1p, rearranged, randomly ordered, or shuffled. Other regions can be added between or within each region. The amino acids in ORF1p and the ORF1p-derived protein can be replaced with corresponding conservative substitutions (e.g., mutual substitutions between Phe, Trp, and Tyr, mutual substitutions between Leu, Ile, and Val, mutual substitutions between Gln and Asn, mutual substitutions between basic amino acids Lys, Arg, and His, mutual substitutions between acidic amino acids Asp and Glu, and mutual substitutions between hydroxyl amino acids Ser and Thr). In addition, the ORF1p-derived protein contains more homologous conserved amino acid sequences between human-derived ORF1p and other genera such as mouse ORF1p (for example, ARR at positions 260-262 in the human-derived ORF1p amino acid sequence, REKG at positions 235-238, and YPAKLS at positions 282-287 (Y at position 282 can be replaced with functionally similar F)), which can enhance the gene editing efficiency of the ORF1p-derived protein. The base sequence in the modified sequence of the ORF1p coding sequence encoding the ORF1p-derived protein can be replaced with a different codon sequence of the same amino acid. As the corresponding sequences on the genome of the sequences upstream and downstream of the target site, sequences containing recombination sites on the genome to be edited (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC), short interspersed elements, partial short interspersed elements, short interspersed element derivatives, long interspersed elements (long interspersed nuclear elements, long interspersed nuclear elements, long interspersed elements, LINEs), partial long interspersed elements and / or long interspersed element derivatives, or other sequences that increase homologous recombination efficiency can be searched for and / or selected to insert the sequence and enhance the efficiency of homologous recombination, thereby enhancing the gene editing effect. In the practical application of the present invention, when the RNA vector containing the RNA frame does not connect the ORF2p functional initiation part downstream of the RNA frame and does not achieve the desired efficiency, or the efficiency of connecting RNA or a fragment thereof with the short interspersed element RNA or its product is not high, it is attempted to increase or decrease the length of the sequence upstream or downstream of the target site on the RNA frame or the sequence to be inserted therein to promote the connection; or, the lasso structure containing the inserted site generated in each prokaryotic or eukaryotic organism is detected based on the following detection method, and the sequence of this lasso structure is the upstream or downstream sequence of the target site or a partial sequence in the upstream or downstream sequence of the target site; or, for example, the upstream or downstream sequence of the target site on the RNA vector containing the RNA frame is appropriately extended to be the sequence to be inserted at the intermediate target site, and RNA is generated; or, for example, a polyA sequence can be added to the 3' site of the RNA vector containing the RNA framework to facilitate the binding with ORF2p. For example, an ORF2p binding sequence, for example a polyA sequence, can be added to a suitable position on the RNA vector containing the RNA framework, or an existing ORF2p binding sequence, for example a polyA sequence, can be extended without affecting the formation of the "Ω" structure of the RNA framework. The ORF2p binding sequence is mainly located at the 3' site or 3' end of the RNA vector containing the RNA framework, for example, each protein expression sequence (such as pan-ORF1p coding sequence or pan-ORF2p coding sequence) on the RNA vector containing the RNA framework, the target site upstream sequence in the RNA framework, the target site downstream sequence in the RNA frame, the short interspersed element RNA, the long interspersed element RNA, the short interspersed element derivative RNA, the long interspersed element derivative RNA, and / or the functional structure of the initiating ORF2p shearing function and reverse transcription or other sequences on the RNA, or in between, before, or after the ORF2p binding sequence, e.g., a multi-A sequence, is added, or its unique ORF2p binding sequence, e.g., a multi-A sequence, is extended, for example, to design a sequence at the 3' site of the RNA vector containing the RNA framework to generate an "Ω" structure, for example, to facilitate ORF2p cleavage of nucleic acid inside the genome. In addition, since the gene editing effect of the present invention is related to the homologous recombination mechanism, the use of recombinases, such as site-specific serine recombinases, in combination with the present invention can increase the efficiency and effectiveness of the present invention. Furthermore, the target region (target site) of gene editing in the present invention may be one or more locations where the region between two or more gene editing target sites can be deleted or replaced with an inserted sequence or partial sequence, when part or all of the gene editing insertion sequence for two or more target sites is the same or similar (high similarity) and the length of the partial sequence is 20 bp or more. When the sequence to be inserted in the present invention is short (100 bp or less), the sequence to be inserted can be inserted into the target site on the genome by homologous recombination and / or other genome repair mechanisms, thereby having high genome insertion efficiency. In the present invention, when the upstream sequence of the target site and / or the downstream sequence of the target site are short (100 bp or less), the sequence to be inserted (the sequence to be inserted) can be inserted into the target site on the genome by homologous recombination and / or other genome repair mechanisms, thereby achieving high genome insertion efficiency. The site to be inserted (insertion target site) described in the present invention is a target site. As shown in Figure 2, which is a schematic diagram of the operation process in the present invention, this gene editing technology can realize technologies such as "RNA-vector-based genome sequence insertion technology", "RNP-vector-based genome sequence insertion technology", "RNA vector and / or RNP vector-mediated genome sequence deletion technology", "genome sequence replacement technology (including sequence replacement, site deletion, site addition, sequence addition, sequence deletion and site replacement)", "inhibition of genome changes caused by transposons, stable genomes and CNVs thereon", and "assistance of other gene editing technologies", which will be described one by one below. 1. Genome sequence insertion technology using RNA vector 1. Genomic sequence insertion mediated by a simple RNA framework using RNA as a vector: Select the upstream and downstream sequences (target site upstream sequence and target site downstream sequence) of the site to be inserted (i.e., the target site), add the sequence to be inserted at the insertion point between the upstream and downstream sequences, and generate the designed sequence-generating RNA as a vector. The RNA is incubated with RNase inhibitors and / or an appropriate amount of Mg 2+ The vector can be incubated in a solution containing 50 mM NaCl (e.g., 6 mmol / L) or other metals or in a "cell solution" to promote correct folding of the RNA and subsequent binding to the corresponding functional proteins, such as ORF2p and / or ORF1p. The vector can then be transferred to in vitro cultured cells, tissues, or organs by conventional means, such as lipid body transfection, or administered to tissues, organs, or organisms via routes such as blood, lymph, cerebrospinal fluid, or local tissue administration, allowing the vector to enter the target cytoplasm and bind to ORF1p and / or ORF2p before entering the nucleus, or ORF1p and / or ORF2p can directly enter the nucleus. After ligating the vector RNA and the short interspersed element RNA of intracellular transcription or its product, it binds to ORF2p of intracellular expression, or binds ORF2p and ORF1p simultaneously, or binds directly to ORF2p expressed in the cell, or binds ORF2p and ORF1p simultaneously (for example, the Ω structure formed by the binding of the target site upstream sequence, target site downstream sequence, and intermediate inserted sequence in the vector to the genome initiates reverse transcription in place of the reverse transcription functional structure in the short interspersed element or its product), and inserts the inserted sequence into the corresponding target site (inserted site) on the genome. Continuing the insertion in the above manner based on the new site generated after insertion will result in sustainable insertion, completing long fragment insertion with no apparent length restriction. If a change in direction is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. As the corresponding sequences on the genome of the sequences upstream and downstream of the target site, sequences containing recombination sites on the genome to be edited (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC), short interspersed elements, partial short interspersed elements, short interspersed element derivatives, long interspersed elements, partial long interspersed elements and / or long interspersed element derivatives, or other sequences that increase homologous recombination efficiency can be searched for and / or selected to insert the sequence and increase the efficiency of homologous recombination, thereby enhancing the gene editing effect. The corresponding gene editing effect can be enhanced by adding recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC), short interspersed elements, partial short interspersed elements and / or short interspersed element derivative sequences in the corresponding sequences on the genome upstream and downstream of the target site. 2. Genomic sequence insertion mediated by RNA as a vector and RNA framework connecting one or more ORF2p functional initiation parts downstream (to minimize the impact on the recipient system, the types of short interspersed element RNA, short interspersed element derivative RNA, long interspersed element RNA, long interspersed element derivative RNA, ORF1p and / or ORF2p in the corresponding recipient system can be selectively adopted): This method does not require connecting the short interspersed element RNA sheared product in vivo to a socket formed from the vector RNA, but connects an RNA frame (framework) consisting of the upstream and downstream sequences of the insertion site (target site) and the intermediate inserted sequence (the upstream and downstream sequences of the insertion site (i.e., the insertion site, target site, target site) (sequences upstream and downstream of the target site (sequences downstream of the target site) (each within 20,000 bp), and the upstream and downstream sequences are added to the intermediate insertion point of the insertion sequence (sequence to be inserted) (within 20,000 bp)) directly downstream with one or more ORF2p functional initiation parts and / or one or more other related sequences, and generates RNA generated from the designed sequence as a vector. The RNA is treated with RNase inhibitors and / or an appropriate amount of Mg 2+(e.g., 6 mmol / L) or other metals or in the cell solution to promote correct folding of the RNA and subsequent binding to the corresponding functional proteins, such as ORF2p and / or ORF1p. Then, the vector is transferred into in vitro cultured cells, tissues, or organs by conventional means such as lipid body transfection, or administered to tissues, organs, or organisms by blood, lymph, cerebrospinal fluid, or local tissue administration, the vector is introduced into the target cytoplasm and then combined with ORF1p and / or ORF2p, or ORF1p and / or ORF2p can directly introduce the vector into the nucleus. After the vector RNA and the short interspersed element RNA or its product of intracellular transcription are linked, they are combined with ORF2p of intracellular expression, ORF2p and ORF1p are combined simultaneously, or they are directly combined with ORF2p expressed in the cell, or ORF2p and ORF1p are combined simultaneously (for example, the Ω structure formed by the combination of the target site upstream sequence, the target site downstream sequence, and the intermediate inserted sequence in the vector with the genome initiates reverse transcription in place of the reverse transcription functional structure in the short interspersed element or its product), and the inserted sequence is inserted into the corresponding target site (inserted site) on the genome. Continuing the insertion in the above manner based on the new sites generated after insertion will result in sustainable insertions, completing the insertion of long fragments with no apparent length limit. Because this method does not require mechanisms specific to eukaryotic systems, such as the splicing machinery, this method is applicable to systems that do not have the eukaryotic pre-mRNA shearing machinery and cannot generate fesostructures, e.g., prokaryotes such as bacteria, as well as to eukaryotes that have the pre-mRNA shearing machinery. If reorientation is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. 3. RNA is used as a vector, and the simple RNA framework simultaneously contains a pan-ORF1p coding sequence and / or a pan-ORF2p coding sequence, or the ORF2p functional initiation portion connecting one or more downstream of the RNA framework simultaneously contains a pan-ORF1p coding sequence and / or a pan-ORF2p coding sequence, mediated by the genome sequence insertion (to minimize the impact on the recipient system, the type of short interspersed element RNA, short interspersed element derivative RNA, long interspersed element RNA, long interspersed element derivative RNA, ORF1p and / or ORF2p in the corresponding recipient system can be selectively adopted): The above two methods add a pan-ORF1p coding sequence and / or a pan-ORF2p coding sequence located before, between, after, or within each component upstream of the upstream sequence of the target site, downstream of the downstream sequence of the target site, upstream or downstream of each ORF2p functional initiation portion (as long as the RNA framework does not affect the formation of "Ω" at the target site) (add sequences upstream and downstream of the insertion site (i.e., the target site) (within 20,000 bp each), and sequences to be inserted at the intermediate insertion point of the upstream and downstream sequences (within 20,000 bp), and generate the above-mentioned RNA as a vector. RNA should be treated with RNase inhibitors and / or an appropriate amount of Mg 2+ (e.g., 6 mmol / L) or other metals or in the cell solution to promote correct folding of the RNA and subsequent binding to the corresponding functional proteins, such as ORF2p and / or ORF1p. The vector is then transferred to in vitro cultured cells, tissues, or organs by conventional means such as lipid body transfection, or administered to a tissue, organ, or organism via blood, lymph, cerebrospinal fluid, or local tissue administration. After the vector enters the target cytoplasm, it is expressed and binds to ORF1p and / or ORF2p, or it binds to the expressed ORF1p and / or ORF2p in the body and enters the cell nucleus, or ORF1p and / or ORF2p can directly introduce the vector into the nucleus. After ligating the vector RNA and the short interspersed element RNA of intracellular transcription or its product, it binds to ORF2p that is self-expressed or encoded by a vector in the cell, or binds ORF2p and ORF1p simultaneously, or directly binds to ORF2p that is self-expressed or encoded by a vector in the cell, or binds ORF2p and ORF1p simultaneously (for example, an Ω structure formed by binding the target site upstream sequence in the vector, the target site downstream sequence, and the intermediate inserted sequence to the genome initiates reverse transcription in place of the reverse transcription functional structure in the short interspersed element or its product), or inserts the inserted sequence into the corresponding target site (inserted site) on the genome by binding one or more ORF2p functional initiation parts connected downstream of the RNA framework to ORF2p that is self-expressed or encoded by a vector in the cell. Continuing the insertion in the above manner based on the new site generated after insertion will result in sustainable insertion, completing long fragment insertion with no apparent length restriction. If a change in direction is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. 4. RNA and / or RNPs containing one or more ORF2p functional start parts, one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences, and / or DNA for expressing one or more ORF2p functional start parts, ORF1p, ORF2p, ORF1p-derived proteins, and / or ORF2p-derived proteins, RNA vectors of simple RNA frameworks, RNA vectors connecting one or more ORF2p functional start parts downstream of the RNA framework, and / or one or more pan-ORF1p coding sequences downstream of the RNA framework, and / or one or more pan-ORF The RNA vectors simultaneously containing one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences, or one or more ORF2p functional start parts connected downstream of the RNA framework, are given to the target system in the same vector and / or in different vectors (to minimize the influence on the recipient system, the type of short interspersed element RNA, short interspersed element derivative RNA, long interspersed element RNA, long interspersed element derivative RNA, ORF1p and / or ORF2p in the corresponding recipient system can be selectively adopted): The RNA vector in "1-3" in the above "1. Genomic sequence insertion technology using RNA as a vector" provides a target system and plays a role, which can be present on the same vector or a different vector as one or more ORF2p functional initiation portions, one or more pan-ORF1p coding sequences, and / or RNA and / or RNP of one or more pan-ORF2p coding sequences, and / or DNA expressing one or more ORF2p functional initiation portions, ORF1p, ORF2p, ORF1p-derived proteins and / or ORF2p-derived proteins, respectively. "RNA containing one or more ORF2p functional start portions" or the corresponding RNA expressing the DNA expression of one or more ORF2p functional start portions may be connected to various RNA vectors and / or RNA frameworks that may or may not be cleaved in vivo, and perform the above-mentioned roles and insert the inserted sequence into the target site. The corresponding proteins expressing RNA comprising one or more long interspersed elements, one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences, or the corresponding proteins expressing DNA expression of the long interspersed elements, ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins, can function as described above and insert the sequence to be inserted into the target site. Continuing the insertion in the above manner based on the new site generated after insertion will result in sustainable insertion, completing long fragment insertion with no apparent length restriction. If a change in direction is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. RNA should be treated with RNase inhibitors and / or an appropriate amount of Mg 2+ (e.g., 6 mmol / L) or other metals or in the cell solution to promote correct folding of the RNA and subsequent binding to corresponding functional proteins, such as ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins. 2. RNP-vectored genomic sequence insertion technology (in order to minimize the impact on the recipient system, the types of short interspersed element RNA, short interspersed element derivative RNA, long interspersed element RNA, long interspersed element derivative RNA, ORF1p and / or ORF2p in the corresponding recipient system can be selectively adopted): First, various RNA vectors in each method described in "Scheme 1" above are prepared, and simultaneously expressed in vitro via a eukaryotic or prokaryotic system, and purified proteins ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins are extracted. The prepared RNA vectors are mixed in vitro with cytoplasm containing ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins, or with physiological fluids containing ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins, and incubated (at an appropriate temperature, which may be room temperature or 37°C, for 48 hours or less). In order to promote accurate folding of the secondary and higher structures of the RNA carrier, Mg 2+ (e.g., metal ion concentration can be appropriately increased) to obtain the RNP vector. The RNP vector is then transferred to in vitro cultured cells, tissues, or organs by conventional means such as lipid body transfection, or administered to tissues, organs, or organisms via routes such as blood, lymph, cerebrospinal fluid, or local tissue administration; the vector or expression sequence carrying ORF2p, ORF1p, ORF2p-derived protein, and / or ORF1p-derived protein may express ORF2p, ORF1p, ORF2p-derived protein, and / or ORF1p-derived protein even after entering the target cytoplasm, and continue to bind if in vitro binding is insufficient, or enter the cell nucleus after binding with ORF2p, ORF1p, ORF2p-derived protein, and / or ORF1p-derived protein expressed in vivo (which may also bind simultaneously with ORF2p, ORF1p, ORF2p-derived protein, and / or ORF1p-derived protein), or the carrier may be mediated by ORF1p and / or ORF2p to directly enter the cell nucleus. Various RNA vectors can still function in vivo if binding to ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins is insufficient in vitro. After ligating the vector RNA and the intracellular transcriptional short interspersed element RNA or its product, it is bound to ORF2p that is self-expressed or encoded by a vector in the cell, or ORF2p and ORF1p are bound simultaneously, or it is directly bound to ORF2p that is self-expressed or encoded by a vector in the cell, or ORF2p and ORF1p are bound simultaneously, or the sequence to be inserted is inserted into the corresponding target site (insertion site) on the genome by binding one or more ORF2p functional initiation parts connected downstream of the RNA framework to ORF2p that is self-expressed or encoded by a vector in the cell. Continuing the insertion in the above manner based on the new site generated after insertion will result in sustainable insertion, completing long fragment insertion with no apparent length restriction. If a change in direction is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. 3. RNA vector and / or RNP vector mediated genome sequence deletion technology 1. Deletion of any region on the genome: The sequence to be inserted in the RNA and / or RNP vector designed in the above-mentioned insertion technology ("1. Genome sequence insertion technology using RNA as a vector" and "2. Genome sequence insertion technology using RNP as a vector") is inserted into a certain sequence (20,000 bp or less) upstream or downstream of the insertion point (up to 100,000 bp) of the insertion point. bp) (if the inserted sequence and its upstream sequence undergo homologous recombination, any sequence can also be inserted between the sequence to be inserted on the genome and the corresponding sequence of the upstream sequence of the target site, without affecting the results or promoting subsequent homologous recombination and / or its action; if the inserted sequence and its downstream sequence undergo homologous recombination, any sequence can also be inserted between the sequence to be inserted on the genome and the corresponding sequence of the downstream sequence of the target site, without affecting the results or promoting subsequent homologous recombination and / or its action), and after inserting this sequence by the RNP or RNA-mediated genome sequence insertion technology described in this invention ("First, genome sequence insertion technology using RNA as a vector" and "Second, genome sequence insertion technology using RNP as a vector"), the sequence between the two same sequences can be removed by homologous recombination. A sequence containing a recombination site (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC) can be selected and inserted to increase the efficiency of subsequent homologous recombination. If a change of direction is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. Furthermore, if the sequence to be removed is 600 bp or less, the corresponding fragment can be deleted by homologous recombination and / or other genome repair mechanisms with high efficiency. 