RNA frameworks for gene editing and methods of gene editing

Abstract

The application provides an RNA framework for gene editing and a gene editing method, the RNA framework comprising a sequence upstream of a target site, a sequence to be inserted, and a sequence downstream of the target site in the 5' to 3' direction. The gene editing method is based on the inherent mechanism of eukaryotes, uses RNP or RNA (which can be prepared in vitro) and related proteins as carriers, and is transferred into the cytoplasm and nucleus to realize gene editing on a specific sequence or site on the genome of a target system, such as insertion, deletion, replacement and site replacement of a specific sequence, and has high targeting accuracy. The application is more suitable for further clinical application than other prior art because no exogenous system or substance such as protein derived from prokaryotes is introduced, and no double-strand break is generated.

Description

Technical Field

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

[0002] Currently, the main gene editing technologies in the biological field include TALEN, ZFN, Targetron, and CRISPR / Cas9. These technologies have been used for some time and are relatively mature, but they still have significant shortcomings.

[0003] ZFN technology can only recognize sequences as short as 9 bp, resulting in poor targeting accuracy. Furthermore, its complexity, high off-target rate, and significant cytotoxicity hinder its practical application. While TALEN technology is simpler than ZFN and can recognize longer sequences, its complexity still impedes its further application in various fields. CRISPR / Cas9 technology is currently the mainstream gene editing technology and is relatively easy to operate, but it still has significant off-target effects, and the risks posed by the resulting DNA double-strand breaks hinder its further clinical application. Targetron technology uses class II introns to introduce exogenous sequences into specific sites in the genome, but this invention introduces class II introns into the genome, creating "scars," and its performance is only good in bacterial gene editing, making it difficult to apply to other more advanced organisms.

[0004] All three technologies face various obstacles to their application, such as off-target effects, technical complexity, and unknown risks caused by double-strand breaks. Furthermore, these technologies inevitably introduce genetic material and proteins not belonging to the receiving system, leading to unpredictable consequences and severely hindering their clinical application. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an RNA framework for gene editing, which enables the insertion, deletion, sequence substitution, and site substitution of DNA in any region of the genome.

[0006] Another object of the present invention is to provide an RNP.

[0007] A third objective of this invention is to provide a DNA sequence.

[0008] The fourth object of the present invention is to provide a DNA vector.

[0009] The fifth objective of this invention is to provide a gene editing method.

[0010] A sixth object of the present invention is to provide the application of the above-described RNA framework for gene editing.

[0011] To achieve the above objectives, the present invention provides an RNA framework for gene editing, which includes an upstream sequence of the target site, a sequence to be inserted, and a downstream sequence of the target site along the 5′→3′ direction;

[0012] The upstream sequence of the target site or its complementary sequence on the RNA framework is used to hybridize with the upstream sequence of the target site or its complementary sequence on the target site in the eukaryotic or prokaryotic genome. The downstream sequence of the target site or its complementary sequence on the RNA framework is used to hybridize with the downstream sequence of the target site or its complementary sequence on the target site in the eukaryotic or prokaryotic genome. The upstream and downstream sequences of the target site on the RNA framework are directly linked in their corresponding sequences in the genome. The upstream and downstream sequences of the target site in the genome are the target sites.

[0013] As described above, the RNA framework for gene editing further includes: directly or indirectly linking one or more ORF2p functional start portions downstream of the target site downstream sequence; or replacing or partially replacing the target site downstream sequence of the RNA framework for gene editing with one or more ORF2p functional start portions; wherein the multiple ORF2p functional start portions are directly or indirectly linked.

[0014] As described above, one or more generic ORF1p encoding sequences and / or one or more generic ORF2p encoding sequences are further inserted inside the ORF2p function start portion; wherein, when a generic ORF1p encoding sequence or a generic ORF2p encoding sequence is inserted inside the ORF2p function start portion, the ORF2p function start portion is directly or indirectly connected to the generic ORF1p encoding sequence or the generic ORF2p encoding sequence; when a) multiple generic ORF1p encoding sequences, or b) multiple generic ORF1p encoding sequences, or c) the sum of the number of generic ORF1p encoding sequences and generic ORF2p encoding sequences is greater than or equal to two, the generic ORF1p encoding sequences are directly or indirectly connected to the generic ORF2p encoding sequences, the generic ORF1p encoding sequences are directly or indirectly connected to each other, the generic ORF2p encoding sequences are directly or indirectly connected to each other, and the ORF2p function start portion is directly or indirectly connected to the generic ORF1p encoding sequence or the generic ORF2p encoding sequence.

[0015] As described above, the RNA framework further includes one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences directly or indirectly connected upstream of the target site, and / or inside the upstream sequence of the target site, and / or inside the downstream sequence of the target site, and / or downstream of the downstream sequence of the target site.

[0016] As described above, when one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are located upstream of the upstream sequence of the target site, inside the upstream sequence of the target site, inside the downstream sequence of the target site, or downstream of the downstream sequence of the target site, and when the sum of the number of a) multiple pan-ORF1p coding sequences at the same position is greater than or equal to two, or b) the sum of the number of multiple pan-ORF2p coding sequences is greater than or equal to two, or c) the sum of the number of pan-ORF1p coding sequences and pan-ORF2p coding sequences is greater than or equal to two, the pan-ORF1p coding sequences are directly or indirectly connected to the pan-ORF2p coding sequences, the pan-ORF1p coding sequences are directly or indirectly connected to each other, and the pan-ORF2p coding sequences are directly or indirectly connected to each other.

[0017] As described above, the RNA framework for gene editing further includes one or more ORF2p functional initiation portions directly or indirectly connected downstream of the target site sequence.

[0018] As described above, when 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, inside the target site upstream sequence, or inside the target site downstream sequence, and at the same position a) the sum of the number of multiple pan-ORF1p coding sequences is greater than or equal to two, or b) the sum of the number of multiple pan-ORF2p coding sequences is greater than or equal to two, or c) the sum of the number of pan-ORF1p coding sequences and pan-ORF2p coding sequences is greater than or equal to two, the pan-ORF1p coding sequences are directly or indirectly connected to the pan-ORF2p coding sequences, the pan-ORF1p coding sequences are directly or indirectly connected to each other, and the pan-ORF2p coding sequences are directly or indirectly connected to each other.

[0019] As described above, when the one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences are located downstream of the target site downstream sequence:

[0020] a) When there are one or more ORF2p functional start portions and one or more generalized ORF1p coding sequences, the one or more ORF2p functional start portions are located before or after the one or more generalized ORF1p coding sequences, or the ORF2p functional start portions and the generalized ORF1p coding sequences are arranged alternately, the ORF2p functional start portions and the generalized ORF1p coding sequences are directly or indirectly connected, multiple generalized ORF1p coding sequences are directly or indirectly connected, and multiple ORF2p functional start portions are directly or indirectly connected; or

[0021] b) When there are one or more ORF2p functional start parts and one or more generalized ORF2p coding sequences, the one or more ORF2p functional start parts are located before or after the one or more generalized ORF2p coding sequences, or the ORF2p functional start parts and the generalized ORF2p coding sequences are arranged alternately, the ORF2p functional start parts and the generalized ORF2p coding sequences are directly or indirectly connected, multiple generalized ORF2p coding sequences are directly or indirectly connected, and multiple ORF2p functional start parts are directly or indirectly connected; or

[0022] c) When there are one or more ORF2p function start portions, one or more generalized ORF1p encoding sequences, and one or more generalized ORF2p encoding sequences, the ORF2p function start portion is located before or after the one or more generalized ORF1p encoding sequences, or before or after the one or more generalized ORF2p encoding sequences, or the one or more generalized ORF1p encoding sequences are located before or after the one or more generalized ORF2p encoding sequences, or the ORF2p function start portion, the generalized ORF1p encoding sequence, and / or the generalized ORF2p encoding sequence. The 2p encoding sequences are arranged at intervals; the starting part of the ORF2p function and the generalized ORF1p encoding sequence are directly or indirectly connected, the starting part of the ORF2p function and the generalized ORF2p encoding sequence are directly or indirectly connected, and multiple generalized ORF1p encoding sequences are directly or indirectly connected; multiple generalized ORF2p encoding sequences are directly or indirectly connected, multiple starting parts of the ORF2p function are directly or indirectly connected, and the generalized ORF1p encoding sequence and the generalized ORF2p encoding sequence are directly or indirectly connected.

[0023] As described above, in the RNA framework, when one or more ORF2p functional start portions, wherein a single ORF2p functional start portion is further directly or indirectly connected to one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, wherein, 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 connected to the pan-ORF1p coding sequence or the pan-ORF2p coding sequence; when the ORF2p functional start portion is directly or indirectly connected to the pan-ORF1p coding sequence or the pan-ORF2p coding sequence, the ORF2p functional start portion is directly or indirectly connected to the pan-ORF1p coding sequence or the pan-ORF2p coding sequence; When the starting part can be inserted into a) multiple generalized ORF1p encoding sequences, or b) multiple generalized ORF2p encoding sequences, or c) the sum of the number of generalized ORF1p encoding sequences and generalized ORF2p encoding sequences is greater than or equal to two, the generalized ORF1p encoding sequences and generalized ORF2p encoding sequences are directly or indirectly connected, the generalized ORF1p encoding sequences are directly or indirectly connected, the generalized ORF2p encoding sequences are directly or indirectly connected, or the starting part of the ORF2p function is directly or indirectly connected to the generalized ORF1p encoding sequence or the generalized ORF2p encoding sequence.

[0024] As described above, the downstream sequence of the target site in the RNA framework is replaced or partially replaced with one or more ORF2p functional start portions; wherein, when there are multiple ORF2p functional start portions, each ORF2p functional start portion is directly or indirectly connected to the others.

[0025] As described above, the sequence of the ORF2p functional initiation portion is a short interstitial element RNA, a long interstitial element RNA, a short interstitial element derivative RNA, a long interstitial element derivative RNA, or a sequence of a functional structure that initiates ORF2p splicing and reverse transcription.

[0026] As described above, the generalized ORF1p encoding sequence is an ORF1p encoding sequence or a modified ORF1p encoding sequence, and the generalized ORF2p encoding sequence is an ORF2p encoding sequence or a modified ORF2p encoding sequence.

[0027] As described above, the RNA framework is obtained through prokaryotic transcription, eukaryotic transcription, or chemical synthesis.

[0028] As described above, the RNA framework is either a linear RNA or located within a linear RNA, or the RNA framework is either a circular RNA or located within a circular RNA.

[0029] As described above, the linear RNA or circular RNA in which the RNA framework is located is obtained through prokaryotic transcription, eukaryotic transcription, or chemical synthesis.

[0030] This transcription process can occur in vitro or in prokaryotes or eukaryotes, within tissues, organs, or cells.

[0031] As described above, the prokaryotic transcription is transcribed by prokaryotic RNA polymerase; the eukaryotic transcription is transcribed by eukaryotic RNA polymerase I, eukaryotic RNA polymerase II, or eukaryotic RNA polymerase III.

[0032] The present invention also provides an RNP, wherein the RNP is obtained by binding the above-mentioned RNA framework for gene editing with ORF1p, ORF2p, ORF1p-derived protein and / or ORF2p-derived protein, or the RNP is obtained by binding the linear RNA or circular RNA in which the RNA framework is located to ORF1p, ORF2p, ORF1p-derived protein and / or ORF2p-derived protein in the above-mentioned RNA framework for gene editing.

[0033] The present invention also provides a DNA sequence for transcription into an RNA framework for gene editing as described above.

[0034] The present invention also provides a DNA sequence for transcription into linear RNA or circular RNA containing the RNA framework described above for gene editing.

[0035] As described above, the DNA sequence is further directly or indirectly linked to a prokaryotic promoter or a eukaryotic promoter upstream, downstream, and / or internally.

[0036] As described above, the prokaryotic promoter is T7, T3, T7lac, Sp6, araBAD, trp, lac, Ptac, pL, LacUV5, Tac, pBAD, or pR.

[0037] 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 and GAL10, GAL4, GAL80, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, human U6, or mouse U6.

[0038] The present invention further provides a DNA vector having the DNA sequence described above.

[0039] This invention provides a gene editing method, comprising the following steps:

[0040] 1) Select the target site to be edited in the genome, and determine the upstream and downstream sequences of the target site on both sides of the target site;

[0041] 2) Prepare the RNA framework for gene editing as described above; and / or prepare the linear RNA or circular RNA in which the RNA framework for gene editing is located as described above; and / or prepare the RNP as described above; and / or prepare the DNA vector as described above.

[0042] 3a) Transform or transfect the RNA framework described above into cells, tissues, organs or organisms to achieve gene editing;

[0043] Or 3b) Transform or transfect the linear RNA or circular RNA in which the RNA framework is located into cells, tissues, organs or organisms to achieve gene editing;

[0044] Or 3c) Transform or transfect the RNP into cells, tissues, organs or organisms to achieve gene editing;

[0045] Or 3d) Transform or transfect the DNA vector into cells, tissues, organs or organisms to achieve gene editing;

[0046] Or 3e) co-transform or co-transfect multiple RNAs, including the RNA framework, the linear RNA or circular RNA in which the RNA framework is located, the RNP, and the DNA vector, into cells, tissues, organs, or organisms to achieve gene editing;

[0047] Or 3f) co-transform or co-transfect 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, ORF1p-derived protein and / or ORF2p-derived protein into cells, tissues, organs or organisms to achieve gene editing.

[0048] This invention also provides a gene editing method, comprising the following steps:

[0049] 1) Select the target site to be edited in the genome, and determine the upstream and downstream sequences of the target site on both sides of the target site;

[0050] 2) Prepare the RNA framework for gene editing as described above; and / or prepare the linear RNA or circular RNA in which the RNA framework for gene editing is located as described above; and / or prepare the RNP as described above; and / or prepare the DNA vector as described above.

[0051] 3) Prepare one or more helper RNAs containing an ORF2p functional start sequence, one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, and / or helper RNPs obtained by combining the helper RNA with ORF1p, ORF2p, ORF1p-derived proteins and / or ORF2p-derived proteins, and / or helper DNA vectors that transcribe the ORF2p functional start sequence, pan-ORF1p coding sequence and / or pan-ORF2p coding sequence;

[0052] 4a) Co-transform or co-transfect the RNA framework and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing;

[0053] Or 4b) co-transform or co-transfect 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 cells, tissues, organs or organisms to achieve gene editing;

[0054] Or 4c) co-transform or co-transfect the RNP and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing;

[0055] Or 4d) co-transform or co-transfect the DNA vector and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing;

[0056] Or 4e) co-transform or co-transfect the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, multiple components of the DNA vector, and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing.

[0057] Alternatively, in step 4f), one or more of the following can be co-transformed or co-transfected into cells, tissues, organs, or organisms to achieve gene editing: the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, the DNA vector, the helper RNA, helper RNP, helper DNA vector prepared in step 3), and one or more of ORF1p, ORF2p, ORF1p-derived protein, and ORF2p-derived protein.

[0058] As described above, the transformation, transfection, co-transformation, or co-transfection into cells, tissues, organs, or organisms, 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. When there is only one RNA framework, one linear RNA or circular RNA in which the RNA framework for gene editing is located, one RNP, or one DNA vector, single-site editing on the genome is achieved. When the sum 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 greater than or equal to two, and the upstream and / or downstream sequences 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, multiple-site editing on the genome is achieved.

[0059] This invention provides the use of the RNA framework for gene editing as described above, or the linear RNA or circular RNA in which the RNA framework for gene editing is located, or the RNP or DNA vector as described above, as a medicament for the prevention and / or treatment of cancer, gene-related diseases, or neurodegenerative diseases.

[0060] As described above, the cancers include 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 adenoma, germ cell tumor, meningioma, meningeal carcinoma, glioblastoma, various astrocytomas, various oligodendrogliomas, astrocytodesmoma, various ependymomas, choroid plexus papilloma, choroid plexus carcinoma, chordoma, various gangliocytomas, olfactory neuroblastoma, sympathetic nervous system neuroblastoma, pineal cell tumor, pineal blastoma, medulloblastoma, retinoblastoma, trigeminal schwannoma, facial and auditory nerve tumor, glomus jugulare tumor, hemangioblastoma, craniopharyngioma, or granular cell tumor.

[0061] As described above, the gene-related diseases are Huntington's disease, Fragile X syndrome, phenylketonuria, Duchenne muscular dystrophy, 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 plus syndrome, albinism, red-green color blindness, achondroplasia, alkaptonuria, congenital deafness, thalassemia, and sickle cell anemia. Diseases, hemophilia, epilepsy related to gene alterations, myoclonus, dystonia, stroke and schizophrenia, vitamin D-resistant rickets, familial adenomatous polyposis, 21-hydroxylase deficiency, arginase deficiency, Alport syndrome, Angelman's syndrome, Renner syndrome, atypical hemolytic uremic syndrome, autoimmune encephalitis, autoimmune hypophysitis, autoimmune insulin receptor disease, β-ketothiolase deficiency, biotinase deficiency, cardiac ion channelopathies, primary carnitine deficiency, Castleman's disease, peroneal muscular atrophy, citrullinemia, congenital adrenal dysplasia, congenital hyperinsulinemia, congenital myasthenia gravis, non-malnutrition myotonia syndrome, congenital Scoliosis, coronary artery dilatation, congenital pure red cell aplasia, 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 (type I, type II), hemophilia, Wilson's disease, hereditary angioedema, hereditary epidermolysis bullosa, hereditary fructose intolerance, hereditary hypomagnesemia, hereditary multiple cerebral infarction dementia, hereditary spastic paraplegia, holocarboxylase synthase deficiency, homocysteineemia, homozygous familial hypercholesterolemia, HHH syndrome, hyperphenylalanineemia, hypoalkaline phosphataseemia, hypophosphatemia Rickets, idiopathic cardiomyopathy, idiopathic hypogonadotropic hypogonadism, idiopathic pulmonary hypertension, idiopathic pulmonary fibrosis, IgG4-related diseases, congenital bile acid synthesis disorders, isovaleric acidemia, Kallmann syndrome, Langerhans histiocytosis, Leren's syndrome, Leber hereditary optic neuropathy, long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, lymphangioleiomyomatosis, lysineuria protein intolerance, lysosomal acid lipase deficiency, maple syrup urine disease, Marfan syndrome, McCune-Albrigh syndrome, medium-chain acyl-CoA dehydrogenase deficiency, methylmalonic acidemia, multifocal motor neuropathy, multiple acyl-CoA dehydrogenase deficiencies, multiple sclerosis, myotonic dystrophy.N-acetylglutamate synthase deficiency, neonatal diabetes mellitus, neuromyelitis optica, Niemann-Pick disease, nonsyndromic deafness, Noonan syndrome, ornithine carbamoyltransferase deficiency, osteogenesis imperfecta, juvenile Parkinson's disease, early-onset Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Peutz-Jeghers syndrome, POEMS syndrome, porphyria, Prader-Willi syndrome, primary combined immunodeficiency, primary hereditary dystonia, primary light chain amyloidosis, progressive familial intrahepatic cholestasis, progressive muscular dystrophy, propionic acidemia, pulmonary alveolar proteinosis, cystic fibrosis, retinitis pigmentosa, severe congenital agranulocytosis, severe myoclonic epilepsy in infants, Dravet syndrome, Silver-Russe syndrome Ill syndrome, sitosterolemia, spinal bulbar muscular atrophy, spinal muscular atrophy, spinocerebellar ataxia, systemic sclerosis, tetrahydrobiopterin deficiency, tuberous sclerosis, primary tyrosinemia, very long chain acyl-CoA dehydrogenase deficiency, Williams syndrome, eczematous thrombocytopenic purpura syndrome, X-linked agammaglobulinemia, X-linked adrenoleukodystrophy, X-linked lymphoproliferative disorders, arteriosclerotic cerebral small vessel disease, cerebral amyloid angiopathy, overt cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy, covert cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy, cathepsin A-related arteriosclerosis with stroke and leukoencephalopathy, pyridoxine-dependent epilepsy, AADC enzyme deficiency in serotonin metabolism, AADC deficiency, or hereditary nephritis.

[0062] As stated above, the neurodegenerative diseases are 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, Lewy body dementia, or progressive supranuclear palsy.

[0063] This invention provides the application of the RNA framework for gene editing as described above, or the linear RNA or circular RNA in which the RNA framework for gene editing as described above, or the RNP or DNA vector in described above, as a tool for inserting, deleting, replacing, deleting, adding, replacing, inverting, and / or correcting inversions of target sequences.

[0064] The present invention provides an RNA framework for gene editing as described above, or a linear RNA or circular RNA in which the RNA framework for gene editing is located, or an RNP or DNA vector as described above, for generating or amplifying a DNA template containing the RNA framework sequence described above.

[0065] This invention provides the application of the RNA framework for gene editing as described above, or the linear RNA or circular RNA in which the RNA framework for gene editing is located, or the RNP, or the DNA vector, or the DNA template as described above, as a tool to improve the gene editing efficiency of TALEN, ZFN, Targetron, Prime Editor, Twin Prime Editor, CRISPR, or CRISPR / Cas9 gene technologies.

[0066] This invention contains or can generate RNA, ssDNA, and / or dsDNA containing upstream sequences of the target site, sequences to be inserted, downstream sequences of the target site, and / or other sequences such as short sporadic elements or partial short sporadic elements. These components can assist technologies such as TALEN, ZFN, Targetron, CRISPR, and CRISPR / Cas9 in homologous recombination or insert corresponding sequences into the target site, promoting greater RNAization, devirulent transfection, and improving the efficiency of genome sequence insertion in these technologies (RNA-transduced cells do not need to enter the nucleus; they can enter the nucleus during non-dividing phase under the binding and action of corresponding proteins such as ORF1p and / or ORF2p).

[0067] The RNA framework of this invention includes downstream connections to short sporadic elements, partially short sporadic elements, long sporadic elements, partially long sporadic elements, and functional structures that initiate ORF2p cleavage and reverse transcription. This allows long sporadic elements, particularly those encoding the human LINE-1-encoded ORF2p, to bind to these structures, cleaving the target genome single strand and using it as a primer for reverse transcription. Ultimately, dsDNA is formed, and the desired sequence is inserted into the target site on the genome via homologous recombination. Because this invention only cleaves the single strand of the genome and does not cause double-strand breaks, it has high safety.

[0068] This invention utilizes RNA and RNA with specific functions bound to human endogenous proteins (also known as RNPs) as the main functional components to perform gene editing on the genome. RNA-based gene editing is safer than DNA-based methods. Simultaneously, in vitro synthesis and transcription of RNA, especially prokaryotic transcription, facilitates the production of RNA in vitro, making further industrial production and commercialization easier.

[0069] In this invention, ORF1p and / or ORF2p can bind to the RNA framework, protecting the RNA framework while assisting in the transport of RNA to the cell nucleus. Furthermore, 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 or / and RNP) to the cleavage site (target site) only when the upstream sequence of the target site on the vector completely matches the corresponding complementary sequence of the upstream sequence of the target site on the genome. This allows for the cleavage of single strands on the genome and further mediates the formation of vector dsDNA, resulting in high targeting accuracy and significantly avoiding the widespread non-specific generation of dsDNA in the cell nucleus that could adversely affect the genome. Theoretically, this makes its safety and accuracy superior to other existing gene editing technologies. Simultaneously, using RNA or RNP as a vector effectively solves the problem of DNA's difficulty in entering the nucleus, facilitating gene editing in cells with low DNA transfection efficiency.

[0070] The beneficial effects of this invention are as follows:

[0071] This invention provides an RNA framework for gene editing. Based on the inherent mechanisms of eukaryotes, this framework uses RNPs or RNA (which can be prepared in vitro) and related proteins as carriers to be transferred into the cytoplasm and nucleus. This enables gene editing of specific sequences or sites on the genome of target systems (such as cells, tissues, organs, or organisms), including insertion, deletion, substitution, and site replacement, while maintaining high targeting accuracy. Because this invention does not introduce exogenous systems or substances such as prokaryotic proteins and does not produce double-strand breaks, it is more suitable for further practical applications, such as clinical applications, compared to other existing technologies. Furthermore, the RNA can be obtained in vivo or in vitro via prokaryotic or eukaryotic promoter transcription or chemical synthesis. Prokaryotic promoter transcription, in particular, has high efficiency, produces longer RNA products, and avoids the damage to the integrity of the target RNA caused by splicing mechanisms in eukaryotic systems. Simultaneously, proteins ORF2p and / or ORF1p can be synthesized in vitro, facilitating industrial-scale production and commercialization. Attached Figure Description

[0072] Figure 1 This is a schematic diagram illustrating the principle of gene editing provided by the present invention.

[0073] Figure 2 This is a schematic diagram of the operation process of the present invention.

[0074] Figure 3 This is a schematic diagram of the basic structure of the RNA framework for gene editing provided by the present invention.

[0075] Figure 4 This is a schematic diagram of the first improved structure of the RNA framework for gene editing provided by the present invention.

[0076] Figure 5 This is a schematic diagram of a second improved structure of the RNA framework for gene editing provided by the present invention.

[0077] Figure 6 This is a schematic diagram of a third improved structure of the RNA framework for gene editing provided by the present invention.

[0078] Figure 7 This is a schematic diagram of the fourth improved structure of the RNA framework for gene editing provided by the present invention.

[0079] Figure 8 This is a schematic diagram of the fifth improved structure of the RNA framework for gene editing provided by the present invention.

[0080] Figure 9 This is a schematic diagram of the sixth improved structure of the RNA framework for gene editing provided by the present invention.

[0081] Figure 10 This is a schematic diagram of the seventh improved structure of the RNA framework for gene editing provided by the present invention.

[0082] Figure 11 This is a schematic diagram of the eighth improved structure of the RNA framework for gene editing provided by the present invention.

[0083] Figure 12 This is a schematic diagram of the ninth improved structure of the RNA framework for gene editing provided by the present invention.

[0084] Figure 13 This is a schematic diagram of the tenth improved structure of the RNA framework for gene editing provided by the present invention.

[0085] Figure 14 This is a schematic diagram of the eleventh improved structure of the RNA framework for gene editing provided by the present invention.

[0086] Figure 15 This is a schematic diagram of the twelfth improved structure of the RNA framework for gene editing provided by the present invention.

[0087] Figure 16 This is a schematic diagram of the thirteenth improved structure of the RNA framework for gene editing provided by the present invention.

[0088] Figure 17 This is a schematic diagram of the fourteenth improved structure of the RNA framework for gene editing provided by the present invention.

[0089] Figure 18 This is a schematic diagram of the fifteenth improved structure of the RNA framework for gene editing provided by the present invention.

[0090] Figure 19This is a schematic diagram of the sixteenth improved structure of the RNA framework for gene editing provided by the present invention.

[0091] Figure 20 This is a schematic diagram of the seventeenth improved structure of the RNA framework for gene editing provided by the present invention.

[0092] Figure 21 This is a schematic diagram of the eighteenth improved structure of the RNA framework for gene editing provided by the present invention. Detailed Implementation

[0093] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0094] Existing technologies such as CRISPR can cause double-strand breaks, easily introduce random sequences and mutations, and are inefficient at introducing the target sequence.

[0095] The RNA framework for gene editing provided by this invention is based on the transposon mechanism, which is widely found in eukaryotes, and the reconstructive mechanism mediated by it that modifies components of the genome, such as repetitive sequences and gene copies. This mechanism may cause deletions or additions of pathogenic triplet nucleotide repeats in some central nervous system degenerative diseases such as Huntington's disease and Fragile X syndrome, promote the amplification of the HIV genome in some proliferating immune cells onto the human genome, and lead to specific increases or decreases in gene copy numbers during embryonic development and tumorigenesis.

[0096] The RNA framework and corresponding RNPs provided by this invention do not cause double-strand breaks and integrate the genome through homologous recombination, which is safer and easier to apply in practice.

[0097] The RNA framework for gene editing provided by this invention uses RNA or RNP as a vector. The sequence to be inserted into a selected gene site (target site) on the genome is accurately located at the target site on the genome via the upstream and downstream sequences of the target site on both sides of the sequence to be inserted 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). Simultaneously, it utilizes short interspersed element (SINE) RNA, long interspersed element (LINE) RNA, short interspersed element derivative RNA, long interspersed element derivative RNA, and / or functional structures that initiate ORF2p cleavage and reverse transcription, as well as the proteins expressed by the long interspersed elements: ORF2p (open reading frame 2 protein, also known as L1 endonuclease) and / or ORF1p (open reading frame 1 protein). The ORF1p and / or ORF2p vectors precisely insert the desired sequence into the target site on the genome. ORF1p and / or ORF2p can bind to the RNA vector, protecting it while assisting in the transport of RNA to the cell nucleus. Furthermore, 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 or / and RNP) to the cleavage site (target site) only when the upstream sequence of the target site on the vector completely matches the corresponding complementary sequence of the upstream sequence of the target site on the genome. This allows for the cleavage of single strands on the genome and further mediates the formation of vector dsDNA, resulting in high targeting accuracy and significantly avoiding the widespread non-specific generation of dsDNA in the cell nucleus that could adversely affect the genome. Theoretically, this makes its safety and accuracy superior to other existing gene editing technologies. Simultaneously, using RNA or RNP as a vector effectively solves the problem of DNA's difficulty in entering the nucleus, facilitating gene editing in cells with low DNA transfection efficiency.

[0098] Downstream of the aforementioned RNA framework, short interstitial element RNA, long interstitial element RNA, short interstitial element derivative RNA, long interstitial element derivative RNA, and / or functional structures that initiate ORF2p cleavage and reverse transcription are linked. This allows the ORF2p encoded by the long interstitial element (LINE), particularly human LINE-1, to bind to it, cleaving the single strand of the target genome. Using this as a primer, reverse transcription is performed, ultimately forming dsDNA, which inserts the desired sequence into the target site on the genome via homologous recombination. Because this invention only cleaves the single strand of the genome and does not cause double-strand breaks, it has high safety.

[0099] This invention can generate RNA through expression in vivo or in vitro in eukaryotic or prokaryotic systems, cells, tissues, organs, and organisms, and generate the required proteins ORF1p and / or ORF2p in or outside the target system (in vitro). The RNA or RNP vector is introduced into the target system, such as cells, tissues, organs, and organisms, to achieve the goal of gene editing, which is convenient for industrial mass production and commercialization.

[0100] Furthermore, since the prokaryotic system or in vitro expression lacks the splicing mechanism of the precursor mRNA in the eukaryotic system, the RNA framework and downstream connectable short interstitial RNA, long interstitial RNA, short interstitial RNA derivative RNA, long interstitial RNA derivative RNA and / or functional structures that initiate ORF2p splicing and reverse transcription can be expressed without hindrance, without suffering potential splicing risks, thus improving the production efficiency and gene editing effect of the present invention.

[0101] Furthermore, this invention, based on the targeted insertion of desired sequences into the genome, can perform relatively accurate deletion, replacement, and substitution of genomic fragment sequences through mechanisms such as homologous recombination or genome repair within the receiving editing system, such as prokaryotes or eukaryotes. Simultaneously, based on the technical principles of this invention, it is understood that vectors can be designed for further insertion at new sites formed after the sequence to be inserted using this invention. This progressive insertion theoretically allows for an unlimited length of inserted sequences into the genome, and can accomplish various types and forms of gene editing purposes, including sequence insertion, deletion, replacement, and site substitution, offering flexible usage. Moreover, this invention can also be used to edit single or multiple CNVs and their ends on the genome, stabilizing, lengthening, shortening, or altering their expression sequences, thereby achieving the purpose of changing or stabilizing gene expression and the state of cells, tissues, organs, or organisms.

[0102] The sequence to be inserted into the RNA framework provided by this invention can be either exogenous or endogenous, with a single insertion length of 1 bp to 20,000 bp. Multiple insertions can achieve genomic insertion of DNA sequences of any length. The upstream nucleotide sequence of the target site can be 1 bp to 20,000 bp in length, and the downstream nucleotide sequence of the target site can also be 1 bp to 20,000 bp in length.

[0103] This invention relates to short interspersed elements (SINEs), long interspersed elements, and related proteins such as ORF1p, ORF2p, and other types of open reading frame proteins (ORFp). Short interspersed elements (SINEs) mainly include Alu elements (such as 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.) and SVA elements in primates (including humans); mammalian-wide interspersed repeat elements (MIRs) such as MIR and MIR3, which are common in various mammals; Mon-1 in monotremes; B1 and B2 elements in rodents; C-element in rabbits; the HE1 family in zebrafish; SINE SmaI in salmon; and Anolis SINE2 and Sauria in reptiles. SINE, IdioSINE1, IdioSINE2, SepiaSINE, Sepioth-SINE1, Sepioth-SINE2A, Sepioth-SINE2B and OegopSINE in invertebrates such as squid, and p-SINE1 in plants such as rice, etc. Sentient elements mainly include various LINE-1 (L1) structures found in different types of organisms, such as L1 (L1RE1 (L1.2, LRE1) and LRE2) in humans, various LINE-2 (L2) and LINE-3 (L3) structures, Ta elements, and six LINE classes: R2, RandI, L1, RTE, I, and Jockey, as well as other LINE types such as LINE-1 in rodents, LINE UnaL2 in eels, LINE R2 in insects, LINE ZfL2-1 and ZfL2-2 in zebrafish, L1 in algae, LINE SART1 in silkworms, L1 in monocotyledons, Tad1 in fungi, L2 in fish, and RTE in some mammals. In addition, LINEs also include subfamilies such as L1s, L1spa, L1Orl, L1.2, and the F and TF subfamilies within L1. These SINEs and LINEs are widely distributed throughout the genomes of various animals and plants, and each organism has its specific SINE and LINEs corresponding to its function. The corresponding DNA sequence of the Alu element is denoted as the Alu sequence.

