Reporting system capable of being used for screening drugs aiming at RNA repetitive sequences

Abstract

The invention provides a report system for screening drugs aiming at pathogenic RNA repetitive sequences, and particularly provides a report system simultaneously containing RNA repetitive sequences with different lengths, which can be used for screening the drugs aiming at the pathogenic RNA repetitive sequences and related basic research and development work. Specifically, the invention provides a nucleic acid construct group for screening drugs for treating diseases related to long RNA repetitive sequences, and a carrier and a screening system based on the nucleic acid construct group. The nucleic acid construct group, the vector and the screening system can be used for screening or determining drugs for treating diseases related to long RNA repetitive sequences in a high-throughput manner.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a reporter system that can be used to screen drugs targeting RNA repeat sequences. Background Art

[0002] RNA repeat sequences refer to repetitive sequences present in RNA molecules, which are widely present in the human transcriptome. In some cases, the length of these repeat sequences may reach a certain extent and lead to the occurrence of specific RNA repeat sequence-related diseases associated with diseases. This disease is usually caused by the inconsistency of the length of the RNA repeat sequences carried by the two alleles in the human body, which is the result of abnormal expansion of microsatellite sequences in the genome. One of the genes with short repeat sequences is a gene with normal function, while the other gene with abnormal repeat sequence length may cause disease.

[0003] Huntington's disease is an inherited neurodegenerative disease, the onset of which is associated with the expansion of the CAG long repeat sequence in the HTT gene. The length of CAG repeats in the population is within different ranges, with 35 CAGs as the critical value. When it is greater than 35 CAGs, the disease is prone to occur. This disease not only leads to deterioration in many aspects such as intelligence, movement and spirit, but there is currently no cure. Myotonic Dystrophy Type 1 (DM1) is a hereditary muscle disease caused by the abnormal length of CUG long repeat sequences in the 3'UTR region of the DMPK gene. DM1 patients have severe abnormalities in muscles, heart, endocrine and nervous systems, and their treatment is extremely challenging. However, there is currently no effective treatment for such RNA repeat sequence-related diseases, which has caused great distress to patients.

[0004] There are some commonalities in the pathogenesis of these RNA repeat sequence-related diseases, including corresponding RNA protein complexes, ribosomes and other elements. There are also some specific mechanisms that require more in-depth research and understanding.

[0005] In the current drug screening system for such diseases, only the clearance effect on the long repeat sequence of pathogenic RNA is considered, while the off-target effect on the short repeat sequence of normal RNA is not considered at the same time. The best system should be able to characterize / screen drugs with strong clearance ability on the long repeat sequence of pathogenic RNA while also characterizing its off-target effect on the short repeat sequence of normal RNA, thereby speeding up the screening process and making the resulting candidate drugs or molecules more selective, bringing better therapeutic effects to patients.

[0006] Therefore, there is an urgent need in the art to develop a screening or reporting system that can quickly and accurately screen out potential drugs for treating RNA repeat sequence-related diseases. Summary of the invention

[0007] The purpose of the present invention is to provide a screening or reporting system that can quickly and accurately screen out potential drugs for treating diseases related to RNA repeat sequences.

[0008] In a first aspect of the present invention, a nucleic acid construct set is provided, the nucleic acid construct set comprising: a first nucleic acid construct and a second nucleic acid construct;

[0009] Furthermore, the first nucleic acid construct contains a first expression cassette, the first expression cassette expresses a long RNA repeat sequence, and the first expression cassette comprises a repeat sequence unit of 3-6 nucleotides, the repeat sequence is repeated 41-2000 times, preferably 45-1500 times, more preferably 50-1000 times;

[0010] The second nucleic acid construct contains a second expression cassette, which expresses a short RNA repeat sequence, and the second expression cassette contains a repeat sequence unit of 3-6 nucleotides corresponding to the first expression cassette, and the repeat sequence is repeated 2-40 times, preferably 5-40 times.

[0011] In another preferred example, the first expression cassette further comprises a first promoter and a coding sequence of a first detectable marker.

[0012] In another preferred example, the second expression cassette further comprises a second promoter and a coding sequence of a second detectable marker.

[0013] In another preferred embodiment, the repeating sequence unit is selected from the following group: CAG, GCG, CGG, CTG, CCG, GAA, CCCCGCCCCGCG, ATTCT / ATTGT, CCTG, TGGAA, GGCCTG, GGGGCC, CCCTCT, ATTTC, AAGGG, GCA, and different equivalent sequences (for example, CAG repeats and AGC repeats, CTG repeats and TGC repeats, etc. are equivalent sequences), or a combination thereof.

[0014] In another preferred embodiment, the corresponding refers to:

[0015] The first expression cassette contains a repetitive sequence unit CTG, and the second expression cassette contains a repetitive sequence unit CTG;

[0016] The first expression cassette contains a repeating sequence unit CCTG, and the second expression cassette contains a repeating sequence unit CCTG;

[0017] The first expression cassette contains a repeating sequence unit CAG, and the second expression cassette contains a repeating sequence unit CAG;

[0018] The first expression cassette contains a repeating sequence unit TGGAA, and the second expression cassette contains a repeating sequence unit TGGAA;

[0019] The first expression cassette contains a repeating sequence unit GGGGCC, and the second expression cassette contains a repeating sequence unit GGGGCC;

[0020] The first expression cassette contains a repeating sequence unit CGG, and the second expression cassette contains a repeating sequence unit CGG;

[0021] The first expression cassette contains a repeating sequence unit GCG, and the second expression cassette contains a repeating sequence unit GCG;

[0022] The first expression cassette contains a repeating sequence unit CCG, and the second expression cassette contains a repeating sequence unit CCG;

[0023] The first expression cassette contains a repeating sequence unit GAA, and the second expression cassette contains a repeating sequence unit GAA;

[0024] The first expression cassette contains a repeating sequence unit GGCCTG, and the second expression cassette contains a repeating sequence unit GGCCTG;

[0025] The first expression cassette contains a repeating sequence unit ATTTC, and the second expression cassette contains a repeating sequence unit ATTTC;

[0026] The first expression cassette contains a repeating sequence unit AAGGG, and the second expression cassette contains a repeating sequence unit AAGGG;

[0027] The first expression cassette contains a repeating sequence unit GCA, and the second expression cassette contains a repeating sequence unit GCA;

[0028] The first expression cassette contains the repeating sequence unit ATTCT / ATTGT, and the second expression cassette contains the repeating sequence unit ATTCT / ATTGT;

[0029] The first expression cassette contains the repeating sequence unit CCCCGCCCCGCG, and the second expression cassette contains the repeating sequence unit CCCCGCCCCGCG.