2. Deletion from the end of CNV: In physiological situations, copy number variation (CNV) is similar to a copy of the original complete gene. Through the above-mentioned mechanism, based on the original complete gene, the CNV as a copy can be continuously extended, and the protein expression and various states of cells, tissues and organisms can be constantly changed. The CNV end is composed of an upstream gene portion and a downstream short interspersed element portion (ORF2p functional start portion), but a short sequence fragment formed by the junction of the lariat structure and the short interspersed element (ORF2p functional start portion) is inserted between these two portions to extend the CNV. During early embryo development, transcription of long interspersed elements is significantly increased, and short interspersed elements on the genome, such as Alu sequences, show significant demethylation. At the same time as the extension of initiation-correlated gene copy number variations (CNVs) through long interspersed element-mediated 3' transduction (based on the right monomer deletion of the short interspersed element upstream of the promoter and the complete short interspersed element structure downstream), the demethylated short interspersed element sequences undergo homologous recombination with each other, deleting (initializing) most of the previously extended CNVs. After that, the thoroughly initialized embryonic cells will regain the hypermethylation state again, and the short interspersed elements at the ends of the CNVs will mediate the gradual extension of the ends of the CNVs, thereby changing the expression context and state of each cell, and the gene expression context of each cell will also inversely affect the changes of the CNVs through the lariat structure, thereby causing changes in the genome and gradually inducing differentiation, which is consistent with the universal appearance of CNVs changes in embryos and the differences of CNVs in different tissues. The extension of different gene CNVs is generally present in various tumor cells and shows a positive correlation with clinical classification. At the same time, the expression level of proto-oncogenes and tumor suppressor genes is also proportional to the length of CNVs, so the formation and progression of tumors should be related to the disruption of CNVs of proto-oncogenes or tumor suppressors. In addition, some irreversible diseases related to external stimuli such as diabetes are also related to the disruption of CNVs. Many resistances are related to the expression changes of corresponding proteins due to long-term external stimuli, so they can be related to the CNV changes of the corresponding genes, which can be improved or inhibited by the present invention. CNV ends in cells or tissues are detected by sequencing and comparison (sequence alignment) (binding sites to gene sequences and partial short interspersed elements), and the gene portion (within 2000 bp) in the CNV end to be processed and / or the 3' partial sequence of a lasso that can be formed within a certain range (within 200,000 bp) downstream in the complete gene at the end (lasso that can be formed downstream can be predicted or detected by the method described below) are selected (this end can also be directly selected and cut out within the downstream range (within 200,000 bp) in the complete gene to replace the above-mentioned 3' partial sequence), the corresponding RNA sequence can also be directly selected as the upstream sequence of the target site, and the sequence adjacent to the upstream (within 100,000 bp) of the sequence to be truncated (100,000 bp upstream of the sequence to be truncated) can be selected. Any sequence can be inserted between the adjacent sequence within 1 bp and the part that serves as the upstream sequence of the target site, and it can have no effect on the results or can promote subsequent homologous recombination and / or its action) and the corresponding RNA sequence of the target site is connected as the inserted sequence, and then a complete short interspersed element RNA, partial short interspersed element RNA or similar short interspersed element RNA (i.e., ORF2p functional start portion) (by the different insertion methods described above) (ORF1p and ORF2p coding sequences can be connected after the above) is connected as the downstream sequence of the target site, synthesis is performed, and the adjacent sequence upstream of the sequence to be deleted at the end is inserted between the gene part of the actual CNV end and the partial short interspersed element sequence (the short interspersed element sequence used on the vector is the same as or close to the short interspersed element sequence around the insertion point, which increases the efficiency) via an RNA vector or an RNP vector by one of the gene insertion methods described above, and then the sequence to be deleted is deleted by homologous recombination between the same sequences. Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC) can be selected and inserted to increase the efficiency of subsequent homologous recombination. If direction reversal is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. Multiple corresponding gene editing RNAs, RNPs and / or DNAs can be simultaneously administered to simultaneously delete the CNV ends of multiple genes or multiple different CNV ends of a gene. This method can change gene expression and cell appearance, thereby modifying the cell state, such as the tumor state, such as the tumor grade, or the differentiation state of the cell. 3. Continuous deletion of CNV ends: By continuing to provide the corresponding RNA, RNP, and / or DNA having the gene editing effect of the present invention while deleting the CNV ends or thereafter according to the CNV end deletion method, the CNV ends generated successively can be deleted, and the continuous action of this process allows the CNV of the corresponding gene to be continuously deleted. Corresponding RNAs, RNPs and / or DNAs with multiple gene editing effects can be administered simultaneously to delete the CNV ends of multiple genes or multiple different CNV ends of a gene simultaneously or sequentially. This method can alter gene expression and cell appearance and therefore modify the state of a cell, for example the state of a tumor, for example the tumor grade, or the differentiation status of a cell. 4. RNA vector and / or RNP vector mediated genomic CNV terminal extension or CNV addition technology 1. Addition of new CNVs on genome: Any position of genome can be used as target site, and one or more copies of genes can be added according to the gene editing method of the present invention. Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC) on genome, short interspersed element sequences, partial short interspersed element sequences, and / or short interspersed element derivatives (ORF2p functional start portion) can be searched or (and) selected, and gene copies can be inserted as corresponding sequences on genome of upstream and downstream sequences of target site, and the corresponding gene editing action can be improved by increasing the efficiency of homologous recombination. Increasing the sequence containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC) in the upstream and downstream sequences of the target site in the RNA framework, short interspersed element RNA, partial short interspersed element RNA, and / or short interspersed element derivative RNA can increase the corresponding gene editing effect. The end of the newly added gene copy (i.e., the newly generated CNV end) can continue gene editing relative to it and be extended or shortened. It is possible to search for sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC and / or CCAGC), short interspersed element sequences, partial short interspersed element sequences, and / or short interspersed element derivatives (DNA sequences corresponding to the functional start portion of ORF2p) on the genome, and insert new gene copies through these sequences as the corresponding sequences on the genome of the downstream sequence of the target site, thereby increasing efficiency, in particular, downstream sequences that target partial short interspersed element RNA are closer to the downstream sequence of the CNV end in natural state. Multiple corresponding RNAs, RNPs and / or DNAs having gene editing effects can be administered simultaneously to add CNVs of multiple genes or CNVs of multiple different lengths and states of a gene simultaneously or sequentially. This method can alter gene expression and cell appearance and therefore modify the state of a cell, for example the state of a tumor, for example the tumor grade, or the differentiation status of a cell. 2. Adding and extending the CNV end on the genome: Adding and extending the existing CNV end on the genome. According to the above-mentioned sequence insertion method of the present invention, the upstream part (gene part) of the CNV end is the corresponding sequence on the genome of the upstream sequence of the target site, the downstream part (short interspersed element sequence (ORF2p functional start part)) of the CNV end is the corresponding sequence on the genome of the downstream sequence of the target site, and the downstream sequence in the complete gene sequence of the upstream part (gene part) of the CNV end is the corresponding sequence on the genome of the sequence to be inserted, thereby extending the corresponding gene CNV end. Multiple corresponding gene editing RNAs, RNPs and / or DNAs can be administered simultaneously to simultaneously add and / or extend the CNV ends of multiple genes or multiple different CNV ends of a gene. This method can alter gene expression and cell appearance and therefore modify the state of a cell, for example the state of a tumor, for example the tumor grade, or the differentiation status of a cell. 5. Genome sequence replacement technology (including sequence replacement, site deletion, site addition, sequence addition, sequence deletion, and site replacement) The sequence to be inserted in the vector designed in the insertion technique is a replacement sequence and a sequence surrounding the sequence to be replaced on the genome (i.e., a sequence to be deleted after homologous recombination between the DNA sequence of the inserted replacement sequence and the sequence on the genome, and whether it is located 3' or 5' of the replacement sequence during vector construction depends on whether the insertion position is upstream or downstream of the replacement sequence on the genome) (the DNA sequence of the replacement sequence should be homologous or similar to the sequence to be replaced on the genome) (when the inserted sequence undergoes homologous recombination with its upstream sequence, any sequence can also be inserted between the corresponding sequence of the inserted sequence on the genome and the corresponding sequence of the upstream sequence of the target site, without affecting the result or subsequent homologous recombination and / or or facilitate its action, and when the sequence to be inserted undergoes homologous recombination with its downstream sequence, any sequence can also be inserted between the sequence to be inserted on the genome and the downstream sequence of the target site, without affecting the results or promoting subsequent homologous recombination and / or its action), and the replacement sequence and the surrounding sequences of the sequence to be replaced on the genome are inserted upstream or downstream of the sequence to be replaced on the genome by the gene editing insertion method, and when homologous recombination occurs between the inserted replacement sequence and the sequence to be replaced on the genome, the sequence to be replaced on the genome is replaced with the inserted replacement sequence that is homologous to it, and the surrounding sequence portion of the sequence to be replaced that was deleted by homologous recombination is reinserted together with the replacement sequence at the time of sequence insertion. Genome replacement includes sequence replacement and site replacement, where sequence replacement, i.e. the replacement sequence to be inserted does not match the corresponding sequence on the genome, such as by having one or several sequences; site replacement, where the replacement sequence to be inserted does not match the corresponding sequence on the genome, such as by having one or several sites; site deletion, where the replacement sequence to be inserted is missing one or several sites compared to the corresponding sequence on the genome; site addition, where the replacement sequence to be inserted has one or several more sites compared to the corresponding sequence on the genome; sequence addition, where the replacement sequence to be inserted has one or more additional sequences compared to the corresponding sequence on the genome; and sequence deletion, where the replacement sequence to be inserted is missing a portion of the sequence, such as one or more sequences, compared to the corresponding sequence on the genome. The smaller the difference between the replacement sequence to be inserted and the corresponding homologous sequence on the genome, the higher the efficiency. In order to increase efficiency, mismatches between the replacement sequence to be inserted and the corresponding homologous sequence on the genome should be avoided as much as possible from being located at or adjacent to both ends or both sides of the replacement sequence to be inserted. If reorientation is required, the outer wrap of the vector can be modified. Note that the whole process avoids RNA degradation. 6. RNA vector and / or RNP vector mediated deletion of genomic sequences is accompanied by sequence insertion, sequence replacement or site replacement. By adding a sequence that needs to be inserted into the genome after a certain sequence (within 20,000 bp) upstream or downstream (within 100,000 bp) of the insertion site in the above-mentioned "RNA vector and / or RNP vector mediated genome sequence deletion technology", it is possible to delete the target sequence and at the same time insert the sequence to be inserted into the genome upstream or downstream of the deleted sequence. In the above-mentioned "RNA vector and / or RNP vector-mediated genome sequence removal technology" operation, if a certain sequence within 20,000 bp upstream or downstream (within 100,000 bp) of the insertion site (target site) is replaced with a sequence at the site or partial sequence that is different from the certain sequence within 20,000 bp upstream or downstream (within 100,000 bp) of the insertion site (target site) (the different site and / or partial sequence is the site and / or sequence to be replaced), then a site or partial sequence at the upstream or downstream sequence of the deleted sequence can be replaced with the site and / or partial sequence to be replaced. Seventh, inhibiting genomic changes caused by translocations and stabilizing the genome and the CNVs thereon (i.e., this gene editing technology inserts a sequence that is not identical or homologous to the genome or the gene part of the CNV end and the upstream, downstream or upstream / downstream sequence of this gene part in the complete gene into the region between the gene part of the CNV end and the partial interspersed element sequence or other region, thereby inhibiting further extension of the CNV. The CNV end is defined as the partial short interspersed element sequence or other related sequence (corresponding DNA sequence of the ORF2p functional start portion) to which the gene sequence is directly bound, where the gene can be extended, and the specific sequence of each specific CNV end gene sequence and partial short interspersed element sequence or other related sequence (corresponding DNA sequence of the ORF2p functional start portion) can be obtained by molecular biology means such as gene sequencing or gene chip) (lipid-soluble substances and cell transfection ability Each vector is packaged in a substance having the above structure, for example, liposomes, or other conventional transfection means, and then transferred to in vitro cultured cells, tissues, or organs, or transferred to tissues, organs, or living organisms via blood, lymph, cerebrospinal fluid, or other routes, or via local tissue administration, or other methods.) (The RNA used below can be replaced with a DNA vector capable of expressing each RNA, which is expressed in each target system to generate RNA, and / or RNP formed by binding the generated RNA to ORF1p, ORF2p, ORF1p-derived protein, and / or ORF2p-derived protein generated by the DNA vector; the RNA used below can also be replaced with RNP formed by binding the corresponding RNA to ORF1p, ORF2p, ORF1p-derived protein, and / or ORF2p-derived protein, if possible or necessary.) 1. Intervene in a specific CNV (the upstream sequence used for insertion is the gene part at the CNV end of a specific gene): Select the CNV that needs to be manipulated, set up an insertion point (target site) at the boundary between the 3' end of the gene part and a short interspersed element or other related sequence (the corresponding DNA sequence of the ORF2p functional start part), the corresponding RNA sequence at the 3' end (within 20000 bp) of the gene part at the CNV end is set as the upstream sequence of the insertion site (target site) in the above-mentioned insertion method, and the downstream sequence is a partial short interspersed element RNA or other related sequence (ORF2p functional start part) (so the short interspersed element RNA, partial short interspersed element RNA, or short interspersed element derivative RNA (ORF2p functional start part) connected after the downstream sequence of the target site in the above-mentioned method can be omitted), the sequence to be inserted is any sequence (within 20000 bp) non-homologous to the genome or the gene part at its CNV end and the upstream and downstream sequences in its complete gene. After the vector construction is completed, it is transferred into corresponding cells, biological tissues or organisms via the above-mentioned RNA vector and / or RNP vector pathway, and the heterologous sequence is inserted into the corresponding CNV end.Since the heterologous sequence does not exist downstream of the corresponding CNV end gene sequence in the complete gene, the CNV end cannot be further extended based on the complete gene sequence, thereby inhibiting further changes of the CNV end. 2. Intervention in widespread CNVs in the genome (the sequence upstream of the target site used for insertion must include the gene portions of all possible CNV ends): (1) Genome disruption sequence method: Cells from the organism, tissue, or cell system that needs to be manipulated are cultured in vitro, or the genome is extracted directly, and after ultrasonic disruption, the cells are amplified using random primers and PCR. A short random sequence (within 20 bp) is designed and synthesized, and a partial short interspersed element sequence is connected downstream. The genomic fragments obtained by amplification are connected to the synthesized short random sequence connecting part short interspersed element sequence fragments, and the connections and amplification are performed by PCR to obtain different genomic fragment sequence connecting random sequences, and then the partial short interspersed element connecting sequence or other related sequences (corresponding DNA sequence of the ORF2p functional start part), the resulting fragments are constructed to generate corresponding RNA, and transferred to the corresponding cells, biological tissues or organisms via the above-mentioned RNA vectors and / or RNP vectors, targeting all CNV ends on the genome through the genomic fragment (fragment) sequence, and the non-homologous sequence is inserted between the gene part of the CNV end and the partial short interspersed element (i.e., the short random sequence or the partial short random sequence is not a part of the gene fragment, but is also non-homologous to the local gene sequence of the corresponding gene fragment), and the non-homologous sequence does not exist downstream of the corresponding CNV end gene fragment sequence in the complete gene, thus inhibiting further changes of the CNV end. (2) Random sequence method: Generate random sequences (within 100 bp) containing appropriate length (including all possible sequence combinations and excluding combinations that are close to short interspersed element sequences) connecting arbitrary and genomic non-homologous sequences (within 20,000 bp), and then connect partial short interspersed element RNA or other related sequences (ORF2p functional start part), or include random sequences (within 100 bp) connecting partial short interspersed element RNA or other related sequences (ORF2p functional start part), and add arbitrary genome non-homologous sequences (within 2,000 bp) after the intermediate natural shearing site. It can also be produced by synthesizing RNA connected to any genomic heterologous sequence (hereinafter referred to as a lasso) after the random sequence, and at the same time, a vector for transcribing short interspersed element RNA and / or partial short interspersed element RNA can be constructed, and a DNA sequence or RNA sequence of a long interspersed element sequence or its protein coding sequence corresponding to the short interspersed element function (or the short interspersed element and / or partial short interspersed element RNA can be directly introduced into the target system) can be combined or additionally expressed downstream of the short interspersed element RNA. Then, the above vector is transferred into the corresponding cell, living tissue or organism, and all CNV ends on the genome are targeted by the above mechanism and method through the random sequence, and the heterologous sequence is inserted into the corresponding CNV end, and the heterologous sequence does not exist downstream of the gene part of the corresponding CNV end in the complete gene, thereby inhibiting further changes of the CNV end. (3) For all lariat species (inserting a small segment of random sequence (within 100 bp) that is non-homologous to the genome into a short interspersed element, the expressed short interspersed element RNA is still sheared normally into a partial short interspersed element (i.e., the insertion position of this non-homologous sequence is downstream of the natural shearing site of the short interspersed element, not at the shearing site), constructing a plasmid that can express this modified short interspersed element RNA, introducing amplified cells from the corresponding engineered organism or a cell line to extract the corresponding species (taking the genome of the corresponding measured species, and arranging the whole genome into a short interspersed element that is long in length (more than 200 bp) and overlaps with each other to some extent (10 bp or more overlap), construct a vector, and overexpress in in vitro cells of the corresponding species via RNA polymerase II), after a while, extract the corresponding nucleic acid based on the sequence specificity of the non-homologous sequence inserted into the short interspersed element RNA, perform sequencing, and detect the part where the non-homologous sequence is inserted (obtain the sequence information of various generated lassos connected to the short interspersed element), and / or simultaneously predict the lassos sequence based on the sequence rules of forming lassos based on pre-mRNA (for example, often ends with AG), obtain all the lassos sequence information of that species or individual, by the lassos end sequencing method. Each lassos 3' sequence (within 20000 bp) is respectively connected to the genome and a non-homologous sequence (within 20000 bp) to synthesize RNA containing the short interspersed element RNA (according to the above, the short interspersed element function can then be combined with the corresponding long interspersed element or its protein coding sequence to increase efficiency), and jointly introduce the target system. The resulting 3' sequence per lasso generates RNA that connects with the genomic non-homologous sequence (within 2000 bp) and then connects with a short interspersed element (as mentioned above, the short interspersed element function can be followed by a long interspersed element or its protein coding sequence to increase efficiency) (the SINE sequence is preferably the same as or similar to the SINE sequence in the gene with the socket 3' sequence linked to it to increase efficiency). The above RNP vector or RNA vector is used to introduce the corresponding cell, tissue, or organism to edit the CNV ends within the entire genome. (4) Short interspersed element sequence modification method: In other words, the short interspersed element RNA modified by additional administration inserts a sequence that is non-homologous to the gene portion in the genome or CNV end and the upstream and downstream sequences in the complete gene of the gene portion into each CNV end, thereby inhibiting the extension of the end. A short sequence (that does not match the 3' sequence of the lasso that occurs in normal cases, and can be a sequence of one small step spanning the short interspersed element natural shearing site (within 100 bp)) is added before the natural shearing site of the short interspersed element RNA, and the short interspersed element transcript (RNA) can be naturally sheared in this novel region as well to generate a complete short interspersed element sequence RNA (to increase efficiency, a long interspersed element or its protein coding sequence corresponding to the corresponding type of short interspersed element function can be added thereafter). Alternatively, any genome non-homologous sequence (within 200 bp) is added after the natural shearing site of the short interspersed element transcript, and a complete short interspersed element RNA (to increase efficiency, a long interspersed element or its protein coding sequence corresponding to the corresponding type of short interspersed element function can be added thereafter) or an RNA containing that RNA sequence can be produced and administered to the corresponding cell, living tissue, or organism. The short interspersed element sequence used must cover all short interspersed elements of the species or individual as much as possible in order to accurately correct all CNV ends on the entire genome (can be obtained by methods such as sequencing or array chips). The whole genome can also be cut into a certain overlapping length fragment (the overlapping length is equal to or greater than the length of one lariat structure) and the RNA of this long fragment is generated and transferred into an in vitro cell system of the corresponding species to generate a lariat structure, and then the corresponding RNA of the modified short interspersed element sequence produced above (downstream, a long interspersed element sequence or its protein coding sequence corresponding to the corresponding type of short interspersed element function can be added, and mediated through the RNA pathway) is transferred, and then the single-stranded RNA ribonucleoprotein complex (RNP) or RNA having the biological activity of the partial short interspersed element (generated from the modified short interspersed element) to which the lariat is bound produced is isolated and purified by properties such as sequence specificity and conventional means, and then acted on by the corresponding RNA or RNP pathway. 