[0104] The main characteristics of SINEs are that they are relatively short transposons distributed throughout the genome, containing an internal RNA polymerase III promoter and ending with an A- or T-rich tail or a short simple repeat sequence, and achieving reverse transcription via LINEs. The right half of their transcript contains a reverse transcription functional structure. LINEs, on the other hand, are transposons widely distributed throughout the genome containing reverse transcriptase coding sequences. Both SINEs and their corresponding LINEs in their respective species reconstruct the genome through similar mechanisms. The basic principle of this mechanism is to connect the lasso structure produced by the organism's treatment of pre-mRNA with the right half of the reverse transcription functional structure produced by cleaving the SINE transcript. The RNA sequence remaining as the right half of the reverse transcription functional structure after cleaving the complete SINE transcript at the middle site is called a partial short interstitial element RNA (MIRNA). To distinguish it, its corresponding coding DNA sequence is called a partial short interstitial element sequence. The cleavage site varies among different short interstitial element RNAs in different species. The natural splicing site of short, sporadic element RNA is generally located in the middle to the front of the full length. For short, sporadic element RNAs with a full length of about 100-400 nt, the natural splicing site is usually located at 100-250 nt. For example, for an Alu element with a full length of about 300 bp, the splicing site of its transcript RNA (i.e., the scAlu splicing site or natural splicing site mentioned below, which is generally located before the middle poly-A sequence of the Alu transcript, but may vary in actual cases) is located at 118 nt. The spliced ​​product contains the right Alu monomer, and may also contain the middle adenosine repeat sequence of the Alu element transcript other than the right A monomer, or together with its upstream 2-3 bases, and the 3′ poly-A repeat sequence after the right monomer. This can be called a partial Alu, and for distinction, its corresponding coding DNA sequence is called a partial Alu sequence. For the transcript RNA of various MIRs with a full length of about 260 nt, splicing sites can be observed in the range of 100-150 nt. In fact, regardless of the location of the site, as long as the remaining right portion of the transcript after cleavage contains a complete reverse transcription functional structure, it can perform its function. The secondary structure of the reverse transcription functional structure forms a special structure, usually Ω-shaped. Its primary structure is characterized by containing two sequences separated by an intermediate spacer sequence. These two sequences can bind to the complementary sequence of the corresponding sequence on the genome that does not contain an intermediate spacer sequence and directly connects the two sequences. The LINE-encoded ORF2p can bind to the sequence located at the 3′ of the transcript in the ORF2p functional initiation structure and cleave the single strand of the genome at the genomic site corresponding to the gap between the two sequences, initiating reverse transcription. In addition, the corresponding transcript (RNA sequence) of the Alu element is denoted as the complete Alu.

[0105] A DNA sequence containing a reverse transcription functional structure that can initiate reverse transcription but differs from conventional short sporadic elements in sequence (e.g., short sporadic elements with partial mutations but still possessing special structures and functions) is called a short sporadic element-like (SINE-like) sequence, and its corresponding RNA sequence is called a short sporadic element-like RNA. Furthermore, an RNA sequence containing a reverse transcription functional structure and an ORF2p binding sequence that initiates ORF2p endonuclease and reverse transcription is called a functional structure that binds ORF2p (e.g., has a poly-A sequence, usually located on the right leg of the "Ω" structure of the reverse transcription functional structure) and initiates ORF2p splicing and reverse transcription. This can be abbreviated as "functional structure that initiates ORF2p splicing and reverse transcription" or "ORF2p initiation structure." The transcription product of this functional structure can form an "Ω" secondary structure due to internal or external factors. ORF2p can bind to and initiate ORF2p splicing. The "Ω" structure formed by the functional and reverse transcription structures (located next to the notch at the 3' position of the "Ω" structure) allows RNA to be converted into double-stranded DNA and bound to complementary, identical, or similar sequences on the genome via proteins ORF1p and ORF2p expressed by corresponding LINEs, such as LINE-1 corresponding to the Alu element and LINE-2 corresponding to various MIR elements. The RNA (transcription product) formed by transcription is converted into single-stranded DNA through reverse transcription, and the double-stranded DNA generated by single-stranded DNA using the genomic sequence as a primer is the transformation product of the transcription product. Through the formation of a specific "Ω" structure, homologous recombination completes the insertion into the genome. Furthermore, LINEs can also complete the above-mentioned RNA-to-double-stranded DNA conversion and genome insertion by transcribing their downstream sequences (i.e., 3' transduction) and binding to complementary sequences on the genome to form an Ω structure. Taking the Alu element and its corresponding LINE-1, which assists its function, as an example: the pre-mRNA produced after gene expression can be spliced ​​to produce overlapping lasso structures. This can occur in any region of the pre-mRNA, the difference being the splicing strength that produces these lassos. Due to the generation of upstream and downstream lassos (lasso sequences without exons), their sequence-difference-based shearing strength is higher than that of surrounding lasso structures (such as those containing exons), making it easier for exons to be completely cleaved during pre-mRNA processing and inhibiting the generation of other lassos. Simultaneously, the ORF1p generated by LINE-1 can protect the nucleic acids it binds to. Both ORF1p and ORF2p, also generated by LINE-1, can localize the bound nucleic acids to the nucleus and transport them into the nucleus. Furthermore, ORF2p can bind to the specific Ω secondary structure of Alu element transcripts and mediate subsequent single-strand cleavage, reverse transcription, and integration of the accessory genome.As mentioned earlier, the transcript of the Alu element can be cleaved at specific sites to produce partial Alu. The lasso structure generated by the cleavage of pre-mRNA can connect to the remaining part of the cleaved Alu element transcript containing the reverse transcription function (i.e., partial Alu) from its 3′ end. ORF2p can recruit via ORF2p binding sequences, such as A-rich sequences, and bind to the 3′ leg of the Ω structure formed by the partial Alu secondary structure. It recognizes the sequence on the genome that matches the sequence on the two legs of the Ω (mainly UU / AAAA, where U and A are discontinuous, i.e., the gap). It cleaves the single strand of the genome at the genomic site directly opposite the Ω gap and unwinds the complementary sequence on the genome as a primer for reverse transcription. This process is called target-primed reverse transcription. Reverse transcription (TPRT); ORF2p moves to the 3′ end of the resulting single-stranded DNA during reverse transcription. The resulting single-stranded DNA sequence can bind to a complementary sequence on the genome and form an Ω structure at the corresponding insertion site. Since the insertion sequence does not exist at the corresponding insertion site on the genome, but the flanking sequences of the insertion sequence on the single-stranded DNA exist on both sides of the insertion site, ORF2p can slide along the matching sequence in a 3′ to 5′ direction to the Ω structure, recognizing a 6-nucleotide sequence on the genome complementary to the sequence located on both sides of the 5′ end gap, mainly 4 nucleotides at 3′ and 2 nucleotides at 5′. A similar process is then used to form double-stranded DNA. Note that only a perfectly matched sequence allows ORF2p to slide to the cleavage site, ensuring its targeting accuracy. The resulting double-stranded DNA, again in an "Ω" shape, binds to the two ends of the corresponding insertion sites with matched sequences. When ORF2p recognizes six nucleotides (primarily four nucleotides at the 3′ end and two nucleotides at the 5′ end), the Ω-shaped gap (where the middle is discontinuous) can be filled by ORF2p endonucleases, creating two single-stranded gaps on the corresponding gene and its own other strand. The central circular portion is then inserted into the genome via homologous recombination. During this process, ORF1p promotes the formation of functional secondary and higher-level structures of the RNA used and facilitates the binding and interaction between functional RNA and the interacting genome. By altering the inserted sequence, deletion or substitution can be achieved through homologous recombination. In this process, the annealing and deconstruction functions of ORF1p, also encoded by LINE, play a supporting role, helping to stabilize the secondary structures generated by nucleic acids during genome reconstruction and their binding to the genome, as well as promoting the separation of nucleic acids from the genome after binding and interaction. Furthermore, ORF1p has high RNA affinity and nuclear localization capabilities. Because ORF2p can only cut one strand of the genome double helix and cannot cause double-strand breaks, it has high safety.Similar mechanisms also apply to other SINE and LINE combinations. Changes in local copy number variations during pathophysiological processes such as embryonic development and tumorigenesis, and the preference for short sporadic element sequences in the insertion of HIV-1 genome deletions into the human genome, may be a manifestation of this mechanism in nature. It has been reported that transcribed mRNA sequences can be integrated into the genome with the assistance of ORF1p and ORF2p, but because the transcription template is a purely exogenous non-homologous sequence, it cannot target specific sites in the genome and does not connect to fragments with reverse transcription functional structures, resulting in low efficiency, randomness, and difficulty in control. This invention redesigns the transcription sequence and connects it to sequences with reverse transcription functional structures, such as various short sporadic element RNAs or partial short sporadic element RNAs, through various active or passive means to achieve more precise and efficient gene editing effects. Furthermore, ORF2p and ORF1p can also bind to long sporadic element RNAs, mediating transposition activity and trunculating the 3' portion of long sporadic element RNA that can form special first-order or higher structures, such as the corresponding transcript (RNA sequence) of the 3' UTR portion, which is called partial long sporadic element RNA. Some long, dispersed RNA elements can be attached to corresponding positions, such as downstream of the RNA framework, to perform their functions according to the above principle. A schematic diagram illustrating the above basic principle is shown below. Figure 1 Based on the above principles, the upstream sequence of the target site in the RNA framework should avoid other sequences, such as sequences that are not homologous to the upstream sequence of the target site. 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, in order to improve gene editing efficiency.

[0106] In this invention, the RNA sequence of the transcript of a naturally occurring (including naturally occurring mutations and other variations) short sporadic element RNA is called short sporadic element RNA, and the RNA sequence of the transcript of a naturally occurring (including naturally occurring mutations and other variations) long sporadic element RNA is called long sporadic element RNA. The short sporadic element derivative RNA or long sporadic element derivative RNA in this invention refers to a combination of changes made by adding other sequences, truncating parts of the sequence, adding, deleting, or rearranging functional structural sequences, generating similar or nearly similar sequences with similar functions, or mixing two element sequences, especially the functional parts, on the basis of short sporadic element RNA or long sporadic element RNA. Short sporadic element derivative RNA or long sporadic element derivative RNA shares at least 50% similarity with short sporadic element RNA or long sporadic element RNA in any consecutive 10 bp or longer sequence. Short sporadic element derivative RNA includes some short sporadic element RNA and other sequences modified based on the natural short sporadic element RNA sequence; long sporadic element derivative RNA includes some long sporadic element RNA and other sequences modified based on the natural long sporadic element RNA sequence. In addition, 7SLRNA, which has a high similarity to short interstitial element RNA, also belongs to the category of short interstitial element derivative RNA. Functional structures that initiate ORF2p splicing and reverse transcription include one or more of the following: short interstitial element RNA, long interstitial element RNA, short interstitial element derivative RNA, long interstitial element derivative RNA, and short interstitial element-like RNA.

[0107] Short discrete element RNA, long discrete element RNA, short discrete element derivative RNA, long discrete element derivative RNA, and / or the functional structure that initiates ORF2p splicing and reverse transcription are collectively referred to as the ORF2p functional initiation part.

[0108] In this invention, the ORF1p coding sequence refers to the RNA sequence of the natural coding sequence of ORF1p, which is scattered throughout elements in the genome. The ORF2p coding sequence refers to the RNA sequence of the natural coding sequence of ORF2p, which is scattered throughout elements in the genome. The modified ORF1p coding sequence in this invention can be obtained by modifying the natural ORF1p coding sequence, or a natural ORF1p sequence containing various variations or mutations. Similarly, the modified ORF2p coding sequence can be obtained by modifying the natural ORF2p coding sequence, or a natural ORF2p sequence containing various variations and mutations. Related modifications include adding other sequences to the natural ORF1p coding sequence or the natural ORF2p coding sequence, truncating parts of the sequence, deleting or rearranging functional structural sequences, generating similar or nearly identical sequences with similar functions, or combining all or part of the sequences of one or more other proteins (including ORF1p and ORF2p) with all or part of the sequences of the ORF1p coding sequence and / or ORF2p coding sequence, especially the mutual mixing (fusion) of their functional sequences to form corresponding fusion protein coding sequences, or combinations of the above modifications; proteins generated from the ORF1p coding sequence are called ORF1p, and proteins generated from the ORF2p coding sequence are called ORF2p; proteins generated from modified sequences of the ORF1p coding sequence or modified sequences of the ORF2p coding sequence are respectively called ORF1p-derived proteins or ORF2p-derived proteins. The modified sequences of the ORF1p coding sequence or the modified sequences of the ORF2p coding sequence and the ORF1p-derived proteins or ORF2p-derived proteins they express should still retain the above-mentioned functions and characteristics. The modified sequences of ORF1p and ORF1p coding sequences are collectively referred to as generalized ORF1p coding sequences, and the modified sequences of ORF2p and ORF2p coding sequences are collectively referred to as generalized ORF2p coding sequences.

[0109] ORF2p mainly contains several functional domains that are currently well-defined, including the endonuclease region (aa: 1-239), the cyptic 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).

[0110] Since the implementation of this invention requires an endonuclease mechanism, adding an endonuclease region, a partial endonuclease region, or a modified structure with more than 50% amino acid homology to the endonuclease region sequence in the ORF2p-derived protein can enhance the role of the ORF2p-derived protein in this invention.

[0111] Since this invention requires a reverse transcription mechanism, adding a reverse transcriptase region, a partial reverse transcriptase region, or a modified structure with more than 50% amino acid homology to the reverse transcriptase region sequence in the ORF2p-derived protein can enhance the role of the ORF2p-derived protein in this invention.

[0112] The role of the undetermined region has been found to reduce the cytotoxicity of endonuclease regions and increase the nuclear localization of the protein or polypeptide fragment in which it resides. In this invention, greater nuclear localization can enhance gene editing efficiency, and lower cytotoxicity is also beneficial for the practical application of this invention. Therefore, adding or adding a longer undetermined region to the ORF2p-derived protein can, to some extent, promote the gene editing efficiency of this invention.

[0113] The role of cysteine-rich regions has been found to promote the binding of ORF2p-derived proteins to nucleic acids. Since the endocytosis of ORF2p requires the assistance of special nucleic acids and their secondary structures to be initiated, in this invention, adding cysteine-rich regions, partially cysteine-rich regions, or modified structures with more than 50% amino acid homology to the cysteine-rich regions in natural O2 to the ORF2p-derived protein can improve the gene editing efficiency of ORF2p or is necessary.

[0114] The Z region can serve as a PCNA binding motif, which can enhance the role of ORF2p in this invention. Therefore, in this invention, adding a Z region, a portion of the Z region, or a modified structure with more than 50% amino acid homology to the Z region sequence in natural ORF2p to the ORF2p-derived protein can improve the gene editing efficiency of the ORF2p-derived protein or is necessary.

[0115] Besides the regions mentioned above, the addition or partial addition of other regions in the natural ORF2p to the ORF2p-derived protein, or the addition or partial addition of other regions of the modified natural ORF2p that maintain more than 50% homology with the original sequence to the ORF2p-derived protein, may also improve the gene editing efficiency of the present invention to a certain extent.

[0116] The regions in an ORF2p-derived protein can be arranged according to the natural ORF2p sequence or in a scrambled order. Other regions can be added between or within each region. Amino acids in ORF2p and the ORF2p-derived protein can be replaced with corresponding conserved substitutions (e.g., substitutions between Phe, Trp, and Tyr; substitutions between Leu, Ile, and Val; substitutions between Gln and Asn; substitutions between basic amino acids Lys, Arg, and His; substitutions between acidic amino acids Asp and Glu; substitutions between hydroxyl amino acids Ser and Thr). Furthermore, containing more homologous conserved amino acid sequences between human ORF2p and other species, such as mouse ORF2p, in the ORF2p-derived protein may improve the gene editing efficiency of the ORF2p-derived protein. The base sequences in the modified ORF2p coding sequence encoding the ORF2p-derived protein can be replaced with different codon sequences of the same amino acid.

[0117] Current research has identified ORF1p as containing the following functional domains: N-terminal domain, coiled coil domain, RNA recognition motif, and C-terminal domain.

[0118] Since higher nucleic acid binding affinity and nucleic acid chaperone activity can enhance the effectiveness of the present invention, and the RNA recognition motif and C-terminal region in ORF1p have the above-mentioned functions in proteins, ORF1p-derived proteins containing RNA recognition motifs and / or C-terminal regions, partial RNA recognition motifs and / or C-terminal regions, or modified structures with more than 30% amino acid homology to the RNA recognition motif or C-terminal region sequence in natural ORF1p can enhance the role of ORF1p-derived proteins in the present invention;

[0119] The coiled-coil region in ORF1p plays a role in the formation of ORF1p trimers, which enhances nucleic acid binding affinity and promotes transposition. Therefore, ORF1p-derived proteins containing coiled-coil regions, partially coiled-coil regions, or modified structures with more than 30% amino acid homology to the coiled-coil regions in natural ORF1p can enhance the role of ORF1p-derived proteins in this invention.

[0120] The N-terminal region also plays a role in the normal function of ORF1p. Therefore, ORF1p-derived proteins containing an N-terminal region, a partial N-terminal region, or a modified structure with more than 30% amino acid homology to the N-terminal region of natural ORF1p can enhance the role of ORF1p-derived proteins in this invention.

[0121] Besides the regions mentioned above, the addition or partial addition of other regions in the natural ORF1p to the ORF1p-derived protein, or the addition or partial addition of other regions of the modified natural ORF1p that maintain more than 50% homology with the original sequence to the ORF1p-derived protein, may also improve the gene editing efficiency of the present invention to a certain extent.

[0122] Since protein phosphorylation plays a role in the normal function of ORF1p, the addition of a conserved proline-directed protein kinase (PDPK) site in ORF1p-derived proteins may enhance the role of ORF1p-derived proteins in this invention.

[0123] The positional distribution of regions in ORF1p-derived proteins can follow the natural ORF1p arrangement or be scrambled. Other regions can be added between or within each region. Amino acids in ORF1p and ORF1p-derived proteins can be replaced with corresponding conserved substitutions (e.g., substitutions between Phe, Trp, and Tyr; substitutions between Leu, Ile, and Val; substitutions between Gln and Asn; substitutions between basic amino acids Lys, Arg, and His; substitutions between acidic amino acids Asp and Glu; substitutions between hydroxyl amino acids Ser and Thr). Containing more homologous conserved amino acid sequences between human ORF1p and other species such as mouse ORF1p in ORF1p-derived proteins (e.g., ARR at positions 260-262, REKG at positions 235-238, and YPAKLS at positions 282-287 in the human ORF1p amino acid sequence (where Y at position 282 can be replaced with a functionally similar F)) can improve the gene editing efficiency of ORF1p-derived proteins. The base sequence in the modified sequence encoding the ORF1p-derived protein can be replaced with a different codon sequence of the same amino acid.

[0124] Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC), short interstitial elements, partially short interstitial elements, short interstitial element derivatives, long interstitial elements (LINE, long interstitial nuclear elements), partially long interstitial elements and / or long interstitial element derivatives, or other sequences that can improve homologous recombination efficiency, can be searched or selected as the corresponding sequences of upstream and downstream sequences of the target site on the genome for sequence insertion, thereby improving gene editing by increasing homologous recombination efficiency.

[0125] In practical applications of this invention, if the RNA vector containing the RNA framework itself does not connect the ORF2p functional initiation portion downstream of the RNA framework and does not achieve the expected efficiency, or if the ligation efficiency of the RNA or its partial fragments with short, scattered element RNA or its products is low, one can try increasing or decreasing the length of the upstream and downstream sequences of the target site on the RNA framework or the sequence to be inserted to promote ligation; or detect the lasso structure containing the insertion site generated in the corresponding prokaryote or eukaryote according to the detection method described below, and use the sequence of the lasso structure as the upstream and downstream sequence of the target site or a partial sequence of the upstream and downstream sequence of the target site, or appropriately extend the upstream and downstream sequences of the target site on the RNA vector containing the RNA framework, with the intermediate target site being the sequence to be inserted to generate RNA; or add a poly-A sequence at the 3' position of the RNA vector containing the RNA framework to promote ORF2p binding; without affecting the formation of the "Ω" structure of the RNA framework, one can add the sequence at a suitable position on the RNA vector containing the RNA framework. Adding an ORF2p binding sequence, such as a polyA sequence, or extending an existing ORF2p binding sequence, such as a polyA sequence, can improve gene editing efficiency. ORF2p binding sequences are primarily located at the 3' region or 3' end of RNA vectors containing RNA frames. They can be added to protein expression sequences (such as pan-ORF1p or pan-ORF2p coding sequences), upstream or downstream sequences of target sites within RNA frames, short interstitial element RNA, long interstitial element RNA, short interstitial element derivative RNA, long interstitial element derivative RNA, and / or functional structural sequences that initiate ORF2p splicing and reverse transcription, or other sequences on the RNA, either between, before, or after these sequences. Alternatively, sequences can be designed at the 3' region of RNA vectors containing RNA frames to generate an "Ω" structure to promote ORF2p endogenous genome editing.

[0126] Furthermore, since the gene editing function of this invention involves homologous recombination, the combined use of recombinases such as site-specific serine recombinases with this invention may increase the efficiency and effectiveness of this invention.

[0127] Furthermore, the target region (target site) for gene editing in this invention can be one or more sites. When the inserted sequences for gene editing targeting two or more target sites are partially or entirely identical or similar (highly similar) and the length of the partial sequence is 20 bp or more, the region between the two or more gene editing target sites can be deleted or replaced with the inserted sequence or part of the sequence.

[0128] When the sequence to be inserted in this invention is short (100 bp and below), the sequence to be inserted may be inserted into the target site on the genome through homologous recombination and / or other genome repair mechanisms, thereby achieving high genome insertion efficiency.

[0129] When the upstream and / or downstream sequences of the target site in this invention are short (100 bp or less), the sequence to be inserted may be inserted into the target site on the genome through homologous recombination and / or other genome repair mechanisms, thereby achieving high genome insertion efficiency.

[0130] It should be noted that the insertion site described in this invention is the target site.

[0131] like Figure 2 The diagram shown illustrates the operation process of this invention. This gene editing technology can achieve "RNA-based genome sequence insertion technology", "RNP-based genome sequence insertion technology", "RNA 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)", "blocking genome changes caused by transposons and stabilizing the genome and its CNVs", and "assisting other gene editing technologies", which will be described one by one below.

[0132] I. RNA-based genome sequence insertion technology

[0133] 1. RNA-based genome sequence insertion mediated by a simple RNA framework: Select upstream and downstream sequences (upstream and downstream of the target site) at the insertion point between the upstream and downstream sequences. The designed sequence is then used to generate RNA for vector production. This RNA can be placed in a container containing RNase inhibitors and / or an appropriate amount of Mg2+. 2+In solutions containing (e.g., 6 mmol / L) or other metals or in cell solutions, RNA folding is promoted, facilitating subsequent binding to corresponding functional proteins such as ORF2p and / or ORF1p. Subsequently, the vector is transferred into in vitro cultured cells, tissues, or organs via conventional methods such as liposome transfection, or administered to tissues, organs, or organisms via blood, lymph, cerebrospinal fluid, or local tissue administration. This allows the vector to enter the target cell cytoplasm, bind to ORF1p and / or ORF2p, and then enter the nucleus; alternatively, ORF1p and / or ORF2p can mediate direct nuclear entry of the vector. After the vector RNA is ligated to the short, scattered element RNA or its product transcribed within the cell, it binds to the intracellularly expressed ORF2p or simultaneously binds to both ORF2p and ORF1p; or it directly binds to the intracellularly expressed ORF2p or simultaneously binds to both ORF2p and ORF1p (e.g., when the upstream and downstream sequences of the target site in the vector, along with the intermediate sequence to be inserted, bind to the genome to form an Ω structure that replaces the reverse transcription functional structure in the short, scattered element or its product, initiating reverse transcription). The sequence to be inserted is then inserted into the corresponding target site on the genome (the insertion site). If insertion continues according to the new site generated after insertion, insertion can be continuous, allowing for the insertion of long fragments without significant length limitations. If directional transfer is required, modifications can be made to the outer coating of the vector. Care should be taken to avoid RNA degradation throughout the entire process. Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC), short elements, partial short elements, and / or short element derivatives on the genome to be edited can be identified or selected as the corresponding sequences of the upstream and downstream sequences of the target site in the genome for sequence insertion, thereby improving gene editing efficiency by increasing homologous recombination efficiency. Increasing the number of recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC), short elements, partial short elements, and / or short element derivative sequences in the corresponding sequences of the upstream and downstream sequences of the target site in the genome may also enhance the corresponding gene editing effect.

[0134] 2. Genomic sequence insertion mediated by RNA as a vector and ligated downstream of one or more ORF2p functional initiation sites (to minimize impact on the receiving system, short interstitial element RNA, short interstitial element derivative RNA, long interstitial element RNA, long interstitial element derivative RNA, ORF1p, and / or ORF2p types can be selected from the corresponding receiving system): This method does not require in vivo ligation of a lasso formed by the vector RNA after short interstitial element RNA cleavage. Instead, the RNA framework (upstream and downstream sequences of the target site (each within 20,000 bp), with the target sequence added at the insertion point within 20,000 bp)) consisting of upstream and downstream sequences of the target site and the intermediate sequence to be inserted is directly ligated downstream to one or more ORF2p functional initiation sites and / or one or more other related sequences. The designed sequence generates RNA for vector production. This RNA can be placed in a container containing RNase inhibitors and / or an appropriate amount of Mg. 2+ In solutions containing (e.g., 6 mmol / L) or other metals or in cell solutions, RNA is promoted to fold correctly and subsequently bind to the corresponding functional proteins ORF2p and / or ORF1p.

[0135] The vector is then transferred into in vitro cultured cells, tissues, or organs via conventional methods such as liposome transfection, or administered to tissues, organs, or organisms via blood, lymph, cerebrospinal fluid, or local tissue administration. This allows the vector to enter the target cell cytoplasm, bind to ORF1p and / or ORF2p, and then enter the nucleus, or ORF1p and / or ORF2p mediate direct nuclear entry. The vector RNA is then linked to intracellularly transcribed short, discrete element RNA or its product, and binds to intracellularly expressed ORF2p or simultaneously to both ORF2p and ORF1p, or directly to intracellularly expressed ORF2p or simultaneously to both ORF2p and ORF1p (e.g., when the "Ω" structure formed by the upstream and downstream sequences of the target site and the intermediate sequence to be inserted (binding to the genome) in the vector serves as the reverse transcription functional structure, replacing the short, discrete element or its product to initiate reverse transcription). The sequence to be inserted is then inserted into the corresponding target site on the genome (the insertion site). If insertion continues using the method described above based on the new sites generated after insertion, continuous insertion can be achieved, resulting in long fragment insertions without significant length limitations. Because this method does not require mechanisms specific to eukaryotic systems, such as splicing, it is suitable for systems lacking eukaryotic pre-mRNA splicing mechanisms to produce lasso structures, such as prokaryotes like bacteria, and also for eukaryotes with pre-mRNA splicing mechanisms. For targeted transfer, modifications can be made to the outer coating of the vector. Care should be taken to avoid RNA degradation throughout the process.

[0136] 3. Genomic sequence insertion mediated by RNA as a vector and consisting of a simple RNA framework containing both pan-ORF1p and / or pan-ORF2p coding sequences, or one or more ORF2p functional initiation portions connected downstream of the RNA framework containing both pan-ORF1p and / or pan-ORF2p coding sequences (to minimize the impact on the receiving system, short interstitial element RNA, short interstitial element derivative RNA, long interstitial element RNA, long interstitial element derivative RNA, ORF1p and / or ORF2p types in the corresponding receiving system may be selected).

[0137] The two methods described above involve adding pan-ORF1p and / or pan-ORF2p coding sequences upstream of the target site, downstream of the target site, and upstream or downstream of the functional initiation portion of each ORF2p sequence. These sequences are positioned before, between, after, or within each component (ensuring the RNA framework forms an "Ω" shape at the target site). The insertion sequence (within 20,000 bp) is added to the upstream and downstream sequences of the target site, and at the insertion point between the upstream and downstream sequences. This produces the aforementioned RNA, which serves as a vector. This RNA can be placed in a container containing RNase inhibitors and / or an appropriate amount of Mg. 2+In solutions containing (e.g., 6 mmol / L) or other metals or in cell solutions, the RNA is promoted to fold correctly and subsequently bind to the corresponding functional proteins (e.g., ORF2p and / or ORF1p). The vector is then transferred into cultured cells, tissues, or organs via conventional methods such as liposome transfection, or administered to tissues, organs, or organisms via blood, lymph, cerebrospinal fluid, or local tissue administration. This allows the vector to enter the target cell cytoplasm, express and produce ORF1p and / or ORF2p, and bind to them; or bind to previously expressed ORF1p and / or ORF2p before entering the nucleus; or ORF1p and / or ORF2p mediate direct nuclear entry of the vector. After the vector RNA is ligated to the short, discrete element RNA or its product transcribed within the cell, it binds to ORF2p expressed within the cell or encoded by the vector, or simultaneously binds to both ORF2p and ORF1p. Alternatively, it binds directly to ORF2p expressed within the cell or encoded by the vector, or simultaneously binds to both ORF2p and ORF1p (e.g., when the upstream and downstream sequences of the target site in the vector, along with the intermediate sequence to be inserted, bind to the genome to form an Ω structure that replaces the reverse transcription functional structure in the short, discrete element or its product, initiating reverse transcription). Or, it uses one or more ORF2p functional initiation portions already linked downstream of the RNA framework to bind to ORF2p expressed within the cell or encoded by the vector, inserting the sequence to be inserted into the corresponding target site on the genome (the insertion site). If insertion continues according to the new site generated after insertion, insertion can be continuous and long fragments without significant length limitations can be inserted. If directional transfer is required, modifications can be made to the outer coating of the vector. Care should be taken to avoid RNA degradation throughout the process.

[0138] 4. RNA and / or RNP containing one or more ORF2p functional start sequences, one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences, and / or DNA expressing one or more ORF2p functional start sequences, ORF1p, ORF2p, ORF1p-derived proteins, and / or ORF2p-derived proteins, and a simple RNA framework RNA vector, RNA framework downstream linker RNA vector containing one or more ORF2p functional start sequences, and / or RNA framework downstream linker RNA vector containing one or more pan-ORF1p coding sequences and / or one or more pan-ORF2p coding sequences, or RNA framework downstream linker RNA vector containing one or more ORF2p functional start sequences, and / or one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences. The RNA vector of the sequence is administered to the target system in the same vector and / or different vectors (to minimize the impact on the receiving system, short interstitial element RNA, short interstitial element derivative RNA, long interstitial element RNA, long interstitial element derivative RNA, ORF1p and / or ORF2p types in the corresponding receiving system can be selected): The RNA vectors in "1-3" of "I. Genomic Sequence Insertion Technology Using RNA Vectors" above can be administered to the target system in the same vector or different vectors with one or more ORF2p functional start portions, one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences of RNA and / or RNP and / or DNA expressing one or more ORF2p functional start portions, ORF1p, ORF2p, ORF1p-derived proteins and / or ORF2p-derived proteins. RNA containing one or more ORF2p initiation sites, or corresponding RNA expressed by DNA expressing one or more ORF2p initiation sites, can be linked in vivo, with or without cleavage, to various RNA vectors and / or RNA frameworks, and perform the aforementioned function to insert the desired sequence into the target site. Corresponding proteins expressed by RNA containing one or more long interstitial element RNAs, one or more pan-ORF1p coding sequences, and / or one or more pan-ORF2p coding sequences, or corresponding proteins expressed by DNA expressing long interstitial element RNAs, ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins, can function as described above to insert the desired sequence into the target site. If insertion continues according to the new sites generated after insertion, insertion can be continuous and long fragments without significant length limitations can be inserted. For directed transfer, the outer coating of the vector can be modified. Care should be taken to avoid RNA degradation throughout the process. The RNA vector can be placed in an environment containing RNase inhibitors and / or an appropriate amount of Mg. 2+In solutions containing (e.g., 6 mmol / L) or other metals or in cell solutions, RNA is promoted to fold correctly and subsequently bind to the corresponding functional proteins (ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins).

[0139] II. Genome sequence insertion technology using RNP as a vector (to minimize the impact on the receiving system, short sporadic elements, short sporadic element derivatives, long sporadic elements, long sporadic element derivatives, ORF1p and / or ORF2p types from the corresponding receiving system can be selected).

[0140] First, prepare the various RNA vectors described in "Scheme 1" above, and simultaneously express and purify the proteins ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins in vitro using eukaryotic or prokaryotic systems. Mix the prepared RNA vectors in vitro with cytoplasm containing ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins, or with physiological fluid containing ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins, and incubate (at a suitable temperature, room temperature or 37°C is acceptable; incubation should be within 48 hours; Mg2+ can be appropriately increased). 2+ (Equal metal ion concentrations are used to promote the correct folding of secondary and higher-level structures of RNA vectors) to obtain RNP vectors.