[0030] In another preferred example, the first promoter and the second promoter each independently include a eukaryotic promoter or a prokaryotic promoter.

[0031] In another preferred embodiment, the first promoter and the second promoter each independently include a constitutive promoter or an inducible promoter.

[0032] In another preferred embodiment, the first promoter and the second promoter are each independently selected from the following group: CMV promoter, CAG promoter, PGK promoter, EF-1α promoter, SV40 promoter, chicken beta-actin promoter, HSV-TK promoter, Ubc promoter, TRE promoter, UAS promoter, Ac5 promoter, Polyhedrin promoter, CaMKIIa promoter, IL-2 promoter, RSV promoter, GAL1 promoter, TEF1 promoter, TDH3 promoter, ADH1 promoter, T7 promoter, T3 promoter, Sp6 promoter, araBAD promoter, trp promoter, lac promoter, pL promoter, or a combination thereof.

[0033] In another preferred embodiment, the first detectable label and the second detectable label are independently selected from the following group: fluorescent groups, luciferase, luminescent markers, quantum dots, β-galactosidase LacZ, or a combination thereof.

[0034] In another preferred embodiment, the fluorescent group is selected from the following group: mCherry, GFP and its derivative EGFP, dTomato, BFP, YFP, or other fluorescent protein combinations.

[0035] In another preferred embodiment, the luciferase is selected from the following group: Firefly luciferase (FLuc), Renilla luciferase (RLuc), Gaussia luciferase (GLuc), Nano-Glo luciferase (NLuc), Cypridina luciferase (CLuc), or other luciferase combinations.

[0036] In another preferred embodiment, the first detectable marker and the second detectable marker are different.

[0037] In another preferred embodiment, the first nucleic acid construct and the second nucleic acid construct are connected via a connecting element.

[0038] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the expression vector of the same nucleic acid construct.

[0039] In another preferred embodiment, the first nucleic acid construct has a structure of Formula I or I':

[0040] Z1-P1-Z2-pA1 (I)

[0041] pA1-Z2-P1-Z1 (I')

[0042] in,

[0043] Z1 is the first promoter;

[0044] P1 is the coding sequence of the first detectable marker;

[0045] Z2 is a repeating sequence unit of 3-6 nucleotides, and the number of repetitions of the repeating sequence is 41-2000 times, preferably 45-1500 times, and more preferably 50-1000 times;

[0046] pA1 is one or more nucleic acid fragments containing poly(A) signal elements or other nucleic acid fragments for transcription termination, preferably, selected from the following group: CaMV poly(A) signal element, EF-1α poly(A) signal element, BGH (bovine growth hormone)-poly(A) signal element, hGH poly(A) signal, SV40-poly(A) signal element, humanβ-globin poly(A) signal element, or a combination thereof;

[0047] Each “-” represents no or a nucleotide linking sequence.

[0048] In another preferred embodiment, the second nucleic acid construct has a structure of Formula II or Formula II':

[0049] Z3-P2-Z4-pA2 (II)

[0050] pA2-Z4-P2-Z3 (II')

[0051] in,

[0052] Z3 is the second promoter;

[0053] P2 is the coding sequence of the second detectable marker;

[0054] Z4 is a repeating sequence unit of 3-6 nucleotides corresponding to Z2, and the repeating number of the repeating sequence is 2-40 times, preferably, 5-40 times;

[0055] pA2 is one or more nucleic acid fragments containing poly(A) signal elements or other nucleic acid fragments for transcription termination, preferably, selected from the following group: CaMV poly(A) signal element, EF-1α poly(A) signal element, BGH (bovine growth hormone)-poly(A) signal element, hGH poly(A) signal, SV40-poly(A) signal element, humanβ-globin polyadenylation signal element, or a combination thereof;

[0056] Each “-” represents no or a nucleotide linking sequence.

[0057] In another preferred embodiment, pA1 and pA2 may be the same or different.

[0058] In another preferred embodiment, the first promoter and the second promoter are the same or their expression strengths are similar.

[0059] In another preferred embodiment, the close expression intensity means that the ratio (T1 / T2) of the expression intensity T1 of the first promoter to the expression intensity T2 of the second promoter is 0.8-1.2.

[0060] In another preferred embodiment, the first promoter is selected from the following group: CMV promoter, CAG promoter, PGK promoter, EF-1α promoter, SV40 promoter, chicken beta-actin promoter, HSV-TK promoter, Ubc promoter, TRE promoter, UAS promoter, Ac5 promoter, Polyhedrin promoter, CaMKIIa promoter, IL-2 promoter, RSV promoter, GAL1 promoter, TEF1 promoter, TDH3 promoter, ADH1 promoter, T7 promoter, T3 promoter, Sp6 promoter, araBAD promoter, trp promoter, lac promoter, pL promoter, or a combination thereof.

[0061] In another preferred embodiment, the second promoter is selected from the following group: CMV promoter, CAG promoter, PGK promoter, EF-1α promoter, SV40 promoter, chicken beta-actin promoter, HSV-TK promoter, Ubc promoter, TRE promoter, UAS promoter, Ac5 promoter, Polyhedrin promoter, CaMKIIa promoter, IL-2 promoter, RSV promoter, GAL1 promoter, TEF1 promoter, TDH3 promoter, ADH1 promoter, T7 promoter, T3 promoter, Sp6 promoter, araBAD promoter, trp promoter, lac promoter, pL promoter, or a combination thereof.

[0062] In another preferred embodiment, the first detectable marker and the second detectable marker are different.

[0063] In another preferred embodiment, the first detectable marker and the second detectable marker can be excited by lasers with different excitation wavelengths to emit fluorescence of different colors.

[0064] In another preferred example, the first detectable marker and the second detectable marker can emit light of different colors by adding different substrates.

[0065] In another preferred embodiment, the repeating sequence unit of Z2 is CTG, and the repeating sequence unit of Z4 is CTG; or

[0066] The repeating sequence unit of Z2 is CCTG, and the repeating sequence unit of Z4 is CCTG; or

[0067] The repeating sequence unit of Z2 is CAG and the repeating sequence unit of Z4 is CAG; or

[0068] The repeating sequence unit of Z2 is TGGAA, and the repeating sequence unit of Z4 is TGGAA; or

[0069] The repeating sequence unit of Z2 is GGGGCC, and the repeating sequence unit of Z4 is GGGGCC; or

[0070] The repeating sequence unit of Z2 is CGG, and the repeating sequence unit of Z4 is CGG.