3. Modifying the ORF2p functional start site on the genome: A non-coding region associated with the transcription of a short interspersed element, short interspersed element derivative, or functional structure of the initiated ORF2p shearing function and reverse transcription on the genome, such as a cis-element such as a promoter, enhancer, regulatory sequence or inducible element, a transcribed region therein, a natural shearing site of the transcript, or other sequences on the functional structure of the initiated ORF2p shearing function and reverse transcription, and / or a non-coding region associated with the transcription of a long interspersed element, long interspersed element derivative, such as a cis-element such as a promoter, enhancer, regulatory sequence or inducible element, a transcribed region therein, a protein coding sequence or other sequences, by inserting any sequence (within 500 bp) according to the present invention, the short interspersed element sequence, short interspersed element derivative sequence, functional structure of the initiated ORF2p shearing function and reverse transcription on the genome cannot be sheared after transcription, and / or the long interspersed element sequence or long interspersed element derivative cannot be transcribed or cannot produce a protein with normal function. First, the sequences of short interspersed elements, long interspersed elements, short interspersed element derivatives, long interspersed element derivatives, functional structure sequences of the activating ORF2p shearing function and reverse transcription, and the cis element sequences of the relevant regions related to the transcription of the corresponding sequences, such as promoters, enhancers, regulatory sequences or inducible elements, are measured in the whole genome of the individual to be prepared for the operation, and the corresponding sequences are obtained. The non-coding regions related to the transcription, such as cis elements such as promoters, enhancers, regulatory sequences or inducible elements, transcribed regions therein, natural shearing sites of the transcripts, protein-coding sequences or other sequences are selected as the target sites, and the upstream and downstream sequences of the target sites in the invention are short interspersed elements, long interspersed elements, short interspersed element derivatives, long interspersed element derivatives, and functional structure sequences of the activating ORF2p shearing function and reverse transcription, and the sequences to be inserted are any sequences. By the above insertion method, any sequence is inserted into the corresponding site of the short interspersed element, long interspersed element, short interspersed element derivative, long interspersed element derivative, or functional structure of the starting ORF2p shearing function and reverse transcription on the genome. Also, by the above gene editing method, the short interspersed element, long interspersed element, short interspersed element derivative, long interspersed element derivative, or functional structure of the starting ORF2p shearing function and reverse transcription on the genome may be replaced (sequence replacement, site deletion, site addition, sequence addition, sequence deletion and / or site replacement) or deleted to inactivate or reduce the function. 4. Fixing the CNV end while deleting it: Select the CNV end that needs to be manipulated, and set the target site at the boundary between the 3' end of the gene part and the partial short interspersed element or other related sequence (the corresponding DNA sequence of the ORF2p functional start part), the 3' end (within 2000 bp) of the CNV end gene part is the target site upstream sequence in the above insertion method, and the target site downstream sequence is the partial short interspersed element RNA or other related sequence (the ORF2p functional start part) (therefore, the partial short interspersed element connected after the downstream sequence of the target site in the above method can be omitted), the sequence to be inserted is the corresponding RNA sequence that connects an arbitrary sequence (within 20000 bp) different from the genome sequence after the sequence adjacent to the upstream of the sequence to be deleted (within 100000 bp) on the genome (an arbitrary sequence can also be inserted between the corresponding sequence of the sequence to be inserted on the genome and the corresponding sequence of the upstream sequence of the target site, which does not affect the results or can promote subsequent homologous recombination and / or its action). After producing the above RNA or RNP vector, it is transferred into the corresponding cell, tissue or organism through the above RNA or RNP pathway, and a non-homologous sequence is inserted into the corresponding CNV end after the sequence adjacent to the upstream of the sequence to be deleted on the genome, and the intermediate sequence is deleted after homologous recombination occurs between the two identical sequences. The non-homologous sequence simultaneously inhibits further extension of the CNV. 5. Intrinsic mechanism suppression method: The intrinsic CNV elongation mechanism of a cell or organism can also be directly suppressed. For example, by RNA interference, the transcription of short interspersed elements, long interspersed elements, short interspersed element derivatives, long interspersed element derivatives, or other related sequences (corresponding DNA sequences of ORF2p functional activation parts), and the production of their RNA and encoded proteins, such as ORF1p, ORF2p, ORF1p-derived proteins, or ORF2p-derived proteins, can be suppressed, and the function of a complex of specific proteins and proteins related to this CNV elongation mechanism, such as ORF1p, ORF2p, ORF1p-derived proteins, or ORF2p-derived proteins, or spliceosomes, can be suppressed to inhibit its function. By combining with functional structures, the above-mentioned gene editing technology can be used to modify short interspersed elements in the genome, such as Alu and various MIRs, short interspersed element derivatives, various long interspersed elements and long interspersed element derivatives, and the corresponding protein coding sequences therein, to inactivate or reduce their activity, thereby suppressing the function of related proteins in homologous recombination or mismatch repair, or administering modified nucleoside substances to inhibit the progress of reverse transcription, thereby suppressing the endogenous CNV elongation mechanism, thereby inhibiting genomic changes and stabilizing CNVs. Eight, editing the genome by administering long fragment RNA or RNP: The intracellular splicing mechanism of RNA in eukaryotic cells can generate lariat structures that overlap each other in sequence, so theoretically, these lariat structures that overlap each other in sequence are multiple RNA frames that contain the corresponding upstream sequence of the target site, the downstream sequence of the target site, and the sequence to be inserted. The downstream sequence of the target site of the RNA framework contained in one lariat structure is the upstream sequence of the target site of the RNA framework contained in the other lariat structure that overlaps in sequence. Therefore, if there is already a partial Alu sequence that connects to the upstream sequence of the sequence to be inserted in the RNA or RNP that you want to give to the genome. After administering RNA or RNP with a long sequence, the sequence information can be gradually inserted into the genome, and finally, the above-mentioned gene editing tasks, such as genome insertion, genome deletion, genome sequence replacement, genome site replacement, sequence insertion, sequence replacement or site replacement simultaneously with genome sequence deletion, are performed, so as to inhibit genome changes caused by transposons, stabilize the genome and the CNVs thereon, and complete genome sequence insertion, genome deletion, genome sequence replacement, genome site replacement, sequence insertion, sequence replacement or site replacement simultaneously with genome sequence deletion, etc., for the CNV ends on the genome. 9. Support other gene editing technologies: The present invention can produce RNA, single-stranded and double-stranded DNA in the target system, and these RNA, single-stranded and double-stranded DNA can provide templates for gene editing (e.g., homologous recombination and other actions) for other gene editing technologies, and can provide DNA templates for gene editing such as inserting foreign sequences (sequences to be inserted) after gene editing for other gene editing technologies, such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas 9, to support and promote the corresponding gene editing technologies. In the present invention, the upstream and downstream sequences of a target site on single-stranded or double-stranded DNA produced by an RNA, RNP and / or DNA vector having gene editing activity are complementarily bound to the corresponding sequences on the genome, and the sequence to be inserted is inserted into the single-stranded or double-stranded DNA cut produced by cleaving the target site on the genome using technologies such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas 9, thereby inserting the sequence to be inserted into the target site on the genome, thereby assisting gene editing by technologies such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas 9 or increasing its efficiency. In the present invention, RNA containing other sequences such as the upstream sequence of the target site, the sequence to be inserted, the downstream sequence of the target site, and / or other sequences such as short interspersed element RNA, partial short interspersed element RNA, etc., ORF2p function activation portion, and ssDNA and / or dsDNA generated by its reverse transcription, these components can assist homologous recombination in technologies such as TALEN, ZFN, Targetron, CRISPR, CRISPR / Cas 9, etc., or insert appropriate sequences into the target site, promote the application of RNA, non-viral transfection (RNA-introduced cells do not need to be introduced into the nucleus, and can be introduced into the nucleus at the non-dividing stage under the binding and action of corresponding proteins such as ORF1p and / or ORF2p), and improve the efficiency of genome sequence insertion in the corresponding technology. In addition, the DNA vector expressing the RNA framework (including other sequences such as the target site upstream sequence, the sequence to be inserted, the target site downstream sequence, and the ORF2p function activation part such as short interspersed element RNA, partial short interspersed element RNA, etc.) can continuously produce RNA containing the RNA framework, and the RNA can be converted into single-stranded DNA or double-stranded DNA by the reverse transcription functional structure on the RNA; other gene editing technologies, such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas 9, cut the genome and then perform gene editing (such as homologous recombination or other actions), such as inserting an exogenous sequence (the sequence to be inserted); the present invention provides a DNA template that can continuously assist and promote the action of the corresponding gene editing technology. At the same time, RNA containing an RNA framework (including target site upstream sequence, sequence to be inserted, target site downstream sequence, and other sequences such as ORF2p function activation part such as short interspersed element RNA, partial short interspersed element RNA, etc.) binds ORF2p and / or ORF1p in vitro, and then is administered to the target, so that ORF2p and / or ORF1p convert the RNA into single-stranded DNA and / or double-stranded DNA in the target body, and other gene editing technologies such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas 9 cut the genome, and then perform gene editing (such as homologous recombination and other actions), such as inserting an exogenous sequence (sequence to be inserted), and the present invention provides a DNA template that can sustainably assist and promote the action of the corresponding gene editing technology. At the same time, after expressing ORF2p and / or ORF1p in the target body, RNA containing an RNA framework (including target site upstream sequence, sequence to be inserted, target site downstream sequence, and other sequences such as ORF2p function activation part such as short interspersed element RNA, partial short interspersed element RNA, etc.) is given to the target, and after ORF2p and / or ORF1p bind with RNA in the target body, the RNA is converted into single-stranded DNA and / or double-stranded DNA, and other gene editing technologies such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas 9 cut the genome, and then perform gene editing (such as homologous recombination and other actions), such as inserting exogenous sequences (sequences to be inserted), and the present invention provides a DNA template that can be sustained. Assists and promotes the action of the corresponding gene editing technology. The RNA used in the present invention may be linear or circular, and the circular RNA can be obtained by adding complementary sequences, such as Alu element sequences or intron sequences, longer than 5 bp on both sides of the RNA framework to generate circular RNA in vitro or in vivo and exert the corresponding action in the present invention. The sequences on both sides of the RNA framework are designed to allow intron self-shearing to occur so that circular RNA containing the RNA framework can be generated in vivo or in vitro. The RNA binding protein (RBP) binding sites on both sides of the RNA framework can also generate circular RNA containing the RNA framework in vivo or in vitro. By applying the technology related to the present invention, the copy number variation and its terminal part in each gene can be edited, the terminal position can be changed, or the terminal can be stabilized, which determines the gene expression and thereby achieves the purpose of stabilizing or changing each state of cells and organisms, and therefore it can be said to "modify the genes and states of cells, tissues and organisms, modify the genome of organisms such as humans to improve function, modify the genome of organisms such as humans to treat gene-related genetic diseases such as Huntington's disease and fragile X syndrome, delay or stop the change of genes or states of cells or organisms, change the genes or states of cells or organisms, and improve tissue and organ reconstructing and regenerating organisms, transforming somatic cells into germline assisted reproduction by introducing transcription factors, preventing or delaying neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple atrophy, primary lateral sclerosis, spinocerebellar ataxia, Pick's disease, frontal lobe dementia, Louisiana dementia, progressive supranuclear palsy, inhibiting the metabolic activity of tumor cells, simultaneously slowing the proliferation rate and production and improving their malignancy, as well as the research and treatment of all other diseases associated with gene and CNVs changes such as diabetes, and other fields of physiology, pathology and pathophysiology research. The present invention can prevent the occurrence of, inhibit the proliferation of, inhibit the increase in the level and progression of, or reverse the nature of, glioma, breast cancer, cervical cancer, lung cancer, stomach cancer, colorectal cancer, duodenal cancer, leukemia, prostate cancer, endometrial cancer, thyroid cancer, lymphoma, pancreatic cancer, liver cancer, melanoma, skin cancer, pituitary tumor, germ cell tumor, meningioma, meningeal cancer, glioma, astrocytoma, oligodendrocyte tumor, astrocytoma, ventricular tumor, choroid plexus papilloma, choroid plexus carcinoma, chordoma, ganglioneuroma, olfactory neuroma, sympathetic neuroma, pinealoma, pineal tumor, medulloblastoma, trigeminal neurinoma, facet neuroma, jugular neurinoma, hemangioplexoma, craniopharyngeal tumor or granular cell tumor and its metastasis. Prevent, delay or ameliorate resistance to drugs such as insulin, levodopa, various tumor chemotherapy drugs and targeted drugs, delay or stop genetic and state changes of cells and organisms, regeneration of tissues and organs and biological regeneration. In the present invention, a certain sequence or site (e.g., a sequence to be inserted and a sequence upstream of a target site on the genome, a sequence downstream of the target site, or sequences on both sides of the target site) (this sequence is DNA or RNA) is defined along the 5'→3' direction, with upstream being before the 5' end of the determined sequence or site, downstream being after the 3' end of the determined sequence or site, an upstream sequence being the sequence before the 5' end of the determined sequence or site, and a downstream sequence being the sequence following the 3' end of the determined sequence or site. When designing an RNA vector or RNP vector sequence, software (e.g., PCFOLD or RNAFOLD) can be used to simulate the secondary structure of the RNA vector or RNP vector, making the target site upstream sequence, especially the free end or free portion of the target site upstream sequence, more single-stranded free, and creating fewer complementary sequences inside the RNA vector or RNP vector sequence, especially the free end, free portion, or part close to the free end of the target site upstream sequence, thereby improving gene editing efficiency. Furthermore, by designing the sequence, the secondary structure of the designed inserted sequence RNA can be made closer to the secondary structure of partial short interspersed element RNA such as partial Alu (such as forming a complementary double-stranded structure on both sides of the bottom notch of the Ω structure formed by the designed sequence, and / or other stem ring structures or protrusion structures in the secondary structure that mimic partial short interspersed element RNA (such as partial Alu)), thereby improving the efficiency of gene editing, such as increasing the efficiency of action of ORF2p. The circular secondary structure and higher structures in the "Ω" structure necessary for the function of ORF2p in the RNA framework and its improved form RNA are preferably simulated to improve efficiency by being close to the circular secondary structure and higher structures in the "Ω" structure in the natural SINE and LINE transcription products corresponding to the ORF2p used, including the stalk ring, protrusion, and the AA bond adjacent to the base of both legs of the two "Ω" structures and the formed double-stranded complementary structures, and including the shape, length, and relative position of the corresponding secondary and higher structures to the base of both legs of the "Ω" structure and sequence similarity of the corresponding positions. The sequence of the right leg of the "Ω" structure, the secondary and higher structures can also be simulated to improve efficiency by being close to the sequence of the right leg of the natural SINE and LINE transcription products corresponding to the ORF2p used, including the stalk ring, protrusion, and double-stranded complementary structures, and including the shape, length, relative position (calculated in the 3' direction from the start of the right leg) of the corresponding secondary and higher structures, and sequence similarity of the corresponding positions. At the same time, the two bases closest to both legs on the open ring structure between the legs in the "Ω" structure can be designed as a pair of bases with adenine (A) or other mismatched weak bonds, which can stabilize the "Ω" structure to a certain extent and improve the gene editing efficiency. LINEs are also classified into stringent and relaxed types, and the 3'UTR portion of the transcripts of stringent LINEs can form a relatively conservative secondary structure with a special structure, which forms a stalk-ring structure at a specific position, characterized by an asymmetric ring or protrusion 4-6 bp from the central ring, which promotes the binding or function of ORF2p in the corresponding species. On the other hand, relaxed types generally do not form this structure, but in some cases may form a similar structure (ring length 5-7 bp, 8-10 bp stalk and one protrusion 4-6 bp away from the ring) which may promote the binding and function of ORF2p. LINEs in humans and most mammals are relaxed, while LINEs in eels (LINE UnaL2), insects (LINE R2), zebrafish (LINE ZfL2-1 and ZfL2-2), algae (L1), silkworms (LINE SART1), monocots (L1), fungi (Tad1), fish (L2) and some mammals (RTE) are strict. For the application of ORF2p corresponding to (generated from) strict LINEs, adding the above stem loop structure to the ORF2p function activation part can increase the binding or activity efficiency of the corresponding ORF2p, while for relaxed LINEs, adding the above stem loop structure (e.g., the "UCCCGCCUGGGCCACAGAGCGAGA" sequence in the Alu element) to the ORF2p function activation part can also increase the binding or activity efficiency of the corresponding ORF2p. The target site on which the present invention operates may be one or more. When there are multiple target sites, the corresponding genomic single strands of ORF2p and / or ORF2p-derived protein excision genome generating primers corresponding to different target sites may be located on the same strand on the same chromosome, complementary strands on the same chromosome, or on different chromosomes. By designing the sequence, the transcribed RNA sequence can have two inverted repeat sequences (e.g., Alu elements, other SINEs, or other inverted repeat sequences) or mutually complementary sequences, and when the two inverted repeat sequences or complementary sequences on the RNA are bound, the part between the two sequences can be made into a circular RNA. In addition, when designing the sequence, RNA shearing signals (sites) can be added to the sequence to promote the formation of circular RNA from linear RNA. In order to promote the progress of DNA homologous recombination, Lig4, DNA-PK, and XRCC6 are specifically suppressed by means of sgRNA, ASO, siRNA, or specific antibodies, thereby improving the efficiency of gene editing in the present invention. The basic structure of the RNA framework for gene editing provided by the present invention includes a target site upstream sequence, a sequence to be inserted, and a target site downstream sequence along the 5'→3' direction, as shown in Figure 3. In order to better understand the form of further change in the RNA framework, several different connection formats are listed for understanding. "Connection" refers to direct connection, and when other sequences are inserted in between, it is called "indirect connection". The intermediate insertion sequence in the "indirect connection" may be any sequence, where the any sequence is a sequence associated or unassociated with the transcription of the RNA framework provided by the present invention, such as the pan-ORF1p coding sequence, the pan-ORF2p coding sequence, long interspersed elements, short interspersed elements, etc., associated with the transcription of the RNA framework, or other coding or non-coding sequences unassociated with the transcription of the RNA framework. In the present invention, "intermediate" means between two sequences, and these two sequences are still intact, whereas "internal" refers to a sequence that is within a sequence, and when one sequence is inserted inside another sequence, it divides the other sequence into two parts. In the present invention, "interval sequence" refers to a number of different sequences, and when each sequence appears once or multiple times, it refers to the arrangement between the different sequences. For example, when sequence A and sequence B appear repeatedly, ABA, ABAB, ABBA, ABBABB, etc. are all different interval sequence forms of sequence A and sequence B. For example, when sequence A, sequence B, and sequence C appear repeatedly, ABCABC, ABBCA, CCABA, etc. are all different interval sequence forms of sequence A, sequence B, and sequence C, and so on for more sequences. (Figure 4) This shows that one ORF2p functional start portion (ORF2p functional activation portion) is further connected downstream of the basic structure of the RNA framework. (Figure 5) Downstream of the basic structure of the RNA framework, multiple ORF2p function-initiating parts are further connected. (Figure 6) In the basic structure of the RNA framework, the downstream sequence of the target site is replaced with the ORF2p function-initiating portion. (Figure 7) In the basic structure of the RNA framework, the downstream sequence of the target site is replaced with multiple ORF2p function-activating segments. (Figure 8) The ORF2p function-activating portion is connected downstream of the basic structure of the RNA framework, and the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence is connected downstream of the ORF2p function-activating portion. (Figure 9) The ORF2p function activation portion is connected downstream of the basic structure of the RNA framework, and the pan-ORF1p coding sequence and / or pan-ORF2p coding sequence are further indirectly connected downstream of the ORF2p function activation portion, thereby representing an indirect connection. (Figure 10) An ORF2p function-activating portion is connected downstream of the basic structure of the RNA framework, and multiple pan-ORF1p coding sequences and / or pan-ORF2p coding sequences are further connected downstream of the ORF2p function-activating portion. (Figure 11) The ORF2p functional activation portion is connected downstream of the basic structure of the RNA framework, and a pan-ORF1p coding sequence and / or a pan-ORF2p coding sequence is inserted within the downstream sequence of the target site. (Figure 12) The pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence are connected downstream of the basic structure of the RNA framework, and the ORF2p function activation portion is connected further downstream. (Figure 13) The RNA framework infrastructure is indirectly connected to the ORF2p functional activation portion. (Figure 14) The RNA framework base structure and ORF2p functional activation portion are each located on different RNA vectors and / or RNPs. (Figure 15) Two ORF2p function-initiating parts are connected downstream of the basic structure of the RNA framework, and two ORF2p function-initiating parts are indirectly connected. (Figure 16) The ORF2p function-initiating portion is connected downstream of the basic structure of the RNA framework and is located on an RNA vector and / or in an RNP different from other ORF2p function-initiating portions. (Figure 17) The ORF2p function activation portion is connected downstream of the basic structure of the RNA framework, and the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence are directly connected downstream, and the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence are indirectly connected to the downstream ORF2p function activation portion. (Figure 18) The ORF2p function-activating portion is connected downstream of the basic structure of the RNA framework, and the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence are connected downstream, and are located on an RNA vector and / or in an RNP different from the other ORF2p function-activating portions. (Figure 19) Downstream of the RNA framework basic structure are spaced pan-ORF1p coding sequences and / or pan-ORF2p coding sequences and the ORF2p functional activation portion. (Figure 20) The ORF2p function activating portion is connected downstream of the basic structure of the RNA framework, and the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence are connected within the ORF2p function activating portion. (Figure 21) The ORF2p functional activation portion is connected downstream of the RNA framework basic structure to form a circular RNA form. In the following examples, the material used is a human-derived cell, so the short interspersed element used is Alu Ya5 in the primate-specific short interspersed element Alu element. The complete sequence of the Alu Ya5 element is shown in Seq ID No. 1, and the partial Alu Ya5 sequence is shown in Seq ID No. 2. When the material is of other species, the short interspersed element can be replaced with the short interspersed element of the corresponding species to improve gene editing efficiency. material 1. pBudORF1-CH plasmid was purchased from Addgene, with the plasmid number 51290. pBudORF2-CH plasmid was purchased from Addgene, with the plasmid number 51289. pBS-L1PA1-CH-mneo plasmid was purchased from Addgene, with the plasmid number 51288. After purchasing pBudORF 1-CH plasmid, pBudORF 2-CH plasmid, and pBS-L 1 PA 1-CH-mneo plasmid, we asked Beijing Synthetic Biological Science and Technology Co., Ltd. to amplify them. 2. CD 293 medium was purchased from Thermofisher, number: 11913019. 