[0141] Subsequently, the RNP vector is transferred into cultured cells, tissues, or organs via conventional methods such as liposome transfection, or administered to tissues, organs, or organisms via blood, lymph, cerebrospinal fluid, or local tissue administration. The vector carries expression sequences for ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins. After entering the target cytoplasm, it can still express and produce ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins, and continue to bind to them even if they are not fully bound in vitro. Alternatively, it can continue to bind to previously expressed ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins (or simultaneously bind to ORF2p, ORF1p, ORF2p-derived proteins, and / or ORF1p-derived proteins) before entering the nucleus. Alternatively, ORF1p and / or ORF2p can mediate direct nuclear entry of the vector. If it binds to ORF2p, ORF1p, ... If the binding of ORF2p-derived proteins and / or ORF1p-derived proteins is insufficient, different RNA vectors can still function in vivo. If the vector RNA is ligated to short, scattered element RNA transcribed within the cell or its product, it binds to ORF2p expressed within the cell or encoded by the vector, or binds to both ORF2p and ORF1p simultaneously; or it binds directly to ORF2p expressed within the cell or encoded by the vector, or binds to both ORF2p and ORF1p simultaneously; or it binds to ORF2p expressed within the cell or encoded by the vector via one or more ORF2p functional initiation sites already linked downstream of the RNA framework, the sequence to be inserted is inserted into the corresponding target site on the genome (the insertion site). If insertion continues according to the above method based on the new site generated after insertion, continuous insertion can be achieved, completing long fragment insertions without significant length limitations. If directional transfer is required, modifications can be made to the outer coating of the vector. Care should be taken to avoid RNA degradation throughout the entire process.

[0142] III. RNA vector and / or RNP vector-mediated genome sequence deletion techniques

[0143] 1. Deletion of any region in the genome: The sequence to be inserted in the RNA and / or RNP vectors designed in the above insertion techniques ("Scheme 1, genome sequence insertion technology using RNA as a vector" and "Scheme 2, genome sequence insertion technology using RNP as a vector") is replaced with a sequence (within 20,000 bp) upstream or downstream of the insertion site (within 100,000 bp). (If homologous recombination occurs between the inserted sequence and its upstream sequence, any sequence can be inserted between the sequence to be inserted and the corresponding sequence upstream of the target site in the genome without affecting the results or promoting subsequent homologous recombination and / or The effects it produces: If the inserted sequence undergoes homologous recombination with its downstream sequence, any sequence can be inserted between the sequence to be inserted and the corresponding sequence downstream of the target site on the genome, without affecting the result or promoting subsequent homologous recombination and / or its effects. Through the RNP or RNA-mediated genome sequence insertion pathways described in this invention ("Scheme 1, RNA-based genome sequence insertion technology" and "Scheme 2, RNP-based genome sequence insertion technology"), the sequence between two identical sequences can be removed with a certain efficiency through homologous recombination after the insertion of the sequence. Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC) can be selected for insertion to improve the efficiency of subsequent homologous recombination. If directional transfer is required, the outer coating of the vector can be modified. Note that RNA degradation should be avoided throughout the process. Furthermore, if the sequence to be removed is 600 bp or less, the corresponding fragment can be deleted through homologous recombination and / or other genome repair mechanisms with high efficiency.

[0144] 2. Delete from the end of CNV:

[0145] Under physiological conditions, copy number variation (CNV) is similar to a copy of the original complete gene. Through the mechanism described above, CNVs, which are copies of the original complete gene, can be continuously extended, resulting in continuous changes in protein expression and various states of cells, tissues, and organisms. The CNV terminus consists of an upstream gene portion and a downstream short sporadic element (ORF2p functional initiation portion). Short sequence fragments formed by the connection of a lasso structure with the short sporadic element (ORF2p functional initiation portion) are continuously inserted between these two portions to extend the CNV. In early embryonic development, transcription of long sporadic elements increases significantly, while short sporadic elements on the genome, such as the Alu sequence, show significant demethylation. While long sporadic element-mediated 3′ transduction (based on the right monomer deletion of the short sporadic element upstream of the promoter and the complete short sporadic element structure downstream) initiation-related gene copy number variations (CNVs) are elongated, the demethylated short sporadic element sequences undergo homologous recombination, deleting (initializing) most of the previously extended CNVs. Subsequently, the fully initialized embryonic cells regained a hypermethylated state, and the ends of CNVs gradually lengthened, mediated by some short, scattered elements at the CNV ends. This altered the expression and state of each cell, and the gene expression of each cell, in turn, influenced CNV changes through the lasso structure, thereby causing changes in the genome and gradually inducing differentiation. This is consistent with the common CNV changes in embryos and the differences in CNVs in various tissues.

[0146] The prolongation of CNVs (Channel Variants) of different genes is prevalent in various tumor cells and is positively correlated with clinical grade. Simultaneously, the expression levels of proto-oncogenes and tumor suppressor genes are also directly proportional to the length of CNVs; therefore, tumor formation and progression should be related to the dysregulation of CNVs in proto-oncogenes or tumor suppressor genes. Furthermore, some irreversible diseases related to external stimuli, such as diabetes, may also be associated with CNV dysregulation. Since most drug resistance is related to long-term external stimuli leading to changes in the expression of corresponding proteins, this invention can involve alterations in the CNVs of the corresponding genes and can be improved or inhibited.

[0147] CNV ends in cells or tissues are detected by sequencing and alignment (alignment to the junction of gene sequence and some short, scattered elements). The gene portion (within 2000 bp) of the CNV end to be treated and / or the 3′ portion of the lasso that can be formed within a range (within 200000 bp) downstream of the end in the intact gene (the downstream lasso can be predicted or detected by the following methods) (or the corresponding RNA sequence of the downstream portion (within 200000 bp) of the end in the intact gene can be directly selected, cut, and replaced with the above-mentioned 3′ portion sequence) are used as upstream sequences of the target site. These are then linked to the sequence immediately upstream (within 100000 bp) of the sequence to be deleted (intercalation is also possible between the sequence immediately upstream (within 100000 bp) of the sequence to be deleted and the portion serving as the upstream sequence of the target site). An RNA sequence that can be inserted without affecting the result or promoting subsequent homologous recombination and / or its effects is used as the target sequence. Then, a complete short element RNA, a partial short element RNA, or a short element-like RNA (i.e., the ORF2p functional initiation part) is ligated (depending on the different insertion methods mentioned above) as the downstream sequence of the target site (as mentioned above, it can be followed by ORF1p and ORF2p coding sequences). The sequence is synthesized and inserted via one of the above gene insertion methods using an RNA vector or RNP vector. The insertion occurs between the gene portion at the end of the actual CNV and a partial short element sequence on the genome (the short element sequence used on the vector should be the same as or closer to the short element sequences around the insertion site to improve efficiency). The target sequence is then deleted via homologous recombination between identical sequences. Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC) can be selected for insertion to improve efficiency. If directional transfer is required, the outer coating of the vector can be modified. Care should be taken to avoid RNA degradation throughout the process. This method allows for the simultaneous administration of multiple gene-editing RNAs, RNPs, and / or DNAs to delete CNV ends of multiple genes or multiple different CNV ends of a single gene. Because it can alter gene expression and cell appearance, it can modify cellular states, such as tumor stages (e.g., grade) or cell differentiation.

[0148] 3. Continuous deletion of CNV ends: If, during or after the aforementioned CNV end deletion, the corresponding RNA, RNP, and / or DNA with gene-editing function described in this invention are administered to delete secondary CNV ends, the continuous action of this process allows the CNVs of the corresponding genes to be continuously and persistently deleted. Multiple corresponding RNAs, RNPs, and / or DNAs with gene-editing function can be administered simultaneously to delete the CNV ends of multiple genes or multiple different CNV ends of a single gene, either simultaneously or sequentially. This method, because it can alter gene expression and cell epigeneity, can modify the state of cells, such as the tumor stage or cell differentiation.

[0149] IV. RNA vector and / or RNP vector-mediated genome CNV end extension or CNV addition technology

[0150] 1. Addition of new CNVs to the genome: One or more gene copies can be added using the gene editing method described in this invention, with any location on the genome as the target site. Sequences containing recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC), short element sequences, partial short element sequences, and / or short element derivative sequences (ORF2p functional start portion) on the genome to be edited can be searched or selected as upstream and downstream sequences of the target site for gene copy insertion. This increases homologous recombination efficiency and enhances the corresponding gene editing effect. Adding recombination sites (GCAGA[A / U]C, CCCA[C / G]GAC / or CCAGC), short element RNA, partial short element RNA, and / or short element derivative RNA in the upstream and downstream sequences of the target site within the RNA framework can also increase the corresponding gene editing effect. The ends of the newly added gene copies are the newly generated CNV ends, which can be extended or shortened for further gene editing. Recombination sites (GCAGA[A / T]C, CCCA[C / G]GAC / or CCAGC), short fragmented element sequences, partial short fragmented elements, and / or short fragmented element derivatives (corresponding DNA sequences of the ORF2p functional initiation region) can be identified on the genome and inserted into new gene copies as downstream sequences of the target sites to improve efficiency. In particular, using partial short fragmented element RNA as downstream sequences of the target sites is more consistent with the downstream sequences of the CNV ends in their natural state. Multiple gene-editing RNAs, RNPs, and / or DNAs can be simultaneously administered to add CNVs of multiple genes or multiple CNVs of a single gene of different lengths and states, either simultaneously or sequentially. This method can modify cell states, such as tumor grade or cell differentiation, by altering gene expression and cellular epigeneity.

[0151] 2. Addition and extension of CNV ends in the genome: This method involves adding and extending existing CNV ends in the genome. Following the sequence insertion method described above, the upstream portion (gene portion) of the CNV end serves as the corresponding upstream sequence of the target site in the genome, the downstream portion (partial short scattered elements (ORF2p functional initiation portion)) of the CNV end serves as the corresponding downstream sequence of the target site in the genome, and the downstream sequence of the upstream portion (gene portion) of the CNV end within the complete gene sequence (in most cases, partial short scattered element sequences) serves as the corresponding sequence of the to-be-inserted sequence in the genome, thus extending the CNV ends of the corresponding genes. Multiple gene-editing RNAs, RNPs, and / or DNAs can be simultaneously administered to add and extend CNV ends of multiple genes or multiple different CNV ends of a single gene. This method can alter gene expression and cell epigeneity, thus modifying cell states, such as tumor stages or cell differentiation.

[0152] V. Genomic sequence substitution techniques (including sequence substitution, site deletion, site addition, sequence addition, sequence deletion, and site substitution)

[0153] The insertion sequence in the vector designed in the above insertion technique is replaced with a replacement sequence and a sequence surrounding the replacement sequence on the genome (i.e., the sequence that will be deleted after homologous recombination between the DNA sequence of the replacement sequence and the sequence on the genome; whether it is located at 3′ or 5′ of the replacement sequence during vector construction depends on whether the insertion site is upstream or downstream of the replacement sequence on the genome). (The DNA sequence of the replacement sequence should be homologous to the replacement sequence on the genome.) (If the inserted sequence undergoes homologous recombination with its upstream sequence, any sequence can be inserted between the corresponding sequence of the insertion sequence 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 effects.) The effects produced; if the inserted sequence undergoes homologous recombination with its downstream sequence, any sequence can 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 effects). Through the above gene editing insertion method, 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. When the inserted replacement sequence undergoes homologous recombination with the sequence to be replaced on the genome, the sequence to be replaced on the genome is replaced by the inserted replacement sequence that is homologous to it. At the same time, the surrounding sequence portion of the sequence to be replaced that was deleted due to homologous recombination is re-inserted along with the replacement sequence during insertion. Genome substitutions include sequence substitution and site substitution. Sequence substitution occurs when the inserted replacement sequence differs from the corresponding sequence in the genome in some parts (e.g., one or more sequences). Site substitution occurs when the inserted replacement sequence differs from the corresponding sequence in the genome in some sites (e.g., one or more sites). Site deletion occurs when the inserted replacement sequence is missing some sites compared to the corresponding sequence in the genome (e.g., one or more sites). Site addition occurs when the inserted replacement sequence is added to some sites compared to the corresponding sequence in the genome (e.g., one or more sites). Sequence addition occurs when the inserted replacement sequence is added to some sites compared to the corresponding sequence in the genome (e.g., one or more sequences). Sequence deletion occurs when the inserted replacement sequence is deleted from some sites compared to the corresponding sequence in the genome (e.g., one or more sequences). The smaller the difference between the inserted replacement sequence and the corresponding homologous sequence in the genome, the higher the efficiency. Inconsistencies between the inserted replacement sequence and the corresponding homologous sequence in the genome should be avoided as much as possible, preferably located at or near the ends or sides of the inserted replacement sequence, to improve efficiency. If directional transfer is required, modifications can be made to the outer coating of the vector. Care should be taken to avoid RNA degradation throughout the entire process.

[0154] VI. RNA vector and / or RNP vector-mediated genomic sequence deletion simultaneously with sequence insertion, sequence substitution, or site substitution.

[0155] By adding the desired genome sequence after a sequence within 20,000 bp upstream or downstream (within 100,000 bp) of the insertion point in the above-mentioned "RNA vector and / or RNP vector-mediated genome sequence deletion technology" operation, the desired genome sequence can be inserted upstream of the deleted sequence while simultaneously deleting the target sequence.

[0156] By replacing a sequence within 20,000 bp upstream or downstream (within 100,000 bp) of the insertion site (target site) in the above-mentioned "RNA vector and / or RNP vector-mediated genome sequence deletion technology" operation with a sequence that differs from a sequence within 20,000 bp upstream or downstream (within 100,000 bp) of the insertion site (target site) at a different site or part of the sequence (the different site and / or part of the sequence is the site and / or sequence to be replaced), the target sequence can be deleted while simultaneously replacing a site or part of the upstream sequence of the deleted sequence with the site and / or part of the sequence to be replaced.

[0157] VII. Preventing genomic changes caused by transposons and stabilizing the genome and its CNVs (i.e., sequences inserted into the genome or the genome, or sequences upstream, downstream, or in the intact genome, at the end of the CNV and between the gene portion and some short element sequences or other regions using this gene editing technology, thus preventing further extension of the CNV; the CNV end is defined as the location where the gene sequence directly connects to the short element sequence or other related sequences (the corresponding DNA sequence of the ORF2p functional initiation portion), where the gene can be extended. The specific sequences of the gene sequence and the short element sequence or other related sequences (the corresponding DNA sequence of the ORF2p functional initiation portion) at each specific CNV end can be determined by molecular biology techniques such as gene sequencing or gene chips. (Segment Acquisition) (Transfected into in vitro cultured cells, tissues, or organs by encapsulating the corresponding vector with lipid-soluble substances or cell-transfecting substances such as liposomes, or by administration via blood, lymph, cerebrospinal fluid, or local tissue administration) (The RNA used below can be replaced with a DNA vector that can express the corresponding RNA, so that it is expressed in the corresponding target system to produce RNA and / or the expressed RNA binds with the ORF1p, ORF2p, ORF1p-derived protein and / or ORF2p-derived protein expressed by the DNA vector to form an RNP; the RNA used below can also be replaced with the corresponding RNA bound to the ORF1p, ORF2p, ORF1p-derived protein and / or ORF2p-derived protein to form an RNP, if possible or necessary)

[0158] 1. Intervention on a specific CNV (where the upstream sequence used for insertion is the gene portion at the end of the CNV of a specific gene): Select the CNV to be operated on, and set the junction of the 3′ end of its gene portion with a partial short element sequence or other related sequences (the corresponding DNA sequence of the ORF2p functional start part) as the insertion site (target site). Set the upstream sequence of the insertion site (target site) in the above insertion method as the corresponding RNA sequence (within 20,000 bp) at the 3′ end of the gene portion at the end of the CNV, and the downstream sequence is the partial short element RNA or other related sequences (ORF2p functional start part) (therefore, the short element RNA, partial short element RNA or short element-like RNA (ORF2p functional start part) connected after the downstream sequence of the target site in the above method can be omitted). The sequence to be inserted is any sequence (within 20,000 bp) that is not homologous to the genome or to the gene portion at the end of the CNV and its upstream and downstream sequences in the complete gene. After the vector is constructed, it is transferred into the corresponding cells, living tissues, or organisms via the aforementioned RNA vector and / or RNP vector pathways, causing a non-homologous sequence to be inserted at the corresponding CNV end. Since the non-homologous sequence does not exist downstream of the corresponding CNV end gene sequence in the complete gene, it is impossible to further extend the CNV end based on the complete gene sequence, thus hindering further changes at the CNV end.

[0159] 2. Intervene in a wide range of CNVs across the genome (the upstream sequence of the target site used for insertion must contain the gene portion at the ends of all possible CNVs):

[0160] (1) Genome fragmentation method: Take cells from the organism, tissue or cell line to be operated on and culture them in vitro, or directly extract the genome, sonicate them and enrich them by random primers and PCR; design and synthesize short random sequences (within 20 bp) and short scattered element sequences in the downstream linker. The enriched genomic fragments are ligated with synthetic short random sequences and then linked to short, scattered element sequence fragments via PCR. Different genomic fragment sequences are ligated with random sequences and then linked to short, scattered element sequences or other relevant sequences (the corresponding DNA sequence of the ORF2p functional initiation region). The resulting fragments are used to construct corresponding RNA, which is then transformed into corresponding cells, living tissues, or organisms via the aforementioned RNA vector and / or RNP vector. The genomic fragment sequences target all CNV ends on the genome, causing non-homologous sequences (i.e., short random sequences or partial short random sequences whose parts are not homologous to the gene fragments, and are non-homologous to the local gene sequence of the corresponding gene fragment) to be inserted between the gene portion and the short, scattered element at the CNV end. Since the non-homologous sequences are not present downstream of the corresponding CNV end gene portion sequence in the complete gene, further changes at the CNV end are prevented.

[0161] (2) Random Sequence Method: Generate a random sequence of appropriate length (within 100 bp) (containing all possible permutations, excluding combinations similar to short sporadic element sequences), ligate any non-homologous sequence (within 20,000 bp) to the genome, and then ligate a portion of the short sporadic element's RNA or other relevant sequences (ORF2p functional initiation region); or ligate a random sequence (within 100 bp) to the natural splice site in the middle of the short sporadic element (e.g., for Alu transcripts, the splice site in the middle that can be spliced ​​to produce scAlu and part of Alu), and then add any non-homologous sequence to the genome. The RNA or other related sequences (ORF2p functional initiation portion) of short, sporadic elements with homologous sequences (within 2000 bp) can be used. Alternatively, random sequences can be synthesized and linked to any non-homologous sequence in the genome (which is subsequently expressed as a lasso). Simultaneously, vectors for transcribing short, sporadic element RNA and / or partially short, sporadic element RNA can be constructed. Downstream of the short, sporadic element RNA, a long, sporadic element sequence or its protein-coding sequence corresponding to the function of the short, sporadic element can be attached or additionally expressed (or the RNA of short, sporadic elements and / or partially short, sporadic elements can be directly introduced into the target system) or its RNA sequence. These vectors are then transferred into corresponding cells, living tissues, or organisms. Using random sequences, the vectors target all CNV ends in the genome according to the aforementioned mechanism and method, causing the insertion of non-homologous sequences at the corresponding CNV ends. Since non-homologous sequences do not exist downstream of the corresponding CNV end gene sequence in the complete gene, further changes at the CNV ends are prevented.

[0162] (3) According to the lasso end sequence method: Detect all lasso species (insert a short, non-homologous random sequence (within 100 bp) into the short sporadic element sequence, and the expressed short sporadic element RNA can still be normally cleaved into a partial short sporadic element (i.e., the insertion position of the non-homologous sequence is downstream of the natural cleavage site of the short sporadic element, and not located at the cleavage site), and construct a plasmid that can express the modified short sporadic element RNA, and transform it into cells amplified from the corresponding organism to be operated on or a cell line of the corresponding species (or the genome of the corresponding species to be tested can be taken, and the whole genome can be truncated into a longer (2) Fragments (over 10 bp or more) with a certain degree of overlap (overlap exceeding 10 bp) are constructed and overexpressed in in vitro cells of the corresponding species using RNA polymerase II. After a period of time, the corresponding nucleic acids are extracted and sequenced using sequence-specific analysis of the non-homologous sequences inserted into the short fragment RNA. This yields the sequence information of various lassos generated by the short fragments linked to the integrated non-homologous sequences. Alternatively, the lasso sequences can be predicted based on the sequence patterns of the lassos formed by pre-mRNA (e.g., often ending with AG), thus obtaining all lasso sequence information for that species or individual. Synthesize 3′ sequences (within 20,000 bp) containing all lasso sequences, and ligate them with any non-homologous sequence (within 20,000 bp) from the genome. Simultaneously generate RNA containing short sporadic elements (which, as described above, can be followed by a long sporadic element sequence corresponding to the function of the short sporadic element or its protein-coding sequence to increase efficiency) and introduce them into the target system. Alternatively, generate 3′ sequences of all obtained lasso sequences, ligate them with any non-homologous sequence (within 2000 bp) from the genome, and then ligate them with a portion of short sporadic elements (which, as described above, can be followed by a long sporadic element sequence corresponding to the function of the short sporadic 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 containing the ligated lasso 3′ sequence to increase efficiency). Transform these RNA sequences into the corresponding cells, tissues, or organisms using the aforementioned RNP vector or RNA vector, and edit the CNV ends across the entire genome.

[0163] (4) Short sporadic element sequence modification method: that is, by giving additional modified short sporadic element RNA, a sequence that is not homologous to the genome or to the gene portion at the end of the CNV and its upstream and downstream sequences in the complete gene is inserted into the end of each CNV, thus preventing the end from extending. Generate RNA containing a short sequence (not necessarily the conventionally generated lasso 3′ sequence, but a short segment spanning the short element's natural splicing site, less than 100 bp) that allows the short element's transcript (RNA) to be naturally spliced ​​in this newly added region, representing a complete short element sequence (a long short element sequence or its protein-coding sequence corresponding to the function of the corresponding short element species can be added after it to increase efficiency); or generate RNA containing a complete short element sequence (less than 200 bp) with any non-homologous sequence to the genome added after the natural splicing site of the short element transcript (a long short element sequence or its protein-coding sequence corresponding to the function of the corresponding short element species can be added after it to increase efficiency) or RNA containing this RNA sequence, and administer it to the appropriate cells, living tissues, or organisms. The short element sequences used should ideally cover all short element sequences of the species or individual (obtainable through sequencing or array microarrays) to allow for precise modification of all CNV ends across the entire genome.

[0164] Alternatively, the entire genome can be segmented into long fragments with a certain degree of overlap (the overlap length is greater than the length of a lasso structure) and RNA from these long fragments can be generated. This RNA can be transferred into the in vitro cell line of the corresponding species to generate a lasso structure. Subsequently, the RNA corresponding to the modified short sporadic element sequence (which can be mediated via the RNA pathway after adding a long sporadic element sequence or its protein-coding sequence corresponding to the function of the short sporadic element of the corresponding species downstream) can be transferred in. Then, the biologically active single-stranded RNA ribonucleoprotein complex (RNP) or RNA connected to the short sporadic element that generates the lasso (generated by the modified short sporadic element) can be isolated and purified using sequence specificity and other properties and conventional methods. It then functions through the corresponding RNA or RNP pathway.

[0165] 3. Modification of the ORF2p functional initiation region on the genome: This invention inserts arbitrary sequences (within 500 bp) into the transcriptional non-coding regions of short scattered elements, short scattered element derivatives, or functional structures initiating ORF2p splicing and reverse transcription, such as promoters, enhancers, regulatory sequences, or inducible elements (cis-acting elements), their transcriptional regions, natural splicing sites of transcripts, or other sequences on short scattered elements, short scattered element derivatives, or functional structures initiating ORF2p splicing and reverse transcription, and / or long scattered elements, long scattered element derivatives, their transcriptional regions, protein-coding sequences, or other sequences within cis-acting elements, protein-coding sequences, or other sequences. This prevents the transcription of short scattered element sequences, short scattered element derivative sequences, and functional structure sequences initiating ORF2p splicing and reverse transcription, or prevents post-transcriptional splicing, and / or prevents the transcription of long scattered element sequences or long scattered element derivatives, or produces proteins with normal function. First, the sequences of short sporadic elements, long sporadic elements, short sporadic element derivatives, long sporadic element derivatives, functional structural sequences initiating ORF2p splicing and reverse transcription, and related regions such as transcription-related non-coding regions such as promoters, enhancers, regulatory sequences, or inducible elements (cis-acting elements) are obtained through sequencing. Transcription-related non-coding regions (such as promoters, enhancers, regulatory sequences, or inducible elements), their transcriptional regions, natural splicing sites of transcription products, protein-coding sequences, or other sequences are selected as target sites. The upstream and downstream sequences of the target sites in this invention are the upstream and downstream sequences relative to the target sites on the short sporadic elements, long sporadic elements, short sporadic element derivatives, long sporadic element derivatives, and functional structural sequences initiating ORF2p splicing and reverse transcription. The inserted sequence is arbitrary. Using the above insertion method, the arbitrary sequence is inserted into the corresponding site on the short sporadic elements, long sporadic elements, short sporadic element derivatives, long sporadic element derivatives, or functional structures initiating ORF2p splicing and reverse transcription in the genome. In addition, the gene editing methods described above can be used to replace (sequence substitution, site deletion, site addition, sequence addition, sequence deletion and / or site substitution) or delete short sporadic elements, long sporadic elements, short sporadic element derivatives, long sporadic element derivatives or functional structural sequences that initiate ORF2p splicing and reverse transcription on the genome, thereby inactivating or reducing their function.

[0166] 4. Deletion and fixation of CNV ends: Select the CNV end to be operated on, and set the junction of its 3′ end of the gene portion and a short, scattered element or other related sequence (the corresponding DNA sequence of the ORF2p functional start portion) as the target site. Set the upstream sequence of the target site in the above insertion method as the 3′ end of the CNV end gene portion (within 2000 bp). The downstream sequence of the target site is the short, scattered element RNA or other related sequence (ORF2p functional start portion) (therefore, the short, scattered element connected after the downstream sequence in the above gene editing method can be omitted). The sequence to be inserted is the sequence immediately upstream of the sequence to be deleted (within 100000 bp) on the genome (within 20000 bp) followed by any sequence (within 20000 bp) that is not from the genome sequence (any sequence can also be inserted between the sequence to be inserted on the genome and the upstream sequence of the target site without affecting the result or promoting subsequent homologous recombination and / or its effects). After generating the aforementioned RNA or RNP vector, it is transferred into the corresponding cells, living tissues, or organisms via the aforementioned RNA or RNP pathway. This causes the corresponding CNV to be inserted into the sequence immediately upstream of the sequence to be deleted on the genome, followed by a non-homologous sequence. When homologous recombination occurs between two identical sequences, resulting in the deletion of the intermediate sequence, the non-homologous sequence will simultaneously prevent the further extension of the CNV.

[0167] 5. Inhibition of Intrinsic Mechanisms: This method can also directly inhibit the inherent CNV elongation mechanisms of cells or organisms, such as inhibiting the transcription of short sporadic elements, long sporadic elements, short sporadic element derivatives, long sporadic element derivatives, or other related sequences (the corresponding DNA sequence of the ORF2p functional initiation region) or their RNA and encoded proteins, such as ORF1p, ORF2p, ORF1p-derived proteins, or ORF2p-derived proteins, through specific proteins related to this CNV elongation mechanism, such as ORF1p, ORF2p, ORF1p-derived proteins, or... By binding the functional structure of ORF2p-derived proteins or splice variants or complexes to inhibit their function, gene editing techniques can be used to modify short sporadic elements on the genome, such as Alu and various MIRs, as well as their derivatives, long sporadic elements and their derivatives, and the corresponding protein-coding sequences therein, to inactivate or reduce their activity. This can also inhibit the function of proteins related to homologous recombination or mismatch repair mechanisms, or introduce modified nucleoside analogs to inhibit reverse transcription. In this way, by inhibiting the intrinsic CNV elongation mechanism, the effects of inhibiting genomic changes and stabilizing CNVs can be achieved.

[0168] 8. Genome editing by administering long RNA fragments or RNPs:

[0169] Because RNA splicing in eukaryotic cells can produce overlapping lasso structures, theoretically these overlapping lasso structures are multiple RNA frames containing upstream sequences, downstream sequences, and the sequence to be inserted at the target site. The downstream sequence of the target site in one lasso structure is the upstream sequence of the target site in another overlapping lasso structure. Therefore, when a portion of the Alu sequence already exists upstream of the desired RNA or RNP sequence, the sequence information can be gradually inserted into the genome after a longer RNA or RNP sequence is given. This ultimately completes various gene editing tasks, such as genome insertion, genome deletion, genome sequence replacement, genome site replacement, simultaneous genome sequence deletion and insertion, sequence replacement, or site replacement, inhibiting genomic changes caused by transposons and stabilizing the genome and its CNVs, and performing genome sequence insertion, deletion, replacement, site replacement, or simultaneous genome sequence deletion and insertion, sequence replacement, or site replacement at the ends of CNVs.

[0170] IX. Assisting other gene editing technologies

[0171] This invention generates RNA, single-stranded DNA, and double-stranded DNA within the target system. These RNA, single-stranded DNA, and double-stranded DNA can provide templates for other gene editing technologies, such as TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9, after the genome is cut, for gene editing (e.g., homologous recombination or other functions), such as inserting exogenous sequences (sequences to be inserted), thus assisting and promoting the function of the corresponding gene editing technologies. Through the complementary binding of upstream and downstream sequences of the target site on the single-stranded or double-stranded DNA generated by the gene-editing RNA, RNP, and / or DNA vectors of this invention with the corresponding sequences on the genome, the sequence to be inserted is inserted into the single-stranded or double-stranded DNA cut site generated by TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9 technologies, thereby inserting the sequence to be inserted into the target site on the genome, assisting or improving the efficiency of gene editing technologies such as TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9. This invention contains or can generate RNA containing upstream sequences of the target site, sequences to be inserted, downstream sequences of the target site, and / or other sequences such as short interstitial RNA, partial short interstitial RNA, and other ORF2p functional initiation portions, as well as ssDNA and / or dsDNA reverse transcribed from them. These components can assist technologies such as TALEN, ZFN, Targetron, CRISPR, and CRISPR / Cas9 in homologous recombination or insert corresponding sequences into the target site, promoting greater RNAization and devirulent transfection of the corresponding technologies (RNA-transduced cells do not need to enter the nucleus and can enter the nucleus during non-dividing phase under the binding and action of corresponding proteins such as ORF1p and / or ORF2p) and improving the genomic sequence insertion efficiency of the corresponding technologies.

[0172] Furthermore, DNA vectors expressing RNA frameworks (containing upstream sequences of the target site, sequences to be inserted, downstream sequences of the target site, and other sequences such as short fragmented element RNA, partial short fragmented element RNA, and other ORF2p functional initiation portions) can continuously generate RNA containing RNA frameworks. These RNAs can then be converted into single-stranded or double-stranded DNA using reverse transcription structures on the RNA. This provides a continuous DNA template for other gene editing technologies such as TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9 after genome cutting (e.g., homologous recombination or other actions), such as inserting foreign sequences (sequences to be inserted), thus assisting and promoting the effects of these gene editing technologies. Meanwhile, RNA containing an RNA framework (containing upstream sequences of the target site, the sequence to be inserted, downstream sequences of the target site, and other sequences such as short interstitial RNA and some short interstitial RNA, etc., the ORF2p functional initiation portion) can be delivered to the target after binding to ORF2p and / or ORF1p in vitro. In the target body, ORF2p and / or ORF1p convert the RNA into single-stranded DNA and / or double-stranded DNA, continuously providing DNA templates for other gene editing technologies such as TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9 to perform gene editing (such as homologous recombination or other functions) after cutting the genome, such as inserting foreign sequences (the sequence to be inserted), thus assisting and promoting the function of the corresponding gene editing technologies. Simultaneously, after expressing ORF2p and / or ORF1p in the target organism, RNA containing an RNA framework (containing the upstream sequence of the target site, the sequence to be inserted, the downstream sequence of the target site, and other sequences such as short interstitial RNA, partial short interstitial RNA, etc., the ORF2p functional initiation portion) can be given to the target. After ORF2p and / or ORF1p bind to the RNA in the target organism, the RNA is converted into single-stranded DNA and / or double-stranded DNA. This continuously provides a DNA template for other gene editing technologies such as TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9 to perform gene editing after cutting the genome (such as homologous recombination or other functions), such as inserting foreign sequences (the sequence to be inserted), thus assisting and promoting the function of the corresponding gene editing technologies.

[0173] The RNA used in this invention can be linear or circular. Circular RNA can be obtained by adding complementary sequences longer than 5 bp, such as Alu element sequences or intron sequences, to both sides of the RNA framework, generating circular RNA in vitro or in vivo to perform the corresponding functions in this invention. Designing the sequences on both sides of the RNA framework to allow intron self-cleavage can also generate circular RNA containing the RNA framework in vivo or in vitro. Containing 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.

[0174] The techniques of this invention can be used to edit copy number variations and their terminal portions in various genes, changing the position of the ends or stabilizing them. Since these determine gene expression, they can be used to stabilize or alter various states of cells and organisms. Therefore, this can be applied to modifying the genes and states of cells, tissues, and organisms; modifying the genome of organisms such as the human genome to improve function; modifying the genome of organisms such as the human genome to treat various gene-related genetic diseases such as Huntington's disease and Fragile X syndrome; delaying or stopping changes in the genes and states of cells and organisms; altering the genes and states of cells or organisms; and regenerating tissues and organs. Research and treatment of various diseases, including gene and biological regeneration, conversion of somatic cells into germ cells through the introduction of transcription factors to assist reproduction, prevention or delay of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple system atrophy, primary lateral sclerosis, spinocerebellar ataxia, Pick's disease, frontotemporal dementia, Lewy body dementia and progressive supranuclear palsy, inhibition of tumor cell metabolic activity, proliferation rate and production, while delaying its deterioration and improving its malignancy, and research and treatment of all other diseases related to gene and CNV alterations such as diabetes, as well as other physiological, pathological and pathophysiological research fields.