[0071] The repeating sequence unit of Z2 is GCG, and the repeating sequence unit of Z4 is GCG;

[0072] The repeating sequence unit of Z2 is CCG, and the repeating sequence unit of Z4 is CCG;

[0073] The repeating sequence unit of Z2 is GAA, and the repeating sequence unit of Z4 is GAA;

[0074] The repeating sequence unit of Z2 is GGCCTG, and the repeating sequence unit of Z4 is GGCCTG;

[0075] The repeating sequence unit of Z2 is ATTTC, and the repeating sequence unit of Z4 is ATTTC;

[0076] The repeating sequence unit of Z2 is AAGGG, and the repeating sequence unit of Z4 is AAGGG;

[0077] The repeating sequence unit of Z2 is GCA, and the repeating sequence unit of Z4 is GCA;

[0078] The repeating sequence unit of Z2 is ATTCT / ATTGT, and the repeating sequence unit of Z4 is ATTCT / ATTGT;

[0079] The repeating sequence unit of Z2 is CCCCGCCCCGCG, and the repeating sequence unit of Z4 is CCCCGCCCCGCG.

[0080] In another preferred embodiment, the length of the first nucleic acid construct and the second nucleic acid construct independently comprises 10 repeating units to 1000 repeating units, preferably, 15 repeating units to 500 repeating units, and more preferably, 20 repeating units to 500 repeating units.

[0081] In another preferred embodiment, the construct group has a structure of formula III (or III' or III" or IIII"' or III"" or III""' or III""" or III"""'):

[0082] Z1-P1-Z2-pA1-Z3-P2-Z4-pA2(III);

[0083] Z3-P2-Z4-pA2-Z1-P1-Z2-pA1(III');

[0084] Z1-P1-Z2-pA1-pA2-Z4-P2-Z3(III”);

[0085] Z3-P2-Z4-pA2-pA1-Z2-P1-Z1(III”');

[0086] pA1-Z2-P1-Z1-Z3-P2-Z4-pA2(III””);

[0087] pA2-Z4-P2-Z3-Z1-P1-Z2-pA1(III””’);

[0088] pA1-Z2-P1-Z1-pA2-Z4-P2-Z3(III”””);

[0089] pA2-Z4-P2-Z3-pA1-Z2-P1-Z1(III"""').

[0090] In the formula, the definitions of each element are as described above.

[0091] In another preferred embodiment, the nucleic acid construct group comprises 10 repeating units to 1000 repeating units, preferably, 15 repeating units to 500 repeating units, more preferably, 20 repeating units to 500 repeating units.

[0092] The second aspect of the present invention provides a recombinant vector, which comprises the nucleic acid construct set as described in the first aspect of the present invention.

[0093] In another preferred embodiment, the vector includes a DNA vector, an RNA vector, a viral vector, a plasmid, a transposon, other gene transfer systems, or a combination thereof; preferably, the vector is a plasmid or a viral vector.

[0094] In another preferred embodiment, the vector is circular or linear.

[0095] The third aspect of the present invention provides a genetically engineered cell, wherein the genetically engineered cell contains the nucleic acid construct set described in the first aspect of the present invention, or contains the recombinant vector described in the second aspect of the present invention.

[0096] In another preferred embodiment, the genetically engineered cells include eukaryotic cells.

[0097] In another preferred embodiment, the genetically engineered cells include mammalian cells.

[0098] In another preferred embodiment, the genetically engineered cells include human embryonic kidney cells HEK293 cells, human skeletal muscle cells, human HeLa cells, human corneal endothelial cells, mouse NIH 3T3 cell line, monkey COS-7 cell line and other commonly used mammalian cell lines.

[0099] The fourth aspect of the present invention provides a kit, which comprises: a container and the nucleic acid construct set according to the first aspect of the present invention located in the container.

[0100] In another preferred embodiment, the kit is used to screen drugs for treating diseases associated with long RNA repeat sequences.

[0101] A fifth aspect of the present invention provides a method for screening drugs for treating diseases associated with long RNA repeat sequences, comprising the steps of:

[0102] (a) introducing the nucleic acid construct set described in the first aspect of the present invention and the drug to be screened into cells together,

[0103] (b) judging whether the drug to be tested is a drug for treating a disease associated with a long RNA repeat sequence by the intensity change of the first detectable marker and the second detectable marker in the nucleic acid construct group.

[0104] In another preferred embodiment, the cells include mammalian cells.

[0105] In another preferred embodiment, the cells include human skeletal muscle cells, human neural stem cells, human fibroblast stem cells, human myoblasts, human heart cells, human renal epithelial cells, human corneal endothelial cells, human tumor cells, human lung cells, human liver cells, human spleen cells, human colon cells, human embryonic stem cells, human bone marrow cells, human thyroid cells, human pancreatic cells, human lymphocytes, human T cells, human colon gland cells, human retinal cells, human gastric cells or other human cells.

[0106] In another preferred embodiment, the cells include HEK293T, Hela, u2os, N2a, U87, NIH3T3, B4G12, COS-7, SY5Y, A549 or other human stable cell lines.

[0107] In another preferred embodiment, the cells are cells cultured in vitro.

[0108] In another preferred example, when the expression level or intensity of the first detectable marker is significantly reduced, and the expression level or intensity of the second detectable marker changes slightly or remains essentially unchanged, it indicates that the drug to be screened can be used as a candidate drug for treating diseases associated with long RNA repeat sequences.

[0109] In another preferred embodiment, when the decrease in the expression amount or intensity of the first detectable marker is small, it indicates that the drug to be screened is not suitable for treating diseases associated with long RNA repeat sequences.

[0110] In another preferred embodiment, the significant decrease in the expression level or intensity of the first detectable marker means that the decrease in the expression level or intensity of the first detectable marker is ≥25%, preferably ≥50%, and more preferably ≥75%.

[0111] In another preferred embodiment, the small reduction in the expression level or intensity of the first detectable marker means that the reduction in the expression level or intensity of the first detectable marker is ≤25%, preferably ≤20%, and more preferably ≤15%.