3. PEI transfection reagent was purchased from Serochem, product number: Prime-AQ 100-100 ML. 4. SMS 293-SUPI was purchased from Sino Biological Inc, part number: M 293-SUPI-100. 5. Potassium acetate was purchased from Sigma-Aldrich, part number: P 1190. 6. Tris-HCl (pH 7.5) was purchased from SHANGBAO (Shanghai) Biotech, product number: T 16588. 7. Glycerin was purchased from Sigma-Aldrich, part number: G 5516. 8. Triton X-100 was purchased from Sigma-Aldrich, part number: T 8787. 9. PMSF protease inhibitor was purchased from Thermofisher, part number: 36978. 10. Ni affinity chromatography column (HISTRAP HP) was purchased from Cytiva. 11. Imidazole was purchased from Sigma-Aldrich, part number: I 5513. 12. Rabbit anti-his was purchased from Sigma-Aldrich, product number: SAB 1306082. 13. BSA was purchased from Sigma-Aldrich, product number: A 1933. 14. Anti-rabbit IgG (whole molecule)-alkaline phosphatase goat antibody was purchased from Sigma-Aldrich, product number: A 3687. 15. pcDNA(TM) 3.1(+) was purchased from Invitrogen, product number: V79020. 16. NheI was purchased from ThermoFisher and the formulation of 10x enzyme cut buffer is: 330 mM Tris-acetate, 100 mM magnesium acetate, 660 mM potassium acetate, 1 mg / mL BSA. 17. T4 DNA ligase and 10× ligation buffer required for the application were purchased from Promega. 18. MEGAscript TM The T7 Transcription Kit was purchased from Thermofisher, product number: AM 1333. 19. Opti-MEM TM I medium was purchased from Thermofisher, product number: A 4124802. 20. RNase inhibitor was purchased from Thermofisher, product number: AM 2694. 21. RNAiMAX transfection reagent was purchased from Thermofisher, product number: 13778030. 22. pcDNA 3.1(+)eGFP was purchased from Addgene, product number: 129020. 23. KOD One TM PCR Master Mix was purchased from DONGYANGFANG (Shanghai) Biotech, product number: KMM-201 S. 24. Hieff Clone® Plus One Step Cloning Kit was purchased from YISHENG (Shanghai) Biotech, product number: 10911 ES 20. 25. Complete medium is made of 90% DMEM medium + 10% fetal bovine serum, of which DMEM medium was purchased from Thermofisher, product number: 11965092, and fetal bovine serum was purchased from Thermofisher, product number: 10100147. 26. Entranster-H 4000 transfection reagent was purchased from ENGREEN (Beijing) Biotech. 27. Blood / cell / tissue genomic DNA extraction kit was purchased from Tiangen(Beijing)Tech, product catalog number: DP 304. 28. MEGAscript TM The SP6 Transcription Kit was purchased from Thermofisher, product number: AM 1330. 29. SuperReal Premix Plus (SYBR Green) was purchased from Tiangen (Beijing) Tech, product catalog number: FP 205. 30. Chemical synthesis of primers and sequences was completed by BioSune (Shanghai) Biotech or ABIOCENTER (Jiangsu) Biotech. Example 1: Production of ORF1p and ORF2p 1. Preparation of ORF1p in human LINE-1 (LRE1) Since a commercial plasmid, pBudORF 1-CH, is available for the expression of ORF1p in human LINE-1 (LRE1) (abbreviated as hLRE1-ORF1p), hLRE 1-ORF1p can be obtained by directly expressing it using the pBudORF 1-CH plasmid. 1) Transfect and express pBudORF 1-CH plasmid HEK 293 cells were cultured in CD 293 medium, subcultured, and then transfected with pBudORF 1-CH plasmid according to the PEI transfection reagent instructions. SMS 293-SUPI supplement was added at the dosages specified in the instructions on the 1st, 3rd, and 5th days after transfection. HEK 293 cells were cultured in flasks under the following flask culture conditions: 5% CO2, temperature 37°C, and rocking bed rotation speed 175 rpm. Reactor culture conditions: pH 7.2, temperature 37°C, agitation speed 150 rpm, and dissolved oxygen 40%. HEK 293 cells were incubated in a rocking bed culture box, and 7 days after transfection, the cells were harvested by centrifugation at 3000 g for 5 min. The SMS 293-SUPI supplement added here can promote cell survival and increase protein yield. 2) Extraction of hLRE1-ORF1p Cell lysis solution (100 mM potassium acetate, 50 mM Tris-HCl (pH 7.5), 5% glycerol, 0.3% TritonX-100) was prepared and pre-cooled, and the protease inhibitor PMSF was added before use to make the PMSF concentration in the cell lysis solution 1 mM. The cell lysis solution was added to the cells, and the cells were lysed by adding 40 ml of cell lysis solution per liter of cells (the lysis process can also use RIPA lysis solution or other types of cell lysis solution placed in it). Then, the cells were blown with a pipette to disintegrate them sufficiently, avoiding foaming and vortexing. Then, the processing of the cells was continued using a glass homogenizer, followed by three cycles of ultrasonication (15 s operation, 15 s interval). The cells were centrifuged at 30000 g for 25 min at 4 °C, and the supernatant was retained and filtered using a 0.22 um filtration membrane. The sample after filtration was taken and protein purification was performed by Ni affinity chromatography column (HISTRAP HP). The protein purification steps are as follows: 1. Wash the Ni affinity chromatography column with 5 Ni affinity chromatography column volumes of deionized water; 2. Place pH 7.4 PBS buffer and equilibrate the Ni affinity chromatography column with 5 to 10 column volumes of PBS buffer; 3. The prepared supernatant was passed through a Ni affinity chromatography column at a rate of 0.5 ml / min. 4. Re-equilibrate the Ni affinity chromatography column with the buffer provided; 5. Prepare solutions containing 20 mM imidazole, 50 mM imidazole, 100 mM imidazole, 250 mM imidazole and 500 mM imidazole with 0.5 M NaCl, respectively. 6. Elute the protein samples on the nickel column with solutions containing 20 mM imidazole, 50 mM imidazole, 100 mM imidazole, 250 mM imidazole and 500 mM imidazole, respectively, and collect the corresponding eluted samples. The collected eluted samples were dialyzed overnight at 4°C, and the composed PBS solution was used as the buffer. Finally, the sample obtained after dialysis was concentrated by ultrafiltration (select the appropriate ultrafiltration tube), and the obtained target protein was examined using SDS-PAGE (Fig antibody used: anti-His rabbit antibody 1:1500 (5%Milk + 0.1%BSA), secondary antibody: goat anti-rabbit IgG basic phosphatase 1:6000 (5%Milk) to confirm the protein concentration, and then hLRE1-ORF1p was extracted, purified, and freeze-dried. 2. Construction of ORF2p in Human LINE-1 (LRE1) Since a commercial plasmid pBudORF2-CH is available for expression of ORF2p in human LINE-1 (LRE1) (abbreviated as hLRE1-ORF2p), hLRE1-ORF2p can be obtained by directly expressing it using the pBudORF2-CH plasmid. The plasmid pBudORF1-CH expressing hLRE1-ORF1p was replaced with the plasmid pBudORF2-CH expressing hLRE1-ORF2p, and purified lyophilized hLRE1-ORF2p was produced according to the method for producing hLRE1-ORF1p. 3. Construction of ORF1p in Human LINE-1(LRE2) Since there is no commercially available plasmid capable of directly expressing ORF1p (abbreviated as hLRE2-ORF1p) in human LINE-1 (LRE2), an expression plasmid was constructed to express it. The DNA sequence of the coding sequence of ORF1p in human LINE-1(LRE2) is shown in Seq ID No. 3: The nucleotide sequence after the modification was as shown in Seq ID No. 4: taa at the end of the DNA sequence of ORF1p in human LINE-1 (LRE 2) was deleted, followed by the addition of coding sequences for Myc and His tags (wavy line), tga (italic bold), and finally the addition of NheI enzyme cut sites and protective bases (CTAGCTAGCTAG) at both ends of the sequence. CTAGCTAGCTAGGAACAAAAACTCATCTCAGAAGAGGATCTGAATATGCATACCGGTCATCATCACCATCACCAT TGA CTAGCTAGCTAG . The nucleotide sequence of Seq ID No.4 was obtained by chemical synthesis, and then the inserted sequence was cut with NheI enzyme. At the same time, the pcDNA(TM)3.1(+) plasmid was also cut with NheI enzyme to obtain a linear plasmid. The inserted sequence and the linear plasmid were then recovered by electrophoresis. A specific enzymatic cleavage reaction system is shown in Table 1: Table 1 Enzyme cleavage reaction system TIFF2025510714000002.tif75170Enzyme cleavage reaction conditions were: incubation at 37°C for 3 hours, followed by heating to 80°C and incubation for 10 minutes to inactivate endonucleases. The sequence to be inserted after enzyme digestion and recovery was ligated to the linearized plasmid, electrophoresed, and recovered to obtain the plasmid pcDNA(TM)3.1(+)-hLRE2-ORF1p for expressing ORF1p in human LINE-1 (LRE 2). Specific ligation reaction systems are shown in Table 2: Table 2. Ligation reaction system TIFF2025510714000003.tif66170The ligation reaction conditions were: incubation at 16°C for 16 h, then heating to 70°C and incubation for 10 minutes to inactivate the ligase. Preparation of ORF 1 protein in human LINE-1 (LRE 2) (abbreviation: hLRE 2-ORF1p): According to the preparation method of hLRE 1-ORF1p, the plasmid pBudORF 1-CH was replaced with pcDNA(TM)3.1(+)-hLRE2-ORF1p, and the plasmid was transfected, expressed, purified and lyophilized to obtain hLRE 2-ORF1p. 4. Construction of ORF2p in Human LINE-1 (LRE2) In human LINE-1 (LRE 2), since there is no commercially available plasmid capable of directly expressing ORF2p (abbreviated as hLRE 2-ORF2p), an expression plasmid was constructed to express it. The nucleotide sequence encoding ORF2p in human LINE-1 (LRE 2) as shown in Seq ID No. 5 was prepared according to the method for preparing ORF1p in human LINE-1 (LRE 2), expressed, purified and lyophilized to obtain hLRE 2-ORF2p. 5. Production of Mouse ORF1p The DNA sequence of the coding sequence of mouse ORF1p, as shown in Seq ID No. 6, was prepared according to the method for preparing ORF1p in human LINE-1 (LRE2), and then lyophilized, expressed and purified to obtain mouse ORF1p, which was named mORF1p. 6. Production of Mouse ORF2p The DNA sequence of the coding sequence of mouse ORF2p, as shown in Seq ID No. 7, was prepared according to the method for preparing ORF1p in human LINE-1 (LRE2), and then lyophilized, expressed and purified to obtain mouse ORF 2p, which was named mORF2p. Example 2: Detection of the effect of producing RNA by in vitro transcription, then transforming the target system with or without ORF1p and / or ORF2p bound outside the target system, and performing gene editing. Mutations in the Lman1 gene cause congenital combined factor V and VIII deficiency (F5F8D), which leads to reduced levels of human FV and FVIII and can cause spontaneous bleeding episodes in patients. A 405 bp sequence in the gene Lman1 in the human genome was selected and is shown in Seq ID No. 8 below: GGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT*CCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCA GAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAGAACG; where * is the selected insertion site (target site), before the insertion site is the upstream sequence of the insertion site in the Lman1 gene (target site upstream sequence), and after the insertion site is the downstream sequence of the insertion site in the Lman1 gene (target site downstream sequence). At *, the exogenous sequence is inserted, which is the sequence to be inserted as shown in Seq ID No. 9. AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG. The sequence of the inserted fragment is shown in Seq ID No. 10: GGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGGCCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where the underlined sequence is the sequence to be inserted as shown in Seq ID No. 9. The T7 promoter sequence shown in Seq ID No. 11 (Seq ID No. 11:TAATACGACTCACTATA) was added upstream of the sequence shown in Seq ID No. 10, and the partial Alu sequence shown in Seq ID No. 2 was added downstream, resulting in the following sequence as shown in Seq ID No. 12: TAATACGACTCACTATAGGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG CCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG ACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCACGCCGCTGCACTCCACCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATTAATAACTGCTGGAGATC , Here, the underlined portion indicates the inserted sequence shown in Seq ID No. 9, the italics indicate the T7 promoter sequence shown in Seq ID No. 11, and the wavy lines indicate the partial Alu sequence shown in Seq ID No. 2. This sequence was obtained by chemical synthesis and was named RNA+partial Alu precursor DNA. The T7 promoter sequence shown in Seq ID No. 11 was added upstream of the sequence shown in Seq ID No. 10, resulting in the following sequence as shown in Seq ID No. 13: TAATACGACTCACTATAGGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG CCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where the underlined sequence is the inserted sequence shown in Seq ID No. 9, and the bolded italics are the T7 promoter sequence shown in Seq ID No. 11, which was obtained by chemical synthesis and named the precursor DNA of RNA. Kit MEGAscript TM According to the instructions of the T7 Transcription Kit, linear RNA + partial Alu precursor DNA or RNA precursor DNA was transcribed to obtain the corresponding RNA, and then the residual DNA was degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration was measured with an ultraviolet spectrophotometer, and RNase-free water was continued to be added to prepare an RNA + partial Alu solution or RNA solution with a concentration of 100 ng / μL. The RNA+Alu portion obtained by the above transcription belongs to the RNA framework shown in Figure 4, and the beginning of the ORF2p function is a short interspersed element sequence-RNA. The RNA obtained by the above transcription belongs to the RNA framework shown in Figure 3. The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were each resuspended in a 500 ng / μL hLRE1-ORF1p solution or hLRE1-ORF2p solution using an Opti-MEM solution to which 1 U / μL of RNAse inhibitor had been added in advance. Prepare RNPs in which RNA or RNA + partial Alu binds to ORF1p and / or ORF2p. Here, the RNPs in which the RNA+part Alu is bound to hLRE1-ORF1p and hLRE1-ORF2p are called RNA+part Alu+hLRE1-ORF1p and RNA+part Alu+hLRE1-ORF2p, respectively, and the reaction system is shown in Table 3: Table 3 Reaction system TIFF2025510714000004.tif38170The amounts of hL1-ORF1p solution and hL1-ORF2p solution added differ because they bind to RNA in different amounts. After gently mixing each component, the reaction system was incubated at room temperature (25° C.) for 10 minutes to obtain RNA+partial Alu+hLRE 1-ORF1p and RNA+partial Alu+hLRE 1-ORF2p, respectively. The preparation of RNA+hLRE1-ORF1p+hLRE1-ORF2p and RNA+partial Alu+hLRE1-ORF1p+hLRE1-ORF2p is carried out as follows: Prepare the reaction system shown in Table 4: Table 4 Reaction system TIFF2025510714000005.tif34170 After thoroughly mixing each component, the reaction system was incubated at room temperature (25°C) for 10 minutes to obtain an RNA + hLRE 1-ORF2p solution and an RNA + partial Alu + hLRE 1-ORF2p solution, respectively. Next, the RNA+hLRE 1-ORF2p solution and the RNA+partial Alu+hLRE 1-ORF2p solution were mixed according to the system shown in Table 5. Table 5. Reaction system TIFF2025510714000006.tif39170After mixing each component flexibly, the reaction system was incubated at room temperature (25°C) for 10 minutes to obtain RNA + hLRE 1-ORF1p + hLRE 1-ORF2p solution and RNA + partial Alu + hLRE 1-ORF1p + hLRE 1-ORF2p solution, respectively. An hLRE1-ORF1p+hLRE1-ORF2p solution containing only hLRE1-ORF1p and hLRE1-ORF2p without RNA or RNA+partial Alu was prepared as a negative control, and the reaction system is shown in Table 6. Table 6 Reaction system TIFF2025510714000007.tif35170 After mixing each component flexibly, the reaction system was incubated at room temperature (25°C) for 10 minutes to obtain a hLRE 1-ORF1p+hLRE 1-ORF2p solution. The transfection solution was made up as shown in Table 7: Table 7. Transfection solution system TIFF2025510714000008.tif27170 The transfection solution was added in equal volume ratios to the hLRE 1-ORF1p + hLRE 1-ORF2p solution, the RNA + partial Alu + hLRE 1-ORF1p solution, the RNA + partial Alu + hLRE 1-ORF2p solution, the RNA + hLRE 1-ORF1p + hLRE 1-ORF2p solution, and the RNA + partial Alu + hLRE 1-ORF1p + hLRE 1-ORF2p solution, respectively, and after gentle mixing, incubated at room temperature (25°C) for 20 minutes to obtain the corresponding transfection solutions. A complex between liposomes contained in the transfection solution and RNA, RNP or protein in the solution, i.e., hLRE 1-ORF1p+hLRE 1-ORF2p-liposome complex, RNA+partial Alu+hLRE 1-ORF1p-liposome complex, RNA+partial Alu+hLRE 1-ORF2p-liposome complex, RNA+hLRE 1-ORF1p+hLRE 1-ORF2p-liposome complex, RNA+partial Alu+hLRE 1-ORF1p+hLRE 1-ORF2p-liposome complex, RNA+partial Alu+hLRE 1-ORF1p+hLRE 1-ORF2p-liposome complex. A liposome complex transfection solution of RNA + partial Alu without hLRE1-ORF1p or hLRE1-ORF2p was prepared, and the reaction system is shown in Table 8: Table 8 Reaction system TIFF2025510714000009.tif25170After gently mixing each component, the reaction system was incubated at room temperature (25°C) for 10 minutes, and then the transfection solutions shown in Table 7 were added in equal volume ratios, gently mixed, and then incubated at room temperature (25°C) for 20 minutes to obtain an RNA + partial Alu-liposome complex. Construction of direct transfection plasmid pcDNA3.1(+)eGFP+RNA+partial Alu The sequence shown in Seq ID No. 10 is followed by the sequence shown in Seq ID No. 2 to obtain the sequence shown in Seq ID No. 14: GGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG CCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG ACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCACGCCGCTGCACTCCACCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATTAATAACTGCTGGAGATC ; Here, the underscores indicate the sequence to be inserted as shown in Seq ID No. 9, and the wavy lines indicate the partial Alu sequence as shown in Seq ID No. 2. This sequence was named RNA+partial Alu and was obtained by chemical synthesis. The sequence shown in Seq ID No. 14 was chemically synthesized and constructed into the pcDNA3.1(+)eGFP vector by homologous recombination ligation so that it was directly ligated downstream of the CMV promoter in the vector, with no other sequences between it and the CMV promoter. The resulting plasmid was named pcDNA3.1(+)eGFP+RNA+partial Alu. The specific steps are: 1. Primers designed to amplify the sequence shown in Seq ID No. 14, the forward primer sequence is 5'-CTATATAAGCAGAGCTGGGTAGAGATTCACTG-3' as shown in Seq ID No. 15, and the reverse primer sequence is 5'-CTCTAGTTAGCCAGAGGATCTCCAGCAGTTAT-3' as shown in Seq ID No. 16, to amplify the sequence shown in Seq ID No. 14 by PCR, and the reaction system is as shown in Table 9: Table 9. Reaction system TIFF2025510714000010.tif73150Amplification conditions were: 94°C 2 min, (98°C 10 sec, 60°C 10 sec, 68°C 2 sec) 40 cycles, 68°C 5 min. The synthetic sequence was flanked on both sides by sequences homologous to the pcDNA3.1(+)eGFP vector, and the amplified product was obtained using standard gel recovery and purification methods. 2. Design PCR primers to amplify pcDNA3.1(+)eGFP vector, among which the forward primer is shown in Seq ID No.17: 5'-AATAACTGCTGGAGATCCTCTGGCTAACTAGAG-3', the reverse primer sequence is shown in Seq ID No.18: 5'-CAGTGAATCTCTACCCAGCTCTGCTTATATAG-3', PCR amplify pcDNA3.1(+)eGFP vector, the reaction system is shown in Table 10: Table 10 Reaction system TIFF2025510714000011.tif71153Amplification conditions were: 94°C 2 min, (98°C 10 sec, 60°C 10 sec, 68°C 6 sec) 40 cycles, 68°C 5 min. Gel recovery and purification was carried out using conventional methods to obtain the pcDNA3.1(+)eGFP plasmid vector, which has sequences at both ends that are homologous to the synthetic sequence. 3. Use the One-Step Rapid Cloning Kit to connect the amplified product with the amplified pcDNA3.1(+)eGFP vector. The specific steps are according to the kit's instructions. The reaction system is as shown in Table 11: Table 11. Binding reaction system TIFF2025510714000012.tif471224. The recombinant product was transferred into responsive cells (DH5α), and the cells were plated to determine the sequence. After the sequence was determined to be correct, the plasmid was extracted to obtain the plasmid pCDNA3.1(+)eGFP-RNA+partial Alu. Transfection First, HeLa cells were subcultured, spread on a 24-well plate, and cultured in complete medium. The day after subculture, the HeLa cells had grown to 60% confluence, and then were cultured in Opti-MEM. TM The medium was replaced with I medium, and according to the RNAiMAX transfection reagent specifications, hLRE1-ORF1p + hLRE1-ORF2p-liposome complex, RNA + partial Alu + hLRE1-ORF1p-liposome complex, RNA + partial Alu + hLRE1-ORF2p-liposome complex, RNA + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex, RNA + partial Alu + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex, RNA + partial Alu-liposome complex were added to HeLa cells and transfected, with three parallels set up for each treatment. 6 h after transfection, the medium was replaced with complete medium. The cultured cells were grown to about 90% and fused, then subcultured again, and after the cells had grown to about 60% and fused after subculture, transfection was repeated once, and after the cells had grown to about 90% and fused again, subsequent operations were performed. For cells transfected with hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, the cells were passaged again after confluence to about 90%, and after passage, the partially complete medium was aspirated to the remaining 0.5 ml, and the control plasmid (pBS-L1PA1-CH-mneo) was transfected. Transfection was performed using Entranster-H4000 transfection reagent. For cells transfected with hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, the cells were passaged again after confluence to about 90%, and after passage, after confluence to about 60%, the partially complete medium was aspirated to the remaining 0.5 ml, and transfection of the control group plasmid (pBS-L1PA1-CH-mneo) was performed. Transfection was performed using Entranster-H4000 transfection reagent. The amount of cell transfection plasmid pBS-L 1 PA 1-CH-mneo per plate was 19.2 μg. The required transfected plasmid was diluted with 600 μL of serum-free DMEM solution and mixed thoroughly. At the same time, 48 μL of Entranster-H 4000 reagent was diluted with 600 μL of serum-free DMEM solution, mixed thoroughly and homogenously, and then left at room temperature for 5 minutes. The two prepared liquids were then mixed together, thoroughly mixed to homogenize, and left to stand at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to the transfected hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, and transfected into a 24-well plate containing 0.5 ml of culture medium per well in which HeLa cells were cultured. When the cells grew to about 90% and were fused, they were passaged again, and the above-mentioned operations were repeated after passage, and the subsequent operations were performed after the cells grew to about 90% and were fused again, and these cells were used as negative controls. Direct transfection of plasmids The constructed plasmid pcDNA3.1(+)eGFP+RNA+partial Alu was cotransfected into HeLa cells with the plasmid pBS-L1PA1-CH-mneo expressing ORF1p and ORF2p (LINE-1), and each group had three parallels, each of which was a 24-well plate cultured with HeLa cells. Transfection step: Hela cells were passaged and laid in a 24-well plate. The day after passage, transfection was performed using Entranster-H 4000 transfection reagent. Two types of plasmids were transfected, 19.2 μg per plasmid, and a total of 38.4 μg was used for transfection per plate cell. The required transfected plasmid was diluted with 600 μL of serum-free DMEM and mixed thoroughly. At the same time, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, mixed thoroughly, and then left at room temperature for 5 minutes. Then, the two prepared liquids were mixed, mixed thoroughly, and left at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to a 24-well plate containing 0.5 ml of Opti-MEM culture medium per well in which Hela cells were cultured, and transfected. When the cells have grown to about 90% and become fused, they are passaged, and after passage, when the cells have grown to about 60% and become fused, the above-mentioned operations are repeated, and when the cells have grown to about 90% and become fused, the subsequent operations are performed. Experimental grouping: There were seven groups in total: hLRE1-ORF1p+hLRE1-ORF2p-liposome complex and plasmid pBS-L1PA1-CH-mneo transfected group was the control group, plasmid direct transfection group was experimental group 1, transfection RNA+hLRE1-ORF1p+hLRE1-ORF2p group was experimental group 2, transfection RNA+partial Alu group was experimental group 3, transfection RNA+partial Alu+hLRE1-ORF1p group was experimental group 4, transfection RNA+partial Alu+hLRE1-ORF2p group was experimental group 5, transfection RNA+partial Alu+hLRE1-ORF1p+hLRE1-ORF2p group was experimental group 6, and each group had three parallels, each of which was a 24-well plate in which HeLa cells were cultured. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Designed primer pair 1, the upstream primer sequence is shown in Seq ID No. 21: 5'-GACTTATCCATGTGCCTGTT-3', the downstream primer sequence is 5'-TTGGCTACGAAATGCTTG-3', as shown in Seq ID No. 22. The upstream primer sequence of primer pair 1 is located in the complete Lman 1 gene, is located upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, is not present in the prepared RNA sequence, and is present only in the cell genome, and the downstream primer sequence of primer pair 1 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 12. Table 12 qPCR reaction system TIFF2025510714000013.tif78153Cellular DNA templates were DNA extracted from cells transfected with each of the seven groups described above. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 1: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 49°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 13. The sequencing verification of the PCR products was correct. Table 13 Results of primer pair 1 (n=3, x±s) TIFF2025510714000014.tif250170Table 10 shows that the relative copy number of experimental group 1 compared with the control group (N / A is calculated based on 40.00) is obviously higher than that of the control group, and is statistically significant (P<0.05), so that the sequence to be inserted can be inserted into the genome target site through the plasmid (DNA) administration receptor system (receptor system) containing the target site upstream sequence, the sequence to be inserted, and the target site downstream sequence (experimental group 1). Meanwhile, the relative copy number of experimental group 6 compared with experimental group 1 is obviously higher than that of experimental group 1, and is statistically significant (P<0.05), which shows that because there is an RNA splicing mechanism in eukaryotic bodies (cells), this mechanism can reduce the efficiency of the production of RNA with gene editing function by plasmid transcription, so that the efficiency of plasmid direct transfection (experimental group 1) is lower than that of directly introducing RNA or RNP containing the target site upstream sequence, the sequence to be inserted, and the target site downstream sequence (experimental group 6) into the receptor system under similar conditions. In addition, compared with the control group (N / A calculated based on 40.00), the relative copy number of experimental group 2 was significantly higher than that of the control group, which was statistically significant (P<0.05). RNA containing only the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site or RNP binding ORF1p and / or ORF2p (experimental group 2) also exerts a gene editing effect, but the effect is weak. RNA containing only the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site or RNP binding ORF1p and / or ORF2p also achieves the purpose of gene editing. On the other hand, compared with the control group (N / A was calculated based on 40.00), the relative copy number of experimental group 3 was significantly higher than that of the control group, which was statistically significant (P<0.05). Even without binding ORF1p and / or ORF2p, the gene editing effect could be achieved after adding partial Alu to the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site (experimental group 3), and the gene editing effect (compared to the relative copy number of the control group) was also significantly higher than that of experimental group 2, which explained that the addition of partial Alu can improve the gene editing efficiency.The relative copy number of experimental groups 4-6 was significantly higher than that of the control group (N / A was calculated based on 40.00), and statistical significance was observed (P<0.05), which demonstrated that the RNA containing the upstream sequence of the target site, the sequence to be inserted, the downstream sequence of the target site, and partial Alu can be bound to ORF1p, ORF2p, or ORF1p and ORF2p to produce gene editing effect. The results also showed that the gene editing effect of experimental groups 4-6 was gradually improved, and the effect of binding ORF2p on improving gene editing efficiency was superior to ORF1p, and at the same time, the binding ORF2p and ORF1p were superior to single binding ORF1p or ORF2p. We compared RNA retransfection by in vitro prokaryotic transcription with direct DNA transfection and compared the gene editing efficiency of both methods. Table 14 Results of primer pair 1 (n=3, x±s) TIFF2025510714000015.tif89170As can be seen from Table 14, the relative amount of copy number in experimental group 6 compared with experimental group 1 is obviously higher than that in experimental group 1, which is statistically significant (P<0.05), which explains the gene editing by producing specific RNA or RNP reintroduced in vitro through prokaryotic promoters or other methods, and in some cases, the efficiency of gene editing is higher than that of introducing DNA to produce RNA in the receptor system, which may be because producing specific RNA or RNP in vitro avoids the shearing or splicing effect on the corresponding RNA in the eukaryotic system. This reflects that producing specific RNA or RNP in vitro and then introducing it into the receptor system such as cells, tissues, organs, and organisms is sometimes more advantageous than directly introducing the corresponding DNA into the receptor system. Example 3: After RNA production by in vitro transcription, the efficiency of gene editing is measured by binding ORF1p and ORF2p to the target system after transfer into the target system. The plasmid pBS-L1PA1-CH-mneo was selected to express ORF1p and ORF2p in vivo. The plasmid contains ORF1 and ORF2 of codon-optimized human L1RP and can express hLRE1-ORF1 and hLRE1-ORF2 in cells. pBS-L1PA1-CH-mneo plasmid transfection First, Hela cells were passaged and spread on a 24-well plate. The day after passage, when the cells grew to 60% confluence, transfection was performed using Entranster-H4000 transfection reagent. Cells transfected pBS-L1PA1-CH-mneo plasmid 19.2 μg per plate. pBS-L1PA1-CH-mneo plasmid was diluted with 600 μL of serum-free DMEM and mixed thoroughly. At the same time, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, mixed thoroughly, and then left at room temperature for 5 minutes. Then, the two prepared liquids were mixed thoroughly and left at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to a 24-well plate containing 0.5 ml of Opti-MEM culture medium per well in which Hela cells were cultured, and transfection was performed. When the cells grow to about 90% and become confluent, they are passaged. After passage, HeLa cells are placed on a 24-well plate, and the day after passage, the cells grow to 60% and become confluent, and further RNA transfection is performed. Three parallel plates are set up, and each parallel plate is a 24-well plate in which HeLa cells transfected with pBS-L1PA1-CH-mneo plasmid are cultured. RNA transfection The RNA + partial Alu solution prepared in Example 2 was mixed according to the system shown in Table 8, and then added to the transfection solution system shown in Table 7 in an equal volume ratio. After gently mixing, the mixture was incubated at room temperature (25°C) for 20 minutes to obtain an RNA + partial Alu-liposome complex. HeLa cells transfected with pBS-L1PA1-CH-mneo plasmid were cultured and grown to 60% confluence, then resuspended in Opti-MEM TMThe medium was replaced with I medium, and the RNA + partial Alu-liposome complex was added to the cells and transfected according to the instructions of the RNAiMAX transfection reagent, and three parallel sets were set up. The cultured cells were grown to about 90% and fused, then re-passaged, and the transfection was repeated once after passage, and the cells were grown again to about 90% and fused, and then the cell DNA was extracted and the subsequent operations were performed, and this experiment was the experimental group. In the same manner, the RNA + partial Alu-liposome complex was transfected into HeLa cells that had not been transfected with the pBS-L1PA1-CH-mneo plasmid as a control, and three parallel runs were similarly set up. Extraction of post-transfection cellular DNA in the experimental and control groups: After aspirating off the cell medium, the cells were washed twice with PBS, and then digested by adding an appropriate amount of 0.25% trypsin, digested at 37°C for 20 min, and blown 15 times every 5 min. After the cells were suspended, the reaction was stopped by adding complete medium containing serum. Then, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured by ultraviolet spectrophotometer. qPCR Testing: GAPDH gene is used as the internal gene, the upstream primer sequence is Seq ID No. 19, the downstream primer sequence is Seq ID No. 20, the upstream primer sequence of primer pair 1 is Seq ID No. 21, the downstream primer sequence is Seq ID No. 18, and qPCR testing is performed. The qPCR reaction system is shown in Table 9. These cellular DNA templates were DNA extracted from cells transfected in the control or experimental groups described above. The above reaction system was prepared on ice, and after preparation, the reaction tube was covered, mixed uniformly, and then centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times. qPCR reaction cycle: Primer pair 1: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 49°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 15. The sequencing verification of the PCR products was correct. Table 15 Results of primer pair 1 (n=3, x±s) TIFF2025510714000016.tif94170Table 15 shows that the experimental group compared with the control group, the relative copy number of the experimental group is obviously higher than that of the control group, with statistical significance (P<0.05), and the gene editing efficiency of producing ORF1p and / or ORF2p in the target system and co-administering specific RNA is higher than that of administering specific RNA alone.This shows that the gene editing effect of the transferred specific RNA can be supported by generating ORF1p and / or ORF2p in the target system, thereby improving the gene editing efficiency. Example 4: After producing specific RNA (3' site is completely Alu) by in vitro transcription, the efficiency of gene editing is measured by transferring the bound / unbound ORF1p and ORF2p outside the target system into the target system. The GALT gene encodes galactose-1-phosphate uridyl transferase, and mutations in it can cause type I galactosemia in humans. Select a 360 bp sequence in the GALT gene and identify the sequence by Seq ID As shown in No. 23: GGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTG*ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG; where * is the selected insertion site (target site), before the insertion site is the upstream sequence of the insertion site in the GALT gene (upstream sequence of the target site), and after the insertion site is the downstream sequence of the insertion site in the GALT gene (downstream sequence of the target site). *Insert exogenous sequence at position *, where exogenous sequence is the sequence to be inserted, as shown in Seq ID No. 24. TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA The sequence after insertion is as shown in Seq ID No. 25: GGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTGTTG TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG The inserted sequence is shown in Seq ID No. 24 in underlined form. The T7 promoter sequence shown in Seq ID No. 11 was added upstream of the sequence shown in Seq ID No. 25, and the Alu sequence shown in Seq ID No. 1 was added downstream, resulting in the sequence shown in Seq ID No. 26: TAATACGACTCACTATAGGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTG TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG GGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCACGCCGCTGCACTCCACCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATTAATAACTGCTGGAGATC Here, the underlined sequence is the sequence to be inserted shown in Seq ID No. 24, the italicized bold text is the T7 promoter sequence shown in Seq ID No. 11, and the wavy line is the Alu sequence shown in Seq ID No. 1. This sequence was obtained by chemical synthesis and named the precursor DNA of RNA+Alu. The T7 promoter sequence shown in Seq ID No. 11 was added upstream of the sequence shown in Seq ID No. 25, and the resulting sequence is shown in Seq ID No. 27: TAATACGACTCACTATAGGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTG TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG, where the underlined sequence is the sequence to be inserted shown in Seq ID No. 24, and the italicized bolded sequence is the T7 promoter sequence shown in Seq ID No. 11, which is obtained by chemical synthesis and is designated as the precursor DNA of RNA. Kit MEGAscript TM According to the instructions of the T7 Transcription Kit, the linear RNA+Alu precursor DNA or RNA precursor DNA was transcribed to obtain the corresponding RNA, then the residual DNA was degraded using the DNase in the kit, resuspended in RNase-free water, the RNA concentration was measured by UV spectrophotometer, and the RNA+Alu solution or RNA solution was continued with the addition of RNase-free water to adjust the concentration to 100 ng / μL. The RNA+Alu obtained by the above transcription belongs to the RNA framework shown in Figure 4, and the ORF2p functional initiation part is a (complete) short interspersed element sequence RNA. The RNA obtained by the above transcription belongs to the RNA framework shown in Figure 3. The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were each resuspended in a 500 ng / μL hLRE1-ORF1p solution or a 500 ng / μL hLRE1-ORF2p solution using an Opti-MEM solution to which 1 U / μL of an RNAse inhibitor had been added in advance. RNPs of RNA or RNA+Alu bound to ORF1p and ORF2p: RNA+hLRE1-ORF1p+hLRE1-ORF2p, RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p, following the procedure below. Prepare the reaction system shown in Table 16: Table 16 Reaction system TIFF2025510714000017.tif34170Each component was gently mixed to homogenize, and the reaction system was then incubated at room temperature (25°C) for 10 minutes to obtain an RNA+hLRE1-ORF1p solution and an RNA+Alu+hLRE1-ORF1p solution, respectively. Next, the RNA+hLRE1-ORF1p solution and the RNA+Alu+hLRE1-ORF1p solution were mixed according to the system shown in Table 17. Table 17 Reaction system TIFF2025510714000018.tif40170 After gently mixing each component, the reaction system was incubated at room temperature (25°C) for 10 minutes to obtain an RNA + hLRE1-ORF1p + hLRE1-ORF2p solution and an RNA + Alu + hLRE1-ORF1p + hLRE1-ORF2p solution, respectively. The RNA+hLRE1-ORF1p+hLRE1-ORF2p solution or the RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p solution was mixed in the same volume ratio as the transfection solution system prepared in Table 7, gently mixed to make homogenous, and then incubated at room temperature (25°C) for 20 minutes to obtain the RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex or the RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex. A transfection solution of the RNA+Alu liposome complex not containing hLRE1-ORF1p and hLRE1-ORF2p was prepared, and the reaction system is shown in Table 18: Table 18 Reaction system TIFF2025510714000019.tif26170Each component was gently mixed to homogenize, and the reaction system was then incubated at room temperature (25°C) for 10 minutes. Next, an equal volume of the transfection solution shown in Table 7 was added, gently mixed to homogenize, and then incubated at room temperature (25°C) for 20 minutes to obtain an RNA+Alu-liposome complex. Construction of direct transfection plasmid pcDNA3.1(+)eGFP+RNA+Alu The sequence shown in Seq ID No. 25 is joined to the sequence shown in Seq ID No. 1 to obtain the sequence shown in Seq ID No. 28: GGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTGTTG TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG GGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCACGCCGCTGCACTCCACCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATTAATAACTGCTGGAGATC , where the underscores represent the insertion sequence shown in Seq ID No. 24, and the wavy lines represent the Alu sequence shown in Seq ID No. 1. The sequence shown in Seq ID No. 28 was chemically synthesized and constructed into the pcDNA3.1(+)eGFP vector by homologous recombination, so that the sequence was directly connected downstream of the CMV promoter in the vector, with no other sequence between this sequence and the CMV promoter, and the plasmid was named pcDNA3.1(+)eGFP+RNA+Alu. The specific steps are: 1. Primers designed to amplify the sequence shown in Seq ID No. 28, the forward primer sequence is 5'-CTATATAAGCAGAGCTGGGGTTCGGCCCT-3' as shown in Seq ID No. 29, and the reverse primer sequence is 5'-CTCTAGTTAGCCAGAGGATCTCCAGCAGTTAT-3' as shown in Seq ID No. 16, to amplify the sequence shown in Seq ID No. 28 by PCR, the reaction system is as shown in Table 19: Table 19 Reaction system TIFF2025510714000020.tif73150Amplification conditions were: 94°C 2 min, (98°C 10 sec, 60°C 10 sec, 68°C 2 sec) 40 cycles, 68°C 5 min. The synthetic sequence was flanked on both sides by sequences homologous to the pcDNA3.1(+)eGFP vector, and the amplified product was obtained using standard gel recovery and purification methods. 2. Design PCR primers to amplify pcDNA3.1(+)eGFP vector, among which the forward primer is shown in Seq ID No.17: 5'-AATAACTGCTGGAGATCCTCTGGCTAACTAGAG-3', the reverse primer sequence is shown in Seq ID No.30: 5'-AGGGCCGAACCCCAGCTCTGCTTATATAG-3', PCR amplify pcDNA3.1(+)eGFP vector, the reaction system is shown in Table 20: Table 20 Reaction system TIFF2025510714000021.tif71153Amplification conditions were: 94°C 2 min, (98°C 10 sec, 60°C 10 sec, 68°C 6 sec) 40 cycles, 68°C 5 min. Gel recovery and purification was carried out using conventional methods to obtain the pcDNA3.1(+)eGFP plasmid vector, which has sequences at both ends that are homologous to the synthetic sequence. 3. Use the One-Step Rapid Cloning Kit to connect the amplified product with the amplified pcDNA3.1(+)eGFP vector. The specific steps are according to the kit's instructions. The reaction system is as shown in Table 11: Table 11. Binding reaction system TIFF2025510714000022.tif471224. The recombinant product was transferred into sensitive cells (DH5α), and the cells were plated and sequenced after transfer. After the sequence was confirmed to be correct, the plasmid was extracted to obtain the plasmid pCDNA3.1(+)eGFP-RNA+Alu. Transfection Experimental group in which the RNA vector binds / does not bind ORF1p and ORF2p in vitro: First, human glioma cells U251 were subcultured in a 24-well plate and cultured in complete medium. The day after subculture, the human glioma cells U251 were grown to 60% confluence, and then Opti-MEM TM The medium was replaced with I medium, and transfection was performed by adding RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, and RNA+Alu-liposome complex to human glioma cells U251 according to the RNAiMAX transfection reagent instructions, and three parallel sets were set up for each. The cultured cells were grown to about 90% and fused, then re-passaged, and transfection was repeated once after passage, and the cells were re-grown to about 90% and fused, after which subsequent operations were performed. Control group: Using the above method, the hLRE1-ORF1p+hLRE1-ORF2p-liposome complex prepared in Example 2 was transfected into U251 cells, and the cells were passaged again after growing to about 90% and fusing. After growing to about 60% after passage, a part of the complete medium was aspirated to 0.5 ml, and a control group plasmid (pBS-L1PA1-CH-mneo) transfection was performed. Transfection was performed using Entranster-H4000 transfection reagent. Plasmid pBS-L1PA1-CH-mneo was taken at 19.2 μg for each plate cell transfection. The required transfected plasmid was diluted with 600 μL of serum-free DMEM solution and mixed thoroughly. At the same time, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM solution, mixed thoroughly, and then left to stand at room temperature for 5 minutes. The two liquids were then thoroughly mixed and left to stand at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to a 24-well plate containing human glial cells U 251 cultured in 0.5 ml of culture medium per well, and transfected. When the cells had grown to about 90% and were fused, they were passaged again, and the above-mentioned operations were repeated after passage, and the subsequent operations were performed after the cells had grown to about 90% and were fused again. Transfection of plasmid direct transfection group: The constructed plasmid pcDNA3.1(+)eGFP+RNA+Alu was transfected into human glial cells U251 together with the plasmid pBS-L1PA1-CH-mneo expressing ORF1p and ORF2p, and each group had three parallels, each of which was a 24-well plate in which human glial cells U251 were cultured. In the transfection step, human glioma cells U251 were passaged and laid on a 24-well plate. After the cells had grown to about 60% and fused the day after passage, transfection was performed using Entranster-H4000 transfection reagent. For each plate cell transfection, two types of plasmids were transfected, and 19.2 μg of each plasmid was collected, for a total of 38.4 μg. The required transfected plasmid was diluted with 600 μL of serum-free DMEM and mixed thoroughly. At the same time, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, mixed thoroughly, and then left at room temperature for 5 minutes. After that, the two prepared liquids were thoroughly mixed and left at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to a "24-well plate containing 0.5 ml of Opti-MEM culture medium per well in which human glial cells U251 were cultured" and transfected. When the cells grow to about 90% and become fused, they are passaged again, and the above-mentioned operations are repeated after passage. After the cells grow to about 90% and become fused again, the subsequent operations are carried out. Experimental grouping: There are a total of five groups: The group transfected with hLRE1-ORF1p + hLRE1-ORF2p and plasmid pBS-L1PA1-CH-mneo was the control group, the group directly transfected with plasmid was the experimental group 1, the transfected RNA + hLRE1-ORF1p + hLRE1-ORF2p group was the experimental group 2, the transfected RNA + Alu group was the experimental group 3, and the transfected RNA + Alu + hLRE1-ORF1p + hLRE1-ORF2p group was the experimental group 4. Each group had three parallels, and each parallel was one 24-well plate of cultured human glial cells U251. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Design primer pair 2, the upstream primer sequence of which is shown in Seq ID No. 31: 5'-CCCCAGTACGATAGCACC-3', and the downstream primer sequence is 5'-GACATAACCGAATCAGAATT-3', as shown in Seq ID No. 32. The upstream primer sequence of primer pair 2 is located in the complete GALT gene, more upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, which is not present in the prepared RNA sequence, but only present in the cell genome, and the downstream primer sequence of primer pair 2 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 21. Table 21 qPCR reaction system TIFF2025510714000023.tif79153Cellular DNA templates were DNA extracted from cells transfected with each of the five groups described above. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 2: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 46°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 22. The sequencing verification of the PCR products was correct. Table 22 Results of primer pair 2 (n=3, x±s) TIFF2025510714000024.tif202170 Table 22 shows that the relative copy number of experimental group 1 is significantly higher than that of the control group (N / A is calculated based on 40.00), and is statistically significant (P<0.05), so that the sequence to be inserted can be inserted into the genome target site through the plasmid (DNA) containing the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site (experimental group 1). Meanwhile, the relative copy number of experimental group 4 is significantly higher than that of experimental group 1, and is statistically significant (P<0.05), which shows that because there is an RNA splicing mechanism in eukaryotic organisms (in cells), this mechanism can reduce the efficiency of the production of RNA with gene editing function by plasmid transcription, so that the efficiency of direct plasmid transfection (experimental group 1) is lower than that of direct introduction of RNA or RNP containing the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site (experimental group 4) into the recipient system under similar conditions. In addition, compared with the control group (N / A calculated based on 40.00), the relative copy number of experimental group 2 was significantly higher than that of the control group, which was statistically significant (P<0.05). RNA containing only the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site or RNP binding ORF1p and / or ORF2p (experimental group 2) also exerts a gene editing effect, but the effect is weak. RNA containing only the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site or RNP binding ORF1p and / or ORF2p also achieves the purpose of gene editing. On the other hand, compared with the control group (N / A was calculated based on 40.00), the relative copy number of experimental group 3 was significantly higher than that of the control group, which was statistically significant (P<0.05). Even without binding ORF1p and / or ORF2p, the gene editing effect could be achieved after adding complete Alu to the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site (experimental group 3), and the gene editing effect (compared to the relative copy number of the control group) was also significantly higher than that of experimental group 2, which explained that the addition of complete Alu can improve the gene editing efficiency.The relative copy numbers of experimental group 4 were all significantly higher than those of the control group (N / A was calculated based on 40.00), with statistical significance (P<0.05), demonstrating that the RNA containing the upstream sequence of the target site, the sequence to be inserted, the downstream sequence of the target site, and the complete Alu can be bound to ORF1p, ORF2p, or ORF1p and ORF2p to produce gene editing effects. The above results also show that the complete Alu sequence can effectively enhance the gene editing effect of the present invention. We generated RNA by in vitro prokaryotic transcription, bound the RNA to ORF1p and ORF2p, and then transfected it to compare with direct DNA transfection, and compared the gene editing efficiency of both methods. Table 23 Results of primer pair 2 (n=3, x±s) TIFF2025510714000025.tif83170As can be seen from Table 23, the relative amount of copy number in experimental group 4 compared with experimental group 1 is obviously higher than that in experimental group 1, which is statistically significant (P<0.05), which explains the gene editing by producing specific RNA or RNP reintroduced in vitro through prokaryotic promoters or other methods, and in some cases, the efficiency of gene editing is higher than that of introducing DNA to produce RNA in the receptor system, which may be because producing specific RNA or RNP in vitro avoids the shearing or splicing effect on the corresponding RNA in the eukaryotic system. This reflects that producing specific RNA or RNP in vitro and then introducing it into the receptor system such as cells, tissues, organs, and organisms is sometimes more advantageous than directly introducing the corresponding DNA into the receptor system. Example 5: Measuring the efficiency of gene editing into the target system after the out-of-target system bound / unbound ORF1p and ORF2p after in vitro transcription to generate RNA (the 3' portion is an RNA sequence corresponding to the 3' UTR of a long interspersed element (partial long interspersed element RNA)) A 400 bp sequence in the gene Lman1 in the human genome was selected and is shown in Seq ID No. 33 below: GAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTTCCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAA*AGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where * is the selected insertion site (target site), before the insertion site is the upstream sequence of the insertion site in the Lman 1 gene (upstream sequence of the target site), and after the insertion site is the upstream sequence of the insertion site in the Lman 1 gene (upstream sequence of the target site). The sequence Seq ID No. 33 is the downstream sequence of the insertion site in gene 1 (the downstream sequence of the target site). The sequence Seq ID No. 33 has 5 bp less at the 5' end than Seq ID No. 8 in order to increase the transcription efficiency of the Sp6 promoter. *Insert the exogenous sequence, which is the sequence to be inserted as shown in Seq ID No. 9. The sequence of the inserted fragment is shown in Seq ID No. 34: GAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTTCCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAA AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG AGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where the underlined sequence is the sequence to be inserted, such as Seq ID No. 9. The Sp6 promoter sequence (Seq ID No. 35: ATTTAGGTGACACTATA) is added upstream of the sequence shown in Seq ID No. 34, and the LINE-3'UTR sequence shown in Seq ID No. 36 is added downstream: ACAATGAGATCACATGGACACAGGAAGGGGAATATCACACTCTGGGGACTGTGGTGGGGTCGGGGGAGGGGGGAGGGGTAGCATTGGGAGATATACCTAATGCTAGATGACACATTAGTGGGTGCAGCGCACCAGCATGGCACATGTATACATATGTAACTAACCTGCACAATGTGCACATGTACCCTAAAACTTAGAGTATAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA, After adding, it will look like Seq ID No.37: ATTTAGGTGACACTATAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTTCCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAA AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGGAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG ACAATGAGATCACATGGACACAGGAAGGGGAATATCACACTCTGGGGACTGTGGTGGGGTCGGGGGAGGGGGGAGGGGTAGCATTGGGAGATATACCTAATGCTAGATGACACATTAGTGGGTGCAGCGCACCAGCATGGCACATGTATACATATGTAACTAACCTGCACAATGTGCACATGTACCCTAAAACTTAGAGTATAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Here, the underlined sequence is the sequence to be inserted, such as Seq ID No. 9, the italicized bold text is the Sp6 promoter sequence, such as Seq ID No. 35, and the wavy line is the LINE-3'UTR sequence, such as Seq ID No. 36. This sequence is obtained by chemical synthesis and is named the precursor DNA of RNA+LINE-3'UTR(RNA). MEGAscript TM According to the instructions of the SP6 transcription kit, the precursor DNA of linear RNA+LINE-3'UTR(RNA) was transcribed to obtain the corresponding RNA, then the residual DNA was degraded using the DNase in the kit, resuspended in RNase-free water, the RNA concentration was measured by UV spectrophotometer, and the RNA+LINE-3'UTR(RNA) solution was continued by adding RNase-free water to adjust its concentration to 100 ng / μL. The RNA+LINE-3'UTR (RNA) obtained by the above transcription belongs to the RNA framework shown in Figure 4, and the beginning of the ORF2p function is a partial long interspersed element RNA. The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in a 500 ng / μL hLRE1-ORF1p solution or hLRE1-ORF2p solution using Opti-MEM solution to which 1 U / μL of RNase inhibitor had been added in advance. An RNP in which RNA+LINE-3'UTR(RNA), ORF1p, and ORF2p are bound: RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p was prepared, and the procedure was carried out according to the following procedure. First, the reaction system shown in Table 24 was prepared: Table 24 Reaction system TIFF2025510714000026.tif32170Each component was gently mixed to homogenize, and the reaction system was then incubated at room temperature (25°C) for 10 minutes to obtain an RNA + LINE-3'UTR (RNA) + hLRE1-ORF2p solution. Next, the RNA + LINE-3'UTR (RNA) + hLRE1-ORF2p solutions were mixed in the systems shown in Table 25. Table 25 Reaction system TIFF2025510714000027.tif34170Each component was gently mixed to homogenize, and the reaction system was then incubated at room temperature (25°C) for 10 minutes to obtain an RNA + LINE-3'UTR (RNA) + hLRE1-ORF1p + hLRE1-ORF2p solution. The RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p solution was mixed with an equal volume ratio of the transfection solution system prepared in Table 7, gently mixed to homogenize, and then incubated at room temperature (25°C) for 20 minutes to obtain the RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex. Transfection First, HeLa cells were subcultured and placed on a 24-well plate, and cultured in complete medium. The day after subculture, HeLa cells grew to 60% confluence, and then Opti-MEM TM The medium was replaced with I medium, and the RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex was added and transfected into HeLa cells according to the RNAiMAX transfection reagent instructions, and each was set up in three parallel cultures. The cultured cells were grown to about 90% and allowed to fuse before being re-passaged, and the transfection was repeated once after passage (according to the procedure described above), and the cells were allowed to regrow to about 90% and allowed to fuse before being subjected to subsequent operations. Experimental grouping: The control group in Example 2 was used as the control group, and transfection RNA + LINE-3'UTR (RNA) + hLRE1-ORF1p + hLRE1-ORF2p was used as the experimental group. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Designed primer pair 1, the upstream primer sequence is shown in Seq ID No. 21: 5'-GACTTATCCATGTGCCTGTT-3', the downstream primer sequence is 5'-TTGGCTACGAAATGCTTG-3', as shown in Seq ID No. 22. The upstream primer sequence of primer pair 1 is located in the complete Lman 1 gene, is located upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, is not present in the prepared RNA sequence, and is present only in the cell genome, and the downstream primer sequence of primer pair 1 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 26. Table 26 qPCR reaction system TIFF2025510714000028.tif79153 Cellular DNA templates were DNA extracted from cells transfected with each of the seven groups described above. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 1: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 49°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 27. The sequencing verification of the PCR products was correct. Table 27 Results of primer pair 1 (n=3, x±s) TIFF2025510714000029.tif88170As can be seen from Table 27, compared with the control group, the relative copy number of the experimental group was obviously higher than that of the control group, which was statistically significant (P<0.05), demonstrating that by linking the corresponding RNA sequence and RNA framework of long interspersed element (LINE-1)-3'UTR, and generating the corresponding specific RNA or RNP in vitro and reintroducing it into the recipient system, gene editing can be performed relatively effectively. In addition, gene editing is possible using the corresponding RNA sequence of the long interspersed element-3'UTR, so the complete long interspersed element RNA (mainly ORF1p and ORF2p base sequences and their corresponding RNA sequence of 3'UTR) is theoretically possible. This example also shows that the Sp6 promoter can be involved in the task of generating RNA in vitro. Example 6: A specific RNA (containing an ORF2p shearing function and a functional structure for initiating reverse transcription (a functional structure for initiating ORF2p shearing function and reverse transcription), and this functional structure portion is included in the downstream sequence of the target site) is generated by in vitro transcription, and then the efficiency of gene editing by transferring the bound / unbound ORF1p and ORF2p outside the target system to the target system is measured. The GALT gene encodes galactose-1-phosphate uridyl transferase, and mutations in it can cause type I galactosemia in humans. The ORF2p shearing function and a functional structure that initiates reverse transcription are constructed, and this functional structural RNA binds to a complementary sequence on the genome to form an "Ω" structure. Here, the 5' portion (left leg) of the "Ω" structure was selected from the downstream sequence (target site downstream sequence) of the sequence to be inserted in the GALT gene having the sequence to be inserted as shown in Seq ID No. 25 in Example 4, as shown in Seq ID No. 38: ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG. The 3' portion (right leg) of the "Ω" structure, as shown in Seq ID No. 39, is composed of the sequence immediately downstream of the sequence shown in Seq ID No. 38 on the genome (the sequence downstream of the target site). AAAACAAAGGTGCCATGATGGGCTGTTCTAACCCCCCCCCACTGCCAGGTAAGGGTGTCAGGGGCTCCAGTGGGTTTCTTGGCTGAGTCTGAGCCAGCACT; The cyclic portion of the "Ω" structure is composed of a randomly generated sequence as shown in Seq ID No. 40. CTGACCATGCTTATACGGACTATCGATTAG. The ring of the "Ω" structure shown in Seq ID No. 40 and the right leg of the "Ω" structure shown in Seq ID No. 39 are connected downstream of the sequence shown in Seq ID No. 25, and the T7 promoter sequence shown in Seq ID No. 11 is added upstream of the sequence shown in Seq ID No. 25 to form the sequence shown in Seq ID No. 41: TAATACGACTCACTATAGGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTG TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG CTGACCATGCTTATACGGACTATCGATTAG AAAACAAAGGTGCCATGATGGGCTGTTCTAACCCCCACCCCCACTGCCAGGTAAGGGTGTCAGGGGCTCCAGTGGGTTTCTTGGCTGAGTCTGAGCCAGCACT Here, the bold italics are the T7 promoter sequence shown in Seq ID No. 11, the wavy line sequence is the circular sequence of the "Ω" structure shown in Seq ID No. 40, the downstream of the wavy line sequence is the right leg of the "Ω" structure shown in Seq ID No. 39, and the space between the upstream of the wavy line sequence and the underline is the left leg of the "Ω" structure (the sequence downstream of the target site) shown in Seq ID No. 38. The sequence shown in Seq ID No. 41 was obtained by chemical synthesis and named as the precursor DNA of the functional structure that has the RNA+ORF2p shearing function and initiates reverse transcription. Kit MEGAscript TM According to the instructions of the T7 Transcription Kit, the precursor DNA of the linear RNA + ORF2p shearing function and functional construct that initiates reverse transcription was transcribed to obtain the corresponding RNA, then the DNase in this kit was used to degrade the residual DNA and resuspended in RNase-free water, the RNA concentration was measured by ultraviolet spectrophotometer, and the RNA + ORF2p shearing function and functional construct solution that initiates reverse transcription was added with RNase-free water to place its concentration at 100 ng / μL. The RNA + ORF2p shearing function obtained by the above transcription and the functional structure that initiates reverse transcription belong to the RNA frame structure in Figure 6, of which the ORF2p shearing function and the functional structure that initiates reverse transcription form an "Ω" structure. The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in 500 ng / μL hLRE1-ORF1p solution or hLRE1-ORF2p solution using Opti-MEM solution to which 1 U / μL RNase inhibitor had been added in advance. Prepare RNP: RNA + activating ORF2p shearing function and reverse transcription functional structure, and ORF1p and ORF2p combined: RNA + activating ORF2p shearing function and reverse transcription functional structure + hLRE1-ORF1p + hLRE1-ORF2p, and perform the following procedure. First, the reaction system shown in Table 28 was prepared: Table 28 Reaction system TIFF2025510714000030.tif37170After gently mixing each component to make it homogenous, the reaction system was incubated at room temperature (25 °C) for 10 minutes to obtain a solution of RNA + ORF2p shearing function and reverse transcription function construct + hLRE1-ORF2p. Next, the RNA + priming ORF2p shearing function and the reverse transcription functional structure + hLRE1-ORF2p solution were mixed in the systems shown in Table 29. Table 29 Reaction system TIFF2025510714000031.tif34170After gently mixing each component to make it homogenous, the reaction system was incubated at room temperature (25 °C) for 10 minutes to obtain a solution of RNA + ORF2p shearing function and reverse transcription function + hLRE1-ORF1p + hLRE1-ORF2p. The RNA + activating ORF2p shearing function and reverse transcription functional structure + hLRE1-ORF1p + hLRE1-ORF2p solution was mixed with an equal volume ratio of the transfection solution system prepared in Table 7, gently mixed to make it homogenous, and then incubated at room temperature (25°C) for 20 min to obtain the RNA + activating ORF2p shearing function and reverse transcription functional structure + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex. Transfection First, human glioma cells U251 were subcultured in a 24-well plate and cultured in complete medium. The day after subculture, the human glioma cells U251 were grown to 60% confluence, and then Opti-MEM TM The medium was replaced with I medium, and RNA + ORF2p shearing function and reverse transcription function construct + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex was added and transfected into human glioma cells U 251 according to the RNAiMAX transfection reagent instructions, and each was set up in three parallel cultures. The cultured cells were grown to about 90% and allowed to fuse, and then re-passaged. After passage, the transfection was repeated once (following the procedure above), and the cells were allowed to grow again to about 90% and allowed to fuse, and then subsequent operations were performed. Experimental grouping: The control group in Example 4 was used as the control group, and the transfection RNA + activating ORF2p shear function and reverse transcription functional structure + hLRE1-ORF1p + hLRE1-ORF2p was used as the experimental group. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Design primer pair 2, the upstream primer sequence of which is shown in Seq ID No. 31: 5'-CCCCAGTACGATAGCACC-3', and the downstream primer sequence is 5'-GACATAACCGAATCAGAATT-3', as shown in Seq ID No. 32. The upstream primer sequence of primer pair 2 is located in the complete GALT gene, more upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, which is not present in the prepared RNA sequence, but only present in the cell genome, and the downstream primer sequence of primer pair 2 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 30. Table 30 qPCR reaction system TIFF2025510714000032.tif79153 Cellular DNA template is DNA extracted from transfected cells in the experimental group. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 2: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 46°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 31. The sequencing verification of the PCR products is correct. Table 31 Results of primer pair 2 (n=3, x±s) TIFF2025510714000033.tif97170 As can be seen from Table 31, compared with the control group, the relative copy number of the experimental group was obviously higher than that of the control group, which was statistically significant (P<0.05), which explains that even if the complete or partial short interspersed element or long interspersed element sequence is not connected, as long as the 3' part of the RNA frame including the target site upstream sequence, the target site downstream sequence and the sequence to be inserted can form a certain secondary structure such as an "Ω"-shaped structure and recruit and bind ORF2p (for example, polyA sequence), the corresponding gene editing purpose can be achieved. Example 7: The efficiency of genome site correction after transcription synthesis of RNA and ORF1p and ORF2p in a eukaryotic target system was detected. The PAH gene codes for phenylalanine hydroxylase (PAH) and is the pathogenic gene for phenylketonuria (PKU). A 250 bp sequence in the gene PAH in the human genome was selected and the sequence is shown in Seq ID No. 42 below: AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCgCTACGACCCATACACC CAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC, where lowercase letters represent the bases to be modified. If this base is changed from G to C, the sequence becomes Seq ID No. 43: AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCcCTACGACCCATACACC CAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC, where lowercase letters represent the modified bases. The uncorrected sequence Seq ID No. 42 is taken as the upstream sequence of the target site, the corrected sequence Seq ID No. 43 is taken as the sequence to be inserted, and the 200 bp sequence adjacent to and downstream of the genic sequence Seq ID No. 42 is taken as the downstream sequence of the target site, resulting in the sequence shown in Seq ID No. 44: AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCGCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCCCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC TTTCCCTACCATCGCCATAGGAAAAATAATAAATTTATTGAAATATTTAATTAAGGAGAAAAGCACCTCCATGTAAGCCATGGGTTCATTGATGGAGAAGAACTTGACAAAAAGGTCAGAATTACCCTTGTGTCCTTTTTCCTTTGACCTTCCTAGATTCCACTCCACCTCCTACCATCATTCCACCTTTCCACACTTGG, Here, the underlined sequence is the sequence to be inserted, Seq ID No. 43, which is upstream of the target site sequence Seq ID No. 42 and downstream of the target site sequence, which is designated the PAH base replacement sequence. A partial Alu sequence was added downstream of the PAH base replacement sequence Seq ID No. 44 to obtain the sequence shown in Seq ID No. 45: AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCGCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCCCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC TTTCCCTACCATCGCCATAGGAAAAATAATAAATTTATTGAAATATTTAATTAAGGAGAAAAGCACCTCCATGTAAGCCATGGGTTCATTGATGGAGAAGAACTTGACAAAAAGGTCAGAATTACCCTTGTGTCCTTTTTCCTTTGACCTTCCTAGATTCCACTCCACCTCCTACCATCATTCCACCTTTCCACACTTGG ACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCACGCCGCTGCACTCCACCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATTAATAACTGCTGGAGATC Here, the underlined sequence is the sequence to be inserted, Seq ID No. 43, and upstream of that is the upstream sequence of the target site, Seq ID No. 42. The wavy line is the partial Alu sequence shown in Seq ID No. 2. Between the sequence to be inserted and the partial Alu sequence is the downstream sequence of the target site, and this sequence is named the PAH base substitution sequence frame + partial Alu sequence. The sequence shown in Seq ID No. 45 was chemically synthesized and constructed into the pcDNA3.1(+)eGFP vector by homologous recombination, so that the sequence was directly connected downstream of the CMV promoter in the vector, with no other sequence between this sequence and the CMV promoter, and the plasmid was named pcDNA3.1(+)eGFP+PAH base replacement sequence frame+partial Alu sequence. The specific steps are: 1. Primers designed to amplify the sequence shown in Seq ID No. 45, the forward primer sequence is 5'-CTATATAAGCAGAGCTAAAATGCCACTGAGAA-3' as shown in Seq ID No. 46, and the reverse primer sequence is 5'-CTCTAGTTAGCCAGAGGATCTCCAGCAGTTAT-3' as shown in Seq ID No. 16, to amplify the sequence shown in Seq ID No. 45 by PCR, the reaction system is as shown in Table 32: Table 32 Reaction system TIFF2025510714000034.tif74150Amplification conditions were: 94°C 2 min, 40 cycles of (98°C 10 sec, 58°C 10 sec, 68°C 2 sec), 68°C 5 min. The synthetic sequence was flanked on both sides by sequences homologous to the pcDNA3.1(+)eGFP vector, and the amplified product was obtained using standard gel recovery and purification methods. 2. Design PCR primers to amplify pcDNA3.1(+)eGFP vector, among which the forward primer is shown in Seq ID No.17: 5'-AATAACTGCTGGAGATCCTCTGGCTAACTAGAG-3', the reverse primer sequence is shown in Seq ID No.47: 5'-GTTCTCAGTGGCATTTTAGCTCTGCTTATATAG-3', PCR amplify pcDNA3.1(+)eGFP vector, the reaction system is shown in Table 33: Table 33 Reaction system TIFF2025510714000035.tif63138Amplification conditions were: 94°C 2 min, (98°C 10 sec, 58°C 10 sec, 68°C 6 sec) 40 cycles, 68°C 5 min. Gel recovery and purification was carried out using conventional methods to obtain the pcDNA3.1(+)eGFP plasmid vector, which has sequences at both ends that are homologous to the synthetic sequence. 3. Use the One-Step Rapid Cloning Kit to connect the amplified product with the amplified pcDNA3.1(+)eGFP vector. The specific steps are performed according to the kit's instructions, and the reaction system is as shown in Table 11. 4. The recombinant product was transferred into sensitive cells (DH5α), and the cells were plated and sequenced. After the sequence was confirmed to be correct, the plasmid was extracted to obtain the plasmid pcDNA3.1(+)eGFP+PAH base substitution sequence frame+partial Alu sequence. The constructed vector pcDNA3.1(+)eGFP+PAH base substitution sequence frame+partial Alu sequence was transfected into HeLa cells together with the plasmid pBS-L1PA1-CH-mneo expressing ORF1p and ORF2p, where the group co-transfected with pcDNA3.1(+)eGFP+PAH base substitution sequence frame+partial Alu sequence and pBS-L1PA1-CH-mneo was the experimental group, and the group co-transfected with plasmid pBS-L1PA1-CH-mneo and plasmid pcDNA3.1(+)eGFP was the control group. Each group had three parallels, and each parallel was a 24-well plate in which HeLa cells were cultured. The transfection experimental procedure for the control group was the same as that of the control group in Example 2. The procedure for co-transfection of the experimental group was as follows: Hela cells were passaged and laid on a 24-well plate. When the cells had grown to around 60% and fused the day after passage, transfection was performed using Entranster-H4000 transfection reagent. For the transfection of each plate cell, two types of plasmids (pcDNA3.1(+)eGFP+PAH base substitution sequence frame+partial Alu sequence and pBS-L1PA1-CH-mneo) were transfected, and 19.2 μg of each plasmid was collected, for a total of 38.4 μg. The required transfected plasmid was diluted with 600 μL of serum-free DMEM and mixed thoroughly. At the same time, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, mixed thoroughly and homogenously, and then left at room temperature for 5 minutes. After that, the two prepared liquids were thoroughly mixed and left at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to a 24-well plate containing 0.5 ml of Opti-MEM medium per well in which HeLa cells had been cultured, and transfected. The cells were grown to about 90% confluent and then re-passaged. After passage, the above-mentioned procedure was repeated, and the cells were grown again to about 90% confluent and then the subsequent procedures were performed. The procedure for the control group co-transfection was as follows: Hela cells were passaged and laid on a 24-well plate. When the cells had grown to around 60% and were fused the day after passage, transfection was performed using Entranster-H4000 transfection reagent. For each plate cell transfection, two types of plasmids (pcDNA3.1(+)eGFP and pBS-L1PA1-CH-mneo) were transfected, and 19.2 μg of each plasmid was collected, for a total of 38.4 μg. The required transfected plasmid was diluted with 600 μL of serum-free DMEM and mixed thoroughly. At the same time, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, mixed thoroughly and homogenously, and then left at room temperature for 5 minutes. After that, the two prepared liquids were thoroughly mixed and left at room temperature for 15 minutes to prepare a transfection complex. The transfection complex was added to a 24-well plate containing 0.5 ml of Opti-MEM medium per well in which HeLa cells had been cultured, and transfected. The cells were grown to about 90% confluent and then re-passaged. After passage, the above-mentioned procedure was repeated, and the cells were grown again to about 90% confluent and then the subsequent procedures were performed. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Design primer pair 3, the upstream primer sequence is shown in Seq ID No. 48: 5'-AGGGAGGTGTCCGTGTTC-3', the downstream primer sequence is 5'-GGGTGTATGGGTCGTAGC-3', as shown in Seq ID No. 49. The upstream primer sequence of primer pair 3 is located in the complete PAH gene, located more upstream of the upstream sequence of the target site, not present in the sequence of the constructed vector, only present in the cell genome, the downstream primer sequence of primer pair 3 is located on the sequence to be inserted, its 3' terminal base matches the unmodified base on the genome, so that when the selected base site on the genome is modified, the PCR product is reduced. All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 34. Table 34 qPCR reaction system TIFF2025510714000036.tif88170 The cellular DNA templates were DNA extracted from the cells transfected in the above two groups. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 2: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 48°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the original site of the modified site and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 35. The sequencing verification of the PCR products is correct. Table 35 Results of primer pair 3 (n=3, x±s) From Table 35 of TIFF2025510714000037.tif121170, the experimental group compared with the control group, the relative copy number of the experimental group was obviously higher than that of the control group, which was statistically significant (P<0.05), which shows that the present invention can achieve the purpose of replacing a specific site on the genome. At the same time, it also shows that sequence replacement, site deletion, site addition, sequence addition and sequence deletion on the genome can be performed by performing homologous recombination on the genome after inserting a specific sequence, as well as site replacement on the genome. Since the purpose of site replacement on the genome can be directly achieved by plasmid transfection, it is understood that, according to another embodiment of the present invention, the corresponding purpose can also be achieved by transcribing the corresponding RNA in vitro and then transfecting it with or without binding ORF1p and / or ORF2p. Both eukaryotic and prokaryotic systems can express RNA and have the ability of homologous recombination, so they are similar to the relevant working mechanisms related to the present invention, and both can realize sequence insertion on genome. However, eukaryotic systems have a splicing mechanism, which may interfere with the synthesis of required specific RNA, causing inconvenience in industrial production. In addition, sequence replacement, site deletion, site addition, sequence addition, sequence deletion and site replacement on genome are achieved by cells themselves through mechanisms such as homologous recombination after the corresponding sequence is inserted by the present invention, so the ability to perform genome modification operations such as sequence replacement, site deletion, site addition, sequence addition, sequence deletion and site replacement on eukaryotic systems means that the corresponding operations on prokaryotic systems can also be performed. Example 8: Detecting the efficiency of separate administration of RNA framework and short interspersed element RNA The T7 promoter sequence shown in Seq ID No. 11 is joined to the Alu sequence shown in Seq ID No. 1 to obtain the sequence shown in Seq ID No. 50: where the bold italicized part is the T7 promoter sequence, and the downstream part of the T7 promoter sequence is the Alu sequence. This partial sequence was obtained by chemical synthesis and named the expression DNA for Alu-RNA. Kit MEGAscript TM According to the instructions of the T7 Transcription Kit, the linear Alu-RNA expression DNA was transcribed to obtain the corresponding Alu-RNA, and then the residual DNA was degraded using the DNase in the kit and resuspended in RNase-free water. The RNA concentration was measured by UV spectrophotometer, and the concentration of the Alu solution was set to 100 ng / μL by adding RNase-free water. Alu-liposome complexes were prepared and the reaction system is shown in Table 36: Table 36 Reaction system TIFF2025510714000038.tif27170After gently mixing each component to homogenize, the reaction system was incubated at room temperature (25°C) for 10 minutes, and then the transfection solutions shown in Table 7 were added in equal volume ratios, gently mixed, and then incubated at room temperature (25°C) for 20 minutes to obtain Alu-liposome complexes. The control group in Example 2 was used as the control group, and the co-transfection of RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex and Alu-liposome complex in Example 2 was used as the experimental group. Hela cells were transfected according to the method in Example 2 (Alu-liposome complex was co-transfected with RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex in equal amounts), and three parallels were set up for each group, and each parallel was a 24-well plate in which Hela cells were cultured. The above transcribed Alu and the RNA in Example 2 form a structure that separates the transcription, as shown in FIG. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Designed primer pair 1, the upstream primer sequence is shown in Seq ID No. 21: 5'-GACTTATCCATGTGCCTGTT-3', the downstream primer sequence is 5'-TTGGCTACGAAATGCTTG-3', as shown in Seq ID No. 22. The upstream primer sequence of primer pair 1 is located in the complete Lman 1 gene, is located upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, is not present in the prepared RNA sequence, and is present only in the cell genome, and the downstream primer sequence of primer pair 1 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 37. Table 37 qPCR reaction system TIFF2025510714000039.tif79152 Cellular DNA templates were DNA extracted from cells transfected with each of the two groups described above. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 1: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 49°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 38. The sequencing verification of the PCR products is correct. Table 38 Results of primer pair 1 (n=3, x±s) From Table 38 of TIFF2025510714000040.tif102170, comparing the experimental group and the control group, the relative copy number of the experimental group was obviously higher than that of the control group, with statistical significance (P<0.05), which explains that the RNA frame including the upstream sequence of the target site, the downstream sequence of the target site and the sequence to be inserted, and the corresponding transcription product RNA of the short interspersed element, partial short interspersed element, long interspersed element and / or partial long interspersed element sequence can be administered to the recipient system at different positions of the same vector or in different vectors, and can be used to insert a specified sequence into a specified gene target site or achieve other gene editing purposes, such as site replacement, sequence replacement, site deletion, site addition, sequence addition and sequence deletion, etc., while expressing the RNA frame including the upstream sequence of the target site, the downstream sequence of the target site and the sequence to be inserted, and the corresponding transcription product RNA of the short interspersed element, partial short interspersed element, long interspersed element and / or partial long interspersed element sequence. The corresponding gene editing objective can also be achieved by arranging the DNA vectors capable of expressing the above-mentioned RNA frames, each including the upstream sequence of the target site, the downstream sequence of the target site, and the sequence to be inserted, on different positions of the same vector or on different vectors, respectively, to provide a receptor system; and the corresponding gene editing objective can also be achieved by arranging the DNA vectors capable of expressing the above-mentioned RNA frames, each including the upstream sequence of the target site, the downstream sequence of the target site, and the sequence to be inserted, on different positions of the same vector or on different vectors, respectively, to administer the receptor system; and the corresponding gene editing objective can also be achieved by arranging the DNA vectors capable of expressing the RNA frames, each including the upstream sequence of the target site, the downstream sequence of the target site, and the sequence to be inserted, on different positions of the same vector or on different vectors, respectively, to administer the receptor system. Example 9 Gene editing effects of ORF1p and ORF2p in human-derived LRE 2 and ORF1p and ORF2p in mouse L1 The ORF1p and ORF2p used in Examples 2 to 8 were derived from LRE1 in human L1, and in this Example, we verified whether ORF1p (hLRE2-ORF1p) and ORF2p (hLRE2-ORF2p) in human LRE2 and ORF1p (mORF1p) and ORF2p (mORF2p) in mouse L1 can still exert gene editing effects after replacing the previous ORF1p and ORF2p in LRE1. The RNA + partial Alu solution prepared in Example 2 was combined with the hLRE2-ORF1p and hLRE2-ORF2p, and mORF1p and mORF2p prepared in Example 1, respectively, to prepare RNA + partial Alu + hLRE2-ORF1p + hLRE2-ORF2p and RNA + partial Alu + mORF1p + mORF2p, following the method for preparing RNA + partial Alu + hLRE1-ORF1p + hLRE1-ORF2p in Example 2. Next, according to the method of Example 2, an RNA+partial Alu+hLRE2-ORF1p+hLRE2-ORF2p-liposome complex and an RNA+partial Alu+mORF1p+mORF2p-liposome complex were prepared. The RNA + partial Alu + hLRE2-ORF1p + hLRE2-ORF2p-liposome complex and the RNA + partial Alu + mORF1p + mORF2p-liposome complex were each transfected into HeLa cells according to the method of Example 2, and three parallel cultures were set up in each group, each of which was a 24-well plate in which HeLa cells were cultured. Experimental grouping: A total of three groups were divided: the control group in Example 2 was used as the control group in this example, the transfected RNA + partial Alu + hLRE2-ORF1p + hLRE2-ORF2p group was used as experimental group 1, and the transfected RNA + partial Alu + mORF1p + mORF2p group was used as experimental group 2, with three parallel groups in each group, each of which was a 24-well plate in which HeLa cells were cultured. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Designed primer pair 1, the upstream primer sequence is shown in Seq ID No. 21: 5'-GACTTATCCATGTGCCTGTT-3', the downstream primer sequence is 5'-TTGGCTACGAAATGCTTG-3', as shown in Seq ID No. 22. The upstream primer sequence of primer pair 1 is located in the complete Lman 1 gene, is located upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, is not present in the prepared RNA sequence, and is present only in the cell genome, and the downstream primer sequence of primer pair 1 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system is shown in Table 39. Table 39 qPCR reaction system TIFF2025510714000041.tif79153 Cellular DNA templates were DNA extracted from cells transfected with each of the two groups described above. The above reaction system was prepared on ice, and after preparation, the reaction tube was closed, mixed gently and uniformly, and centrifuged briefly to ensure that all components were located at the bottom of the tube. Each 24-well plate cell sample was repeated three times at the same time. qPCR reaction cycle: Primer pair 1: Pre-denatured at 95°C for 15 min, followed by 40 cycles of (denaturation at 95°C for 10 s, annealing at 49°C for 20 s, and extension at 72°C for 20 s). GAPDH primers reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 40. The sequencing verification of the PCR products is correct. Table 40 Results of primer pair 1 (n=3, x±s) TIFF2025510714000042.tif164170Table 40 shows that compared with the control group, the relative copy number of experimental group 1 is obviously higher than that of the control group, with statistical significance (P<0.05), which shows that the ORF1p and ORF2p expressed in LRE2 in human source L1 can also achieve the corresponding gene editing purpose. At the same time, compared with the control group, the relative copy number of experimental group 2 is obviously higher than that of the control group, with statistical significance (P<0.05), which shows that the ORF1p and ORF2p expressed in mouse source L1 can also achieve the corresponding gene editing purpose. According to the description of this embodiment, ORF1p and / or ORF2p expressed by different L1 species in human genome or ORF1p and / or ORF2p expressed by L1 of different species can be applied to gene editing in the present invention, and can achieve the corresponding gene editing purpose.At the same time, ORF1p and ORF2p of human source LRE1, ORF1p and ORF2p of human source LRE2 and ORF1p and ORF2p of mouse source are also modified sequences of other ORF1p and ORF2p coding sequences, this embodiment supports the application of modified sequence of ORF1p coding sequence and modified sequence of ORF2p coding sequence. Example 10: After producing specific RNA (3' site is partially Alu) by in vitro transcription, ORF1p and ORF2p are combined outside the target system, and then introduced into the target system to measure the efficiency of gene editing. The Alu used in Examples 2-9 is Alu Ya 5 among Alu elements, and in this Example, in order to test the effect of other kinds of Alu elements, the sequence of Alu Yb 8 was selected to perform gene editing, and the DNA sequence of Alu Yb 8 is shown in Seq ID No. 51: GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCATGAGGTCAGGAGATCGAGACCATCCTGGCTAACAAGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAAAAAAATTAGCCGGGCGCGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCAGTCCGCAGTCCGGCCTAGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAATGTGGCCAAAAGTGTCAGAAA; A portion of the DNA sequence in Alu Yb 8 was excised, as shown in Seq ID No. 52: AAAAATACAAAAAAAAAAAATTAGCCGGGCGCGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCAGTCCGCAGTCCGGCCTAGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAATGTGGCCAAAAGTGTCAGAAA. A 405 bp sequence in the gene Lman1 in the human genome shown in Seq ID No. 8 was selected and the sequence to be inserted shown in Seq ID No. 9 was inserted to obtain the sequence shown in Seq ID No. 10. The T7 promoter sequence shown in Seq ID No. 11 was added upstream of the sequence shown in Seq ID No. 10, and the partial Alu Yb8 sequence shown in Seq ID No. 52 was added downstream to obtain the sequence shown in Seq ID No. 53: TAATACGACTCACTATAGGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG CCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG AAAAATACAAAAAAAAAAAATTAGCCGGGCGCGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCAGTCCGCAGTCCGGCCTAGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAATGTGGCCAAAAGTGTCAGAAA , Here, the underlined sequence is the sequence to be inserted (sequence to be inserted) shown in Seq ID No. 9, the italicized bold text is the T7 promoter sequence shown in Seq ID No. 11, and the wavy line is the partial Alu Yb8 sequence shown in Seq ID No. 52. This sequence was obtained by chemical synthesis and is named the precursor DNA of RNA + partial Alu Yb8. Linear RNA + partial Alu Yb8 precursor DNA or RNA precursor DNA with the MEGAscript kit TMThe corresponding RNA was obtained by transcription according to the instructions of the T7 Transcription Kit, and then residual DNA was degraded using the DNase in the kit, resuspended in RNase-free water, and the RNA concentration was measured by UV spectrophotometry, followed by the RNA + partial Alu Yb8 solution with the addition of RNase-free water to place its concentration at 100 ng / μL. The RNA+ portion Alu obtained by the above transcription belongs to the RNA framework shown in Figure 4, and the ORF2p functional initiation portion is another portion, a short interspersed element RNA. The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were each resuspended to a 500 ng / μL hLRE1-ORF1p solution or hLRE1-ORF2p solution using an Opti-MEM solution to which 1 U / μL of RNase inhibitor had been added in advance. Prepare the RNP consisting of RNA + partial Alu Yb8, ORF1p and ORF2p: RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p, according to the following procedure: Prepare the reaction system shown in Table 41: Table 41 Reaction system TIFF2025510714000043.tif48170After gentle mixing to homogeneity, the system was incubated at room temperature (25°C) for 10 minutes to obtain an RNA + partial Alu Yb8 + hLRE1-ORF1p solution. Next, the RNA+partial Alu Yb8+hLRE1-ORF1p solution was mixed in the system shown in Table 42. Table 42 Reaction system TIFF2025510714000044.tif40170 After gently mixing each component to homogenize, the reaction system was incubated at room temperature (25°C) for 10 minutes to obtain an RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p solution. The RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p solution was mixed with an equal volume ratio of the transfection solution system prepared in Table 7, gently mixed to homogenize, and then incubated at room temperature (25°C) for 20 minutes to obtain the RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex. Transfection First, HeLa cells were subcultured and placed on a 24-well plate, and cultured in complete medium. The day after subculture, the HeLa cells were allowed to grow to 60% confluence, and then Opti-MEM TM The medium was replaced with I medium, and RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex was added to HeLa cells and transfected according to the RNAiMAX transfection reagent instructions, and three parallel runs were set up. 6 h after transfection, the medium was replaced with complete medium. The cultured cells were grown to about 90% and allowed to fuse, then re-passaged, and the transfection was repeated once after passaging (following the procedure above), and the cells were allowed to regrow to about 90% and allowed to fuse, before the subsequent operations were performed. Experimental grouping: The control group of Example 2 was used as the control group of this example, and the prepared RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p group was used as the experimental group, with three parallel groups in each group, each of which was a 24-well plate cultured with one HeLa cell. Extract the post-transfection cell DNA from each group: After aspirating off the cell medium, wash the cells twice with PBS, then add an appropriate amount of 0.25% trypsin to digest them, digest them at 37°C for 20 min, and blow them 15 times every 5 min. After the cells were suspended, add complete medium containing serum to stop the reaction. Then, extract the cell DNA according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and measure the DNA concentration by ultraviolet spectrophotometer. qPCR Testing: Since the copy number of the GAPDH gene is stable, the GAPDH gene is used as the endogenous gene. The upstream primer sequence for detecting the GAPDH gene is 5'-CACTGCCACCCAGAAGACTG-3' as shown in Seq ID No. 19, and the downstream primer sequence is 5'-CCTGCTTCACCACCTTCTTG-3' as shown in Seq ID No. 20. Designed primer pair 1, the upstream primer sequence is shown in Seq ID No. 21: 5'-GACTTATCCATGTGCCTGTT-3', the downstream primer sequence is 5'-TTGGCTACGAAATGCTTG-3', as shown in Seq ID No. 22. The upstream primer sequence of primer pair 1 is located in the complete Lman 1 gene, is located upstream of the insertion site (target site) upstream sequence (target site upstream sequence) used in the prepared RNA, is not present in the prepared RNA sequence, and is present only in the cell genome, and the downstream primer sequence of primer pair 1 is located on the exogenous sequence to be inserted (sequence to be inserted). All of the above primers were obtained by chemical synthesis. The qPCR reaction system was as shown in Table 9, and the qPCR reaction was carried out according to the qPCR reaction cycle of Example 2. The GAPDH primer reacted under the same conditions. After observing the exponential growth phase of the amplification curves of GAPDH and the inserted sequence and confirming near parallelism, the obtained data were -ΔΔCt The results analyzed by the relative method are shown in Table 43. The sequencing verification of the PCR products is correct. Table 43 Results of primer pair 1 (n=3, x±s) TIFF2025510714000045.tif102170 Table 43 shows that the experimental group and the control group are compared, and the relative copy number of the experimental group is significantly higher than that of the control group, with statistical significance (P<0.05), which shows that the purpose of gene editing can be achieved even if the type of Alu element is exchanged. It is explained that the present invention can be applied to all types of Alu elements and short interspersed elements in all biological species. From the above-mentioned embodiments, it can be seen that the RNA framework provided by the present invention can generate RNA through eukaryotic or prokaryotic systems and cells, tissues, living organisms or in vitro expression, generate the required proteins ORF1p and / or ORF2p in the target system or outside the target system (in vitro), and achieve the goal of gene editing in the target system through the introduction of vectors in the form of RNA or RNP, facilitating industrial mass production and commercialization. In addition, since there is no splicing mechanism of precursor mRNA in eukaryotic systems in prokaryotic systems or in vitro expression, the RNA framework and downstream connectable short interspersed element RNA, short interspersed element derivative RNA, long interspersed element, long interspersed element derivative RNA, and / or functional structures of the initiating ORF2p shearing function and reverse transcription can be expressed without any hindrance without potential splicing risks, improving the production efficiency and gene editing effect of the present invention. Therefore, the present invention can perform precise sequence deletion, sequence replacement and individual site replacement through the homologous recombination or genome repair of the recipient system itself after targeting the insertion of a desired sequence into the genome. At the same time, based on the technical principle of the present invention, the present invention can continue to design a vector through the new site formed after the sequence is inserted by the present invention, and the inserted sequence has no theoretical upper limit in length, and can achieve gene editing purposes such as sequence insertion, deletion, replacement and site replacement in various and various forms, and the usage method is flexible. In addition, the present invention can also perform gene editing on CNV and its ends to make them stable, unchanged, extended, shortened or change their expression sequence, etc., to achieve the purpose of changing or stabilizing the gene expression and self-state of cells or organisms. Since short interspersed elements, long interspersed elements, and the proteins expressed by them are widely present in eukaryotes, the present invention can be used to perform gene editing operations on a wide range of eukaryotes. It can also be applied to the treatment of diseases involving genetic alterations, and to the change and stabilization of cellular and biological conditions associated with genetic alterations. The present invention can also be used for gene editing of various prokaryotes. The genome introduction sequence by other gene editing tools, such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas9 and other technologies, is mainly completed by homologous recombination between template DNA and genome target site and its surrounding region after incision of genome target site, which is easy to introduce random sequence and mutation, and the efficiency of inserting target sequence is low.The RNA framework and corresponding RNP provided by the present invention do not cause double strand breaks, and carry out genome integration by homologous recombination, which is relatively safe and convenient to use. The present invention allows the administration of an exogenous sequence in the form of RNA to a target system and insert it into the genome, thus demonstrating the ability of the RNA to be converted into DNA and generate template DNA. Therefore, when the DNA capable of expressing the RNA framework and / or its improved RNA of the present invention, the RNA framework and / or its improved RNA of the present invention, and the RNP generated by the RNA framework and / or its improved RNA of the present invention binding to ORF2p, ORF1p, ORF2p-derived protein and / or ORF1p-derived protein is administered to a target system, template DNA can be generated without introducing template DNA, or template DNA can be generated (amplified) in large quantities. Therefore, the present invention can also improve the gene editing function of other gene editing tools such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas9. In addition, based on the results and principles of the examples, the RNA required for gene editing in the present invention can be generated in vitro, bound to ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins, and introduced into a prokaryotic or eukaryotic system to generate single-stranded DNA or double-stranded DNA bound to ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins, which can then be introduced into a target system such as a prokaryotic or eukaryotic system to achieve the purpose of gene editing. The above examples only represent some embodiments of the present invention, and the description is more specific and detailed, but therefore cannot be understood as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make some modifications and improvements without departing from the concept of the present invention. All of these belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be in accordance with the appended claims.