[0175] This invention can be used to treat gliomas, 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 adenoma, germ cell tumor, meningioma, meningeal carcinoma, glioblastoma, various astrocytomas, various oligodendrogliomas, astrocytodesmomas, various ependymomas, choroid plexus papillomas, choroid plexus carcinomas, chordomas, various gangliocytomas, olfactory neuroblastomas, and sympathetic nervous system tumors. It is used to prevent the occurrence of neuroblastoma, pineal cell carcinoma, pineal blastoma, medulloblastoma, trigeminal schwannoma, facial and auditory nerve carcinoma, glomus jugulare tumor, hemangioblastoma, craniopharyngioma, or granular cell carcinoma and their metastatic carcinomas; to inhibit their proliferation and prevent their grade from increasing and progressing or to reverse their nature; to prevent, delay or improve resistance to drugs such as insulin, levodopa, various tumor chemotherapy drugs and targeted drugs; and to delay or stop changes in the genes and states of cells and organisms, tissue and organ regeneration and biological regeneration.

[0176] In this invention, a certain sequence or site (such as the sequence to be inserted and the upstream sequence, downstream sequence or flanking sequence of the target site on the genome) (the sequence is DNA or RNA) is defined along the 5′→3′ direction, with the upstream being before the 5′ end of the certain sequence or site and the downstream being after the 3′ end of the certain sequence or site. The upstream sequence is the sequence located before the 5′ end of the certain sequence or site, and the downstream sequence is the sequence after the 3′ end of the certain sequence or site.

[0177] When designing RNA or RNP vector sequences, software (such as PCFOLD or RNAFOLD) can be used to simulate the secondary structure of the RNA or RNP vector. This can improve gene editing efficiency by making the upstream sequence of the target site, especially the free ends or free portions of the upstream sequence, more of it in a single-stranded free state, and reducing complementary sequences within the RNA or RNP vector sequence, especially the free ends, free portions, or portions near the free ends of the upstream sequence. Furthermore, designing sequences that make the secondary structure of the designed RNA to be inserted more closely resemble the secondary structure of some short, scattered element RNAs (such as some Alu) (e.g., forming complementary double-stranded structures on both sides of the bottom notch of the Ω structure formed by the designed sequence, and / or mimicking other stem-loop structures or protrusions in the secondary structure of some short, scattered element RNAs (such as some Alu)).

[0178] The loop-shaped secondary and higher-order structures within the "Ω" structure of the RNA framework and its improved forms, which are required for the ORF2p to function, should ideally approximate (imitate) the loop-shaped secondary and higher-order structures within the "Ω" structure in the natural SINE and LINE transcripts corresponding to the ORF2p used, to increase efficiency. This includes the stem loop, bulge, and AA binding at the root points of the two "Ω" structures, as well as the resulting double-stranded complementary structures, including the shape, length, relative position (calculated from the start of the right leg towards the 3' direction), and sequence similarity of the corresponding secondary and higher-order structures. Similarly, the sequence and secondary and higher-order structures of the right leg of the "Ω" structure can also mimic the sequence and secondary and higher-order structures of the right leg of the natural SINE and LINE transcripts corresponding to the ORF2p used, to increase efficiency. This includes the stem loop, bulge, and double-stranded complementary structures, including the shape, length, relative position (calculated from the start of the right leg towards the 3' direction), and sequence similarity of the corresponding secondary and higher-order structures. Meanwhile, designing the two bases closest to the two legs of the open-loop structure between the two legs of the "Ω" structure as adenine (A) or other base pairs with mismatched weak binding can stabilize the "Ω" structure to a certain extent, thereby improving gene editing efficiency.

[0179] LINEs can be further divided into stringent and relaxed types. In stringent LINEs, the portion of the transcript corresponding to the 3'UTR can form a special, relatively conserved secondary structure, forming a stem-loop structure at a specific location. This structure is characterized by an asymmetric loop or protrusion 4-6 bp from the central loop. This structure promotes the binding and function of ORF2p in the corresponding species. Relaxed LINEs generally do not form this structure, but in some cases, a similar structure may form (a 5-7 bp loop, an 8-10 bp stem, and a protrusion 4-6 bp from the loop), potentially promoting ORF2p binding and function. The LINEs in humans and most mammals are loosely structured, while those in eels (LINE UnaL2), insects (LINE R2), zebrafish (LINE ZfL2-1 and ZfL2-2), algae (L1), silkworms (LINESART1), monocots (L1), fungi (Tad1), fish (L2), and some mammals (RTE) are tightly structured. For the application of ORF2p corresponding to tightly structured LINEs, adding the aforementioned stem-loop structure to the beginning of the ORF2p function can increase the binding or efficiency of the corresponding ORF2p. Similarly, for loosely structured LINEs, adding the aforementioned stem-loop structure (such as the "UCCCGCCUGGGCCACAGAGCGAGA" sequence in the beginning of the ORF2p function may also increase the binding or efficiency of the corresponding ORF2p.

[0180] The target sites acted upon by this invention can be one or more; when there are multiple target sites, the single-stranded genome of the primers generated after the ORF2p and / or ORF2p-derived proteins corresponding to different target sites cleave the genome can be the same strand on the same chromosome, the complementary strand on the same chromosome, or located on different chromosomes.

[0181] The designed sequence contains two inverted repeat sequences (such as Alu elements, other SINEs or other inverted repeat sequences) or complementary sequences on the transcribed RNA sequence. When the two inverted repeat sequences or complementary sequences on the RNA combine, the part between the two sequences can form circular RNA. In addition, adding RNA splicing signals (sites) to the sequence during the design process can also promote the formation of circular RNA from linear RNA.

[0182] The gene editing efficiency in this invention is improved by specifically inhibiting Lig4, DNA-PK, and XRCC6 through methods such as sgRNA, ASO, siRNA, or specific antibodies to promote DNA homologous recombination.

[0183] The basic structure of the RNA framework for gene editing provided by this invention is as follows: Figure 3 As shown, the sequence along the 5′→3′ direction includes the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site. To better understand further variations of the RNA framework, some different connection types are listed for comprehension; where "connection" indicates a direct connection, and "indirect connection" refers to the insertion of other sequences in between.

[0184] In the case of “indirect connection”, the intermediate insertion sequence can be any sequence. Here, any sequence is a sequence that is related to or unrelated to the transcription of the RNA framework provided by the present invention, such as the pan-ORF1p coding sequence, pan-ORF2p coding sequence, long sporadic element, short sporadic element, etc. related to the transcription of the RNA framework, or other coding sequences or non-coding sequences unrelated to the transcription of the RNA framework.

[0185] In this invention, "middle" means between two sequences that are still complete; while "inside" means within a sequence, where when a sequence is inserted into the inside of another sequence, it means that the other sequence is divided into two parts.

[0186] In this invention, "interval arrangement" refers to multiple different sequences. When each sequence appears once or multiple times, the arrangement between multiple different sequences is as follows: when sequence A and sequence B appear repeatedly, ABA, ABAB, ABBA, ABBABB, etc. are all different interval arrangements of sequence A and sequence B; when sequence A, sequence B, and sequence C appear repeatedly, ABCABC, ABBCA, CCABA, etc. are all different interval arrangements of sequence A, sequence B, and sequence C. More sequences are also possible.

[0187] Figure 4 The diagram shows an ORF2p functional initiation component further connected downstream of the basic structure of the RNA framework.

[0188] Figure 5 The diagram shows the further connection of multiple ORF2p functional initiation sites downstream of the basic structure of the RNA framework.

[0189] Figure 6 The diagram shows the replacement of the downstream sequence of the target with an ORF2p functional initiation portion within the basic RNA framework structure.

[0190] Figure 7 The diagram shows how the downstream sequence of the target is replaced with multiple ORF2p functional initiation portions within the basic RNA framework structure.

[0191] Figure 8 The diagram shows the ORF2p functional initiation portion connected downstream of the basic structure of the RNA framework, and then the pan-ORF1p coding sequence and / or pan-ORF2p coding sequence connected downstream of the ORF2p functional initiation portion.

[0192] Figure 9 The diagram shows the connection of the ORF2p functional initiation portion downstream of the basic structure of the RNA framework, followed by the indirect connection of the pan-ORF1p coding sequence and / or pan-ORF2p coding sequence downstream of the ORF2p functional initiation portion, where ... indicates indirect connection.

[0193] Figure 10 The diagram shows the ORF2p functional initiation portion connected downstream of the basic structure of the RNA framework, followed by the connection of multiple pan-ORF1p coding sequences and / or pan-ORF2p coding sequences downstream of the ORF2p functional initiation portion.

[0194] Figure 11 The diagram shows the ORF2p functional initiation portion connected downstream of the basic structure of the RNA framework, and the insertion of pan-ORF1p coding sequences and / or pan-ORF2p coding sequences within the downstream sequence of the target site.

[0195] Figure 12 The diagram shows the connection of pan-ORF1p coding sequences and / or pan-ORF2p coding sequences downstream of the basic structure of the RNA framework, and then the connection of the ORF2p functional initiation portion downstream.

[0196] Figure 13 The diagram shows the indirect connection between the basic structure of the RNA framework and the functional initiation part of ORF2p.

[0197] Figure 14 The diagram shows that the basic structure of the RNA framework and the functional initiation part of ORF2p are located on different RNA vectors and / or in RNPs.

[0198] Figure 15 The diagram shows two ORF2p functional initiation sites connected downstream of the basic structure of the RNA framework, and the two ORF2p functional initiation sites are indirectly connected.

[0199] Figure 16 The diagram shows the ORF2p functional initiation portion linked downstream of the basic structure of the RNA framework, and located on different RNA vectors and / or in RNPs, along with other ORF2p functional initiation portions.

[0200] Figure 17 The diagram shows the ORF2p functional start portion connected downstream of the basic structure of the RNA framework, and then directly connected downstream to the pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence. The pan-ORF1p coding sequence and / or the pan-ORF2p coding sequence are indirectly connected to the downstream ORF2p functional start portion.

[0201] Figure 18The diagram shows an ORF2p functional start-up sequence linked downstream of the basic structure of the RNA framework, followed by a pan-ORF1p coding sequence and / or a pan-ORF2p coding sequence, located on different RNA vectors and / or in RNPs, along with another ORF2p functional start-up sequence.

[0202] Figure 19 The diagram shows the pan-ORF1p coding sequence and / or pan-ORF2p coding sequence arranged downstream of the basic structure of the RNA framework, along with the ORF2p functional initiation portion.

[0203] Figure 20 The diagram shows the ORF2p functional initiation portion connected downstream of the basic structure of the RNA framework. The ORF2p functional initiation portion contains a pan-ORF1p coding sequence and / or a pan-ORF2p coding sequence.

[0204] Figure 21 The diagram shows the ORF2p functional initiation portion being connected downstream of the basic structure of the RNA framework, forming a circular RNA form.

[0205] In the following embodiments, since the material used is human cells, the short sporadic element used is Alu Ya5, a short sporadic element specific to primates. The complete sequence of the Alu Ya5 element is shown in Seq ID No. 1, and a partial Alu Ya5 sequence is shown in Seq ID No. 2. When using materials from other species, the short sporadic element can be replaced with a short sporadic element of the corresponding species to increase gene editing efficiency.

[0206] Material

[0207] 1. pBudORF1-CH plasmid was purchased from Addgene, plasmid number: 51290; pBudORF2-CH plasmid was purchased from Addgene, plasmid number: 51289; pBS-L1PA1-CH-mneo plasmid vector was purchased from Addgene, product number: 51288; pBudORF1-CH plasmid, pBudORF2-CH plasmid, and pBS-L1PA1-CH-mneo plasmid were amplified by Beijing Hesheng Biotechnology Co., Ltd. after purchase.

[0208] 2. CD293 culture medium was purchased from Thermo Fisher Scientific, product number: 11913019.

[0209] 3. PEI transfection reagent was purchased from Serochem, product number: Prime-AQ100-100ML.

[0210] 4. SMS 293-SUPI was purchased from Sino Biological Inc., product number: M293-SUPI-100.

[0211] 5. Potassium acetate was purchased from Sigma-Aldrich, product number: P1190.

[0212] 6. Tris-HCl (pH 7.5) was purchased from Shanghai Shangbao Biotechnology Co., Ltd., product number: T16588.

[0213] 7. Glycerin was purchased from Sigma-Aldrich, product number: G5516.

[0214] 8. The Triton X-100 was purchased from Sigma-Aldrich, product number: T8787.

[0215] 9. The PMSF protease inhibitor was purchased from Thermo Fisher Scientific, product number: 36978.

[0216] 10. Ni affinity chromatography column (HISTRAP HP) was purchased from Cytiva.

[0217] 11. Imidazole was purchased from Sigma-Aldrich, product number: I5513

[0218] 11. Rabbit anti-his was purchased from Sigma-Aldrich, product number: SAB1306082.

[0219] 12. BSA was purchased from Sigma-Aldrich, product number: A1933.

[0220] 13. Anti-rabbit IgG (full molecular weight)-alkaline phosphatase goat anti-anti-rabbit was purchased from Sigma-Aldrich, product number: A3687.

[0221] 14.pcDNA TM 3.1(+) Purchased from Invitrogen, product number: V79020.

[0222] 15. NheI was purchased from ThermoFisher. The 10× enzyme digestion buffer formulation was: 330mM Tris-acetate, 100mM magnesium acetate, 660mM potassium acetate, and 1mg / mL BSA.

[0223] 16. T4 DNA ligase and the 10× ligation buffer required for its application were purchased from Promega.

[0224] 17. MEGAscript TM The T7 Transcription Kit was purchased from Thermo Fisher Scientific, product number AM1333.

[0225] 18. Opti-MEM TM I. Culture medium was purchased from Thermo Fisher Scientific, product number: A4124802.

[0226] 19. The RNase inhibitor was purchased from Thermo Fisher Scientific, product number: AM2694.

[0227] 20. RNAiMAX transfection reagent was purchased from Thermo Fisher Scientific, product number: 13778030.

[0228] 21.pcDNA3.1(+)eGFP was purchased from Addgene, product number: 129020.

[0229] 22. KOD One TM The PCR Master Mix was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., product number: KMM-201S.

[0230] 23. One-step rapid cloning kit (Hieff) The Plus One Step Cloning Kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product number: 10911ES20.

[0231] 24. The complete culture medium was prepared from 90% DMEM medium and 10% fetal bovine serum. The DMEM medium was purchased from Thermo Fisher Scientific, product number: 11965092, and the fetal bovine serum was purchased from Thermo Fisher Scientific, product number: 10100147.

[0232] 25. Entranster-H4000 transfection reagent was purchased from Beijing Engen Biotechnology Co., Ltd.

[0233] 26. The blood / cell / tissue genomic DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd., product catalog number: DP304.

[0234] 27. MEGAscript TMThe SP6 transcription kit was purchased from Thermo Fisher Scientific, product number: AM1330.

[0235] 28. SuperReal PreMix Plus (SYBR Green) was purchased from Tiangen Biotech (Beijing) Co., Ltd., product catalog number: FP205.

[0236] 29. The chemical synthesis of primers and sequences was carried out by Platinum Biotech (Shanghai) Co., Ltd. or Aibosen (Jiangsu) Biotechnology Co., Ltd.

[0237] Example 1: Preparation of ORF1p and ORF2p

[0238] 1. Preparation of ORF1p from human LINE-1 (LRE1)

[0239] The human LINE-1 (LRE1) ORF1p (hLRE1-ORF1p) has a commercially available plasmid pBudORF1-CH for expression. Therefore, hLRE1-ORF1p can be obtained by directly expressing it using the pBudORF1-CH plasmid.

[0240] 1) Transfect pBudORF1-CH plasmid and express

[0241] HEK293 cells were cultured and passaged in CD293 medium, and then the pBudORF1-CH plasmid was transfected into HEK293 cells according to the PEI transfection reagent instructions. SMS 293-SUPI feed solution was added to HEK293 cells on days 1, 3, and 5 post-transfection, according to the instructions. HEK293 cells were cultured in shake flasks under the following conditions: 5% CO2, 37°C, and 175 rpm. Reactor culture conditions were: pH 7.2, 37°C, 150 rpm, and 40% dissolved oxygen. HEK293 cells were incubated in a shaker incubator, and centrifuged at 3000g for 5 min 7 days after transfection to harvest the cells. The added SMS 293-SUPI feed solution promoted cell survival and increased protein production.

[0242] 2) Extraction of hLRE1-ORF1p

[0243] Prepare and pre-cool the cell lysis buffer (100 mM potassium acetate, 50 mM Tris-HCl (pH 7.5), 5% glycerol, 0.3% Triton X-100), and add the protease inhibitor PMSF to a concentration of 1 mM in the buffer before use. Add the cell lysis buffer to the cells at a rate of 40 ml per liter of cells (RIPA lysis buffer or other prepared cell lysis buffers can also be used). Afterward, thoroughly lyse the cells by pipetting, avoiding bubbling and vortexing. Then, continue processing the cells using a glass homogenizer, followed by three cycles of sonication (15 s operation, 15 s interval). Centrifuge at 30000 g at 4°C for 25 min, retain the supernatant, and filter through a 0.22 μm filter membrane. Purify the protein using a Ni affinity chromatography column (HISTRAP HP).

[0244] The protein purification steps are as follows:

[0245] 1. Rinse the Ni affinity chromatography column with 5 times the column volume of deionized water;

[0246] 2. Prepare a PBS buffer solution with a pH of 7.4, and equilibrate the Ni affinity chromatography column with 5–10 column volumes of PBS buffer;

[0247] 3. The prepared supernatant was flowed through a Ni affinity chromatography column at a rate of 0.5 ml / min;

[0248] 4. Re-equilibrate the Ni affinity chromatography column using the prepared buffer solution;

[0249] 5. Prepare solutions containing 20 mM imidazole, 50 mM imidazole, 100 mM imidazole, 250 mM imidazole and 500 mM imidazole respectively using 0.5 M NaCl;

[0250] 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.

[0251] The collected eluted samples were dialyzed overnight at 4°C using prepared PBS solution as buffer. Finally, the dialyzed samples were concentrated by ultrafiltration (using a suitable ultrafiltration tube), and the resulting target protein was detected by SDS-PAGE (primary antibody: rabbit anti-his 1:1500 (5% Milk + 0.1% BSA); secondary antibody: goat anti-rabbit IgG alkaline phosphatase 1:6000 (5% Milk)) to confirm the protein concentration. hLRE1-ORF1p was then extracted, purified, and lyophilized.

[0252] 2. Preparation of ORF2p in human LINE-1 (LRE1)

[0253] The human LINE-1 (LRE1) ORF2p (hLRE1-ORF2p) has a commercially available plasmid pBudORF2-CH for expression. Therefore, hLRE1-ORF2p can be obtained by directly expressing it using the pBudORF2-CH plasmid.

[0254] The plasmid pBudORF1-CH expressing hLRE1-ORF1p was replaced with the plasmid pBudORF2-CH expressing hLRE1-ORF2p, and purified lyophilized hLRE1-ORF2p was prepared according to the preparation method of hLRE1-ORF1p.

[0255] 3. Preparation of ORF1p in human LINE-1 (LRE2)

[0256] There is no commercially available plasmid for directly expressing ORF1p (hLRE2-ORF1p) in human LINE-1 (LRE2), so an expressible plasmid was constructed for expression.

[0257]

[0258] The taa terminus of the ORF1p sequence in human LINE-1 (LRE2) was removed, and then the coding sequences for Myc and His tags (tiled) were added. Next, tga (italicized and bold) was added, and finally, NheI restriction sites and protective bases (CTAGCTAGCTAG) were added to both ends of the sequence. The modified nucleotide sequence is shown in Seq ID No. 4.

[0259]

[0260] The nucleotide sequence of Seq ID No. 4 was obtained through chemical synthesis, followed by NheI digestion to obtain the sequence to be inserted. Simultaneously, the pcDNA was... TM 3.1 The (+) plasmid was also digested with NheI to obtain a linear plasmid; then the insert sequence and the linear plasmid were recovered by electrophoresis.

[0261] The specific enzyme digestion reaction system is shown in Table 1:

[0262] Table 1 Enzyme digestion reaction system

[0263]

[0264] The enzyme digestion reaction conditions were: incubation at 37°C for 3 hours, followed by heating to 80°C for 10 minutes to inactivate the endonuclease.

[0265] The digested and recovered sequence to be inserted was ligated with the LINE-1 plasmid, electrophoresed, and recovered to obtain the plasmid pcDNA for expressing ORF1p in human LINE-1(LRE2). TM 3.1(+)-hLRE2-ORF1p.

[0266] The specific connection reaction system is shown in Table 2:

[0267] Table 2 Connection Reaction System

[0268]

[0269] The ligation reaction conditions were: incubation at 16°C for 16 hours, followed by incubation at 70°C for 10 minutes to inactivate the ligase.

[0270] Preparation of ORF1 protein (hLRE2-ORF1p) from human LINE-1 (LRE2): Following the preparation method of hLRE1-ORF1p, the plasmid pBudORF1-CH was replaced with pcDNA. TM 3.1(+)-hLRE2-ORF1p was transfected, expressed, purified and lyophilized to obtain hLRE2-ORF1p.

[0271] 4. Preparation of ORF2p in human LINE-1 (LRE2)

[0272] There is no commercially available plasmid for directly expressing ORF2p (hLRE2-ORF2p) in human LINE-1 (LRE2), so an expressible plasmid was constructed for expression.

[0273] The nucleotide sequence encoding ORF2p in human LINE-1 (LRE2) is shown in Seq ID No. 5. hLRE2-ORF2p was prepared, expressed, purified and lyophilized according to the preparation method of ORF1p in human LINE-1 (LRE2).

[0274] 5. Preparation of mouse ORF1p

[0275] The DNA sequence encoding mouse ORF1p is shown in Seq ID No. 6. The lyophilized mouse ORF1p was prepared, expressed, and purified according to the preparation method of ORF1p in human LINE-1 (LRE2), and named mORF1p.

[0276] 6. Preparation of mouse ORF2p

[0277] The DNA sequence encoding mouse ORF2p is shown in Seq ID No. 7. The freeze-dried mouse ORF2p was prepared, expressed, and purified according to the preparation method of ORF1p in human LINE-1 (LRE2), and named mORF2p.

[0278] Example 2 examines the effect of gene editing after RNA is generated by in vitro transcription and then transferred into the target system with / without binding to ORF1p and / or ORF2p outside the target system.

[0279] The Lman1 gene is the pathogenic gene for combined deficiency of coagulation factors V and VIII (F5F8D). Mutations in this gene can lead to decreased levels of FV and FVIII in humans, and patients may exhibit spontaneous bleeding symptoms.

[0280] A 405bp sequence from the Lman1 gene in the human genome was selected, and its sequence is shown in Seq ID No. 8 below:

[0281] GGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTCCTGAATTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT*CCTCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGA TTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG; where * represents the selected insertion site (target site). The sequence preceding the insertion site is the upstream sequence of the Lman1 gene (upstream of the target site), and the sequence following the insertion site is the downstream sequence of the Lman1 gene (downstream of the target site). A foreign sequence is inserted at the * position; this foreign sequence is the sequence to be inserted, and its sequence is shown in Seq ID No. 9:

[0282] AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCGGAGG.

[0283] The sequence after insertion is shown as Seq ID No. 10:

[0284] GGGTAGAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTT AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACCTGCTCAGGG GTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGCGCAGGAGCG GAGGCCTCCCCCTTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAAAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where the underlined part is the sequence to be inserted as shown in Seq ID No. 9.

[0285] Upstream of the sequence shown in Seq ID No. 10, add the T7 promoter sequence shown in Seq ID No. 11 (Seq ID No. 11: TAATACGACTCACTATA), and downstream of the sequence shown in Seq ID No. 2, add a portion of the Alu sequence shown in Seq ID No. 12.

[0286] The underlined part is the sequence to be inserted as shown in Seq ID No. 9, the italicized and bold part is the T7 promoter sequence as shown in Seq ID No. 11, and the wavy part is the partial Alu sequence as shown in Seq ID No. 2. This sequence was obtained through chemical synthesis and named RNA+partial Alu precursor DNA.

[0287] Add a T7 promoter sequence, as shown in Seq ID No. 11, upstream of the sequence shown in Seq ID No. 10. The result is shown in Seq ID No. 13.

[0288] The underlined part is the sequence to be inserted, as shown in Seq ID No. 9, and the italicized and bold part is the T7 promoter sequence, as shown in Seq ID No. 11. This sequence was obtained through chemical synthesis and named RNA precursor DNA.

[0289] According to the MEGAscript of the reagent kit TMThe T7 Transcription Kit instructions state that linear RNA + Alu precursor DNA or RNA precursor DNA is transcribed to obtain the corresponding RNA. Then, the residual DNA is degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration is measured with a UV spectrophotometer, and RNase-free water is added to adjust the concentration to 100 ng / μL of RNA + Alu solution or RNA solution.

[0290] The RNA+ portion Alu obtained from the above transcription belongs to Figure 4 The RNA framework structure contains the ORF2p functional initiation region, which consists of short, scattered RNA elements. The RNA transcribed above belongs to... Figure 3 RNA framework structure.

[0291] The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in Opti-MEM solution with 1 U / μL of RNase inhibitor added beforehand to form hLRE1-ORF1p solution or hLRE1-ORF2p solution with a concentration of 500 ng / μL.

[0292] Prepare RNPs that bind RNA or RNA+ Alu to ORF1p and / or ORF2p.

[0293] Among them, the RNPs that bind to hLRE1-ORF1p and hLRE1-ORF2p respectively in the RNA+ Alu region are called RNA+ Alu+hLRE1-ORF1p and RNA+ Alu+hLRE1-ORF2p, respectively. The reaction system is shown in Table 3.

[0294] Table 3 Reaction System

[0295]

[0296]

[0297] Because the amount of RNA bound to hL1-ORF1p solution and hL1-ORF2p solution is different, the amount added is also different.

[0298] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain RNA+ Alu+hLRE1-ORF1p and RNA+ Alu+hLRE1-ORF2p, respectively.

[0299] The preparation of RNA+hLRE1-ORF1p+hLRE1-ORF2p and RNA+partial Alu+hLRE1-ORF1p+hLRE1-ORF2p shall be carried out according to the following steps:

[0300] Configure the reaction system as shown in Table 4:

[0301] Table 4 Reaction System

[0302]

[0303] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain RNA+hLRE1-ORF2p solution and RNA+partial Alu+hLRE1-ORF2p solution, respectively.

[0304] Then, the RNA+hLRE1-ORF2p solution and the RNA+partial Alu+hLRE1-ORF2p solution were mixed according to the systems shown in Table 5.

[0305] Table 5 Reaction System

[0306]

[0307] After gently mixing the components, the reaction system was incubated at room temperature (25℃) for 10 min to obtain RNA+hLRE1-ORF1p+hLRE1-ORF2p solution and RNA+partial Alu+hLRE1-ORF1p+hLRE1-ORF2p solution, respectively.

[0308] A solution containing only hLRE1-ORF1p and hLRE1-ORF2p, without RNA or with RNA+Alu, was prepared as a negative control. The reaction system is shown in Table 6.

[0309] Table 6 Reaction System

[0310]

[0311]

[0312] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain the hLRE1-ORF1p+hLRE1-ORF2p solution.

[0313] Prepare the transfection solutions as shown in Table 7:

[0314] Table 7 Transfection Solution System

[0315]

[0316] The transfection solutions were added in equal volume ratios to hLRE1-ORF1p+hLRE1-ORF2p solution, RNA+ Alu+hLRE1-ORF1p solution, RNA+ Alu+hLRE1-ORF2p solution, RNA+hLRE1-ORF1p+hLRE1-ORF2p solution, and RNA+ Alu+hLRE1-ORF1p+hLRE1-ORF2p solution, respectively. After gentle mixing, the solutions were incubated at room temperature (25℃) for 20 min to obtain the corresponding transfection solutions. The liposomes in the transfection solution will form complexes with RNA, RNPs or proteins in the solution, namely the hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, the RNA+partial Alu+hLRE1-ORF1p-liposome complex, the RNA+partial Alu+hLRE1-ORF2p-liposome complex, the RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, and the RNA+partial Alu+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex.

[0317] Transfection solutions of RNA + partial Alu liposome complexes without hLRE1-ORF1p or hLRE1-ORF2p were prepared. The reaction system is shown in Table 8.

[0318] Table 8 Reaction System

[0319]

[0320] After gently mixing all components, the reaction system was incubated at room temperature (25°C) for 10 min. Then, the transfection solutions shown in Table 7 were added in equal volume ratios, gently mixed, and incubated at room temperature (25°C) for 20 min to obtain the RNA+ Alu-liposome complex.

[0321] Construction of plasmid pcDNA3.1(+)eGFP+RNA+partial Alu by direct transfection

[0322] By concatenating the sequence shown in Seq ID No. 2 after the sequence shown in Seq ID No. 10, we obtain the sequence shown in Seq ID No. 14:

[0323]

[0324] The underlined part is the sequence to be inserted as shown in Seq ID No. 9; the wavy line is the partial Alu sequence as shown in Seq ID No. 2. This sequence is named RNA+ partial Alu and was obtained through chemical synthesis.

[0325] The sequence shown in Seq ID No. 14 was chemically synthesized and constructed into the pcDNA3.1(+)eGFP vector through reverse circular expansion and homologous ligation, so that the sequence was directly linked downstream of the CMV promoter in the vector, and there were no other sequences between the sequence and the CMV promoter. The vector was named plasmid pcDNA3.1(+)eGFP+RNA+partial Alu.

[0326] The specific steps are as follows:

[0327] 1. Design primers to amplify the sequence shown in Seq ID No. 14. The forward primer sequence is shown in Seq ID No. 15: 5'-CTATATAAGCAGAGCTGGGTAGAGATTCACTG-3', and the reverse primer sequence is shown in Seq ID No. 16: 5'-CTCTAGTTAGCCAGAGGATCTCCAGCAGTTAT-3'. Perform PCR amplification on the sequence shown in Seq ID No. 14. The reaction system is shown in Table 9.

[0328] Table 9 Reaction System

[0329]

[0330]

[0331] The amplification conditions were: 94℃ for 2 min; (98℃ for 10 sec, 60℃ for 10 sec, 68℃ for 2 sec) for 40 cycles; 68℃ for 5 min.

[0332] The amplified product was obtained by gel recovery and purification using conventional methods. The amplified product had sequences homologous to the pcDNA3.1(+)eGFP vector added to both sides of the synthesized sequence.

[0333] 2. Design PCR primers for amplifying the pcDNA3.1(+)eGFP vector. The forward primer is shown in Seq ID No. 17: 5'-AATAACTGCTGGAGATCCTCTGGCTAACTAGAG-3', and the reverse primer sequence is shown in Seq ID No. 18: 5'-CAGTGAATCTCTACCCAGCTCTGCTTATATAG-3'. PCR amplification of the pcDNA3.1(+)eGFP vector was performed, and the reaction system is shown in Table 10.

[0334] Table 10 Reaction System

[0335]

[0336] The amplification conditions were: 94℃ for 2 min; (98℃ for 10 sec, 60℃ for 10 sec, 68℃ for 6 sec) for 40 cycles; 68℃ for 5 min.

[0337] The pcDNA3.1(+)eGFP plasmid vector was obtained by gel recovery and purification using conventional methods. The plasmid vector has sequences homologous to the synthesized sequence at both ends.

[0338] 3. The amplified product and the amplified pcDNA3.1(+)eGFP vector were ligated using a one-step rapid cloning kit. The specific steps were performed according to the kit instructions. The reaction system is shown in Table 11.

[0339] Table 11 Connection Reaction System

[0340]

[0341] 4. The recombinant product was transformed into competent cells (DH5α), and bacteria were picked and sequenced on transformed plates. After the sequencing was confirmed to be correct, the plasmid was extracted to obtain plasmid pCDNA3.1(+)eGFP-RNA+partial Alu.

[0342] transfection

[0343] First, HeLa cells were passaged and seeded into 24-well plates using complete culture medium. The day after passage, when the HeLa cells reached 60% confluence, the medium was replaced with Opti-MEM. TM In culture medium I, following the RNAiMAX transfection reagent instructions, 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, and RNA+partial Alu-liposome complex were added to HeLa cells for transfection, with three replicates for each treatment. Six hours after transfection, the culture medium was replaced with complete culture medium. Cells were cultured until approximately 90% confluence was reached, then passaged. After passage, when cells reached approximately 60% confluence, transfection was repeated once more. Once cells again reached approximately 90% confluence, subsequent procedures were performed.

[0344] For cells transfected with the hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, after the cells reached approximately 90% confluence, they were passaged again. After passage, when the cells reached approximately 60% confluence, a portion of the complete culture medium was aspirated down to 0.5 ml, and the control plasmid (pBS-L1PA1-CH-mneo) was transfected. Entranster-H4000 transfection reagent was used for transfection. For each plate of cells, 19.2 μg of plasmid pBS-L1PA1-CH-mneo was used. The desired plasmid was diluted with 600 μL of serum-free DMEM solution and mixed thoroughly. Simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM solution and mixed thoroughly, then incubated at room temperature for 5 min. The two prepared liquids were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. The transfection complex was added to 24-well plates containing HeLa cells that had been transfected with the hLRE1-ORF1p+hLRE1-ORF2p-liposome complex and contained 0.5 ml of culture medium per well. Cells were passaged when they reached approximately 90% confluence. This process was repeated until the cells again reached approximately 90% confluence, at which point further steps were performed. These cells served as a negative control.

[0345] Transfection of plasmids

[0346] The constructed plasmid pcDNA3.1(+)eGFP+RNA+ Alu was co-transfected with plasmid pBS-L1PA1-CH-mneo expressing ORF1p and ORF2p(LINE-1) into HeLa cells. Each group was divided into 3 replicates, and each replicate was a 24-well plate cultured with HeLa cells.