[0112] In another preferred embodiment, the expression amount or intensity of the second detectable marker changes slightly or remains essentially unchanged, which means that the decrease in the expression amount or intensity of the second detectable marker is ≤25%, preferably ≤15%, more preferably ≤5%, and more preferably 0.

[0113] In another preferred embodiment, the introduction method comprises one or more methods selected from the following group: calcium phosphate-mediated transfection, nuclear transfection, electroporation, cationic polymer transfection, viral transduction, virion transfection, viral particle transfection, liposome transfection, cationic liposome transfection, immunoliposome transfection, non-liposomal transfection, dendrimer transfection, heat shock transfection, magnetofection, lipofection, gene gun delivery, impalefection, sonoporation and optical transfection.

[0114] In another preferred embodiment, the drugs to be screened include drugs that can reduce the expression level or intensity of the first detectable marker but do not reduce the expression level or intensity of the second detectable marker.

[0115] In another preferred embodiment, the drug to be screened includes CRISPR reagents.

[0116] In another preferred embodiment, the CRISPR reagent includes a gene editing protein and an optional guide RNA.

[0117] In another preferred embodiment, the gene editing protein is capable of recognizing long RNA repeat sequences and cutting the recognized long RNA repeat sequences.

[0118] In another preferred embodiment, the guide RNA guides the gene editing protein to specifically bind to the DNA portion that can express the long RNA repeat sequence.

[0119] In another preferred embodiment, the gene editing protein is selected from the following group: Cas13a, Cas13b, Cas13d, Cas13x, Cas13y, RNA-targeting Cas9 (RCas9, dead Cas9 from Streptococcus pyogenes), dSaCas9 (dead Cas9 from Staphylococcus aureus), or a combination thereof.

[0120] In another preferred embodiment, the drug to be screened includes an artificial RNA degrading enzyme.

[0121] In another preferred embodiment, the RNA degrading enzyme has a structure shown in Formula IV or IV' from the NC end of the protein:

[0122] D1-A-L1-B-D2 (IV);

[0123] D1-B-L1-A-D2 (IV')

[0124] In the formula, each "-" is independently a connecting peptide or a peptide bond;

[0125] A is the PUF protein fragment;

[0126] B is the full length or active fragment of an RNase or RNA helicase;

[0127] L1 is none or a linker peptide;

[0128] D1 is no or localized peptide;

[0129] D2 is absent or localized peptide.

[0130] In another preferred embodiment, the PUF protein fragment is a fragment of the PUF protein domain that can recognize and bind to RNA.

[0131] In another preferred embodiment, the PUF protein includes PUF protein and homologous proteins thereof.

[0132] In another preferred embodiment, the PUF protein is from a mammal, preferably from a primate, and more preferably from a human.

[0133] In another preferred embodiment, the PUF protein fragment contains n recognition units, each of which is used to recognize an RNA base, wherein n is a positive integer of 5-30.

[0134] In another preferred embodiment, n is 6-24, more preferably 8-20, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24.

[0135] In another preferred embodiment, each recognition unit is an α-helical repeating sequence, and three amino acids at specific positions on the repeating sequence are responsible for binding to RNA bases.

[0136] In another preferred embodiment, the n recognition units are connected in series to form an RNA base recognition region.

[0137] In another preferred embodiment, the PUF protein fragment further comprises optional protection regions respectively located on both sides of the RNA base recognition region, for protecting the RNA base recognition region.

[0138] In another preferred embodiment, the PUF protein fragment includes a domain located upstream of the RNA base recognition region in the PUF protein (excluding the protection zone), but may or may not include a domain located downstream of the RNA base recognition region in the PUF protein (excluding the protection zone).

[0139] In another preferred embodiment, the protection zone is a non-functional repeat sequence at both ends of the RNA base recognition region.

[0140] In another preferred embodiment, the localization peptide sequence includes a nuclear localization signal sequence, a mitochondrial localization signal sequence or a nuclear export signal sequence.

[0141] In another preferred embodiment, the connecting peptide and / or positioning peptide signal are each independently located between the PUF protein fragment and the RNA enzyme or RNA helicase.

[0142] In another preferred embodiment, the RNA is single-stranded.

[0143] In another preferred embodiment, the PUF protein fragment is selected from the following group:

[0144] (a) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1;

[0145] (b) a polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% homology (or identity) with the amino acid sequence of SEQ ID NO: 1, and the polypeptide has the biological function of SEQ ID NO: 1;

[0146] (c) A derivative polypeptide formed by replacing, deleting or adding one or more (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, and retaining the biological function shown in SEQ ID NO: 1.

[0147] In another preferred example, the PUF protein fragment has an amino acid sequence as shown in SEQ ID NO.1.

[0148] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTH PYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG(SEQ ID NO.:1)

[0149] In another preferred embodiment, the full length or active fragment of the RNA enzyme or RNA helicase is selected from the following group: PIN (from SMG6, UNIPROT: Q86US8), ANG (UNIPROT: P03950), RNASEL (UNIPROT: Q05823), RRP44 (UNIPROT: Q9Y2L1), RNASE1 (UNIPROT: P07998), RNASET2 (UNIPROT: O00584) 、SUPV3L1(UNIPROT:Q8IYB8)、PNPT1(UNIPROT:Q8TCS8)、REXO2(UNIPROT:Q9Y3B8)、MRPL44(UNIPROT:Q9H9J2)、LACTB2(UNIPROT:Q53H82)、TSEN15(UNIPROT:Q8WW01)、ZC3H12A(UNIPROT:Q5D1E8), or their active regions and sequence combinations.

[0150] In another preferred embodiment, the full length or active fragment of the RNA enzyme or RNA helicase is selected from the following group: ANG (UNIPROT: P03950), RNASE1 (UNIPROT: P07998), RNASET2 (UNIPROT: O00584), SUPV3L1 (UNIPROT: Q8IYB8), PNPT1 (UNIPROT: Q8TCS8), REXO2 (UNIPROT: Q9Y3B8), or a combination thereof.

[0151] In another preferred embodiment, the RNA enzyme or RNA helicase includes an RNA enzyme or RNA helicase having a homology of at least 80%, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98% or 99% with the above-mentioned RNA enzyme or RNA helicase.

[0152] In another preferred embodiment, the artificial RNA degrading enzyme is selected from the following group:

[0153] (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 95-111, 116-119;

[0154] (b) a polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% homology (or identity) with the amino acid sequence shown in any one of SEQ ID NOs: 95-111, 116-119, and the polypeptide has the biological function shown in any one of SEQ ID NOs: 99-115, 120-123;

[0155] (c) A derivative polypeptide formed by replacing, deleting or adding one or more (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs: 95-111, 116-119, and retaining the biological function shown in any one of SEQ ID NOs: 95-111, 116-119.