Claims

1. In the 5' to 3' direction, it comprises a target site upstream sequence, a sequence to be inserted, and a target site downstream sequence, An RNA framework for gene editing, characterized in that a target site upstream sequence or a complementary sequence of the target site upstream sequence hybridizes with a target site upstream sequence or a complementary sequence of the target site upstream sequence of a target site in a eukaryotic genome or a prokaryotic genome, and a target site downstream sequence or a complementary sequence of the target site downstream sequence hybridizes with a target site downstream sequence or a complementary sequence of the target site downstream sequence of a target site in a eukaryotic genome or a prokaryotic genome, the target site upstream sequence and the target site downstream sequence are directly linked in corresponding sequences in the genome, and a target site is located between the target site upstream sequence and the target site downstream sequence in the genome.

2. The RNA framework for gene editing described in claim 1, characterized in that one or more ORF2p functional initiation portions are directly or indirectly linked downstream of the target site downstream sequence, or the target site downstream sequence is replaced or partially replaced by one or more ORF2p functional initiation portions, and multiple ORF2p functional initiation portions are directly or indirectly linked to each other.

3. Within the ORF2p functional start portion, one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are further inserted, and when a pan-ORF1p coding sequence or a pan-ORF2p coding sequence is inserted within the ORF2p functional start portion, the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or pan-ORF2p coding sequence, and within the ORF2p functional start portion, a) multiple pan-ORF1p coding sequences, or b) multiple pan-ORF2p coding sequences are inserted. The RNA framework for gene editing described in claim 2, characterized in that: a sequence is inserted; or c) if the total number of pan-ORF1p coding sequences and pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequences and the pan-ORF2p coding sequences are directly or indirectly linked, the pan-ORF1p coding sequences are directly or indirectly linked to each other, the pan-ORF2p coding sequences are directly or indirectly linked to each other, and the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or the pan-ORF2p coding sequence.

4. The RNA framework for gene editing described in claim 1, further comprising one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences linked directly or indirectly upstream of the upstream sequence of the target site, and / or within the upstream sequence of the target site, and / or within the downstream sequence of the target site, and / or downstream of the downstream sequence of the target site.

5. The RNA framework for gene editing described in claim 4, characterized in that the one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are located upstream of the target site upstream sequence, within the target site upstream sequence, within the target site downstream sequence, or downstream of the target site downstream sequence, and are at the same position: a) the total number of the multiple pan-ORF1p coding sequences is two or more, or b) the total number of the multiple pan-ORF2p coding sequences is two or more, or c) when the total number of the pan-ORF1p coding sequences and the pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequences and the pan-ORF2p coding sequences are directly or indirectly linked between each other, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other.

6. The RNA framework for gene editing described in claim 4, characterized in that one or more ORF2p functional initiation portions are further linked directly or indirectly downstream of the downstream sequence of the target site.

7. The RNA framework for gene editing described in claim 6, characterized in that the one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are located upstream of the target site upstream sequence, within the target site upstream sequence, or within the target site downstream sequence, and are in the same position: a) the total number of the multiple pan-ORF1p coding sequences is two or more, or b) the total number of the multiple pan-ORF2p coding sequences is two or more, or c) when the total number of the pan-ORF1p coding sequences and the pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequences and the pan-ORF2p coding sequences are directly or indirectly linked, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other.

8. When the one or more pan-ORF1p coding sequences and / or the one or more pan-ORF2p coding sequences are located downstream of a target site downstream sequence, a) when one or more ORF2p functional initiations and one or more panORF1p coding sequences are present, the one or more ORF2p functional initiations are located before or after the one or more panORF1p coding sequences, or the ORF2p functional initiation is spaced apart from the panORF1p coding sequence, the ORF2p functional initiation and the panORF1p coding sequence are directly or indirectly linked, a plurality of the panORF1p coding sequences are directly or indirectly linked to each other, a plurality of the ORF2p functional initiations are directly or indirectly linked to each other, or b) when one or more ORF2p functional initiations and one or more pan-ORF2p coding sequences are present, the one or more ORF2p functional initiations are located before or after the one or more pan-ORF2p coding sequences, or the ORF2p functional initiation is spaced apart from the pan-ORF2p coding sequence, the ORF2p functional initiation and the pan-ORF2p coding sequence are directly or indirectly linked, a plurality of the pan-ORF2p coding sequences are directly or indirectly linked to each other, a plurality of the ORF2p functional initiations are directly or indirectly linked to each other, or c) if one or more ORF2p functional initiations, one or more pan-ORF1p coding sequences, and one or more pan-ORF2p coding sequences are present, the ORF2p functional initiations are located before or after the one or more pan-ORF1p coding sequences, or the one or more pan-ORF2p coding sequences are located before or after the one or more pan-ORF2p coding sequences, or the ORF2p functional initiations, the pan-ORF1p coding sequences, and / or the pan-ORF2p coding sequences are spaced apart; The RNA framework for gene editing described in claim 6, characterized in that the ORF2p functional start portion and the pan-ORF1p coding sequence are directly or indirectly linked, the ORF2p functional start portion and the pan-ORF2p coding sequence are directly or indirectly linked, a plurality of the pan-ORF1p coding sequences are directly or indirectly linked to each other, a plurality of the pan-ORF2p coding sequences are directly or indirectly linked to each other, a plurality of the ORF2p functional start portions are directly or indirectly linked to each other, and the pan-ORF1p coding sequence and the pan-ORF2p coding sequence are directly or indirectly linked.

9. When the internal portion of a single ORF2p functional initiation portion of the one or more ORF2p functional initiation portions is further linked directly or indirectly to one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, The RNA framework for gene editing according to any one of claims 6 to 8, characterized in that when one pan-ORF1p coding sequence or one pan-ORF2p coding sequence is inserted into the ORF2p functional start portion, the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or pan-ORF2p coding sequence, and when a) multiple pan-ORF1p coding sequences, or b) multiple pan-ORF2p coding sequences are inserted into the ORF2p functional start portion, or c) when the total number of pan-ORF1p coding sequences and pan-ORF2p coding sequences is two or more, the pan-ORF1p coding sequence and the pan-ORF2p coding sequence are directly or indirectly linked, the pan-ORF1p coding sequences are directly or indirectly linked to each other, and the pan-ORF2p coding sequences are directly or indirectly linked to each other, and the ORF2p functional start portion is directly or indirectly linked to the pan-ORF1p coding sequence or pan-ORF2p coding sequence.

10. The target site downstream sequence is replaced or partially replaced by one or more functional initiation portions of ORF2p; The RNA framework for gene editing according to any one of claims 4 to 9, characterized in that, when there are multiple ORF2p functional start portions, each of the ORF2p functional start portions is directly or indirectly linked to each other.

11. The RNA framework for gene editing described in any one of claims 2, 3, and 6 to 10, characterized in that the sequence of the ORF2p functional start portion is a short interspersed element RNA, a long interspersed element RNA, a short interspersed element derivative RNA, a long interspersed element derivative RNA, or a sequence of a functional structure for initiating ORF2p cleavage function and reverse transcription.

12. The RNA framework for gene editing according to any one of claims 3 to 11, characterized in that the pan-ORF1p coding sequence is an ORF1p coding sequence or a modified version of the ORF1p coding sequence, and the pan-ORF2p coding sequence is an ORF2p coding sequence or a modified version of the ORF2p coding sequence.

13. An RNA framework for gene editing according to any one of claims 1 to 12, characterized in that it is obtained by prokaryotic transcription, eukaryotic transcription or chemical synthesis.

14. The RNA framework for gene editing according to any one of claims 1 to 12, characterized in that it is a linear RNA or is located in a linear RNA, or it is a circular RNA or is located in a circular RNA.

15. The RNA framework for gene editing according to claim 14, characterized in that the linear RNA on which the RNA framework is located or the circular RNA on which the RNA framework is located is obtained by prokaryotic transcription, eukaryotic transcription or chemical synthesis.

16. The RNA framework for gene editing according to claim 13 or 15, characterized in that the prokaryotic transcription is transcription by a prokaryotic RNA polymerase, and the eukaryotic transcription is transcription by a eukaryotic RNA polymerase I, a eukaryotic RNA polymerase II, or a eukaryotic RNA polymerase III.

17. An RNP, characterized in that the RNP is obtained by binding the RNA framework for gene editing according to any one of claims 1 to 13 to ORF1p, ORF2p, an ORF1p-derived protein and / or an ORF2p-derived protein, or the RNA framework for gene editing according to claim 14 or 15 to a linear RNA in which the RNA framework is located or a circular RNA in which the RNA framework is located is bound to ORF1p, ORF2p, an ORF1p-derived protein and / or an ORF2p-derived protein.

18. A DNA sequence that transcribes an RNA framework for gene editing according to any one of claims 1 to 13.

19. A DNA sequence characterized in that it transcribes a linear or circular RNA in which the RNA framework for gene editing described in claim 14 or 15 is located.

20. 20. The DNA sequence according to claim 18 or 19, further comprising a prokaryotic or eukaryotic promoter linked directly or indirectly upstream, downstream and / or within said DNA sequence.

21. 21. The DNA sequence of claim 20, wherein the prokaryotic promoter is T7, T3, T7lac, Sp6, araBAD, trp, lac, Ptac, pL, LacUV5, Tac, pBAD or pR.

22. 21. The DNA sequence of claim 20, wherein the eukaryotic promoter is CMV, pCMV, EF1a, SV40, human PGK1, mouse PGK1, Ubc, human beta actin, CAG, EFT3, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1, GAL10, GAL1, GAL10, GAL4, GAL80, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, human U6 or mouse U6.

23. A DNA vector comprising the DNA sequence according to any one of claims 18 to 22.

24. Step 1) of selecting a target site to be edited from a genome and determining an upstream sequence of the target site and a downstream sequence of the target site on both sides of the target site; Step 2) of producing an RNA framework for gene editing according to any one of claims 1 to 13, and / or producing a linear RNA or a circular RNA in which the RNA framework for gene editing according to claim 14 or 15 is located, and / or producing an RNP according to claim 17, and / or producing a DNA vector according to claim 23; 3a) transforming or transfecting said RNA framework into a cell, tissue, organ or organism to achieve gene editing; or Step 3b) of transforming or transfecting a cell, tissue, organ or organism with the linear or circular RNA in which the RNA framework is located to achieve gene editing; or 3c), transforming or transfecting the RNP into a cell, tissue, organ, or organism to achieve gene editing; or 3d) transforming or transfecting the DNA vector into a cell, tissue, organ or organism to achieve gene editing; or Step 3e) of co-transforming or co-transfecting a plurality of the RNA frameworks, the linear or circular RNA in which the RNA frameworks are located, the RNPs, and the DNA vectors into a cell, tissue, organ, or organism to achieve gene editing; or and step 3f) of co-transforming or co-transfecting one or more of the RNA framework, the linear RNA or circular RNA in which the RNA framework is located, the RNP, and the DNA vector with ORF1p, ORF2p, an ORF1p-derived protein, and / or an ORF2p-derived protein into a cell, tissue, organ, or organism to achieve gene editing.

25. Step 1) of selecting a target site to be edited from a genome and determining an upstream sequence of the target site and a downstream sequence of the target site on both sides of the target site; Step 2) of producing an RNA framework for gene editing according to any one of claims 1 to 13, and / or producing a linear RNA or a circular RNA in which the RNA framework for gene editing according to claim 14 or 15 is located, and / or producing an RNP according to claim 17, and / or producing a DNA vector according to claim 23; 3) producing an ORF2p functional initiation sequence, one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, and / or a helper RNA obtained by binding said helper RNA to ORF1p, ORF2p, ORF1p-derived protein and / or ORF2p-derived protein, and / or a helper DNA vector transcribing the ORF2p functional initiation sequence, the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence; Step 4a) of co-transforming or co-transfecting the RNA framework with the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4b) of co-transforming or co-transfecting the linear RNA or circular RNA in which the RNA framework for gene editing is located and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4c) of co-transforming or co-transfecting the RNPs with the helper RNA, helper RNPs and / or helper DNA vectors produced in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4d) of co-transforming or co-transfecting the DNA vector with the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into a cell, tissue, organ or organism to achieve gene editing; or Step 4e) of co-transforming or co-transfecting the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector with the helper RNA, helper RNP, and / or helper DNA vector produced in step 3) into a cell, tissue, organ, or organism to achieve gene editing; or and step 4f) of co-transforming or co-transfecting the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and one or more of the DNA vector, the helper RNA, the helper RNP, and the helper DNA vector produced in step 3), and one or more of ORF1p, ORF2p, an ORF1p-derived protein, and an ORF2p-derived protein into a cell, tissue, organ, or organism to achieve gene editing.

26. The RNA framework for transforming, transfecting, cotransforming or cotransfecting a cell, tissue, organ or organism, the linear or circular RNA in which the RNA framework for gene editing is located, the RNP, the DNA vector, are one or more; In the case of a linear or circular RNA in which one of the RNA frameworks or one of the RNA frameworks for gene editing is located, or one of the RNPs or one of the DNA vectors, editing is realized at one location on the genome; The gene editing method of claim 24 or 25, characterized in that when the total number of the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector is two or more, and when the upstream sequence of the target site and / or the downstream sequence of the target site in the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, and the DNA vector are different, editing or manipulation of multiple sites on the genome is realized.

27. Use of an RNA framework for gene editing according to any one of claims 1 to 13, or a linear or circular RNA in which an RNA framework for gene editing according to claim 14 or 15 is located, or an RNP according to claim 17, or a DNA vector according to claim 23, as a drug for preventing and / or treating cancer, a gene-related disease or a neurodegenerative disease.

28. 28. The use according to claim 27, wherein the cancer is selected from the group consisting of glioma, breast cancer, cervical cancer, lung cancer, gastric cancer, colorectal cancer, duodenal cancer, leukemia, prostate cancer, endometrial cancer, thyroid cancer, lymphoma, pancreatic cancer, liver cancer, melanoma, skin cancer, pituitary tumor, germ cell tumor, meningioma, meningeal carcinoma, glioblastoma, various astrocytomas, various oligodendrogliomas, astrocytic oligodendrogliomas, various ependymomas, choroid plexus papilloma, choroid plexus carcinoma, chordoma, various ganglioneuromas, olfactory neuroblastoma, sympathetic nervous system neuroblastoma, pineal cell tumor, pineoblastoma, medulloblastoma, retinoblastoma, trigeminal neurinoma, facial acoustic neuroma, glomus jugular tumor, hemangioreticuloma, craniopharyngioma and granular cell tumor.

29. The diseases associated with the gene include Huntington's disease, fragile X syndrome, phenylketonuria, Duchenne muscular dystrophy, mitochondrial encephalomyopathy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, polysaccharidosis type IIIA, mucopolysaccharidosis type IIIB, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, mucopolysaccharidosis type IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type IX, spinal muscular atrophy, Parkinson's syndrome, albinism, red-green color blindness, achondroplasia, melauria, congenital deafness, thalassemia, sickle cell disease, epilepsy associated with genetic alterations, myelopathy, and the like. Occlonus, dystonia, stroke and schizophrenia, vitamin D-resistant rickets, familial colonic polyposis syndrome, 21-hydroxylase deficiency, arginase deficiency, Alport syndrome, Angelman syndrome, Jeune syndrome, atypical hemolytic uraemic syndrome, autoimmune encephalitis, autoimmune hypophysitis, insulin autoimmune syndrome, beta-ketothiolase deficiency, biotinidase deficiency, channelopathies, primary carnitine deficiency, Castleman disease, Charcot-Marie-Tooth disease, citrullinemia, congenital adrenal hypoplasia, congenital hyperinsulinemia, congenital myasthenia syndrome, nondystrophic myotonia syndrome, congenital scoliosis, coronary artery ectasia, congenital dyserythroplastic anemia, Erdheim-Chester disease, Fabry disease, familial Mediterranean fever, Fanconi anemia, galactosemia, Gaucher disease, generalized myasthenia gravis, Gitelman syndrome, glutaric acidemia type I, glycogen storage disease (types I and II), hemophilia, hepatolenticular degeneration, hereditary angioedema, hereditary epidermolysis bullosa, hereditary fructose intolerance, hereditary hypomagnesemia, hereditary multi-infarct dementia, hereditary spastic paraplegia, holocarboxylase synthase deficiency, hyperhomocytosis, hypercalcaemia ... Steinemia, homozygous familial hypercholesterolemia, HHH syndrome, hyperphenylalaninemia, hypophosphatasia, hypophosphatemic rickets, idiopathic cardiomyopathy, idiopathic hypogonadism, idiopathic pulmonary hypertension, idiopathic pulmonary fibrosis, IgG4-related disease, congenital bile acid metabolism disorder, isovaleric acidemia, Kallmann syndrome, Langerhans histiocytosis, Laron syndrome, Leber's hereditary optic neuropathy, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, lymphangioleiomyomatosis, lysinuric protein intolerance, lysosomal acid lipase deficiency, maple syrup urine disease, Marfan syndrome,McCune-Albright syndrome, medium-chain acyl-CoA dehydrogenase deficiency, methylmalonic acidemia, multifocal motor neuropathy, multiple acyl-CoA dehydrogenase deficiency, multiple sclerosis, myotonic dystrophy, N-acetylglutamate synthetase deficiency, neonatal diabetes mellitus, neuromyelitis optica, Niemann-Pick disease, nonsyndromic hearing loss, Noonan syndrome, ornithine carbamoyltransferase deficiency, osteogenesis imperfecta, juvenile Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Peutz-Jeghers syndrome, POEMS syndrome, porphyria, Prader-Willi syndrome, primary immunodeficiency, primary hereditary dystonia, primary light-chain amyloidosis, progressive familial intrahepatic cholestasis, progressive muscular dystrophy, propionic acidemia, pulmonary alveolar proteinosis, cystic fibrosis, retinitis pigmentosa, congenital agranulocytosis, severe infantile 28. The use according to claim 27, characterized in that the disease is myoclonic epilepsy, Dravet syndrome, Silver-Russell syndrome, sitosterolemia, spinal-bulbar muscular atrophy, spinal muscular atrophy, spinocerebellar ataxia, systemic sclerosis, tetrahydrobiopterin deficiency, tuberous sclerosis, tyrosinemia, very long-chain acyl-CoA dehydrogenase deficiency, Williams syndrome, eczematous thrombocytopenia with immunodeficiency syndrome, X-linked agammaglobulinemia, adrenoleukodystrophy, X-linked lymphoproliferative syndrome, arteriosclerotic cerebral small vessel disease, cerebral amyloid angiopathy, autosomal recessive cerebral arteriopathy with subcortical infarctions and leukoencephalopathy, autosomal dominant cerebral arteriopathy with subcortical infarctions and leukoencephalopathy, cathepsin A-related arteriopathy with stroke and leukoencephalopathy, pyridoxine-dependent epilepsy, serotonin-metabolizing AADC enzyme deficiency, AADC deficiency or hereditary nephritis.

30. 28. The use according to claim 27, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, multiple system atrophy, primary lateral sclerosis, Pick's disease, frontotemporal dementia, dementia with Lewy bodies or progressive supranuclear palsy.

31. Use of an RNA framework for gene editing according to any one of claims 1 to 13, or a linear or circular RNA in which the RNA framework for gene editing according to claim 14 or 15 is located, or an RNP according to claim 17, or a DNA vector according to claim 23, as a tool for inserting a target sequence, deleting a target sequence, replacing a target sequence, deleting a target site, adding a target site, adding a target sequence, replacing a target site, inverting a target gene sequence, and / or correcting an inversion of a target gene sequence.

32. Use of an RNA framework for gene editing according to any one of claims 1 to 13, or a linear or circular RNA in which an RNA framework for gene editing according to claim 14 or 15 is located, or an RNP according to claim 17, or a DNA vector according to claim 23, in the production or amplification of a DNA template containing an RNA framework sequence according to any one of claims 1 to 13.

33. Use of the RNA framework for gene editing according to any one of claims 1 to 13, or the linear RNA or circular RNA in which the RNA framework for gene editing according to claim 14 or 15 is located, or the RNP according to claim 17, or the DNA vector according to claim 23, or the DNA template according to claim 32, as a tool for improving the gene editing efficiency of TALEN, ZFN, Targetron, Prime Editor, Twin Prime Editor, CRISPR or CRISPR / Cas9 gene technology.

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