[0347] The transfection procedure was as follows: HeLa cells were passaged and seeded into 24-well plates. The day after passage, transfection was performed using Entranster-H4000 transfection reagent. Two plasmids were transfected per plate, with 19.2 μg of each plasmid, for a total of 38.4 μg. The desired plasmid was diluted with 600 μL of serum-free DMEM and thoroughly mixed. Simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM and thoroughly mixed, then incubated at room temperature for 5 min. The two prepared liquids were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. The transfection complex was added to each well of a 24-well plate containing 0.5 ml of Opti-MEM medium for transfection. Cells were passaged when they reached approximately 90% confluence. After passage, the above steps were repeated when cells reached approximately 60% confluence, and subsequent procedures were performed when cells reached approximately 90% confluence.

[0348] Experimental Groups:

[0349] The study was divided into seven groups: the control group was transfected with the hLRE1-ORF1p+hLRE1-ORF2p-liposome complex and the plasmid pBS-L1PA1-CH-mneo; the experimental group was directly transfected with the plasmid; the experimental group was transfected with RNA+hLRE1-ORF1p+hLRE1-ORF2p; the experimental group was transfected with RNA+partial Alu; the experimental group was transfected with RNA+partial Alu+hLRE1-ORF1p; the experimental group was transfected with RNA+partial Alu+hLRE1-ORF2p; and the experimental group was transfected with RNA+partial Alu+hLRE1-ORF1p+hLRE1-ORF2p. Each group had three replicates, and each replicate was a 24-well plate cultured with HeLa cells.

[0350] DNA was extracted from transfected cells in each group: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0351] qPCR detection:

[0352] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0353] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19: 5′–CACTGCCACCCAGAAGACTG-3′; the downstream primer sequence is shown in Seq ID No. 20: 5′-CCTGCTTCACCACCTTCTTG-3′.

[0354] Primer pair 1 was designed with the upstream primer sequence shown in Seq ID No. 21: 5′-GACTTATCCATGTGCCTGTT-3′; and the downstream primer sequence shown in Seq ID No. 22: 5′-TTGGCTACGAAATGCTTG-3′. The upstream primer sequence of primer pair 1 is located in the complete Lman1 gene, further upstream of the insertion site (target site) upstream sequence used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 1 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0355] All of the above primers were obtained through chemical synthesis.

[0356] The qPCR reaction system is shown in Table 12.

[0357] Table 12 qPCR reaction system

[0358]

[0359]

[0360] The cellular DNA templates were extracted from the transfected cells in the above seven groups.

[0361] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0362] qPCR reaction cycle:

[0363] Primer pair 1: Pre-denaturation at 95℃ for 15 min; (denaturation at 95℃ for 10 s, annealing at 49℃ for 20 s, extension at 72℃ for 20 s) for 40 cycles. GAPDH primers were reacted under the same conditions.

[0364] After observing the exponential growth phase in the GAPDH and the amplification curves for detecting the inserted sequence, and confirming that they are approximately parallel, the process is repeated using 2... -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 13. The PCR products were verified to be correct by sequencing.

[0365] Table 13 Results for primer pair 1 (n=3, )

[0366]

[0367]

[0368] As shown in Table 10, compared with the control group (N / A calculated as 40.00), the relative copy number of Experiment 1 was significantly higher than that of the control group, which was statistically significant (P < 0.05). Therefore, by giving the plasmid (DNA) containing the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site to the receiving system, the sequence to be inserted can be inserted into the genomic target site (Experiment 1). Compared with Experiment 1, the relative copy number of Experiment 6 was significantly higher than that of Experiment 1, which was statistically significant (P < 0.05). This indicates that due to the RNA splicing mechanism in eukaryotes (intracellular), the efficiency of plasmid transcription to produce gene-editing RNA is reduced. Therefore, the efficiency of direct plasmid transfection (Experiment 1) is lower than that of directly introducing RNA or RNP containing the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site into the receiving system under similar conditions (Experiment 6). Furthermore, compared with the control group (N / A calculated as 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). This indicates that RNA containing only the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site, or RNPs bound to ORF1p and / or ORF2p (experimental group 2), can also exert gene editing effects, but the effect is weaker. This shows that RNA containing only the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site, or RNPs bound to ORF1p and / or ORF2p, can also achieve the purpose of gene editing. Compared with the control group (N / A calculated at 40.00), the relative copy number of experimental group 3 was significantly higher (P < 0.05), indicating that even without binding to ORF1p and / or ORF2p, adding a portion of Alu (experimental group 3) in addition to the upstream, insert, and downstream sequences of the target site can still achieve gene editing. Furthermore, the gene editing effect (relative copy number compared to the control group) was significantly higher than that of experimental group 2, suggesting that the addition of a portion of Alu can improve gene editing efficiency. Compared with the control group (N / A calculated at 40.00), the relative copy numbers of experimental groups 4-6 were all significantly higher (P < 0.05), indicating that binding ORF1p, ORF2p, or both ORF1p and ORF2p to RNA containing the upstream, insert, and downstream sequences of the target site, as well as a portion of Alu, can produce gene editing effects. Furthermore, the results showed that the gene editing effect of experimental groups 4-6 gradually improved, indicating that the effect of combining ORF2p on improving gene editing efficiency was better than that of ORF1p, and the combination of ORF2p and ORF1p was better than the combination of ORF1p or ORF2p alone.

[0369] The gene editing efficiency of in vitro prokaryotic RNA generation followed by transfection and direct DNA transfection were compared.

[0370] Table 14 Results for primer pair 1 (n=3, )

[0371]

[0372] As shown in Table 14, compared with Experiment 1, the relative copy number of Experiment 6 was significantly higher in Experiment 6 than in Experiment 1, which was statistically significant (P < 0.05). This indicates that gene editing by generating specific RNA or RNPs in vitro through prokaryotic promoters or other methods and then introducing them into the receiving system is, in some cases, more efficient than gene editing by generating RNA in the receiving system after introducing DNA. This may be because generating specific RNA or RNPs in vitro avoids the cleavage or splicing of the RNA produced by the eukaryotic system. This reflects that generating specific RNA or RNPs in vitro and then introducing them into the receiving system, such as cells, tissues, organs, or organisms, has advantages in certain situations compared to directly introducing the corresponding DNA into the receiving system.

[0373] Example 3: Detecting the efficiency of RNA generated from in vitro transcription, which binds to ORF1p and ORF2p and performs gene editing within the target system.

[0374] The plasmid pBS-L1PA1-CH-mneo was selected for expressing ORF1p and ORF2p in vivo. This plasmid contains codon-optimized human L1RP ORF1 and ORF2, and can express hLRE1-ORF1 and hLRE1-ORF2 in cells.

[0375] pBS-L1PA1-CH-mneo plasmid transfection

[0376] First, HeLa cells were passaged and seeded in 24-well plates. The day after passage, when the cells reached 60% confluence, they were transfected using Entranster-H4000 transfection reagent. Each plate of cells was transfected with 19.2 μg of pBS-L1PA1-CH-mneo plasmid. The pBS-L1PA1-CH-mneo plasmid was diluted with 600 μL of serum-free DMEM and mixed thoroughly. Simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM and mixed thoroughly, then incubated at room temperature for 5 min. The two solutions were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. The transfection complex was added to each well of a 24-well plate containing 0.5 ml of Opti-MEM medium and cultured with HeLa cells for transfection. When the cells reached approximately 90% confluence, they were passaged. After passage, HeLa cells were seeded into 24-well plates. The day after passage, when the cells reached 60% confluence, further RNA transfection was performed. Three replicates were set up, each consisting of one 24-well plate containing HeLa cells transfected with the pBS-L1PA1-CH-mneo plasmid.

[0377] RNA transfection

[0378] The RNA+ Alu solution prepared in Example 2 was mixed according to the system in Table 8, and then added to the transfection solution system in Table 7 in an equal volume ratio. After gentle mixing, it was incubated at room temperature (25°C) for 20 min to obtain the RNA+ Alu-liposome complex.

[0379] HeLa cells transfected with pBS-L1PA1-CH-mneo plasmid were cultured to 60% confluence, and then replaced with Opti-MEM. TM In culture medium I, RNA+ Alu-liposome complex was added to cells according to the RNAiMAX transfection reagent instructions for transfection, with three replicates per cell. Cells were cultured until approximately 90% confluence was reached, then passaged. Transfection was repeated once more, and cells were allowed to confluence again to approximately 90% confluence. Cell DNA was then extracted for further processing. This group served as the experimental group.

[0380] Using the same method, the RNA+ portion of the Alu-liposome complex was transfected into HeLa cells that were not transfected with the pBS-L1PA1-CH-mneo plasmid as a control group, and three replicates were also set up.

[0381] DNA was extracted from transfected cells in both the experimental and control groups: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cellular DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0382] qPCR detection:

[0383] The GAPDH gene was used as an internal reference gene. The upstream primer sequence is shown in Seq ID No. 19, and the downstream primer sequence is shown in Seq ID No. 20. For primer pair 1, the upstream primer sequence is shown in Seq ID No. 21, and the downstream primer sequence is shown in Seq ID No. 18. qPCR detection was performed.

[0384] The qPCR reaction system is shown in Table 9.

[0385] The cellular DNA templates used were extracted from transfected cells in the control or experimental groups.

[0386] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0387] qPCR reaction cycle:

[0388] Primer pair 1: Pre-denaturation at 95℃ for 15 min; (denaturation at 95℃ for 10 s, annealing at 49℃ for 20 s, extension at 72℃ for 20 s) for 40 cycles. GAPDH primers were reacted under the same conditions.

[0389] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 15. The PCR products were verified to be correct by sequencing.

[0390] Table 15 Results for primer pair 1 (n=3, )

[0391]

[0392] Table 15 shows that, compared with the control group, the relative copy number of the experimental group was significantly higher, which was statistically significant (P < 0.05). Compared with the administration of specific RNA alone, the gene editing efficiency was higher when ORF1p and / or ORF2p were generated in the target system in combination with the administration of specific RNA. This indicates that generating ORF1p and / or ORF2p in the target system can assist the gene editing function of the transferred specific RNA and improve the gene editing efficiency.

[0393] Example 4: Detecting the efficiency of gene editing after in vitro transcription of specific RNA (with intact Alu at the 3' region), binding / not binding ORF1p and ORF2p outside the target system, and then transfecting it into the target system.

[0394] The GALT gene encodes galactose-1-phosphate uridine transferase, and mutations in it can lead to human type I galactosemia.

[0395] A 360bp sequence from the GALT gene was selected, as shown in Seq ID No. 23: GGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTG*ATGCATGGGCCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCC CTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCACAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG; where * represents the selected insertion site (target site). The sequence preceding the insertion site is the upstream sequence of the insertion site in the GALT gene (upstream sequence of the target site), and the sequence following the insertion site is the downstream sequence of the insertion site in the GALT gene (downstream sequence of the target site). A foreign sequence is inserted at the * position; this foreign sequence is the sequence to be inserted, and its sequence is shown in Seq ID No. 24.

[0396] TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAACCTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA

[0397] The sequence after insertion is shown in Seq ID No. 25:

[0398] GGGGTTCGGCCCTGCCCGTAGCACAGCCAAGCCCTACCTCTCGGTTATTCTTTTCTCCCGTCACCACCCAGTAAGGTCATGTGCTTCCACCCCTGGTCGGATGTAACGCTGCCACTCATGTCGGTCCCTGAGATCCGGGCTGTTGTTG TGACTACTGAGATTACTTTGACATGTCCCACTTATTAATATCACCTTAAGTTTGGGTTCGATTAATATTATGTAAC CTGTGAACGAGATAAGATTCTAGAGATTTAATCGAACCTTAATTCTGATTCGGTTATGTCAAAAGGTGTCTTGA ATGCATGGGCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG

[0399] The underlined part is the sequence to be inserted, as shown in Seq ID No. 24.

[0400] Add the T7 promoter sequence shown in Seq ID No. 11 upstream of the sequence shown in Seq ID No. 25, and add the Alu sequence shown in Seq ID No. 1 downstream. The result is shown in Seq ID No. 26. The underlined part is the sequence to be inserted, as shown in Seq ID No. 24; the italicized and bold part is the T7 promoter sequence, as shown in Seq ID No. 11; and the wavy part is the Alu sequence, as shown in Seq ID No. 1. This sequence was obtained through chemical synthesis and named RNA+Alu precursor DNA.

[0401] Add a T7 promoter sequence, as shown in Seq ID No. 11, upstream of the sequence shown in Seq ID No. 25. The result is shown in Seq ID No. 27.

[0402]

[0403] The underlined part is the sequence to be inserted, as shown in Seq ID No. 24, and the italicized and bold part is the T7 promoter sequence, as shown in Seq ID No. 11. This sequence was obtained through chemical synthesis and named RNA precursor DNA.

[0404] According to the MEGAscript kit TM The T7 Transcription Kit instructions state that linear RNA+Alu precursor DNA or RNA precursor DNA is transcribed to obtain the corresponding RNA. Then, the residual DNA is degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration is measured with a UV spectrophotometer, and RNase-free water is added to adjust the concentration to 100 ng / μL of RNA+Alu solution or RNA solution.

[0405] The RNA+Alu obtained from the above transcription belongs to Figure 4 The RNA framework structure contains the ORF2p functional initiation region, which is a (complete) short, scattered element RNA. The RNA transcribed above belongs to... Figure 3 RNA framework structure.

[0406] The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in Opti-MEM solution with 1 U / μL of RNase inhibitor added beforehand to form hL1-ORF1p solution or hL1-ORF2p solution with a concentration of 500 ng / μL.

[0407] To prepare RNPs that bind RNA or RNA+Alu to ORF1p and ORF2p: RNA+hLRE1-ORF1p+hLRE1-ORF2p, RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p, follow these steps.

[0408] Configure the reaction system as shown in Table 16:

[0409] Table 16 Reaction System

[0410]

[0411] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain RNA+hLRE1-ORF1p solution and RNA+Alu+hLRE1-ORF1p solution, respectively.

[0412] Then, the RNA+hLRE1-ORF1p solution and the RNA+Alu+hLRE1-ORF1p solution were mixed according to the systems shown in Table 17.

[0413] Table 17 Reaction System

[0414]

[0415]

[0416] After gently mixing the components, the reaction system was incubated at room temperature (25℃) for 10 min to obtain RNA+hLRE1-ORF1p+hLRE1-ORF2p solution and RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p solution, respectively.

[0417] Mix the RNA+hLRE1-ORF1p+hLRE1-ORF2p solution or the RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p solution with the transfection solution system prepared in Table 7 in equal volume ratio, mix gently, and incubate at room temperature (25℃) for 20 min to obtain the RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex or the RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex.

[0418] The transfection solution of RNA + Alu liposome complex without hLRE1-ORF1p and hLRE1-ORF2p was prepared. The reaction system is shown in Table 18.

[0419] Table 18 Reaction System

[0420]

[0421] After gently mixing all components, the reaction system was incubated at room temperature (25°C) for 10 min. Then, the transfection solutions shown in Table 7 were added in equal volume ratios, and after gentle mixing, the mixture was incubated at room temperature (25°C) for 20 min to obtain the RNA+Alu-liposome complex.

[0422] Construction of plasmid pcDNA3.1(+)eGFP+RNA+Alu by direct transfection

[0423] By concatenating a sequence like Seq ID No. 1 after a sequence like Seq ID No. 25, we obtain a sequence like Seq ID No. 28:

[0424]

[0425] The underlined part is the insertion sequence as shown in Seq ID No. 24; the wavy line is the Alu sequence as shown in Seq ID No. 1.

[0426] The sequence shown in Seq ID No. 28 was chemically synthesized and constructed into the pcDNA3.1(+)eGFP vector via reverse circular expansion and homologous ligation. This resulted in the sequence being directly ligated downstream of the CMV promoter in the vector, with no other sequences between the sequence and the CMV promoter. The vector was named plasmid pcDNA3.1(+)eGFP+RNA+Alu.

[0427] The specific steps are as follows:

[0428] 1. Design primers to amplify the sequence shown in Seq ID No. 28. The forward primer sequence is shown in Seq ID No. 29: 5'-CTATATAAGCAGAGCTGGGGTTCGGCCCT-3', and the reverse primer sequence is shown in Seq ID No. 16: 5'-CTCTAGTTAGCCAGAGGATCTCCAGCAGTTAT-3'. Perform PCR amplification on the sequence shown in Seq ID No. 28. The reaction system is shown in Table 19.

[0429] Table 19 Reaction System

[0430]

[0431] The amplification conditions were: 94℃ for 2 min; (98℃ for 10 sec, 60℃ for 10 sec, 68℃ for 2 sec) for 40 cycles; 68℃ for 5 min.

[0432] The amplified product was obtained by gel recovery and purification using conventional methods. Sequences homologous to the pcDNA3.1(+)eGFP vector were added to both sides of the synthesized sequence.

[0433] 2. Design PCR primers for amplifying the pcDNA3.1(+)eGFP vector. The forward primer is shown in Seq ID No. 17: 5'-AATAACTGCTGGAGATCCTCTGGCTAACTAGAG-3', and the reverse primer sequence is shown in Seq ID No. 30: 5'-AGGGCCGAACCCCAGCTCTGCTTATATAG-3'. PCR amplification of the pcDNA3.1(+)eGFP vector was performed, and the reaction system is shown in Table 20.

[0434] Table 20 Reaction System

[0435]

[0436]

[0437] The amplification conditions were: 94℃ for 2 min; (98℃ for 10 sec, 60℃ for 10 sec, 68℃ for 6 sec) for 40 cycles; 68℃ for 5 min.

[0438] The pcDNA3.1(+)eGFP plasmid vector was obtained by gel recovery and purification using conventional methods. The plasmid vector has sequences homologous to the synthesized sequence at both ends.

[0439] 3. Use a one-step rapid cloning kit to ligate the amplified product and the amplified pcDNA3.1(+)eGFP vector. Follow the kit instructions for specific steps, and prepare the reaction system as shown in Table 11.

[0440] 4. The recombinant product was transformed into competent cells (DH5α), and bacteria were picked from the transformed plates and sequenced. After the sequencing was confirmed to be correct, the plasmid was extracted to obtain the plasmid pCDNA3.1(+)eGFP-RNA+Alu.

[0441] transfection

[0442] RNA vectors in vitro binding / non-binding ORF1p and ORF2p experimental groups:

[0443] First, human glioma cells U251 were passaged and seeded into 24-well plates using complete culture medium. The day after passage, when the U251 cells reached 60% confluence, the medium was replaced with Opti-MEM. TMIn culture medium I, following the RNAiMAX transfection reagent instructions, RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex, and RNA+Alu-liposome complex were added to U251 human glioma cells for transfection, with three replicates for each. Cells were cultured until approximately 90% confluence was reached, then passaged. After passage, transfection was repeated once more. Once the cells again reached approximately 90% confluence, subsequent procedures were performed.

[0444] Control group:

[0445] The hLRE1-ORF1p+hLRE1-ORF2p-liposome complex prepared in Example 2 was transfected into U251 cells using the method described above. After the cells reached approximately 90% confluence, they were passaged again. After passage, when the cells reached approximately 60% confluence, a portion of the complete culture medium was aspirated down to 0.5 ml, and the control plasmid (pBS-L1PA1-CH-mneo) was transfected. Entranster-H4000 transfection reagent was used for transfection. For each plate of cells, 19.2 μg of plasmid pBS-L1PA1-CH-mneo was used. The desired plasmid was diluted with 600 μL of serum-free DMEM solution and mixed thoroughly. Simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM solution and mixed thoroughly, then incubated at room temperature for 5 min. The two prepared liquids were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. The transfection complex was added to 24-well plates containing U251 glioma cells that had been transfected with the hLRE1-ORF1p+hLRE1-ORF2p-liposome complex and had 0.5 ml of culture medium per well. Cells were passaged when they reached approximately 90% confluence. This process was repeated until the cells reached approximately 90% confluence again, at which point further steps were performed.

[0446] Transfection of plasmid direct transfection groups:

[0447] The constructed plasmid pcDNA3.1(+)eGFP+RNA+Alu was co-transfected with plasmid pBS-L1PA1-CH-mneo expressing ORF1p and ORF2p into human glioma cells U251. Each group had 3 replicates, and each replicate was a 24-well plate containing human glioma cells U251.

[0448] The transfection procedure was as follows: Human glioma cells U251 were passaged and seeded into 24-well plates. On the second day after passage, when the cells reached approximately 60% confluence, transfection was performed using Entranster-H4000 transfection reagent. Two plasmids were transfected per plate, with 19.2 μg of each plasmid, for a total of 38.4 μg. The desired plasmid was diluted with 600 μL of serum-free DMEM and thoroughly mixed. Simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM and thoroughly mixed, then incubated at room temperature for 5 min. The two prepared liquids were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. The transfection complex was added to each well of a 24-well plate containing 0.5 ml of Opti-MEM medium and cultured with human glioma cells U251 for transfection. When the cells reach about 90% confluence, passage them. Repeat the above steps after passage. When the cells reach about 90% confluence, proceed with the next steps.

[0449] Experimental Groups:

[0450] The study was divided into five groups: the control group was transfected with hLRE1-ORF1p+hLRE1-ORF2p and plasmid pBS-L1PA1-CH-mneo; the experimental group was directly transfected with plasmid; the experimental group was transfected with RNA+hLRE1-ORF1p+hLRE1-ORF2p; the experimental group was transfected with RNA+Alu; and the experimental group was transfected with RNA+Alu+hLRE1-ORF1p+hLRE1-ORF2p. Each group had three replicates, and each replicate consisted of a 24-well plate containing human glioma cells U251.

[0451] DNA was extracted from transfected cells in each group: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0452] qPCR detection

[0453] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0454] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19; the downstream primer sequence is shown in Seq ID No. 20.

[0455] Primer pair 2 was designed with the upstream primer sequence shown in Seq ID No. 31: 5'-CCCCAGTACGATAGCACC-3'; and the downstream primer sequence shown in Seq ID No. 32: 5'-GACATAACCGAATCAGAATT-3'. The upstream primer sequence of primer pair 2 is located in the complete GALT gene, further upstream of the insertion site (target site) used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 2 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0456] All of the above primers were obtained through chemical synthesis.

[0457] The qPCR reaction system is shown in Table 21.

[0458] Table 21 qPCR reaction system

[0459]

[0460] The cellular DNA templates were extracted from the transfected cells in the above 5 groups.

[0461] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0462] qPCR reaction cycle:

[0463] Primer pair 2: pre-denaturation at 95℃ for 15 min; (denaturation at 95℃ for 10 s, annealing at 46℃ for 20 s, extension at 72℃ for 20 s) for 40 cycles. GAPDH primers were reacted under the same conditions.

[0464] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 22. The PCR products were verified to be correct by sequencing.

[0465] Table 22 Results for primer pair 2 (n=3, )

[0466]

[0467]

[0468] As shown in Table 22, compared with the control group (N / A calculated as 40.00), the relative copy number of Experiment 1 was significantly higher than that of the control group, which was statistically significant (P < 0.05). Therefore, by giving the plasmid (DNA) containing the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site to the receiving system, the sequence to be inserted can be inserted into the genomic target site (Experiment 1). Compared with Experiment 1, the relative copy number of Experiment 4 was significantly higher than that of Experiment 1, which was statistically significant (P < 0.05). This indicates that due to the RNA splicing mechanism in eukaryotes (intracellular), the efficiency of plasmid transcription to produce gene-editing RNA is reduced. Therefore, the efficiency of direct plasmid transfection (Experiment 1) is lower than that of directly introducing RNA or RNP containing the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site into the receiving system under similar conditions (Experiment 4). Furthermore, compared with the control group (N / A calculated as 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). This indicates that RNA containing only the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site, or RNPs bound to ORF1p and / or ORF2p (experimental group 2), can also exert gene editing effects, but the effect is weaker. This shows that RNA containing only the upstream sequence, the sequence to be inserted, and the downstream sequence of the target site, or RNPs bound to ORF1p and / or ORF2p, can also achieve the purpose of gene editing. Compared with the control group (N / A calculated as 40.00), the relative copy number of experimental group 3 was significantly higher (P < 0.05), indicating that even without binding ORF1p and / or ORF2p, adding complete Alu (experimental group 3) in addition to the upstream, insert, and downstream sequences of the target site can still achieve gene editing. Furthermore, its gene editing effect (relative copy number compared to the control group) was significantly higher than that of experimental group 2, demonstrating that the addition of complete Alu can improve gene editing efficiency. Compared with the control group (N / A calculated as 40.00), the relative copy number of experimental group 4 was significantly higher (P < 0.05), indicating that binding ORF1p, ORF2p, or both ORF1p and ORF2p to RNA containing the upstream, insert, and downstream sequences of the target site and complete Alu can produce gene editing. These results also demonstrate that the complete Alu sequence can effectively enhance the gene editing effect of this invention.

[0469] The gene editing efficiency of transfection was compared between in vitro prokaryotic transcription to produce RNA, which was then bound to ORF1p and ORF2p, and direct DNA transfection.

[0470] Table 23 Results for primer pair 2 (n=3, )

[0471]

[0472] As shown in Table 23, compared with Group 1, the relative copy number of Group 4 was significantly higher in Group 4 than in Group 1, which was statistically significant (P < 0.05). This indicates that gene editing by generating specific RNA or RNPs in vitro through prokaryotic promoters or other methods and then introducing them into the receiving system is, in some cases, more efficient than gene editing by generating RNA in the receiving system after introducing DNA. This may be because generating specific RNA or RNPs in vitro avoids the cleavage or splicing of the RNA produced by the eukaryotic system. This reflects that generating specific RNA or RNPs in vitro and then introducing them into the receiving system, such as cells, tissues, organs, or organisms, has advantages in certain situations compared to directly introducing the corresponding DNA into the receiving system.

[0473] Example 5: Detecting the efficiency of gene editing after in vitro transcription of RNA (RNA sequence corresponding to the 3' UTR of a long interstitial element in the 3' portion (partial long interstitial element RNA)) and its binding / non-binding to ORF1p and ORF2p in vitro before being transferred into the target system.

[0474] A 400bp sequence from the Lman1 gene in the human genome was selected, and its sequence is shown in Seq ID No. 33 below:

[0475] GAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTCCTGAATTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTTCCTCCCCTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAA*AGATTA CCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where * represents the selected insertion site (target site). The sequence preceding the insertion site is the upstream sequence of the insertion site in the Lman1 gene (upstream sequence of the target site), and the sequence following the insertion site is the downstream sequence of the insertion site in the Lman1 gene (downstream sequence of the target site). Sequence Seq ID No. 33 is 5 bp shorter at the 5' end than Seq ID No. 8, the purpose of which is to increase the transcription efficiency of the Sp6 promoter.

[0476] Insert an exogenous sequence at the * position. This exogenous sequence is the sequence to be inserted, and its sequence is shown in Seq ID No.9.

[0477] The sequence after insertion is shown in Seq ID No. 34:

[0478] GAGATTCACTGCCTTAGTCTCATGTAGTCTCGTGTAGTCTTTTGAGTAAATAACATAAAGTATCTCAAGACTTTTTCATAACTTGATATTATTTTAGTCTTCCTGAATTTTAAATATTGAAAAGCTGAGTGTCTTGTCTGTTTTCCTCCCCTACACTATAGTGACGGGGCTAGTCAAGCTTTGGCAAGTTGCCAGAGGGACTTCCGCAACAAACCCTATCCTGTCCGAGCAA AGGTGCCTGCACATACTGCATGTGAGAGTCTGGAGACGCCAGACTGTTCTGAGTCCTGACC TGCTCAGGGGTGAGGTCCCTCTGAGCCTGAGCAAGCATTTCGTAGCCAACCATGAATTTCCGGACAGTGGCAGAGC GCAGGAGCGGAGGAGATTACCTATTACCAGAACACACTGACAGTAAGTAACATCTATTTAGAGAGAATCAAATAAACAATGTTACAGTATCACTTTTCATTTTGAATTTTTGATAGAAATTAAATGCACTTAAATTTGGATATGCTTACATACTCTTCATTGTTACTCTAAGAGAACG, where the underlined part is the sequence to be inserted as shown in Seq ID No.9.

[0479] Add the Sp6 promoter sequence (Seq ID No. 35: ATTTAGGTGACACTATA) upstream of the sequence shown in Seq ID No. 34, and add the LINE-3' UTR sequence shown in Seq ID No. 36 downstream: ACAATGAGATCACATGGACACAGGAAGGGGAATATCACACTCTGGGGACTGTGGTGGGGTCGGGGGAGGGGGGAGGGGGTAGCATTGGGAGATATACCTAATGCTAGATGACACATTAGTGGGTGCAGCGCACCAGCATGGCACATGTATACATATGTAACTAACCTGCACAATGTGCACATGTACCCTAAAACTTAGAGTATAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA,

[0480] After adding, it will look like Seq ID No. 37:

[0481] The underlined part is the sequence to be inserted as shown in Seq ID No. 9, the italicized and bold part is the Sp6 promoter sequence as shown in Seq ID No. 35, and the wavy part is the LINE-3'UTR sequence as shown in Seq ID No. 36. This sequence was obtained through chemical synthesis and named the precursor DNA of RNA+LINE-3'UTR (RNA).

[0482] According to MEGAscript TMThe SP6 transcription kit instructions state that the precursor DNA of linear RNA+LINE-3'UTR (RNA) is transcribed to obtain the corresponding RNA. Then, the residual DNA is degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration is measured by UV spectrophotometer, and RNase-free water is added to adjust the concentration to 100 ng / μL RNA+LINE-3'UTR (RNA) solution.

[0483] The RNA+LINE-3'UTR (RNA) obtained from the above transcription belongs to Figure 4 The ORF2p functional initiation part of the RNA framework structure is a partially long, scattered element RNA.

[0484] The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in Opti-MEM solution with 1 U / μL of RNase inhibitor added beforehand to form hLRE1-ORF1p solution or hLRE1-ORF2p solution with a concentration of 500 ng / μL.

[0485] To prepare the RNP that binds RNA+LINE-3'UTR(RNA) to ORF1p and ORF2p: RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p, follow these steps.

[0486] First, prepare the reaction system as shown in Table 24:

[0487] Table 24 Reaction System

[0488]

[0489] After gently mixing all components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain the RNA+LINE-3'UTR(RNA)+hLRE1-ORF2p solution.

[0490] Then, the RNA+LINE-3'UTR(RNA)+hLRE1-ORF2p solutions were mixed according to the systems shown in Table 25.

[0491] Table 25 Reaction System

[0492]

[0493] After gently mixing all components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain the RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p solution.

[0494] Mix the RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p solution with the transfection solution system prepared in Table 7 in equal volume ratio, gently mix, and incubate at room temperature (25℃) for 20 min to obtain the RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex.

[0495] transfection

[0496] First, HeLa cells were passaged and seeded into 24-well plates using complete culture medium. The day after passage, when the HeLa cells reached 60% confluence, the medium was replaced with Opti-MEM. TM In culture medium I, following the RNAiMAX transfection reagent instructions, the RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex was added to HeLa cells for transfection, with three replicates per cell. Cells were cultured until approximately 90% confluence was reached, then passaged. After passage, transfection was repeated once more (following the aforementioned steps). Once cells again reached approximately 90% confluence, subsequent procedures were performed.

[0497] Experimental Groups

[0498] The control group in Example 2 was used as the control group, and RNA+LINE-3'UTR(RNA)+hLRE1-ORF1p+hLRE1-ORF2p was used as the experimental group.

[0499] DNA extraction from transfected cells in the experimental group: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0500] qPCR detection:

[0501] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0502] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19: 5′–CACTGCCACCCAGAAGACTG-3′; the downstream primer sequence is shown in Seq ID No. 20: 5′-CCTGCTTCACCACCTTCTTG-3′.

[0503] Primer pair 1 was designed with the upstream primer sequence shown in Seq ID No. 21: 5′-GACTTATCCATGTGCCTGTT-3′; and the downstream primer sequence shown in Seq ID No. 22: 5′-TTGGCTACGAAATGCTTG-3′. The upstream primer sequence of primer pair 1 is located in the complete Lman1 gene, further upstream of the insertion site (target site) upstream sequence used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 1 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0504] All of the above primers were obtained through chemical synthesis.

[0505] The qPCR reaction system is shown in Table 26.

[0506] Table 26 qPCR reaction system

[0507]

[0508]

[0509] The cellular DNA template was the DNA extracted from the transfected cells in the above experimental groups.

[0510] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0511] qPCR reaction cycle:

[0512] Primer pair 1: Pre-denaturation at 95℃ for 15 min; (denaturation at 95℃ for 10 s, annealing at 49℃ for 20 s, extension at 72℃ for 20 s) for 40 cycles. GAPDH primers were reacted under the same conditions.

[0513] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 27. The PCR products were verified to be correct by sequencing.

[0514] Table 27 Results for primer pair 1 (n=3, )

[0515]

[0516] As shown in Table 27, the relative copy number in the experimental group was significantly higher than that in the control group (P < 0.05), indicating that gene editing can be effectively performed by linking the corresponding RNA sequence of the 3'UTR of the long discrete element (LINE-1) to the RNA framework and generating the corresponding specific RNA or RNP in vitro before introducing it into the receiving system. Furthermore, since the corresponding RNA sequence of the 3'UTR of the long discrete element is possible, the complete long discrete element RNA (mainly the ORF1p and ORF2p coding sequences plus their corresponding RNA sequences in the 3'UTR) can theoretically also be used. This example also demonstrates that the Sp6 promoter can participate in the in vitro RNA generation process.