[0156] In another preferred embodiment, the connecting peptide is a non-flexible peptide or a rigid peptide.

[0157] In another preferred embodiment, the connecting peptide is selected from the following group: XTEN peptide segment, GGGGS or other short peptide segments.

[0158] In another preferred embodiment, the length of the connecting peptide is 0-40 aa, preferably 2-20 aa.

[0159] A sixth aspect of the present invention provides a system for screening drugs for treating diseases associated with long RNA repeat sequences, the system comprising:

[0160] (a) the nucleic acid construct set according to the first aspect of the present invention;

[0161] (b) drugs to be screened;

[0162] Among them, when the expression level or intensity of the first detectable marker in the nucleic acid construct group is significantly reduced, and the expression level or intensity of the second detection marker changes very little or remains basically unchanged, it indicates that the drug to be screened is a candidate or potential drug that can be used to treat long RNA repeat sequence-related diseases.

[0163] In another preferred embodiment, the first detectable marker is driven by a first promoter (such as CMV promoter, CAG promoter, PGK promoter, EF-1α promoter, SV40 promoter, chicken beta-actin promoter, HSV-TK promoter, Ubc promoter, TRE promoter, UAS promoter, Ac5 promoter, Polyhedrin promoter, CaMKIIa promoter, IL-2 promoter, RSV promoter, or other eukaryotic cell promoters) to express the first detectable marker.

[0164] In another preferred embodiment, the second detectable marker is driven by a second promoter (such as CMV promoter, CAG promoter, PGK promoter, EF-1α promoter, SV40 promoter, chicken beta-actin promoter, HSV-TK promoter, Ubc promoter, TRE promoter, UAS promoter, Ac5 promoter, Polyhedrin promoter, CaMKIIa promoter, IL-2 promoter, RSV promoter, or other eukaryotic cell promoters) to express the second detectable marker.

[0165] In another preferred embodiment, the first promoter and the second promoter are the same or have similar expression strengths.

[0166] In another preferred embodiment, the close expression intensity means that the ratio (T1 / T2) of the expression intensity T1 of the first promoter to the expression intensity T2 of the second promoter is 0.8-1.2.

[0167] In another preferred embodiment, when the expression level or intensity of the first detectable marker is significantly reduced, the expression level or intensity of the second detectable marker changes slightly or remains essentially unchanged.

[0168] In another preferred example, the first detectable marker and the second detectable marker are expressed respectively by the same promoter.

[0169] A seventh aspect of the present invention provides a screening composition for screening drugs for treating diseases associated with long RNA repeat sequences, the screening composition comprising:

[0170] (a) drugs to be screened;

[0171] (b) the nucleic acid construct set according to the first aspect of the present invention;

[0172] Among them, when the expression amount or intensity of the first detectable marker in the nucleic acid construct group is significantly reduced, and the expression amount or intensity of the second detection marker changes slightly or remains basically unchanged, it indicates that the drug to be screened is a candidate or potential drug that can be used to treat long RNA repeat sequence-related diseases.

[0173] The eighth aspect of the present invention provides a use of the system described in the sixth aspect of the present invention or the screening composition described in the seventh aspect of the present invention for screening drugs for treating diseases associated with long RNA repeat sequences.

[0174] In another preferred embodiment, the drug for treating long RNA repeat sequence-related diseases includes CRISPR reagents.

[0175] In another preferred embodiment, the CRISPR reagent includes a gene editing protein and an optional guide RNA.

[0176] In another preferred embodiment, the gene editing protein is capable of recognizing long RNA repeat sequences and cutting the recognized long RNA repeat sequences.

[0177] In another preferred embodiment, the guide RNA guides the gene editing protein to specifically bind to the DNA portion that can express the long RNA repeat sequence.

[0178] In another preferred embodiment, the gene editing protein is selected from the following group: Cas13a, Cas13b, Cas13d, Cas13x, Cas13y, RNA-targeting Cas9 (RCas9, dead Cas9 from Streptococcus pyogenes), dSaCas9 (dead Cas9 from Staphylococcus aureus), or a combination thereof.

[0179] In another preferred embodiment, the drug for treating long RNA repeat sequence-related diseases includes an artificial RNA degrading enzyme.

[0180] In another preferred embodiment, the RNA degrading enzyme has a structure shown in Formula IV or IV' from the NC end:

[0181] D1-A-L1-B-D2 (IV);

[0182] D1-B-L1-A-D2 (IV')

[0183] In the formula, each "-" is independently a connecting peptide or a peptide bond;

[0184] A is the PUF protein fragment;

[0185] B is the full length or active fragment of an RNase or RNA helicase;

[0186] L1 is none or a linker peptide;

[0187] D1 is no or localized peptide;

[0188] D2 is absent or localized peptide.

[0189] The ninth aspect of the present invention provides a composition comprising:

[0190] (a) A drug for treating a disease associated with long RNA repeat sequences obtained by the method described in the fifth aspect of the present invention or the system described in the sixth aspect of the present invention, wherein the drug for treating a disease associated with long RNA repeat sequences comprises an artificial RNA degrading enzyme.

[0191] In another preferred embodiment, the RNA degrading enzyme has a structure shown in Formula IV or IV' from the NC end:

[0192] D1-A-L1-B-D2 (IV);

[0193] D1-B-L1-A-D2 (IV')

[0194] In the formula, each "-" is independently a connecting peptide or a peptide bond;

[0195] A is the PUF protein fragment;

[0196] B is the full length or active fragment of an RNase or RNA helicase;

[0197] L1 is none or a linker peptide;

[0198] D1 is no or localized peptide;

[0199] D2 is absent or localized peptide.

[0200] The tenth aspect of the present invention provides a method for preparing an animal model for screening drugs for treating diseases associated with long RNA repeat sequences, the method comprising the following steps:

[0201] (a) providing non-human mammalian cells, and introducing the nucleic acid construct set described in the first aspect of the present invention into the cells, thereby obtaining cells that simultaneously express long RNA repeat sequences and short RNA repeat sequences;

[0202] (b) Using the cells obtained in step (a) that simultaneously express long RNA repeat sequences and short RNA repeat sequences, an animal model is prepared for screening drugs for treating diseases associated with long RNA repeat sequences.

[0203] In another preferred embodiment, the cells include somatic cells, germ cells and / or embryonic stem cells.

[0204] In another preferred embodiment, the embryonic stem cells also include embryos less than 14 days old and / or fertilized eggs.

[0205] In another preferred embodiment, the cells are selected from non-human mammals, preferably rodents or primates, preferably mice, rats, rabbits and / or monkeys, chimpanzees.

[0206] In another preferred embodiment, the cell is a fertilized egg of a non-human mammal.

[0207] The eleventh aspect of the present invention provides a use of a non-human mammal model prepared by the method described in the tenth aspect of the present invention, wherein the model is used to screen or identify substances (therapeutic agents) for treating diseases associated with long RNA repeat sequences.

[0208] A twelfth aspect of the present invention provides a method for screening or identifying a potential therapeutic agent for treating a disease associated with a long RNA repeat sequence, comprising the following steps:

[0209] (a) in a test group, in the presence of a test drug, administering the test drug to a non-human mammal model prepared by the method of the ninth aspect of the present invention, and analyzing the expression amount or intensity of the first detectable marker and the second detectable marker of the animal model of the test group; and in a control group in which the test drug is not administered and other conditions are the same, analyzing the expression amount or intensity of the first detectable marker and the second detectable marker of the animal model of the control group;

[0210] (b) comparing the expression levels or intensities of the first detectable marker and the second detectable marker in the test group and the control group animal models, wherein, if the expression level or intensity of the first detectable marker in the animal model administered with the test drug is significantly reduced compared with the control group, and the expression level or intensity of the second detectable marker remains essentially unchanged, it indicates that the test compound can be used as a potential therapeutic agent for treating diseases associated with long RNA repeat sequences.

[0211] In another preferred embodiment, the long RNA repeat sequence-related diseases include the disease types shown in Table 1 and Table 2.

[0212] In another preferred embodiment, the diseases associated with the long RNA repeat sequence include: frontotemporaldementia (FTD), Huntington disease (HD), myotonic dystrophy Type 1 / 2 (DM1 / 2), amyotrophic lateral sclerosis (ALS), Fuchs endothelial corneal dystrophy (FECD), autosomal-dominant cerebellar ataxias (ADCAs), and fragile X syndrome (FXS).

[0213] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0214] In another preferred embodiment, the method includes step (c): applying the potential therapeutic agent screened or identified in step (b) to the non-human mammal model prepared by the method described in the tenth aspect of the present invention, so as to determine its effect on the expression level or intensity of the first detectable marker and the second detectable marker in the animal model.

[0215] The thirteenth aspect of the present invention provides a non-human mammal model prepared by the method described in the tenth aspect of the present invention.

[0216] A fourteenth aspect of the present invention provides a use of the genetically engineered cells described in the third aspect of the present invention for preparing biological preparations for constructing an animal model for screening drugs for treating long RNA repeat sequence-related diseases in non-human mammals.

[0217] In another preferred embodiment, the biological agent is a liquid preparation.

[0218] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0219] Figure 1 Schematic diagram of the dual reporter gene system with different RNA repeat sequences. (A) CUG repeat sequence; (B) CAG repeat sequence; (C) CCUG repeat sequence; (D) TGGAA repeat sequence.

[0220] Figure 2 .Results of induced expression of the dual reporter gene system with CUG repeat sequences in Flp-In T-REX 293 cells. (A) Expression of reporter gene fluorescent protein in cells without the addition of inducer; (B) Expression of reporter gene fluorescent protein in cells with the addition of 1 μg / mL inducer doxycycline; (C) Expression imaging results of the dual reporter gene system with CUG repeat sequences in cells.

[0221] Figure 3 . Flowchart of an experiment comparing the effectiveness of existing systems that can target RNA repetitive sequences.

[0222] Figure 4 . Flow cytometry results of existing systems that can target RNA repetitive sequences.

[0223] Figure 5 .Existing RT-qPCR detection results of systems that can target RNA repetitive sequences.

[0224] Figure 6Screening of novel artificial RNA degrading enzymes using a dual reporter gene system with different RNA repeat sequences. (A) Schematic diagram of the composition of artificial RNA degrading enzymes; (B) Screening test results of novel artificial RNA degrading enzymes (RT-qPCR).

[0225] Figure 7 .Flow cytometric detection results of the newly constructed artificial RNA degrading enzyme on the RNA repeat sequence dual reporter system.

[0226] Figure 8 Abnormal RNA aggregates generated by fluorescent RNA-labeled RNA repeat sequences in the cell nucleus. (A) Schematic diagram of fluorescent RNA-labeled RNA repeat sequences; (B) The number of abnormal RNA aggregates generated by RNA repeat sequences of different lengths in the cell nucleus; (C) Imaging results of RNA repeat sequences of different lengths in cells; (D) The distribution of fluorescent RNA labeling CUG repeat sequences is consistent with the distribution of fluorescent proteins targeting CUG repeat sequences.

[0227] Fig. 9 .Effects of different systems on the elimination of RNA repeat sequence nuclear aggregation. (A) Regulation of RNA repeat sequence nuclear aggregation by non-PUF system; (B) Regulation of RNA repeat sequence nuclear aggregation by different PUF systems; (C) Statistical results of all systems that can regulate RNA repeat sequence aggregation in cells (n=50).

[0228] Fig.10 .Strategies for constructing mouse models with dual reporter gene systems carrying CUG repeat sequences of different lengths.

[0229] Fig.11 .Strategies for constructing mouse models with dual reporter gene systems carrying CAG repeat sequences of different lengths.

[0230] Fig.12 .Imaging identification results of dual reporter gene mice with different CUG repeat sequence lengths.

[0231] Fig.13 .Comparison of existing systemic effects using dual reporter gene mouse model. (A) Schematic diagram of mouse experimental scheme, control AAV virus particles were injected into the left leg of the mouse, and experimental group AAV virus particles were injected into the right leg of the mouse. The experiment lasted for 2 months. (B) RT-qPCR detection results of the reporter gene with long CUG sequence (mCherry-160CUG). (C) RT-qPCR detection results of the reporter gene with short CUG sequence (EGFP-14CUG).