[0517] Example 6 examines the efficiency of gene editing after in vitro transcription of specific RNA (containing functional structures that initiate ORF2p splicing and reverse transcription, and which are partially contained in the downstream sequence of the target site) binds / does not bind ORF1p and ORF2p outside the target system and is then transferred into the target system.

[0518] The GALT gene encodes galactose-1-phosphate uridine transferase, and mutations in it can lead to human type I galactosemia.

[0519] A functional structure was constructed to initiate ORF2p splicing and reverse transcription. This functional structure RNA binds to its complementary sequence on the genome to form an "Ω" structure.

[0520] The 5' portion (left leg) of the "Ω" structure is selected from the downstream sequence (target site downstream sequence) of the GALT gene containing the sequence to be inserted, as shown in Example 4, such as Seq ID No. 25, as shown in Seq ID No. 38:

[0521] ATGCATGGGCTCAGTCACAGAGGAGCTGGGTGCCCAGTACCCTTGGGTGCAGGTTTGTGAGGTCGCCCCTTCCCCTGGATGGGCAGGGAGGGGGTGATGAAGCTTTGGTTCTGGGGAGTAACATTTCTGTTTCCAGGGTGTGGTCAGGAGGGAGTTGACTTGGTGTCTTTTGGCTAACAGAGCTCCGTATCCCTATCTGATAGATCTTTG.

[0522] The 3' part (right leg) of the "Ω" structure is composed of the downstream sequence immediately following the sequence shown in Seq ID No. 38 (downstream sequence of the target site) on the genome, as shown in Seq ID No. 39:

[0523] AAAACAAAGGTGCCATGATGGGCTGTTCTAACCCCCACCCCCACTGC CAGGTAAGGGTGTCAGGGGCTCCAGTGGGTTTCTTGGCTGAGTCTGAGCC AGCACT;

[0524] The ring-shaped portion of the “Ω” structure is composed of a randomly generated sequence, as shown in Seq ID No. 40:

[0525] CTGACCATGCTTATACGGACTATCGATTAG.

[0526] Connect the loop of the “Ω” structure shown in Seq ID No. 40 and the right leg structure of the “Ω” structure shown in Seq ID No. 39 downstream of the sequence shown in Seq ID No. 25, and add the T7 promoter sequence shown in Seq ID No. 11 upstream of the sequence shown in Seq ID No. 25 to form the sequence shown in Seq ID No. 41:

[0527]

[0528]

[0529] In this sequence, the bold italic text represents the T7 promoter sequence as shown in Seq ID No. 11; the wavy line sequence represents the circular sequence of the “Ω” structure as shown in Seq ID No. 40; downstream of the wavy line sequence is the right leg structure of the “Ω” structure as shown in Seq ID No. 39; and upstream of the wavy line sequence and between the underline and the left leg structure of the “Ω” structure (downstream of the target site) as shown in Seq ID No. 38. The sequence shown in Seq ID No. 41 was obtained through chemical synthesis and named the precursor DNA of the functional structure that initiates ORF2p splicing and reverse transcription via RNA+.

[0530] According to the MEGAscript kit TM The T7 Transcription Kit instructions state that the precursor DNA of the functional structure that initiates ORF2p cleavage and reverse transcription of linear RNA+ is transcribed to obtain the corresponding RNA. Then, the residual DNA is degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration is measured by UV spectrophotometer, and RNase-free water is added to adjust the concentration to 100 ng / μL of the functional structure solution that initiates ORF2p cleavage and reverse transcription of RNA+.

[0531] The RNA+ transcribed above initiates the ORF2p splicing function and the reverse transcription function. Figure 6 The RNA framework structure contains an “Ω” structure, in which the functional structures that initiate ORF2p splicing and reverse transcription form the structure.

[0532] The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in Opti-MEM solution with 1 U / μL of RNase inhibitor added beforehand to form hLRE1-ORF1p solution or hLRE1-ORF2p solution with a concentration of 500 ng / μL.

[0533] To prepare the RNP that binds to ORF1p and ORF2p, the functional structure that initiates ORF2p cleavage and reverse transcription with RNA+ is: RNA+ initiates ORF2p cleavage and reverse transcription +hLRE1-ORF1p+hLRE1-ORF2p, the following steps were performed.

[0534] First, configure the reaction system as shown in Table 28:

[0535] Table 28 Reaction System

[0536]

[0537] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain the RNA+ functional structure that initiates ORF2p cleavage and reverse transcription + hLRE1-ORF2p solution.

[0538] Then, the RNA+ functional structure that initiates ORF2p cleavage and reverse transcription + hLRE1-ORF2p solution was mixed according to the systems shown in Table 29.

[0539] Table 29 Reaction System

[0540]

[0541] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain the RNA+ functional structure that initiates ORF2p cleavage and reverse transcription + hLRE1-ORF1p+hLRE1-ORF2p solution.

[0542] The RNA+-initiating ORF2p cleavage and reverse transcription functional structure +hLRE1-ORF1p+hLRE1-ORF2p solution was mixed with the transfection solution system prepared in Table 7 in an equal volume ratio. After gentle mixing, it was incubated at room temperature (25℃) for 20 min to obtain the RNA+-initiating ORF2p cleavage and reverse transcription functional structure +hLRE1-ORF1p+hLRE1-ORF2p-liposome complex.

[0543] transfection

[0544] First, human glioma cells U251 were passaged and seeded into 24-well plates using complete culture medium. The day after passage, when the U251 cells reached 60% confluence, the medium was replaced with Opti-MEM. TM In culture medium I, following the RNAiMAX transfection reagent instructions, the RNA+ hLRE1-ORF1p+ hLRE1-ORF2p-liposome complex, which initiates ORF2p cleavage and reverse transcription, was added to U251 human glioma cells for transfection, with three replicates for each. Cells were cultured until approximately 90% confluence was reached, then passaged. After passage, transfection was repeated once more (following the above steps). Once cells again reached approximately 90% confluence, subsequent procedures were performed.

[0545] Experimental Groups

[0546] The control group in Example 4 was used as the control group, and the functional structure RNA+ that initiates ORF2p splicing and reverse transcription+hLRE1-ORF1p+hLRE1-ORF2p was used as the experimental group.

[0547] DNA was extracted from transfected cells in each group: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0548] qPCR detection

[0549] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0550] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19; the downstream primer sequence is shown in Seq ID No. 20.

[0551] Primer pair 2 was designed with the upstream primer sequence shown in Seq ID No. 31: 5'-CCCCAGTACGATAGCACC-3'; and the downstream primer sequence shown in Seq ID No. 32: 5'-GACATAACCGAATCAGAATT-3'. The upstream primer sequence of primer pair 2 is located in the complete GALT gene, further upstream of the insertion site (target site) upstream sequence used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 2 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0552] All of the above primers were obtained through chemical synthesis.

[0553] The qPCR reaction system is shown in Table 30.

[0554] Table 30 qPCR reaction system

[0555]

[0556] The cellular DNA template was DNA extracted from transfected cells in the experimental group.

[0557] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0558] qPCR reaction cycle:

[0559] Primer pair 2: 95℃ pre-denaturation for 15 min; (95℃ denaturation for 10 s, 46℃ annealing for 20 s, 72℃ extension for 20 s) 40 cycles. GAPDH primers were reacted under the same conditions.

[0560] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 31. The PCR products were verified to be correct by sequencing.

[0561] Table 31 Results for primer pair 2 (n=3, )

[0562]

[0563]

[0564] As shown in Table 31, the relative copy number of the experimental group was significantly higher than that of the control group, which was statistically significant (P < 0.05). This indicates that even without connecting complete or partial short or long sporadic element sequences, as long as the 3' part of the RNA frame containing the upstream sequence of the target site, the downstream sequence of the target site, and the sequence to be inserted can form a specific secondary structure such as an "Ω" shape and can recruit and bind ORF2p (e.g., through polyA sequences), the corresponding gene editing purpose can be achieved.

[0565] Example 7: Detecting the efficiency of RNA transcription and synthesis, as well as ORF1p and ORF2p, in modifying gene loci on the genome within a eukaryotic target system.

[0566] The PAH gene encodes phenylalanine hydroxylase, which is the pathogenic gene for phenylketonuria.

[0567] A 250bp sequence from the PAH gene in the human genome was selected, as shown in Seq ID No. 42 below:

[0568] AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCgCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC, where lowercase letters represent the bases to be modified.

[0569] The sequence after changing the base from G to C is shown in Seq ID No. 43 below:

[0570] AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCcCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC, where lowercase letters represent modified bases.

[0571] Using the unmodified sequence Seq ID No. 42 as the upstream sequence of the target site, the modified sequence Seq ID No. 43 as the sequence to be inserted, and a 200bp sequence immediately adjacent to Seq ID No. 42 and located downstream of the gene sequence Seq ID No. 42 as the downstream sequence of the target site, the sequence shown in Seq ID No. 44 is obtained:

[0572] AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGTGCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCAGTTCGCTACGACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTAAGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTC AAAATGCCACTGAGAACTCTCTTAAGACTACCTTTCTCCAAATGGT GCCCTTCACTCAAGCCTGTGGTTTTGGTCTTAGGAACTTTGCTGCCACAATACCTCGGCCCTTCTCAGTTCCCTAC GACCCATACACCCAAAGGATTGAGGTCTTGGACAATACCCAGCAGCTTAAGATTTTGGCTGATTCCATTAACAGTA AGTAATTTACACCTTACGAGGCCACTCGGTTTCTCAGTAATCGAAGACTGTCTTTCCCTACCATCGCCATAGGAAAAATAATAAATTTATTGAAATATTTAATTAAGGAGAAAAGCACCTCCATGTAAGCCATGGGTTCATTGATGGAGAAGAACTTGACAAAAAGGTCAGAATTACCCTTGTGTCCTTTTTCCTTTGACCTTCCTAGATTCCACTCCACCTCCTACCATCATTCCACCTTTCCACACTTGG, where the underlined part is the sequence to be inserted, Seq ID No. 43. Its upstream is the upstream sequence of the target site, Seq ID No. 42, and its downstream is the downstream sequence of the target site. This sequence is named the PAH base substitution sequence.

[0573] Adding a portion of the Alu sequence downstream of the PAH base substitution sequence Seq ID No. 44 yields the sequence shown in Seq ID No. 45:

[0574]

[0575] The underlined part is the Seq ID No. 43 of the sequence to be inserted, upstream of which 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. The sequence between the sequence to be inserted and the partial Alu sequence is the downstream sequence of the target site. This sequence is named PAH base substitution sequence framework + partial Alu sequence.

[0576] The sequence shown in Seq ID No. 45 was chemically synthesized and constructed into the pcDNA3.1(+)eGFP vector through reverse loop expansion and homologous ligation. This allowed the sequence to be directly linked downstream of the CMV promoter in the vector, ensuring that there were no other sequences between the sequence and the CMV promoter. The resulting vector was pcDNA3.1(+)eGFP+PAH base substitution sequence frame+partial Alu sequence.

[0577] The specific steps are as follows:

[0578] 1. Design primers to amplify the sequence shown in Seq ID No. 45. The forward primer sequence is shown in Seq ID No. 46: 5'-CTATATAAGCAGAGCTAAAATGCCACTGAGAA-3', and the reverse primer sequence is shown in Seq ID No. 16: 5'-CTCTAGTTAGCCAGAGGATCTCCAGCAGTTAT-3'. Perform PCR amplification on the sequence shown in Seq ID No. 45. The reaction system is shown in Table 32.

[0579] Table 32 Reaction System

[0580]

[0581] The amplification conditions were: 94℃ for 2 min; (98℃ for 10 sec, 58℃ for 10 sec, 68℃ for 2 sec) for 40 cycles; 68℃ for 5 min.

[0582] The amplified product was obtained by gel recovery and purification using conventional methods. Sequences homologous to the pcDNA3.1(+)eGFP vector were added to both sides of the synthesized sequence.

[0583] 2. Design PCR primers for amplifying the pcDNA3.1(+)eGFP vector. The forward primer is shown in Seq ID No. 17: 5'-AATAACTGCTGGAGATCCTCTGGCTAACTAGAG-3', and the reverse primer sequence is shown in Seq ID No. 47: 5'-GTTCTCAGTGGCATTTTAGCTCTGCTTATATAG-3'. PCR amplification of the pcDNA3.1(+)eGFP vector was performed, and the reaction system is shown in Table 33.

[0584] Table 33 Reaction System

[0585]

[0586] The amplification conditions were: 94℃ for 2 min; (98℃ for 10 sec, 58℃ for 10 sec, 68℃ for 6 sec) for 40 cycles; 68℃ for 5 min.

[0587] The pcDNA3.1(+)eGFP plasmid vector was obtained by gel recovery and purification using conventional methods. The plasmid vector has sequences homologous to the synthesized sequence at both ends.

[0588] 3. Use a one-step rapid cloning kit to ligate the amplified product and the amplified pcDNA3.1(+)eGFP vector. Follow the kit instructions for specific steps, and prepare the reaction system as shown in Table 11.

[0589] 4. The recombinant product was transformed into competent cells (DH5α), and bacteria were picked and sequenced after transformation. After the sequencing was confirmed to be correct, the plasmid was extracted to obtain the plasmid pCDNA3.1(+)eGFP+PAH base substitution sequence frame + partial Alu sequence.

[0590] The constructed vector pcDNA3.1(+)eGFP+PAH base substitution sequence framework + partial Alu sequence was co-transfected with plasmid pBS-L1PA1-CH-mneo expressing ORF1p and ORF2p into HeLa cells. The experimental group was co-transfected with pcDNA3.1(+)eGFP+PAH base substitution sequence framework + partial Alu sequence and pBS-L1PA1-CH-mneo, and the control group was co-transfected with plasmid pBS-L1PA1-CH-mneo and plasmid pcDNA3.1(+)eGFP. Each group was divided into 3 replicates, and each replicate was a 24-well plate cultured with HeLa cells.

[0591] The transfection procedure for the control group was the same as that for the control group in Example 2.

[0592] The co-transfection procedure for the experimental group was as follows: HeLa cells were passaged and seeded in 24-well plates. On the second day after passage, when the cells reached approximately 60% confluence, transfection was performed using Entranster-H4000 transfection reagent. For each plate of cells, two plasmids (pcDNA3.1(+)eGFP+PAH base substitution sequence framework + partial Alu sequence and pBS-L1PA1-CH-mneo) were co-transfected, with 19.2 μg of each plasmid, totaling 38.4 μg. The desired plasmid was diluted with 600 μL of serum-free DMEM and thoroughly mixed; simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, thoroughly mixed, and incubated at room temperature for 5 min. The two prepared liquids were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. Transfection complex was added to each well of a 24-well plate containing HeLa cells and 0.5 ml of Opti-MEM medium. Cells were passaged when they reached approximately 90% confluence. This process was repeated after passage, and subsequent steps were performed once cells reached approximately 90% confluence.

[0593] The co-transfection procedure for the control group was as follows: HeLa cells were passaged and seeded in 24-well plates. On the second day after passage, when the cells reached approximately 60% confluence, transfection was performed using Entranster-H4000 transfection reagent. For each plate of cells, two plasmids (pcDNA3.1(+)eGFP and pBS-L1PA1-CH-mneo) were co-transfected, with 19.2 μg of each plasmid, totaling 38.4 μg. The required plasmid was diluted with 600 μL of serum-free DMEM and thoroughly mixed; simultaneously, 48 μL of Entranster-H4000 reagent was diluted with 600 μL of serum-free DMEM, thoroughly mixed, and incubated at room temperature for 5 min. The two prepared liquids were then mixed thoroughly and incubated at room temperature for 15 min to prepare the transfection complex. The transfection complex was added to each well of a 24-well plate containing 0.5 ml of Opti-MEM medium and cultured with HeLa cells for transfection. When the cells reach about 90% confluence, passage them. Repeat the above steps after passage. When the cells reach about 90% confluence, proceed with the next steps.

[0594] DNA was extracted from transfected cells in each group: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0595] qPCR detection:

[0596] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0597] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19: 5′–CACTGCCACCCAGAAGACTG-3′; the downstream primer sequence is shown in Seq ID No. 20: 5′-CCTGCTTCACCACCTTCTTG-3′.

[0598] Primer pair 3 was designed, with the upstream primer sequence shown in Seq ID No. 48: 5′-AGGGAGGTGTCCGTGTTC-3′; and the downstream primer sequence shown in Seq ID No. 49: 5′-GGGTGTATGGGTCGTAGC-3′. The upstream primer sequence of primer pair 3 is located in the complete PAH gene, further upstream of the target site, and is not present in the constructed vector sequence, but only in the cell genome. The downstream primer sequence of primer pair 3 is located on the sequence to be inserted, and its 3' terminal base matches an unmodified base on the genome. Therefore, if the selected base site on the genome is modified, the PCR product will be reduced.

[0599] All of the above primers were obtained through chemical synthesis.

[0600] The qPCR reaction system is shown in Table 34.

[0601] Table 34 qPCR reaction system

[0602]

[0603] The cellular DNA templates were extracted from the transfected cells in the two groups above.

[0604] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0605] qPCR reaction cycle:

[0606] Primer pair 3: 95℃ pre-denaturation for 15 min; (95℃ denaturation for 10 s, 48℃ annealing for 20 s, 72℃ extension for 20 s) 40 cycles. GAPDH primers were reacted under the same conditions.

[0607] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 35. The PCR products were verified to be correct by sequencing.

[0608] Table 35 Results for primer pair 3 (n=3, )

[0609]

[0610] As shown in Table 35, compared with the control group, the relative copy number of the experimental group was significantly higher than that of the control group, which was statistically significant (P < 0.05), indicating that the present invention can achieve the purpose of replacing specific sites on the genome. It also demonstrates that genome-wide sequence replacement, deletion, addition, and deletion, which are based on homologous recombination after the insertion of specific sequences, are also feasible.

[0611] Since direct transfection with plasmids can achieve the purpose of site replacement on the genome, it is known from other embodiments of the present invention that transfection with corresponding RNA transcribed in vitro, with or without ORF1p and / or ORF2p, can also achieve the same purpose.

[0612] Since both eukaryotic and prokaryotic systems can express RNA and possess homologous recombination capabilities, their working mechanisms are similar to those of this invention, enabling sequence insertion into the genome. However, the splicing mechanism in eukaryotic systems may interfere with the synthesis of specific RNAs and cause inconvenience in industrial production. Furthermore, sequence substitution, deletion, addition, and replacement in the genome are achieved by the cell itself through homologous recombination after the insertion of the corresponding sequence according to this invention. Therefore, the feasibility of genome modification operations such as sequence substitution, deletion, addition, and replacement in eukaryotic systems implies that corresponding operations are also feasible in prokaryotic systems.

[0613] Example 8: Testing the efficiency of separately administering RNA frameworks and short, scattered RNA elements.

[0614] Concatenating the T7 promoter sequence shown in Seq ID No. 11 with the Alu sequence shown in Seq ID No. 1 yields the sequence shown in Seq ID No. 50:

[0615]

[0616] The bolded italic section represents the T7 promoter sequence, and downstream of the T7 promoter sequence is the Alu sequence. This part of the sequence was obtained through chemical synthesis and named the Alu-RNA expression DNA.

[0617] According to the MEGAscript kit TM The T7 Transcription Kit instructions state that the linear Alu-RNA expression DNA is transcribed to obtain the corresponding Alu-RNA. Then, the residual DNA is degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration is measured with a UV spectrophotometer, and RNase-free water is added to adjust the concentration to 100 ng / μL of Alu solution.

[0618] The reaction system for preparing the Alu-liposome complex is shown in Table 36.

[0619] Table 36 Reaction System

[0620]

[0621]

[0622] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min. Then, the transfection solutions shown in Table 7 were added in equal volume ratios, gently mixed, and incubated at room temperature (25°C) for 20 min to obtain the Alu-liposome complex.

[0623] Using the control group in Example 2 as the control group, and co-transfecting the RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex and the Alu-liposome complex in Example 2 (experimental group), the cells were transfected into HeLa cells according to the method in Example 2 (the Alu-liposome complex and the RNA+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex were transfected simultaneously in equal amounts). Each group had three replicates, and each replicate was a 24-well plate cultured with HeLa cells.

[0624] The transcribed Alu from the above example, together with the RNA from Example 2, forms the following... Figure 14 The structure shown is a separate transcriptional structure.

[0625] Then, DNA was extracted from transfected cells in each group: after aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with an appropriate amount of 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0626] qPCR detection:

[0627] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0628] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19: 5′–CACTGCCACCCAGAAGACTG-3′; the downstream primer sequence is shown in Seq ID No. 20: 5′-CCTGCTTCACCACCTTCTTG-3′.

[0629] Primer pair 1 was designed with the upstream primer sequence shown in Seq ID No. 21: 5′-GACTTATCCATGTGCCTGTT-3′; and the downstream primer sequence shown in Seq ID No. 22: 5′-TTGGCTACGAAATGCTTG-3′. The upstream primer sequence of primer pair 1 is located in the complete Lman1 gene, further upstream of the insertion site (target site) used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 1 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0630] All of the above primers were obtained through chemical synthesis.

[0631] The qPCR reaction system is shown in Table 37.

[0632] Table 37 qPCR reaction system

[0633]

[0634]

[0635] The cellular DNA templates were extracted from the transfected cells in the two groups above.

[0636] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0637] qPCR reaction cycle:

[0638] Primer pair 1: Pre-denaturation at 95℃ for 15 min; (denaturation at 95℃ for 10 s, annealing at 49℃ for 20 s, extension at 72℃ for 20 s) for 40 cycles. GAPDH primers were reacted under the same conditions.

[0639] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 38. The PCR products were verified to be correct by sequencing.

[0640] Table 38 Results for primer pair 1 (n=3, )

[0641]

[0642] As shown in Table 38, compared with the control group, the relative copy number of the experimental group was significantly higher than that of the control group, which was statistically significant (P < 0.05). This indicates that RNA frameworks containing upstream and downstream sequences of the target site and the sequence to be inserted, as well as short, partial, long, and / or partial long fragmented elements, can be separately administered to the receiving system at different locations within the same vector or on different vectors to achieve the purpose of inserting the specified sequence into the target gene site or to achieve other gene editing purposes such as site substitution, sequence substitution, site deletion, site addition, and sequence deletion. Simultaneously, DNA vectors that can express RNA frameworks containing upstream and downstream sequences of the target site and the sequence to be inserted, as well as short, partial, long, and / or long fragmented elements, can also express RNA frameworks containing upstream and downstream sequences of the target site and the sequence to be inserted, as well as short, partial, long, and / or long fragmented elements. DNA vectors containing partially long, sporadic elements can be separated and administered to the receiving system at different locations within the same vector or on different vectors to achieve the corresponding gene editing purpose. Similarly, RNA frameworks containing upstream and downstream sequences of the target site and the sequence to be inserted, along with DNA vectors expressing short, partially short, long, and / or partially long sporadic elements, can be separated and administered to the receiving system at different locations within the same vector or on different vectors to achieve the corresponding gene editing purpose.

[0643] Example 9: Gene editing effects of ORF1 and ORF2p in human LRE2 and mouse L1.

[0644] The ORF1p and ORF2p used in Examples 2-8 are derived from LRE1 in human L1. In this example, we verify whether ORF1p (hLRE2-ORF1p) and ORF2p (hLRE2-ORF2p) in human LRE2 and ORF1p (mORF1p) and ORF2p (mORF2p) in mouse L1 can still play a gene editing role when replacing the previous ORF1p and ORF2p in LRE1.

[0645] The RNA+ fraction Alu solution prepared in Example 2 was combined with hLRE2-ORF1p and hLRE2-ORF2p, mORF1p and mORF2p prepared in Example 1 to prepare RNA+ fraction Alu+hLRE2-ORF1p+hLRE2-ORF2p and RNA+ fraction Alu+mORF1p+mORF2p, respectively. The method was the same as the preparation method of RNA+ fraction Alu+hLRE1-ORF1p+hLRE1-ORF2p in Example 2.

[0646] Then, RNA+ partial Alu+hLRE2-ORF1p+hLRE2-ORF2p-liposome complex and RNA+ partial Alu+mORF1p+mORF2p-liposome complex were prepared according to the method in Example 2.

[0647] The RNA+ Alu+hLRE2-ORF1p+hLRE2-ORF2p-liposome complex and the RNA+ Alu+mORF1p+mORF2p-liposome complex were transfected into HeLa cells according to the method in Example 2. Each group had three replicates, and each replicate was a 24-well plate cultured with HeLa cells.

[0648] Experimental Groups:

[0649] The study was divided into three groups: the control group in Example 2 was used as the control group in this example; the group transfected with RNA+ Alu+hLRE2-ORF1p+hLRE2-ORF2p was Experiment 1; and the group transfected with RNA+ Alu+mORF1p+mORF2p was Experiment 2. Each group had three replicates, and each replicate was a 24-well plate cultured with HeLa cells.

[0650] DNA was extracted from transfected cells in each group: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0651] qPCR detection:

[0652] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0653] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19: 5′–CACTGCCACCCAGAAGACTG-3′; the downstream primer sequence is shown in Seq ID No. 20: 5′-CCTGCTTCACCACCTTCTTG-3′.

[0654] Primer pair 1 was designed with the upstream primer sequence shown in Seq ID No. 21: 5′-GACTTATCCATGTGCCTGTT-3′; and the downstream primer sequence shown in Seq ID No. 22: 5′-TTGGCTACGAAATGCTTG-3′. The upstream primer sequence of primer pair 1 is located in the complete Lman1 gene, further upstream of the insertion site (target site) upstream sequence used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 1 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0655] All of the above primers were obtained through chemical synthesis.

[0656] The qPCR reaction system is shown in Table 39.

[0657] Table 39 qPCR reaction system

[0658]

[0659] The cellular DNA templates were extracted from the transfected cells in the above groups.

[0660] The above reaction system was prepared on ice. After preparation, the reaction tube was capped, gently mixed, and briefly centrifuged to ensure that all components were at the bottom of the tube. Each 24-well plate cell sample was subjected to three replicates.

[0661] qPCR reaction cycle:

[0662] Primer pair 1: Pre-denaturation at 95℃ for 15 min; (denaturation at 95℃ for 10 s, annealing at 49℃ for 20 s, extension at 72℃ for 20 s) for 40 cycles. GAPDH primers were reacted under the same conditions.

[0663] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 40. The PCR products were verified to be correct by sequencing.

[0664] Table 40 Results for primer pair 1 (n=3, )

[0665]

[0666] As shown in Table 40, compared with the control group, the relative copy number of experimental group 1 was significantly higher than that of the control group, which was statistically significant (P < 0.05), indicating that the ORF1p and ORF2p expressed by LRE2 in human L1 can also achieve the corresponding gene editing purpose. Similarly, compared with the control group, the relative copy number of experimental group 2 was significantly higher than that of the control group, which was statistically significant (P < 0.05), indicating that the ORF1p and ORF2p expressed by mouse L1 can also achieve the corresponding gene editing purpose. This embodiment demonstrates that the ORF1p and / or ORF2p expressed by different L1 species in the human genome or the ORF1p and / or ORF2p expressed by L1 from different species can all be applied to gene editing in this invention to achieve the corresponding gene editing purpose. Meanwhile, the ORF1p and ORF2p of human LRE1, the ORF1p and ORF2p of human LRE2, and the ORF1p and ORF2p of mouse are also modified sequences of other ORF1p and ORF2p coding sequences. Therefore, this embodiment also supports the application of modified sequences of ORF1p coding sequences and modified sequences of ORF2p coding sequences.

[0667] Example 10: Detecting the efficiency of gene editing after in vitro transcription of specific RNA (partial Alu at the 3' site), binding ORF1p and ORF2p outside the target system, and then transfecting it into the target system.

[0668] The Alu used in Examples 2-9 is Alu Ya5 from the Alu element. In this example, to test the effects of other types of Alu elements, the sequence of Alu Yb8 was selected for gene editing. The DNA sequence of Alu Yb8 is shown in Seq ID No. 51: GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCATGAGGTCAGGAGATCGAGACCATCCTGGCTAACAAGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAAAAAAATTAGCCGGGCGCGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCAGTCCGCAGTCCGGCCTAGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAATGTGGCCAAAAGTGTCAGAAA; Alu... Partial DNA sequence in Yb8, such as Seq ID No. 52 shows: AAAAATACAAAAAAAAAAAATTAGCCGGGCGCGGTGGCGGGCGCCTTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAAGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCAGTCCGCAGTCCGGCCTAGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAATGTGGCCAAAAGTGTCAGAAA.

[0669] A 405bp sequence from the Lman1 gene in the human genome, as shown in Seq ID No. 8, was selected and inserted into the sequence to be inserted, as shown in Seq ID No. 9, resulting in the sequence shown in Seq ID No. 10. Upstream of the sequence shown in Seq ID No. 10, the T7 promoter sequence, as shown in Seq ID No. 11, was added, and downstream, a portion of the Alu Yb8 sequence, as shown in Seq ID No. 52, was added, resulting in the sequence shown in Seq ID No. 53.

[0670]

[0671]

[0672] The underlined part is the sequence to be inserted as shown in Seq ID No. 9, the italicized and bold part is the T7 promoter sequence as shown in Seq ID No. 11, and the wavy part is the partial Alu Yb8 sequence as shown in Seq ID No. 52. This sequence was obtained through chemical synthesis and named RNA+partial Alu Yb8 precursor DNA.

[0673] According to the MEGAscript kit TM The T7 Transcription Kit instructions state that the precursor DNA of linear RNA+ AluYb8 or RNA precursor DNA is transcribed to obtain the corresponding RNA. Then, the residual DNA is degraded with the DNase in the kit and resuspended in RNase-free water. The RNA concentration is measured with a UV spectrophotometer, and RNase-free water is added to adjust the concentration to 100 ng / μL of RNA+ AluYb8 solution.

[0674] The RNA+ portion Alu obtained from the above transcription belongs to Figure 4 The RNA framework structure contains an ORF2p functional initiation portion that is another type of short, scattered element RNA.

[0675] The hLRE1-ORF1p and hLRE1-ORF2p prepared in Example 1 were resuspended in Opti-MEM solution with 1 U / μL of RNase inhibitor added beforehand to form hLRE1-ORF1p solution or hLRE1-ORF2p solution with a concentration of 500 ng / μL.

[0676] The RNP for preparing the RNA+ portion of Alu Yb8 binding to ORF1p and ORF2p is as follows: RNA+ portion Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p. This is performed according to the following steps:

[0677] Configure the reaction system as shown in Table 41:

[0678] Table 41 Reaction System

[0679]

[0680] After gently mixing, the system was incubated at room temperature (25°C) for 10 min to obtain the RNA+ fraction Alu Yb8+hLRE1-ORF1p solution.

[0681] Then, the RNA+ portion of the Alu Yb8+hLRE1-ORF1p solution was mixed according to the systems shown in Table 42.

[0682] Table 42 Reaction System

[0683]

[0684]

[0685] After gently mixing the components, the reaction system was incubated at room temperature (25°C) for 10 min to obtain the RNA+ fraction AluYb8+hLRE1-ORF1p+hLRE1-ORF2p solution.

[0686] The RNA+ fraction Alu Yb8+hLRE1-ORF1p+hLRE1-ORF2p solution was mixed with the transfection solution system prepared in Table 7 in equal volume ratio. After gentle mixing, the mixture was incubated at room temperature (25℃) for 20 min to obtain the RNA+ fraction Alu Yb8+hLRE1-ORF1p+hLRE1-ORF2p-liposome complex.

[0687] transfection

[0688] First, HeLa cells were passaged and seeded into 24-well plates using complete culture medium. The day after passage, when the HeLa cells reached 60% confluence, the medium was replaced with Opti-MEM. TM In culture medium 1, following the RNAiMAX transfection reagent instructions, add the RNA + partial Alu Yb8 + hLRE1-ORF1p + hLRE1-ORF2p-liposome complex to HeLa cells for transfection, with three replicates per cell. Six hours after transfection, replace the medium with complete medium. Continue culturing until the cells reach approximately 90% confluence, then passage. Repeat the transfection process once more (following the aforementioned steps). Once the cells again reach approximately 90% confluence, proceed with subsequent steps.

[0689] Experimental Groups:

[0690] The study was divided into two groups: the control group in Example 2 was used as the control group in this example, and the RNA+AluYb8+hLRE1-ORF1p+hLRE1-ORF2p group was used as the experimental group. Each group had three parallels, and each parallel was a 24-well plate cultured with HeLa cells.

[0691] DNA extraction from transfected cells: After aspirating the cell culture medium, the cells were washed twice with PBS, and then digested with 0.25% trypsin at 37°C for 20 minutes, with 15 pipetting cycles every 5 minutes. Once the cells were resuspended, the reaction was terminated by adding complete culture medium containing serum. Subsequently, cell DNA was extracted according to the product instructions of the blood / cell / tissue genomic DNA extraction kit, and the DNA concentration was measured using a UV spectrophotometer.

[0692] qPCR detection:

[0693] Because the GAPDH gene copy number is stable, the GAPDH gene is set as an internal reference gene.

[0694] The upstream primer sequence for detecting the GAPDH gene is shown in Seq ID No. 19: 5′–CACTGCCACCCAGAAGACTG-3′; the downstream primer sequence is shown in Seq ID No. 20: 5′-CCTGCTTCACCACCTTCTTG-3′.