[0232] Fig.14. Crystal structure of PUF domain binding to target RNA (PDB ID 2YJY) (left). PUF binding to RNA (right). DETAILED DESCRIPTION

[0233] Through extensive and in-depth research, the inventors have developed for the first time a nucleic acid construct group for screening drugs for treating diseases associated with long RNA repeat sequences, as well as a vector and a screening system for the nucleic acid construct group. Using the nucleic acid construct group, vector, and screening system, drugs for treating diseases associated with long RNA repeat sequences can be screened or determined with high throughput. Based on the screening method for RNA repeat sequences developed by the present invention, potential drugs for treating diseases associated with RNA repeat sequences can be screened quickly and accurately. The screening method of the present invention combines technologies from multiple fields such as bioinformatics, and can comprehensively analyze and evaluate the potential therapeutic capabilities of multiple drugs. The present invention was completed on this basis.

[0234] Long RNA repeats

[0235] As used herein, the term "long RNA repeat sequence" or "RNA long repeat sequence" is used interchangeably and refers to a long sequence with an abnormal number of RNA repeat units, which can cause abnormal RNA metabolism in cells, thereby leading to the occurrence of diseases. It is caused by abnormal expansion of microsatellite sequences on the genome.

[0236] The correspondence between the types of diseases caused by long RNA repeat sequences and long RNA repeat sequences is shown in Tables 1 and 2. (Reference: Depienne, C., & Mandel, J.-L. (2021). 30 years of repeat expansion disorders: What have we learned and what are the remaining challenges? The American Journal of Human Genetics, 108 (5), 764–785.)

[0237] Table 1 List of disordered repeat expansion sequences (sorted by discovery year)

[0238]

[0239]

[0240] Disorders: BCCD, brachydactyly and cleidocranial dysplasia; BPES, blepharophimosis, ptosis and epicanthus inversus; CCHS, congenital central hypoventilation syndrome; DRPLA, dentatorubral-pallidoluysian atrophy; EIEE1, early infantile epileptic encephalopathy type 1; HD, Huntington disease; HDL2, Huntington disease-like 2; HFGS, hand-foot-genital syndrome; HPE5, holoprosencephaly type 5, holoprosencephaly type 5; MRGH, mental retardation with isolated growth hormone deficiency; OPMD, oculopharyngeal muscular dystrophy; SBMA, spinal and bulbar muscular atrophy; SPD1, synpolydactyly type 1; SCA, spinocerebellar ataxia.

[0241] Genetic characteristics: AD, autosomal dominant, autosomal dominant; XLR, X-linked recessive, X-linked recessive.

[0242] Pathogenic mechanism: polyA(DN),polyAlanine tract associated with dominant-negative effect; polyA(LoF),polyAlanine tractleading to a loss of function of the protein; polyQ,polyGlutamine protein toxicity; polyG,polyGlycinepeptide toxicity; RNA,RNA toxicity; RNA(foci),RNA focitoxicity.

[0243] Identification method: cl, cloning; CGA, candidate gene analysis; ExScr, expansion screening; L, linkage; RED, repeat expansion detection.

[0244] Table 2 List of non-coding repeat expansion disorder sequences (sorted by discovery year)

[0245]

[0246]

[0247]

[0248]

[0249] Disorders: BSS, Baratela-Scott syndrome; CANVAS, cerebellar ataxia, neuropathy and vestibular areflexia syndrome; DM1, myotonic dystrophy type 1; DM2, myotonic dystrophy type 2; EPM1, progressive myoclonus epilepsy type 1 (Unverricht-Lundborg disease); FAME, familial adult myoclonic epilepsy; FECD3, Fuchsendothelial corneal dystrophy type 3; FRAXE, fragileXE syndrome; FRDA, Friedreich ataxia, hereditary cerebellar ataxia syndrome; FTD / ALS, frontotemporal dementia / amyotrophic lateral sclerosis; FXPOI, Fragile X-associated premature ovarian infertility; FXS, fragile X syndrome; FXTAS, fragile X-associated tremor ataxia syndrome; GDPAG, global developmental delay, progressive ataxia, and elevated glutamine; NIID, neuronalintranuclear inclusion disease; OPDM1, oculopharyngodistal myopathy type 1; OPDM2, oculopharyngodistal myopathy type 2;OPML1, oculopharyngeal myopathy with leukoencephalopathy type 1; SCA, spinocerebellar ataxia; XDP, X-linked dystonia parkinsonism.

[0250] Genetic characteristics: AD, autosomal dominant, autosomal dominant; XL, X-linked, X chromosome linked; XLR, X-linked, recessive, X chromosome linked recessive inheritance; XLD, X-linked, dominant, X chromosome linked dominant inheritance.

[0251] Pathogenic mechanism: EGS, epigenetic gene silencing; Me / GS, genesilencing associated with hypermethylation; PS, protein sequestration; RNA, RNA toxicity; RNA(foci), RNA focitoxicity; RAN, repeat-associated non-AUG translation.

[0252] Identification methods: ADO, allele drop-out; cl, cloning; CGA,

[0253] candidate gene analysis, candidate gene analysis; exp, expression, experiment; ExScr, expansion screening, expansion screening; Fam, clinical analyses in fragile X-affected families, clinical analysis of fragile X-affected families; FS, fragile site, fragile site; GWAS, genome-wide association study, genome-wide association study; K, karyotyping, chromosome karyotyping analysis; L, linkage, gene linkage; LRS, long-read sequencing, long-read sequencing; RED, repeat expansion detection, repeat sequence detection; WGS, whole-genome sequencing, whole genome sequencing.

[0254] Short RNA repeats

[0255] As used herein, the terms "short RNA repeat sequence" or "short RNA repeat sequence" are used interchangeably and refer to short sequences with a normal number of RNA repeat units that do not cause abnormal RNA metabolism in cells.

[0256] Gene editing proteins

[0257] The present invention mainly focuses on CRISPR proteins, and currently Cas13a, Cas13b, Cas13d, Cas13x, Cas13y, SpCas9 and SaCas9 have been discovered and proven to be active.