[0695] Primer pair 1 was designed with the upstream primer sequence shown in Seq ID No. 21: 5′-GACTTATCCATGTGCCTGTT-3′; and the downstream primer sequence shown in Seq ID No. 22: 5′-TTGGCTACGAAATGCTTG-3′. The upstream primer sequence of primer pair 1 is located in the complete Lman1 gene, further upstream of the insertion site (target site) used in the prepared RNA, and is not present in the prepared RNA sequence, but only exists in the cell genome. The downstream primer sequence of primer pair 1 is located on the foreign sequence to be inserted (the sequence to be inserted).

[0696] All of the above primers were obtained through chemical synthesis.

[0697] The qPCR reaction system is shown in Table 9. The qPCR reaction was carried out according to the qPCR reaction cycle of Example 2. The GAPDH primers were reacted under the same conditions.

[0698] After observing the exponential growth phase in the amplification curves of GAPDH and the amplification curves detecting the inserted sequence to confirm approximate parallelism, the following steps were taken: -ΔΔCt The data were analyzed using a relative method, and the results are shown in Table 43. The PCR products were verified to be correct by sequencing.

[0699] Table 43 Results for primer pair 1 (n=3, )

[0700]

[0701] As shown in Table 43, compared with the control group, the relative copy number of the experimental group was significantly higher than that of the control group, which was statistically significant (P < 0.05). This indicates that even if the type of Alu element is changed, the purpose of gene editing can still be achieved. This demonstrates that the present invention is applicable to all types of Alu elements and short sporadic elements in all species.

[0702] As can be seen from the above embodiments, the RNA framework provided by the present invention can generate RNA through eukaryotic or prokaryotic systems and cells, tissues, organisms or in vitro expression, and generate the required proteins ORF1p and / or ORF2p in or outside the target system (in vitro), and introduce them into the target system in the form of RNA or RNP vectors to achieve the goal of gene editing, which is convenient for industrial mass production and commercialization.

[0703] Furthermore, since the prokaryotic system or in vitro expression lacks the splicing mechanism of the precursor mRNA in the eukaryotic system, the RNA framework and downstream connectable short sporadic element RNA, short sporadic element derivative RNA, long sporadic element, long sporadic element derivative RNA, and / or functional structures that initiate ORF2p splicing and reverse transcription can be expressed without hindrance, without suffering potential splicing risks, thus improving the production efficiency and gene editing effect of the present invention.

[0704] Therefore, this invention can accurately delete, replace, and replace individual sites by inserting the desired sequence into the genome, through homologous recombination or genome repair within the editing system itself. Furthermore, based on the technical principles of this invention, it is known that vectors can be designed and inserted again from new sites formed after the sequence was previously inserted using this invention. This progressive insertion theoretically allows for an unlimited length of inserted sequence and can accomplish various types and forms of gene editing, including sequence insertion, deletion, replacement, and site replacement, offering flexible usage. In addition, this invention can also be used to edit CNVs and their ends, stabilizing, lengthening, shortening, or altering their expression sequences, thereby changing or stabilizing gene expression and the state of cells or organisms.

[0705] Since short and long discrete elements and the proteins they express are widely distributed in eukaryotes, this invention can be used for gene editing in a wide range of eukaryotes. Furthermore, it can be applied to the treatment of diseases involving gene alterations and to altering or stabilizing the state of cells or organisms associated with gene changes. In addition, this invention can also be used for gene editing in various prokaryotes.

[0706] Other gene editing tools, such as TALEN, ZFN, Targetron, CRISPR, or CRISPR / Cas9, introduce sequences into the genome primarily through homologous recombination between the template DNA and the target site and its surrounding regions after cutting the genomic target site. This method is prone to introducing random sequences and mutations, and the efficiency of introducing the target sequence is low. The RNA framework and corresponding RNP provided in this invention do not cause double-strand breaks and integrate the genome through homologous recombination, making it safer and easier for practical applications.

[0707] This invention can deliver exogenous sequences to the target system in the form of RNA and insert them into the genome, thus demonstrating that the RNA is converted into DNA and has the ability to generate template DNA.

[0708] Therefore, if DNA expressing the RNA framework and / or its improved form of RNA of this invention, the RNA framework and / or its improved form of RNA of this invention, and RNPs generated by the RNA framework and / or its improved form of RNA of this invention combined with ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins are given to the target system, template DNA can be generated or a large amount of template DNA can be generated (amplified) without the introduction of template DNA. Therefore, this invention can also improve the gene editing function of other gene editing tools such as TALEN, ZFN, Targetron, CRISPR or CRISPR / Cas9 technologies.

[0709] Furthermore, based on the results and principles of the embodiments, RNA required for gene editing in this invention can be generated in vitro and combined with ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins. This RNA can then be introduced into a prokaryotic or eukaryotic system to generate single-stranded or double-stranded DNA bound to ORF2p, ORF1p, ORF2p-derived proteins and / or ORF1p-derived proteins. This DNA can then be introduced into the target system (such as a prokaryotic or eukaryotic system) to achieve the purpose of gene editing.

[0710] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. SEQUENCE LISTING <110> Sui, Yunpeng <120> RNA frameworks and gene editing methods for gene editing <130> DOME <160> 53 <170> PatentIn version 3.3 <210> 1 <211> 333 <212> DNA <213> Homo sapiens <400> 1 gggccgggcg cggtggctca cgcctgtaat cccagcactt tgggaggccg aggcgggcgg 60 atcacgaggt caggagatcg agaccatccc ggctaaaacg gtgaaacccc gtctctacta 120 aaaatacaaa aaattagccg ggcgtggtgg cgggcgcctg tagtcccagc tactcgggag 180 gctgaggcag gagaatggcg tgaacccggg aggcggagct tgcagtgagc cgagatcacg 240 ccgctgcact ccaccctggg cgacagagcg agactccgtc tcaaaaaaaa aaaaaaaaaa 300 aaaaaaaaaa aagattaata actgctggag atc 333 <210> 2 <211> 217 <212> DNA <213> Artificial sequence <400> 2 actaaaaata caaaaaatta gccgggcgtg gtggcgggcg cctgtagtcc cagctactcg 60 ggaggctgag gcaggagaat ggcgtgaacc cgggaggcgg agcttgcagt gagccgagat 120 cacgccgctg cactccaccc tgggcgacag agcgagactc cgtctcaaaa aaaaaaaaaa 180 aaaaaaaaaa aaaaaagatt aataactgct ggagatc 217 <210> 3 <211> 1017 <212> DNA <213> Homo sapiens <400> 3 atggggaaaa aacagaacag aaaaactgga aactctaaaa cgcagagcgc ctctcctcct 60 ccaaaggaac gcaagttcctc accagcaaca gaacaaagct ggatggagaa tgattttgac 120 gagctgagag aagaaggctt cagacgatca aattactctg agctacggga ggacattcaa 180 accaaaggca aagaagttga aaactttgaa aaaaatttag aagaatgtat aactagaata 240 accaatacag agaagtgctt aaaggagctg atggagctga aaaccaaggc tcgagaacta 300 cgtgaagaat gcagaagcct caggagccga tgcgatcaac tggaagaaag ggtatcagca 360 atggagaatg aaatgaatga aatgaagcga gaagggaagt ttagagaaaa aagaataaaa 420 agaaatgagc aaagcctcca agaaatatgg gactatgtga aaagaccaaa tctacgtctg 480 attggtgtac ctgaaagtga tgtggagaat ggaaccaagt tggaaaacac tctgcaggat 540 attatccagg agaacttccc caatctagca aggcaggcca acgttcagat tcaggaaata 600 cagagaacgc cacaaagata ctcctcgaga agagcaactc caagacacat aattgtcaga 660 ttcaccaaag ttgaaatgaa ggaaaaaatg ttaagggcag ccagagagaa aggtcgggtt 720 accctcaaag ggaagcctat cagactaaca gcagatctct cggcagaaac cctacaagcc 780 agaagagagt gggggccaat attcaacatt cttaaagaaa agaattttca acccagaatt 840 tcatttccag ccaaactaag cttcataagt gaaggagaaa gaaaatactt tacagacaag 900 caaatgctga gagattttgt caccaccagg cctaccctaa aagagctcct gaaggaagca 960 ctaaacatgg aaaggaacaa ccggtaccag ccgctgcaaa atcatgccaa aatgtaa 1017 <210> 4 <211> 1104 <212> DNA <213> Artificial sequence <400> 4 ctagctagct agatggggaa aaaacagaac agaaaaactg gaaactctaa aacgcagagc 60 gcctctcctc ctccaaagga acgcagttcc tcaccagcaa cagaacaaag ctggatggag 120 aatgattttg acgagctgag agaagaaggc ttcagacgat caaattactc tgagctacgg 180 gaggacattc aaaccaaagg caaagaagtt gaaaactttg aaaaaaattt agaagaatgt 240 ataactagaa taaccaatac agagaagtgc ttaaaggagc tgatggagct gaaaaccaag 300 gctcgagaac tacgtgaaga atgcagaagc ctcaggagcc gatgcgatca actggaagaa 360 agggtatcag chaatggag tgaatgaat gaatgaagc gagagggaa gtttagagaa 420 aaagaata aaagaatga gcaagccctc aaagaat gggactgt gaaagacca 480 aatctacgtc tgattggtgt acctgaaagt gatgtggaga atggaaccaa gttggaaaac 540 actctgcagg attackatcca ggagaacttc cccaatctag caacgcaggc caacgttcag 600 attcaggaaa tacagagaac gccahaaga tactcctcga gagagcaac tccagacac 660 aatattgtca gattcaccaa agttgaatg aaggaaaaaa tgttaagggc agccagagag 720 aaaggtcggg taccctca agggaagcct atcagacta cagcagatct ctcggcagaa 780 accctacaag ccagagaga gtggggggcca atattcaca ttcttaaga aaagaattttt 840 caacccagaa ttcattcc agccaaacta agctcataa gtgaaggaga aagaaaatac 900 tttacagaca agcaatgct gagagatttt gtcaccacca ggcctaccct aaagagctc 960 ctgaaggaag cactaaacat ggaaggaac aaccggtacc agccgctgca aaatcatgcc 1020 aaaatggaac aaaaactcat ctcagagag gatctgaata tgcataccgg tcatcac 1080 grasshopper gactagctag ctag 1104 <210> 5 <211> 3828 <212> DNA <213> Homo sapiens <400> 5 agacaggat caattcaca cataacata ttactttaa atataatgg actaaattct 60 gcaattaaaa gatacagact ggcaagttgg ataagagtc aagacccatc agtgtgctgt 120 attcaggaaa cccatctcat gtgcagagac acacataggc tcaaataaa aggatggagg 180 aagatctacc aagcaaatgg aaaacaaaa aaggcagggg ttgcaatcct agtctctgat 240 aaaacagact ttaaccaac aagatcaa agagacaaag aaggccatta cataatggta 300 aagggatcaa ttcacaga ggagctact atcctaata tttagcacc caatacagga 360 gcacccagat tcataagca agtcctgagt gacctacaa gagacttaga ctcccacaca 420 ttaataatgg gagactttaa wolf tcaatattag acgatcaac gagacagaaa 480 gtcaacagg atacccagga attgactca gctctgcacc aagcagacct atagacatc 540 tacagaactc tccaccccaa tacacagaa tatacatttt ttcagcacc tacaccacacc 600 tattccaaaa tcgaccacat agttggaagt aaagctctcc tcagcaaatg taaaagaaca 720. sightseeing ctcagaccac agtgcaatca aactagaact sightseeing aatctcactc aaagccgctc aactcatgg aaactgaaca acctgctcct gaatgactac tgggtacata acgaaatgaa ggcagaaata aagatgttct ttgaaaccaa cgagaacaaa gacaccacat accagaatct ctgggacgca ttcaaagcag tgtgtagagg gaatttata gcactaatg cctacaagag aaagcagga agatccaaaa ttgacaccct aacatcacaa ttaaagaac tagaaagca agagcaaaca cattcaaaag ctagcagaag gcaagaaata actaaaatca gagcagaact gaggaata gagacacaaa aaacccttca aaaaatcaat gaatccagga gctggttttt tgaaaggatc aacaaaattg atagccgct agcaagacta ataaagaaa aaagagagaa gacaaata gacacaata aaaatgataa aggggatatc accaccgatc ccacagaat acaaactacc atcagagat actacaaaca cctctacgca aataactag aaaatctaga agaaatggat acattcctcg acacatacac tctcccaaga ctaaaacagg agaagttga atctctgaat ggaccaataa caggctctga attgtggca 1380 ataatcaata gtttaccac caaaagagt ccaggaccag atggattcac agccgaattc 1440 taccagaggt acaggagga actgtacca ttccttctga aactattcca atcaatagaa 1500 aaagagggaa tccccctaa ctcattttat gaggccagca tcattctgat accaagccg 1560 ggcagagaca caccaaaaa agagaatttt agaccaatat ccttgatgaa cattgatgca 1620 aaaatcctca attaaatact gggcaaccga atccagcagc accaaaaa gcttatccac 1680 catgatcaag tgggttcat ccctgggatg caggctggt tcaatacg caatcaata 1740 aatgtaatcc agcatataaa cagagccaaaaaaacc acatgatt ctcaatagat 1800 gcagaaaaag cctttgacaa aattcaaca cccttcatgc taaaaactct cataaatta 1860 ggtattgatg ggacgtattt caaatata agagctatct atgacaacc cacagccaat 1920 atcatactga atgggcaaa actggaagca ttcccttga aactggcac agacaggga 1980 tgccctctt caccgctcct attcacata gtgttggaag ttctggccag ggcaatcagg 2040 caggagaagg aaataaggg tattcatta ggaaaagagg aagtcaatt gtccctgttt 2100 gcagacgaca tgattgtta tctagaaac cccattgtct cagcccaaaa tctccttaag 2160 ctgataagca acttcagca agtctcagga tacaaatca atgtacaaa atcacaagca 2220 ttcttataca ccaacacag aaaacagag agccaaatca tggtgaact cccattcaca 2280 attgcttca agaggataaa atacctagga atccaactta cagggatgt gaaggaccctc 2340 ttcaaggaga actacaaacc actgctcaag gaaataaag aggacaaaaaatggaag 2400 aacattccat gctcatgggt aggagaatc atatcgtga aaatggccat actgcccaag 2460 gtaatttaca gattcaatgc catccccatc aagctaccaa tgactttctt cacagaattg 2520 gaaaaaacta ctttaaagtt caatggaac CAAAAAAG cccgcattgc caagtcaatc 2580 ctaagccaaa agacaaagc tggaggcatc acactacctt acttcaact attackacaag 2640 gctacagtaa ccaaacagc atggtactgg taccaaaca gagatataga tcaatggaac 2700 agaacagagc cctcagaaat atgccacat atctacaac atctgatctt tgacaaacct 2760 gagaaaaaca agcaatgggg aaaggattcc ctatttaata aatggtgctg ggaaaactgg 2820 ctagccatat gtagaaagct gaaactggat cctttcctta caccttatac aaaaatcaat 2880 tcaagatgga ttaaagattt aaacgttaaa cttaaaacca taaaaccct agaagaaaac 2940 ctaggcatta ccattcagga cataggcgtg ggcaaggact tcatgtccaa aacaccaaaa 3000 gcaatggcaa caaaagacaa aattgacaaa tgggatctaa ttaaactaaa gagcttctgc 3060 acagcaaaag aaactaccat cagagtgaac aggcaaccta caacatggga gaaaattttc 3120 gcaacctact catctgacaa agggctaata tccagaatct acaatgaact caaacaaatt 3180 tacaagaaaa aaacaaaaa ccccatcaaa aagtgggcga aggacatgaa cagacacttc 3240 tcaaaagaag acatttatgc agccaaaaaa cacatgaaga aatgctcatc atcactggcc 3300 atcagagaaa tgcaaatcaa aaccactatg agatatcatc tcacaccagt tagaatggca 3360 atcattaaaa agtcaggaaa caacaggtgc tggagaggat gcggagaaat aggaacactt 3420 ttacactgtt ggtgggactg taaactagtt caaccattgt ggaagtcagt gtggcgattc 3480 ctcagggatc tagataga ataccattt gacccagcca tcccattact gggtatatac 3540 ccagaggact ataaatcatg ctgcttaaa gatacatgca ctcgtatgtt tattgcggca 3600 ctattcacaa tagcaaaac ttggaaccaa cccaaatgtc ccaatgat agactggatt 3660 aagaaaatgt ggcacatata caccatggaa tattatgcag ccataaaaaa tgatgagttc 3720 atatcctttg taggacatg gatgaatttg gaaaccatca ttctcagtaa actatcgcaa 3780 gaacaaaaaa ccaacaccg catattctca ctcataggtg ggaattga 3828 <210> 6 <211> 1115 <212> DNA <213> Muscles <400> 6 atggcgaaag gtaaacggag gatcttact aacaggaacc agaccactc accatcacca 60 gaacccagca cacccactc gcccagtcca gggaacccca acacacctga gaacctagac 120 ctagatttaa aagcatatct catgatgatg gtagagggca tcagaagga ctttaataaa 180 tcacttaag aaatacagga gaacactgct aaagagttac aagtccttaa agaaaaacag 240 gaaaaaaaac attack agaagtcctt ahaaaaaag aggaaaaaac attackacag 300 gtgatggaaa tgaaaaac catactagac ctaaaaaggg aagtagacac aaaaaaaa 360 actcaaagcg agcacact agagatagaa accctaggaa agaatctgg aaccatagat 420 ttgagcatca gcaacagaat acagagatg gagagagaa tctcaggtgc agaacattcc 480 atagagaaca tcggcacaac atcaagaa atgaaag CAAAAGATc ctaaccaaa 540 atatccagga aatccaggac acaatagaa gaccaacgt acggataata ggagtggatg 600 frequency ttttc aaggtccag windtttxtxtxtx 660 acttcccaaa tctaaagaat gagatgcata tgaacataca agaagcctac agaactccaa 720 atagactgga ccagaaaga aattcctcccc gandacataat atcagaca tcaatgcac 780 taaaaaaaggtaaaagtaaaaaaagg tcaaaagg taaaaggca 840 agcctatcag attacacca gatttttcac cagagactat gaagccaga agagcctgga 900 cagatgttat acagacacta agagacaca aactgcagcc caggtacta tacccaccca 960 aactctcaat tatcatagag ggagaaacca aagtattcca cgacaaacc aattcacgc 1020 attatctctc cacgaatcca gccctcaa ggataac agaaaaaac catacaag 1080 acgggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaS <210> 7 <211> 3846 <212> DNA <213> Muscles <400> 7 atgccacctt acaactaa ataacagga agcaacatt acttttcctt atatctctt 60 aacaatcaatg gtctcaactc gccataaaa agacatagac taacaactg gctacacaaa 120 CAGAccCA catttgctg cttacaggaa actcatctca gagaaaaga tagacactac 180 ctcagaatga aaggctggaa aacaatttc caagcaatg gtagagaa acaagcagga 240 gtagccatcc taatatctga taagattgac ttccaaccca aagtcatcaa aaagacaag 300 gagggacact tcattcat aaggtaaa atcctccaag aggactctc aattctgaat 360 atctatgctc caatacaag agcagccaca ttcactaag aaactttagt aaagctcaa 420 gcacacattg cgcctcacac ataatagtg ggagacttca accaccact ttcaccaatg 480 multiplication ggaaacaga actaaaagg multiplication aactaaca multiplication 540 caaatggatc tgacagatat ctacagaaca ttttacctta aaaaaaagg ataccttc 600 ttctcagcac ctcatggtac cttctccaaa attgaccaca taataggtca caatcaggc 660 ctcacagat taaaaaatat tgaaattgtc ccatgtatcc tatcagatca ccatgcacta 720 aggctgatct tcaataca ataaataac agaaagccaa cattcacatg gaactgaac 780 aacactcttc tcaatgatc cttggtcaag gagaata agaagaat taagacttt 840 ttagagttta atgaaaatga agccacaacg tacccaacc ttgggacac atgaaagca 900 tttctaagag ggaactcat agctatgagt gccttcaaga aaaaacggga gagagcacat 960 actagcagct tgacacaca tctaaaagct ctagaaaaaa aggaagcaa ttcaccaag 1020 aggagtagac ggshaggaat atcaactc aggggtgaaa tcaccagt ggshaacaact 1080 agaactattc aagaattta ccaacgagg agttggttct ttgagaaat ccaacata 1140 gataaaccct tagctagact cactaaaggg cacagggaca aaatcctaat taacaaatc 1200 agaaatgaaa agggacaat aaaacagat cctgagaaa tccaaacac catcagatcc 1260 ttctacaaaa ggctatactc aacaaaactg gaaaacctgg acgaaatgga caaatttctg gacagatacc aggtaccaa gttgaatcag gatcaagttg accttctaaa cagtcccata tcccctaaag aaatagaagc agttattaat agtctcccag ccaaaaaag cccaggacca gacgggttta gtgcagagtt ctatcagacc ttcaaagaag atctaactcc agttctgcac aaactttttc acaagataga agtagaaggt attctaccca actcatttta tgaagccact attactctga tacctaacc acagaagat ccaacaaaga tagagaactt cagaccaatt tctcttatga acatcgatgc aaaaatcctt aataaaattc tcgctaaccg aatccaagaa cacattaag caatcatcca tcctgacca gtaggtttta ttccagggat gcagggatgg tttatatac gaaaatccat caatgtaatc cattatata acaaactcaa agacaaaaac cacatgatca tctcgttaga tgcagaaaaa gcatttgaca agatccaaca cccattcatg ataaaagttc tggaagatc aggaattcaa ggccaatacc taaacatgat aaaagcaatc 1980. 1980. 1980. 1980. 1980. 1980. 1980. 1980. 1980. 1980 aaatcaggga ctagacaagg ctgcccactt tctccctacc ttttcaacat agtacttgaa 2040 gtattagcca gagcaattcg acaacaaaag gagatcaagg ggatacaaat tggaaaagag 2100 gaagtcaaaa tatcactttt tgcagatgat atgatagtat atataagtga ccctaaaaat 2160 tccaacagag aactcctaaa cctgataaac agcttcggtg aagtagctgg atataaaatt 2220 aactcaaaca agtcaatggc ctttctctac acaaagaata aacaggctga gaaagaaatt 2280 agggaaacaa cacccttc atagccaca aataatataa aatatctcgg cgtgactcta 2340 acgaaggaag tgaaagatct gtatgataaa aacttcaagt ccctgaagaa agaaattaaa 2400 gaagatctca gaagatggaa agatctccca tgctcatgga ttggcaggac caacattgta 2460 aaaatggcta tcttgccaaa agcaatctac agattcaatg caatccccat taaattcca 2520 actcaattct tcaacgaatt agaaggagca atttgcaaat tcatctggaa taacaaaaaa 2580 ccgaggatag caaaaactct tctcaaggat aaaagaacct ctggtggaat caccatgcct 2640 gacctaaagc tttactacag agcaattgtg ataaaaactg catggtactg gtatagagac 2700 agacaagtag accaatggaa tagaattgaa gacccagaaa tgaacccaca cacctatggt 2760 cacttgatct tcgacaaggg agccaaaacc atccagtgga agaaagacag cattttcaac 2820 aattggtgct ggcacaactg gttgttatca tgtagaagaa tgcgaatcga tccatactta 2880 tctccttgta ctaaggtcaa atctaagtgg atcaaggaac ttcacataaa accagagaca 2940 ctgaaactta tagaggaagaa agtgggggaaa agtcttgaag atatgggcac agggaaaaa 3000 ttcctgaaca gaacagcaat ggcttgtgct gtaagatcga gaattgacaa atgggaccta 3060 atgaaactcc aaagtttctg caaggcaaaa gacactgtct ataagacaaa aagaccacca 3120 acagactggg aaaggatctt tacctatcct aaatcagata ggggactaat atccaacata 3180 tataaagaac tcaagaaggt ggacctcaga aaatcaaata accccccttaa aaaatggggc 3240 tcagaactga acaaagaatt ctcacctgag gaataccgaa tggcagagaa gcacctgaaa 3300 aaatgttcaa catccttaat catcagggaa atgcaaatca aaacaaccct gagattccac 3360 ctcacaccag tgagaatggc taagatcaaa aattcaggtg acagcagatg ctggcgagga 3420 tgtggagaaa gaggaacact cctccattgt tggtggtt gcaggcttgt acaaccactc 3480 tggaaatcag tctggcggtt cctcagaaaa tggacatag tactaccgga ggatccagca 3540 atacctctcc tgggcatata tccagaagaa gcccaactg gtagagagga cacatgctcc 3600 actatgttca tagcagcctt attatata gccagaaact ggaagaacc cagatgcccc 3660 tcacagagg atggataca gaaatgtgg tacatctaca caatggagta ctactcagct 3720 attaaaaga atgaatttat gaaattccta gccaaatgga tggacctgga gagcatcatc 3780 ctgagtgagg taacacaatc aaaggaac tcacacata tgtactcact gataagtgga 3840 tag 3846 <210> 8 <211> 405 <212> DNA <213> Homo sapiens <400> 8 gggtagagat tcactgcctt agtctcatgt agtctcgtgt agtctttga gtaaataaca 60 taaagtatct caacttt tcatacttg atattattt agtctctcctg aatttttaaa 120 tattgaaaag ctgagtgtct tgtctgtttt cctcccctt acactatagt gacggggcta 180 gtcaagcttt ggcaagttgc cagagggact tccgcaacaa accctatcct gtccgagcaa 240 agattaccta ttaccagaac acactgacag taagtaacat ctatttagag agaatcaaat 300 aaacaatgtt acagtatcac ttttcatttt gaatttttga tagaaattaa atgcacttaa 360 atttggatat gcttacatac tcttcattgt tactctaaga gaacg 405 <210> 9 <211> 150 <212> DNA <213> Artificial sequence <400> 9 aggtgcctgc acatactgca tgtgagagtc tggagacgcc agactgttct gagtcctgac 60 ctgctcaggg gtgaggtccc tctgagcctg agcaagcatt tcgtagccaa ccatgaattt 120 ccggacagtg gcagagcgca ggagcggagg 150 <210> 10 <211> 555 <212> DNA <213> Artificial sequence <400> 10 gggtagagat tcactgcctt agtctcatgt agtctcgtgt agtcttttga gtaaataaca 60 taaagtatct caagactttt tcataacttg atattatttt agtcttcctg aatttttaaa 120 tattgaaaag ctgagtgtct tgtctgtttt aggtgcctgc acatactgca tgtgagagtc 180 tggagacgcc agactgttct gagtcctgac ctgctcaggg gtgaggtccc tctgagcctg 240 agcaagcatt tcgtagccaa ccatgaattt ccggacagtg gcagagcgca ggagcggagg 300 cctccccctt acactatagt gacggggcta gtcaagcttt ggcaagttgc cagagggact 360 tccgcaacaa accctatcct gtccgagcaa agattaccta ttaccagaac acactgacag 420 taagtaacat ctatttagag agaatcaaat aaacaatgtt acagtatcac ttttcatttt 480 gaatttttga tagaaattaa atgcacttaa atttggatat gcttacatac tcttcattgt 540 tactctaaga gaacg 555 <210> 11 <211> 17 <212> DNA <213> Bacteriophage T7 <400> 11 taatacgact cactata 17 <210> 12 <211> 789 <212> DNA <213> Artificial Sequence <400> 12 taatacgact cactataggg tagagattca ctgccttagt ctcatgtagt ctcgtgtagt 60 cttttgagta aataacataa agtatctcaa gactttttca taacttgata ttattttagt 120 cttcctgaat ttttaaatat tgaaaagctg agtgtcttgt ctgttttagg tgcctgcaca 180 tactgcatgt gagagtctgg agacgccaga ctgttctgag tcctgacctg ctcaggggtg 240 aggtccctct gagcctgagc aagcattcg tagccaacca tgaatttccg gagtggca 300 gagcgcagga gcggaggcct ccccttaca ctatagtgac ggggctagtc aagctttggc 360 aagttgccag agggacttcc gcaacaacc ctacctgtc cgagcaaga ttacctatta 420 ccagacaca ctgacagtaa gtaacatcta tttagagaga atcaataa caatgttaca 480 gtatcacttt tcattttgaa ttttgatag aaattaaatg cacttaaatt tggatatgct 540 tacatactct tcattgttac tctaagagaa cgactaaaaa tacaaaaaat tagccggggcg 600 tggtggcggg cgcctgtagt cccagctact cgggaggctg aggcaggaga atggcgtgaa 660 cccgggaggc ggagcttgca gtgagccgag atcacgccgc tgcactccac cctggggcgac 720 agagcgagac tccgtctca aaaaaaaaa aaaaaaaaaaaaaaaga ttaatactg 780 ctggagatc 789 <210> 13 <211> 572 <212> DNA <213> Artificial Sequence <400> 13 taatacgact cactataggg tagagattca ctgccttagt ctcatgtagt ctcgtgtagt 60 cttttgagta aataacataa agtatctcaa gactttttca taacttgata ttattttagt 120 cttcctgaat ttttaaatat tgaaaagctg agtgtcttgt ctgttttagg tgcctgcaca 180 tactgcatgt gagagtctgg agacgccaga ctgttctgag tcctgacctg ctcaggggtg 240 aggtccctct gagcctgagc aagcatttcg tagccaacca tgaatttccg gacagtggca 300 gagcgcagga gcggaggcct cccccttaca ctatagtgac ggggctagtc aagctttggc 360 aagttgccag agggacttcc gcaacaaacc ctatcctgtc cgagcaaaga ttacctatta 420 ccagaacaca ctgacagtaa gtaacatcta tttagagaga atcaaataaa caatgttaca 480 gtatcacttt tcattttgaa tttttgatag aaattaaatg cacttaaatt tggatatgct 540 tacatactct tcattgttac tctaagagaa cg 572 <210> 14 <211> 772 <212> DNA <213> Artificial Sequence <400> 14 gggtagagat tcactgcctt agtctcatgt agtctcgtgt agtctttga gtaaataaca 60 taaagtatct caacttt tcatacttg atattattt agtctctcctg aatttttaaa 120 tattgaaaag ctgagtgtct tgtctgtttt aggtgcctgc acatactgca tgtgagagtc 180 tggacgcc agactgttct gagtcctgac ctgctcaggg gtgaggtccc tctgagcctg 240 agcaagcatt tcgtagccaa ccatgaattt ccggacagtg gcagagcgca ggagcggagg 300 cctccccctt acactatagt gacggggcta gtcaagcttt ggcaagttgc cagagggact 360 tccgcaacaa accctacct gtccgagcaa agattaccta ttaccagaac acactgacag 420 taagtacat ctatttagag agaatcaat aaacaatgtt acagtacac tttcatttt 480 gatttttga tagaaattta atgcacttaa atttggatat gcttacatac tcttcattgt 540 tactctaaga gaacgactaa aaatacaaa aattagccgg gcgtggtggc gggcgcctgt 600 agtcccagct actcgggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt 660 gcagtgagcc gagatcacgc cgctgcactc caccctgggc gagagagcga gactccgtct 720 caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agattaataa ctgctggaga tc 772 <210> 15 <211> 32 <212> DNA <213> Artificial sequence <400> 15 ctatataagc agagctgggt agagattcac tg 32 <210> 16 <211> 32 <212> DNA <213> Artificial sequence <400> 16 ctctagttag ccagaggatc tccagcagtt at 32 <210> 17 <211> 33 <212> DNA <213> Artificial sequence <400> 17 aataactgct ggagatcctc tggctaactagag 33 <210> 18 <211> 32 <212> DNA <213> Artificial sequence <400> 18 cagtgaatct ctacccagct ctgcttatat ag 32 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <400> 19 cactgccacc cagaagactg 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <400> 20 cctgcttcac caccttcttg 20 <210> 21 <211> 20 <212> DNA <213> Artificial sequence <400> 21 gacttatcca tgtgcctgtt 20 <210> 22 <211> 18 <212> DNA <213> Artificial sequence <400> 22 ttggctacga aatgcttg 18 <210> 23 <211> 360 <212> DNA <213> Homo sapiens <400> 23 ggggttcggc cctgcccgta gcacagccaa gccctacctc tcggttatct tttctcccgt 60 caccacccag taaggtcatg tgcttccacc cctggtcgga tgtaacgctg ccactcatgt 120 cggtccctga gatccgggct gttgttgatg catgggcctc agtcacagag gagctgggtg 180 cccagtaccc ttgggtgcag gtttgtgagg tcgccccttc ccctggatgg gcagggaggg 240 ggtgatgaag ctttggttct ggggagtaac atttctgttt ccacagggtg tggtcaggag 300 ggagttgact tggtgtcttt tggctaacag agctccgtat ccctatctga tagatctttg 360 <210> 24 <211> 150 <212> DNA <213> Artificial sequence <400> 24 tgactactga gattactttg acatgtccca cttattaata tcaccttaag tttgggttcg 60 tgactactga gattactttg acatgtccca cttattaata tcaccttaag tttgggttcg 60 attaatatta tgtaacctgt gaacgagata agattctaga gatttaatcg aaccttaatt 120 attaatatta tgtaacctgt gaacgagata agattctaga gatttaatcg aaccttaatt 120 ctgattcggt tatgtcaaaa ggtgtcttga 150 ctgattcggt tatgtcaaaa ggtgtcttga 150 <210> 25<210> 25 <211> 510<211> 510 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <400> 25<400> 25 ggggttcggc cctgcccgta gcacagccaa gccctacctc tcggttatct tttctcccgt 60 ggggttcggc cctgcccgta gcacagccaa gccctacctc tcggttatct tttctcccgt 60 caccacccag taaggtcatg tgcttccacc cctggtcgga tgtaacgctg ccactcatgt 120 caccacccag taaggtcatg tgcttccacc cctggtcgga tgtaacgctg ccactcatgt 120 cggtccctga gatccgggct gttgttgtga ctactgagat tactttgaca tgtcccactt 180 cggtccctga gatccgggct gttgttgtga ctactgagat tactttgaca tgtcccactt 180 attaatatca ccttaagttt gggttcgatt aatattatgt aacctgtgaa cgagataaga 240 attaatatca ccttaagttt gggttcgatt aatattatgt aacctgtgaa cgagataaga 240 ttctagagat ttaatcgaac cttaattctg attcggttat gtcaaaaggt gtcttgaatg 300 ttctagagat ttaatcgaac cttaattctg attcggttat gtcaaaaggt gtcttgaatg 300 catgggcctc agtcacagag gagctgggtg cccagtaccc ttgggtgcag gtttgtgagg 360 catgggcctc agtcacagag gagctgggtg cccagtaccc ttgggtgcag gtttgtgagg 360 tcgccccttc ccctggatgg gcagggaggg ggtgatgaag ctttggttct ggggagtaac 420 tcgccccttc ccctggatgg gcagggaggg ggtgatgaag ctttggttct ggggagtaac 420 atttctgttt ccacagggtg tggtcaggag ggagttgact tggtgtcttt tggctaacag 480 atttctgttt ccacagggtg tggtcaggag ggagttgact tggtgtcttt tggctaacag 480 agctccgtat ccctatctga tagatctttg 510 <210> 26 <211> 860 <212> DNA <213> Artificial Sequence <400> 26 taatacgact cactataggg gttcggccct gcccgtagca cagccaagcc ctacctctcg 60 gttatctttt ctcccgtcac cacccagtaa ggtcatgtgc ttccacccct ggtcggatgt 120 aacgctgcca ctcatgtcgg tccctgagat ccgggctgtt gttgtgacta ctgagattac 180 tttgacatgt cccacttatt aatatcacct taagtttggg ttcgattaat attatgtaac 240 ctgtgaacga gataagattc tagagattta atcgaacctt aattctgatt cggttatgtc 300 aaaaggtgtc ttgaatgcat gggcctcagt cacagaggag ctgggtgccc agtacccttg 360 ggtgcaggtt tgtgaggtcg ccccttcccc tggatgggca gggagggggt gatgaagctt 420 tggttctggg gagtaacatt tctgtttcca cagggtgtgg tcaggaggga gttgacttgg 480 tgtcttttgg ctaacagagc tccgtatccc tatctgatag atctttgggg ccgggcgcgg 540 tggctcacgc ctgtaatccc agcactttgg gaggccgagg cgggcggatc acgaggtcag 600 gagatcgaga ccatcccggc taaaacggtg aaaccccgtc tctactaaaa atacaaaaaa 660 ttagccgggc gtggtggcgg gcgcctgtag tcccagctac tcgggaggct gaggcaggag 720 aatggcgtga acccgggagg cggagcttgc agtgagccga gatcacgccg ctgcactcca 780 ccctgggcga cagagcgaga ctccgtctca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 840 attaataact gctggagatc 860 <210> 27 <211> 527 <212> DNA <213> Artificial Sequence <400> 27 taatacgact cactataggg gttcggccct gcccgtagca cagccaagcc ctacctctcg 60 gttatctttt ctcccgtcac cacccagtaa ggtcatgtgc ttccacccct ggtcggatgt 120 aacgctgcca ctcatgtcgg tccctgagat ccgggctgtt gttgtgacta ctgagattac 180 tttgacatgt cccacttatt aatatcacct taagtttggg ttcgattaat attatgtaac 240 ctgtgaacga gataagattc tagagattta atcgaacctt aattctgatt cggttatgtc 300 aaaaggtgtc ttgaatgcat gggcctcagt cacagaggag ctgggtgccc agtacccttg 360 ggtgcaggtt tgtgaggtcg ccccttcccc tggatgggca gggagggggt gatgaagctt 420 tggttctggg gagtaacatt tctgtttcca cagggtgtgg tcaggaggga gttgacttgg 480 tgtcttttgg ctaacagagc tccgtatccc tatctgatag atctttg 527 <210> 28 <211> 843 <212> DNA <213> Artificial Sequence <400> 28 ggggttcggc cctgcccgta gcacagccaa gccctacctc tcggttatct tttctcccgt 60 caccacccag taaggtcatg tgcttccacc cctggtcgga tgtaacgctg ccactcatgt 120 cggtccctga gatccgggct gttgttgtga ctactgagat tactttgaca tgtcccactt 180 attaatatca ccttaagttt gggttcgatt aatattatgt aacctgtgaa cgagataaga 240 ttctagagat ttaatcgaac cttaattctg attcggttat gtcaaaaggt gtcttgaatg 300 catgggcctc agtcacagag gagctgggtg cccagtaccc ttgggtgcag gtttgtgagg 360 tcgccccttc ccctggatgg gcagggaggg ggtgatgaag ctttggttct ggggagtaac 420 atttctgttt ccacagggtg tggtcaggag ggagttgact tggtgtcttt tggctaacag 480 agctccgtat ccctatctga tagatctttg gggccgggcg cggtggctca cgcctgtaat 540 cccagcactt tgggaggccg aggcgggcgg atcacgaggt caggagatcg agaccatccc 600 ggctaaaacg gtgaaacccc gtctctacta aaaatacaaa aaattagccg ggcgtggtgg 660 cgggcgcctg tagtcccagc tactcgggag gctgaggcag gagaatggcg tgaacccggg 720 aggcggagct tgcagtgagc cgagatcacg ccgctgcact ccaccctggg cgacagagcg 780 agactccgtc tcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagattaata actgctggag 840 atc 843 <210> 29 <211> 29 <212> DNA <213> Artificial sequence <400> 29 ctatataagc agagctgggg ttcggccct 29 <210> 30 <211> 29 <212> DNA <213> Artificial sequence <400> 30 agggccgaac cccagctctg cttatatag 29 <210> 31 <211> 18 <212> DNA <213> Artificial sequence <400> 31 ccccagtacg atagcacc 18 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <400> 32 gacataaccg aatcagaatt 20 <210> 33 <211> 400 <212> DNA <213> Homo sapiens <400> 33 gagattcact gccttagtct catgtagtct cgtgtagtct tttgagtaaa taacataaag 60 tatctcaaga ctttttcata acttgatatt attttagtct tcctgaattt ttaaatattg 120 aaaagctgag tgtcttgtct gttttcctcc cccttacact atagtgacgg ggctagtcaa 180 gctttggcaa gttgccagag ggacttccgc aacaaaccct atcctgtccg agcaaagatt 240 acctattacc agaacacact gacagtaagt aacatctatt tagagagaat caaataaaca 300 atgttacagt atcacttttc attttgaatt tttgatagaa attaaatgca cttaaatttg 360 gatatgctta catactcttc attgttactc taagagaacg 400 <210> 34 <211> 550 <212> DNA <213> Artificial sequence <400> 34 gagattcact gccttagtct catgtagtct cgtgtagtct tttgagtaaa taacataaag 60 tatctcaaga ctttttcata acttgatatt attttagtct tcctgaattt ttaaatattg 120 aaaagctgag tgtcttgtct gttttcctcc cccttacact atagtgacgg ggctagtcaa 180 gctttggcaa gttgccagag ggacttccgc aacaaaccct atcctgtccg agcaaaggtg 240 cctgcacata ctgcatgtga gagtctggag acgccagact gttctgagtc ctgacctgct 300 caggggtgag gtccctctga gcctgagcaa gcatttcgta gccaaccatg aatttccgga 360 cagtggcaga gcgcaggagc ggaggagatt acctattacc agaacacact gacagtaagt 420 aacatctatt tagagagaat caaataaaca atgttacagt atcacttttc attttgaatt 480 tttgatagaa attaaatgca cttaaatttg gatatgctta catactcttc attgttactc 540 taagagaacg 550 <210> 35 <211> 17 <212> DNA <213> Bacteriophage SP6 <400> 35 atttaggtga cactata 17 <210> 36 <211> 243 <212> DNA <213> Artificial Sequence <400> 36 acaatgagat cacatggaca caggaagggg aatatcacac tctggggact gtggtggggt 60 cgggggaggg gggaggggta gcattgggag atatacctaa tgctagatga cacattagtg 120 ggtgcagcgc accagcatgg cacatgtata catatgtaac taacctgcac aatgtgcaca 180 tgtaccctaa aacttagagt ataattaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240 aaa 243 <210> 37 <211> 810 <212> DNA <213> Artificial Sequence <400> 37 atttaggtga cactatagag attcactgcc ttagtctcat gtagtctcgt gtagtctttt 60 gagtaaataa cataaagtat ctcaagactt tttcataact tgatattatt ttagtcttcc 120 tgaattttta aatattgaaa agctgagtgt cttgtctgtt ttcctccccc ttacactata 180 gtgacggggc tagtcaagct ttggcaagtt gccagaggga cttccgcaac aaaccctatc 240 ctgtccgagc aaaggtgcct gcacatactg catgtgagag tctggagacg ccagactgtt 300 ctgagtcctg acctgctcag gggtgaggtc cctctgagcc tgagcaagca tttcgtagcc 360 aaccatgaat ttccggacag tggcagagcg caggagcgga ggagattacc tattaccaga 420 acacactgac agtaagtaac atctatttag agagaatcaa ataaacaatg ttacagtatc 480 acttttcatt ttgaattttt gatagaaatt aaatgcactt aaatttggat atgcttacat 540 actcttcatt gttactctaa gagaacgaca atgagatcac atggacacag gaaggggaat 600 atcacactct ggggactgtg gtggggtcgg gggagggggg aggggtagca ttgggagata 660 tacctaatgc tagatgacac attagtgggt gcagcgcacc agcatggcac atgtatacat 720 atgtaactaa cctgcacaat gtgcacatgt accctaaaac ttagagtata attaaaaaaa 780 aaaaaaaaaaaaaaaaaaaaaaaaaaaaa 810 <210> 38 <211> 213 <212> DNA <213> Homo sapiens <400> 38 atgcatgggc ctcagtcaca gaggagctgg gtgcccagta cccttgggtg caggttttgtg 60 aggtcgcccc ttcccctgga tgggcaggga gggggtgatg aagctttggt tctggggagt 120 aacatttctg tttccacagg gtgtggtcag gagggagttg acttggtgtc ttttggctaa 180 cagagctccg tatccctatc tgatagatct ttg 213 <210> 39 <211> 103 <212> DNA <213> Homo sapiens <400> 39 aaaacaaagg tgccatgatg ggctgttcta acccccaccc ccactgccag gtaagggtgt 60 caggggctcc agtgggtttc ttggctgagt ctgagccagc act 103 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <400> 40 ctgaccatgc ttatacggac tatcgattag 30 <210> 41 <211> 660 <212> DNA <213> Artificial Sequence <400> 41 taatacgact cactataggg gttcggccct gcccgtagca cagccaagcc ctacctctcg 60 gttatctttt ctcccgtcac cacccagtaa ggtcatgtgc ttccacccct ggtcggatgt 120 aacgctgcca ctcatgtcgg tccctgagat ccgggctgtt gttgtgacta ctgagattac 180 tttgacatgt cccacttatt aatatcacct taagtttggg ttcgattaat attatgtaac 240 ctgtgaacga gataagattc tagagattta atcgaacctt aattctgatt cggttatgtc 300 aaaaggtgtc ttgaatgcat gggcctcagt cacagaggag ctgggtgccc agtacccttg 360 ggtgcaggtt tgtgaggtcg ccccttcccc tggatgggca gggagggggt gatgaagctt 420 tggttctggg gagtaacatt tctgtttcca cagggtgtgg tcaggaggga gttgacttgg 480 tgtcttttgg ctaacagagc tccgtatccc tatctgatag atctttgctg accatgctta 540 tacggactat cgattagaaa acaaaggtgc catgatgggc tgttctaacc cccaccccca 600 ctgccaggta agggtgtcag gggctccagt gggtttcttg gctgagtctg agccagcact 660 <210> 42 <211> 250 <212> DNA <213> Homo sapiens <400> 42 aaaatgccac tgagaactct cttaagacta cctttctcca aatggtgccc ttcactcaag 60 cctgtggttt tggtcttagg aactttgctg ccacaatacc tcggcccttc tcagttcgct 120 acgacccata cacccaaagg attgaggtct tggacaatac ccagcagctt aagattttgg 180 ctgattccat taacagtaag taatttacac cttacgaggc cactcggttt ctcagtaatc 240 gaagactgtc 250 <210> 43 <211> 250 <212> DNA <213> Homo sapiens <400> 43 aaaatgccac tgagaactct cttaagacta cctttctcca aatggtgccc ttcactcaag 60 cctgtggttt tggtcttagg aactttgctg ccacaatacc tcggcccttc tcagttccct 120 acgacccata cacccaaagg attgaggtct tggacaatac ccagcagctt aagattttgg 180 ctgattccat taacagtaag taatttacac cttacgaggc cactcggttt ctcagtaatc 240 gaagactgtc 250 <210> 44 <211> 700 <212> DNA <213> Artificial Sequence <400> 44 aaaatgccac tgagaactct cttaagacta cctttctcca aatggtgccc ttcactcaag 60 cctgtggttt tggtcttagg aactttgctg ccacaatacc tcggcccttc tcagttcgct 120 acgacccata cacccaaagg attgaggtct tggacaatac ccagcagctt aagattttgg 180 ctgattccat taacagtaag taatttacac cttacgaggc cactcggttt ctcagtaatc 240 gaagactgtc aaaatgccac tgagaactct cttaagacta cctttctcca aatggtgccc 300 ttcactcaag cctgtggttt tggtcttagg aactttgctg ccacaatacc tcggcccttc 360 tcagttccct acgacccata cacccaaagg attgaggtct tggacaatac ccagcagctt 420 aagattttgg ctgattccat taacagtaag taatttacac cttacgaggc cactcggttt 480 ctcagtaatc gaagactgtc tttccctacc atcgccatag gaaaaataat aaatttattg 540 aaatatttaa ttaaggagaa aagcacctcc atgtaagcca tgggttcatt gatggagaag 600 aacttgacaa aaaggtcaga attacccttg tgtccttttt cctttgacct tcctagattc 660 cactccacct cctaccatca ttccaccttt ccacacttgg 700 <210> 45 <211> 917 <212> DNA <213> Artificial Sequence <400> 45 aaaatgccac tgagaactct cttaagacta cctttctcca aatggtgccc ttcactcaag 60 cctgtggttt tggtcttagg aactttgctg ccacaatacc tcggcccttc tcagttcgct 120 acgacccata cacccaaagg attgaggtct tggacaatac ccagcagctt aagattttgg 180 ctgattccat taacagtaag taatttacac cttacgaggc cactcggttt ctcagtaatc 240 gaagactgtc aaaatgccac tgagaactct cttaagacta cctttctcca aatggtgcc 300 ttcactcaag cctgtggttt tggtcttagg aactttgctg ccacaatacc tcggcccttc 360 tcagttccct acgacccata cacccaaagg attgaggtct tggacaatac ccagcagctt 420 aagattttgg ctgattccat taacagtaag taatttacac cttacgaggc cactcggttt 480 ctcagtaatc gaagactgtc tttccctacc atcgccatag gaaaaataat aaatttattg 540 aaatttaa ttaaggagaa aagcacctcc atgtaagcca tgggttcatt gatggagaag 600 aacttgacaa aaaggtcaga attacccttg tgtcctttttt cctttgacct tcctagattc 660 cactccacct cctaccatca ttccaccttt ccacacttgg actaaaaata caaaaaatta 720 gccgggcgtg gtggcgggcg cctgtagtcc cagctactcg ggaggctgag gcaggagaat 780 ggcgtgaacc cgggaggcgg agcttgcagt gagccgagat cacgccgctg cactccaccc 840 tgggcgacag agcgagactc cgtctcaaaa aaaaaaaaa aaaaaaaaa aaaaaagatt 900 aataactgct ggagatc 917 <210> 46 <211> 32 <212> DNA <213> Artificial sequence <400> 46 ctatataagc agagctaaaa tgccactgag aa 32 <210> 47 <211> 33 <212> DNA <213> Artificial sequence <400> 47 gttctcagtg gcattttagc tctgcttata tag 33 <210> 48 <211> 18 <212> DNA <213> Artificial sequence <400> 48 agggaggtgt ccgtgttc 18 <210> 49 <211> 18 <212> DNA <213> Artificial sequence <400> 49 gggtgtatgg gtcgtagc 18 <210> 50 <211> 350 <212> DNA <213> Artificial sequence <400> 50 taatacgact cactataggg ccgggcgcgg tggctcacgc ctgtaatccc agcactttgg 60 gaggccgagg cgggcggatc acgaggtcag gagatcgaga ccatcccggc taaaacggtg 120 aaaccccgtc tctactaaaa atacaaaaaa ttagccgggc gtggtggcgg gcgcctgtag 180 tcccagctac tcgggaggct gaggcaggag aatggcgtga acccgggagg cggagcttgc 240 agtgagccga gatcacgccg ctgcactcca ccctgggcga cagagcgaga ctccgtctca 300 aaaaaaaaaa aaaaaaaaaa aaaaaaaaag attaataact gctggagatc 350 <210> 51 <211> 335 <212> DNA <213> Homo sapiens <400> 51 ggccgggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggccga ggcgggtgga 60 tcatgaggtc aggagatcga gaccatcctg gctaacaagg tgaaaccccg tctctactaa 120 aaatacaaaa aaaaaaaatt agccgggcgc ggtggcgggc gcctgtagtc ccagctactc 180 gggaggctga ggcaggagaa tggcgtgaac ccgggaagcg gagcttgcag tgagccgaga 240 ttgcgccact gcagtccgca gtccggccta ggcgacagag cgagactccg tctcaaaaaa 300 aaaaaaaaaa aaaatgtggc caaaagtgtc agaaa 335 <210> 52 <211> 217 <212> DNA <213> Artificial Sequence <400> 52 aaaaatacaa aaaaaaaaaa ttagccgggc gcggtggcgg gcgcctgtag tcccagctac 60 tcgggaggct gaggcaggag aatggcgtga acccgggaag cggagcttgc agtgagccga 120 gattgcgcca ctgcagtccg cagtccggcc taggcgacag agcgagactc cgtctcaaaa 180 aaaaaaaaaa aaaaaatgtg gccaaaagtg tcagaaa 217 <210> 53 <211> 789 <212> DNA <213> Artificial sequence <400> 53 taatacgact cactataggg tagagattca ctgccttagt ctcatgtagt ctcgtgtagt 60 cttttgagta aataacataa agtatctcaa gactttttca taacttgata ttattttagt 120 cttcctgaat ttttaaatat tgaaaagctg agtgtcttgt ctgttttagg tgcctgcaca 180 tactgcatgt gagagtctgg agacgccaga ctgttctgag tcctgacctg ctcaggggtg 240 aggtccctct gagcctgagc aagcatttcg tagccaacca tgaatttccg gacagtggca 300 gagcgcagga gcggaggcct cccccttaca ctatagtgac ggggctagtc aagctttggc 360 aagttgccag agggacttcc gcaacaaacc ctatcctgtc cgagcaaaga ttacctatta 420 ccagaacaca ctgacagtaa gtaacatcta tttagagaga atcaaataaa caatgttaca 480 gtatcacttt tcattttgaa tttttgatag aaattaaatg cacttaaatt tggatatgct 540 tacatactct tcattgttac tctaagagaa cgaaaaatac aaaaaaaaa aattagccgg 600 gcgcggtggc gggcgcctgt agtcccagct actcgggagg ctgaggcagg agaatggcgt 660 gaacccggga agcggagctt gcagtgagcc gagattgcgc cactgcagtc cgcagtccgg 720 cctaggcgac agagcgagac tccgtctcaa aaaaaaaaa aaaaaaatg tggccaaaag 780 tgtcagaaa 789

Claims

1. An RNA framework for gene editing, characterized in that, The RNA framework includes the upstream sequence of the target site, the sequence to be inserted, and the downstream sequence of the target site along the 5′→3′ direction. The upstream sequence of the target site or its complementary sequence on the RNA framework is used to hybridize with the upstream sequence of the target site or its complementary sequence on the target site in a eukaryotic or prokaryotic genome; the downstream sequence of the target site or its complementary sequence on the RNA framework is used to hybridize with the downstream sequence of the target site or its complementary sequence on the target site in a eukaryotic or prokaryotic genome; the upstream sequence of the target site and the downstream sequence of the target site on the RNA framework are directly linked in their corresponding sequences in the genome; The target site is located between the upstream and downstream sequences of the target site in the genome sequence. The RNA framework for gene editing further includes: directly or indirectly linking one or more ORF2p functional initiation portions downstream of the target site sequence; wherein the multiple ORF2p functional initiation portions are directly or indirectly linked together. One or more ORF1p encoding sequences and / or one or more ORF2p encoding sequences are further inserted inside the ORF2p function start portion; wherein, when the ORF2p function start portion inserts one ORF1p encoding sequence or one ORF2p encoding sequence, the ORF2p function start portion is directly or indirectly connected to the ORF1p encoding sequence or the ORF2p encoding sequence; when the ORF2p function start portion inserts a) multiple ORF1p encoding sequences, or b) multiple ORF2p encoding sequences, or c) the sum of the number of ORF1p encoding sequences and ORF2p encoding sequences is greater than or equal to two, the ORF1p encoding sequences are directly or indirectly connected to the ORF2p encoding sequences, the ORF1p encoding sequences are directly or indirectly connected to each other, the ORF2p encoding sequences are directly or indirectly connected to each other, and the ORF2p function start portion is directly or indirectly connected to the ORF1p encoding sequence or the ORF2p encoding sequence. The sequence of the ORF2p functional initiation portion is either a short sporadic element RNA or a long sporadic element RNA.

2. The RNA framework for gene editing as described in claim 1, characterized in that, The RNA framework further includes one or more ORF1p coding sequences and / or one or more ORF2p coding sequences directly or indirectly connected upstream of the target site, and / or inside the upstream sequence of the target site, and / or inside the downstream sequence of the target site, and / or downstream of the downstream sequence of the target site.

3. The RNA framework for gene editing as described in claim 2, characterized in that, When one or more ORF1p coding sequences and / or one or more ORF2p coding sequences are located upstream of the upstream sequence of the target site, inside the upstream sequence of the target site, inside the downstream sequence of the target site, or downstream of the downstream sequence of the target site, and at the same position a) the sum of the number of multiple ORF1p coding sequences is greater than or equal to two, or b) the sum of the number of multiple ORF2p coding sequences is greater than or equal to two, or c) the sum of the number of ORF1p coding sequences and ORF2p coding sequences is greater than or equal to two, the ORF1p coding sequences are directly or indirectly connected to the ORF2p coding sequences, the ORF1p coding sequences are directly or indirectly connected to each other, and the ORF2p coding sequences are directly or indirectly connected to each other.

4. The RNA framework for gene editing as described in claim 2, characterized in that, The RNA framework for gene editing further includes one or more ORF2p functional initiation portions directly or indirectly connected downstream of the target site sequence.

5. The RNA framework for gene editing as described in claim 4, characterized in that, When one or more ORF1p coding sequences and / or one or more ORF2p coding sequences are located upstream of the target site, inside the target site's upstream sequence, or inside the target site's downstream sequence, and at the same location, a) the sum of the number of multiple ORF1p coding sequences is greater than or equal to two, or b) the sum of the number of multiple ORF2p coding sequences is greater than or equal to two, or c) the sum of the number of ORF1p coding sequences and ORF2p coding sequences is greater than or equal to two, the ORF1p coding sequences are directly or indirectly connected to the ORF2p coding sequences, the ORF1p coding sequences are directly or indirectly connected to each other, and the ORF2p coding sequences are directly or indirectly connected to each other.

6. The RNA framework for gene editing as described in claim 4, characterized in that, When the one or more ORF1p coding sequences and / or one or more ORF2p coding sequences are located downstream of the target site downstream sequence: a) When there are one or more ORF2p function start portions and one or more ORF1p encoding sequences, the one or more ORF2p function start portions are located before or after the one or more ORF1p encoding sequences, or the ORF2p function start portions and ORF1p encoding sequences are arranged alternately, the ORF2p function start portions and the ORF1p encoding sequences are directly or indirectly connected, the multiple ORF1p encoding sequences are directly or indirectly connected, and the multiple ORF2p function start portions are directly or indirectly connected; or b) When there are one or more ORF2p function start portions and one or more ORF2p encoding sequences, the one or more ORF2p function start portions are located before or after the one or more ORF2p encoding sequences, or the ORF2p function start portions and ORF2p encoding sequences are arranged alternately, the ORF2p function start portions and the ORF2p encoding sequences are directly or indirectly connected, the multiple ORF2p encoding sequences are directly or indirectly connected, and the multiple ORF2p function start portions are directly or indirectly connected; or c) When there are one or more ORF2p function start portions, one or more ORF1p encoding sequences, and one or more ORF2p encoding sequences, the ORF2p function start portion is located before or after the one or more ORF1p encoding sequences, or before or after the one or more ORF2p encoding sequences, or the one or more ORF1p encoding sequences are located before or after the one or more ORF2p encoding sequences, or the ORF2p function start portion, the ORF1p encoding sequence, and / or the ORF2p encoding sequence are arranged alternately; the ORF2p function start portion and the ORF1p encoding sequence are directly or indirectly connected, the ORF2p function start portion and the ORF2p encoding sequence are directly or indirectly connected, and the multiple ORF1p encoding sequences are directly or indirectly connected; the multiple ORF2p encoding sequences are directly or indirectly connected, the multiple ORF2p function start portions are directly or indirectly connected, and the ORF1p encoding sequence and the ORF2p encoding sequence are directly or indirectly connected.

7. The RNA framework for gene editing as described in any one of claims 4-6, characterized in that, In the RNA framework, one or more ORF2p functional start portions, wherein a single ORF2p functional start portion is further directly or indirectly connected to one or more ORF1p coding sequences and / or one or more ORF2p coding sequences, wherein, when the ORF2p functional start portion contains one ORF1p coding sequence or one ORF2p coding sequence, the ORF2p functional start portion is directly or indirectly connected to the ORF1p coding sequence or the ORF2p coding sequence; when the ORF2p functional start portion contains a) multiple ORF1p coding sequences, or b) multiple ORF2p coding sequences, or c) the sum of the number of ORF1p coding sequences and ORF2p coding sequences is greater than or equal to two, the ORF1p coding sequences are directly or indirectly connected to the ORF2p coding sequences, the ORF1p coding sequences are directly or indirectly connected to each other, the ORF2p coding sequences are directly or indirectly connected to each other, and the ORF2p functional start portion is directly or indirectly connected to the ORF1p coding sequence or the ORF2p coding sequence.

8. The RNA framework for gene editing as described in any one of claims 1-6, characterized in that, The RNA framework is obtained through prokaryotic transcription, eukaryotic transcription, or chemical synthesis.

9. The RNA framework for gene editing as described in any one of claims 1-6, characterized in that, The RNA framework is either a linear RNA or located within a linear RNA, or the RNA framework is either a circular RNA or located within a circular RNA.

10. The RNA framework for gene editing as described in claim 9, characterized in that, The linear RNA or circular RNA containing the RNA framework is obtained through prokaryotic transcription, eukaryotic transcription, or chemical synthesis.

11. The RNA framework for gene editing as described in claim 10, characterized in that, The prokaryotic transcription is performed by prokaryotic RNA polymerase; the eukaryotic transcription is performed by eukaryotic RNA polymerase I, eukaryotic RNA polymerase II, or eukaryotic RNA polymerase III.

12. An RNP, characterized in that, The RNP is obtained by binding ORF1p and / or ORF2p to an RNA framework for gene editing as described in any one of claims 1-8, or by binding ORF1p and / or ORF2p to the linear RNA or circular RNA in which the RNA framework is located as described in claim 9 or 10 for gene editing.

13. A DNA molecule, characterized in that, Transcription of the RNA framework for gene editing as described in any one of claims 1-8.

14. A DNA molecule, characterized in that, Transcription of the linear RNA or circular RNA in which the RNA framework for gene editing is located, as described in claim 9 or 10.

15. The DNA molecule as described in claim 13 or 14, characterized in that, The DNA sequence is further directly or indirectly linked to a prokaryotic promoter or a eukaryotic promoter upstream, downstream, and / or internally.

16. The DNA molecule as claimed in claim 15, characterized in that, The prokaryotic promoters are T7, T3, T7lac, Sp6, araBAD, trp, lac, Ptac, pL, LacUV5, Tac, pBAD, or pR.

17. The DNA molecule as claimed in claim 15, characterized in that, The eukaryotic promoters are CMV, pCMV, EF1a, SV40, human PGK1, mouse PGK1, Ubc, human beta-actin, CAG, EFT3, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1, GAL10, GAL1 and GAL10, GAL4, GAL80, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, human U6, or mouse U6.

18. A DNA vector, characterized in that, The DNA vector carries the DNA molecule as described in any one of claims 13 to 17.

19. A gene editing method for purposes other than disease diagnosis or treatment, characterized in that, Includes the following steps: 1) Select the target site to be edited in the genome, and determine the upstream and downstream sequences of the target site on both sides of the target site; 2) Prepare the RNA framework for gene editing as described in any one of claims 1 to 8; and / or prepare the linear RNA or circular RNA in which the RNA framework for gene editing is located as described in claim 9 or 10; and / or prepare the RNP as described in claim 12; and / or prepare the DNA vector as described in claim 18; 3a) Transforming or transfecting the RNA framework described above into cells, tissues, organs or organisms to achieve gene editing; Or 3b) Transform or transfect the linear RNA or circular RNA in which the RNA framework is located into cells, tissues, organs or organisms to achieve gene editing; Or 3c) Transform or transfect the RNP into cells, tissues, organs or organisms to achieve gene editing; (or 3d) Transform or transfect the DNA vector into cells, tissues, organs or organisms to achieve gene editing; Or 3e) co-transform or co-transfect multiple RNAs, including the RNA framework, the linear RNA or circular RNA in which the RNA framework is located, the RNP, and the DNA vector, into cells, tissues, organs, or organisms to achieve gene editing; Or 3f) co-transform or co-transfect one or more of the RNA framework, the linear RNA or circular RNA in which the RNA framework is located, the RNP, the DNA vector, and ORF1p and / or ORF2p into cells, tissues, organs or organisms to achieve gene editing.

20. A gene editing method for non-disease diagnosis or treatment purposes, characterized in that, Includes the following steps: 1) Select the target site to be edited in the genome, and determine the upstream and downstream sequences of the target site on both sides of the target site; 2) Prepare the RNA framework for gene editing as described in any one of claims 1 to 8; and / or prepare the linear RNA or circular RNA in which the RNA framework for gene editing is located as described in claim 9 or 10; and / or prepare the RNP as described in claim 12; and / or prepare the DNA vector as described in claim 18; 3) Prepare one or more helper RNAs containing an ORF2p functional start sequence, one or more ORF1p coding sequences and / or one or more ORF2p coding sequences, and / or helper RNPs obtained by binding the helper RNA to ORF1p and / or ORF2p, and / or helper DNA vectors that transcribe the ORF2p functional start sequence, ORF1p coding sequence and / or ORF2p coding sequence. 4a) Co-transform or co-transfect the RNA framework and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing; Or 4b) co-transform or co-transfect 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 cells, tissues, organs or organisms to achieve gene editing; Or 4c) co-transform or co-transfect the RNP and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing; Or 4d) co-transform or co-transfect the DNA vector and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing; Or 4e) co-transform or co-transfect the RNA framework, the linear RNA or circular RNA in which the RNA framework for gene editing is located, the RNP, the DNA vector, and the helper RNA, helper RNP and / or helper DNA vector prepared in step 3) into cells, tissues, organs or organisms to achieve gene editing. Or 4f) co-transform or co-transfect 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, the DNA vector, the helper RNA, helper RNP, helper DNA vector prepared in step 3), and ORF1p and / or ORF2p into cells, tissues, organs or organisms to achieve gene editing.

21. The gene editing method for non-disease diagnosis or treatment purposes as described in claim 19 or 20, characterized in that, The transformation, transfection, co-transfection, or co-transfection into cells, tissues, organs, or organisms 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 may be one or more. When there is only one RNA framework, one linear RNA or circular RNA in which the RNA framework for gene editing is located, one RNP, or one DNA vector, single-site editing on the genome is achieved. When the sum 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 greater than or equal to two, and the upstream and / or downstream sequences 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, multiple sites on the genome can be edited or manipulated.

22. The use of the RNA framework for gene editing as described in any one of claims 1 to 8, or the linear RNA or circular RNA on which the RNA framework for gene editing as described in claim 9 or 10 is located, or the RNP as described in claim 12, or the DNA vector as described in claim 18, as a tool for inserting, deleting, replacing, deleting, adding, replacing, inverting, and / or correcting inversions of target gene sequences, wherein the use is not for disease diagnosis or treatment purposes.

23. The RNA framework for gene editing as described in any one of claims 1 to 8, or the linear RNA or circular RNA to which the RNA framework for gene editing as described in claims 9 or 10 is located, or the RNP as described in claim 12, or the DNA vector as described in claim 18, for generating or amplifying a DNA template containing the RNA framework sequence as described in any one of claims 1 to 8.

24. The use of the RNA framework for gene editing as described in any one of claims 1 to 8, or the linear RNA or circular RNA to which the RNA framework for gene editing as described in claim 9 or 10 is located, or the RNP as described in claim 12, or the DNA vector as described in claim 18, or the DNA template as described in claim 23, as a tool to improve the gene editing efficiency of TALEN, ZFN, Targetron, Prime Editor, Twin Prime Editor, CRISPR, or CRISPR / Cas9 gene technologies, wherein the use is not for disease diagnosis or treatment purposes.

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