[0258] PUF protein

[0259] The Drosophila melanogaster Pumilio and Caenorhabditis elegans fem-3 mRNA binding factors (PUFs) are sequence-specific RNA binding proteins that recognize mRNA targets using repeat structures within the protein. Typical PUF proteins have a C-terminal RNA binding domain (also known as the PUF domain), which contains eight tandem repeats in its protein structure, as well as N- and C-terminal regions. The eight PUF repeats are arranged in an arch shape, and each repeat consists of three α helices. RNA binds to the concave surface of the PUF domain in an antiparallel manner, and each base is specifically recognized by a repeat sequence. The second α helix of each repeat sequence contains a conserved RNA recognition code consisting of 5 amino acid residues, which is represented as "12XX5" (X represents a random residue), where residues 1 and 5 directly interact with RNA bases through polarity, and residue 2 stacks with the base. The stacking residues in each PUF repeat sequence play an important role in binding affinity, while the two polar or charged residues determine the specificity of the PUF repeat. Therefore, there is a set of specific recognition codes in the natural PUF protein: cysteine ​​and glutamine bind to adenine, asparagine and glutamine bind to uracil, and serine and glutamate bind to guanine. In 2011, two independent research groups successfully deciphered the missing cytosine recognition code in the PUF recognition code through the yeast three-hybrid system (serine and arginine bind to cytosine). So far, the PUF protein can recognize any 8-nt RNA by changing its RNA recognition code. Fig.14 shown.

[0260] The unique structure and relatively short length of the PUF domain make it an ideal RNA binding scaffold for artificial proteins to regulate RNA processing. Several artificial PUF regulatory factors with different functional domains have been successfully developed and applied in different fields. Existing artificial PUF factors are being used in biomedical research and new treatments for RNA-related diseases. Despite the success of early applications, there are several limitations to using customized PUF domains as programmable RNA binding scaffolds. One major limitation is that natural PUF proteins recognize 8-nt RNA sequences. However, in some cases, PUF proteins may be required to recognize target sequences of different lengths. For example, when it is necessary to bind a long RNA in the entire transcriptome, PUFs with more repeats will minimize off-target effects. In contrast, shorter recognition sites are more practical for some in vitro applications to bind RNA substrates at multiple sites. The latest research on PUF structures of different lengths shows that when PUFs have 9-10 repeat structures, their recognition ability for RNA reaches the maximum. When the repeat structure is increased or decreased, it still binds to its target sequence, but its affinity is reduced.

[0261] Screening system and method of the present invention

[0262] The present invention constructs for the first time a system for screening drugs for treating diseases associated with long RNA repeat sequences, the system comprising:

[0263] (a) drugs to be screened;

[0264] (b) the nucleic acid construct set according to the first aspect of the present invention;

[0265] Among them, when the expression amount or intensity of the first detectable marker is significantly reduced, and the expression amount or intensity of the second detectable marker changes slightly or remains basically unchanged, it indicates that the drug to be screened is a drug for treating long RNA repeat sequence-related diseases.

[0266] The system of the present invention is mainly composed of plasmids that can express RNA with two different repeat sequence lengths at the same intensity.

[0267] To facilitate screening, two RNAs with different repeat lengths can be linked to different reporter genes, including but not limited to various fluorescent proteins, luciferases, and other reporter genes that can be used to characterize gene expression levels.

[0268] The reporter gene system or screening system reported in the present invention can be used through a variety of detection methods, including but not limited to real-time fluorescence quantitative PCR, RNA-seq, fluorescence microscopy, microplate reader, protein immunoblotting (Western Blotting), RNA immunoblotting (Northern Blotting), flow cytometry (Flow Cytometry), etc., to simultaneously detect the clearance of two different lengths of RNA by different drug molecules.

[0269] The system of the present invention can be used to characterize the clearance effects of different tools on long repetitive sequences at the mammalian cell level, and the clearance of short repetitive sequences by each system can also be observed simultaneously.

[0270] The system of the present invention can be used at the model animal (zebrafish, mouse) level to characterize the clearance effects of different tools on long repetitive sequences, and the clearance of short repetitive sequences by each system can also be observed simultaneously.

[0271] Furthermore, according to the present invention, RNAs with two different repeat sequence lengths can be expressed simultaneously with the same intensity (or similar intensity), which is convenient for subsequent detection and evaluation.

[0272] According to the present invention, the expression methods that can be used include bicistronic expression or dual gene reverse expression, using the same (or similar expression strength) eukaryotic promoter, including but not limited to the commonly used CMV promoter, CAG promoter, PGK promoter, EF-1α promoter, SV40 promoter and other alternative promoters.

[0273] According to the present invention, the two RNAs expressed simultaneously contain two RNA repeat sequences of different lengths, one of which is a normal-length RNA with a shorter RNA repeat sequence, and the other is an abnormal-length RNA with a longer RNA repeat sequence.

[0274] The present invention has developed a reporting system for screening drugs targeting long RNA repeat sequences. Based on the screening method for RNA repeat sequences developed by the present invention, potential drugs for treating diseases related to RNA repeat sequences can be screened quickly and accurately. The screening method of the present invention combines technologies from multiple fields such as bioinformatics and can comprehensively analyze and evaluate the potential therapeutic capabilities of multiple drugs.

[0275] The research and development of the reporter system of the present invention will help to further understand the pathophysiological mechanisms of diseases associated with long RNA repeat sequences, and promote the development and progress of treatment methods for such diseases. At the same time, the drug screening of RNA repeat sequence-related diseases by this reporter system will have broad application prospects in the entire research field, bringing more opportunities and possibilities for the treatment of RNA-related diseases. By further studying the molecular mechanisms and clinical manifestations of RNA repeat sequence-related diseases, we will have the opportunity to design and discover more innovative and therapeutic drugs for RNA repeat sequence-related diseases.

[0276] The present invention also establishes a system that can simultaneously and equally express RNA with two different repeat sequence lengths. The system simulates the in vivo allele expression intensity to the greatest extent possible, but the repeat sequence lengths carried are inconsistent, and is very suitable for drug screening for such diseases.

[0277] The main advantages of the present invention include:

[0278] (1) For the first time, a nucleic acid construct group for screening drugs for treating long RNA repeat sequence-related diseases, as well as a vector and a screening system for the nucleic acid construct group have been developed. The nucleic acid construct group, vector and screening system can be used to screen or identify drugs for treating long RNA repeat sequence-related diseases at a high throughput.

[0279] (2) Based on the screening method for RNA repeat sequences developed by the present invention, potential drugs for treating diseases related to RNA repeat sequences can be screened quickly and accurately. The screening method of the present invention combines technologies from multiple fields such as bioinformatics and can comprehensively analyze and evaluate the potential therapeutic abilities of multiple drugs. The present invention is completed on this basis.

[0280] (3) The system of the present invention can be used to characterize or screen drugs targeting long repeat sequences while also characterizing their effects on short repeat sequences. And because the same promoter element is used, the expression intensity of RNA of these two lengths is close, which is similar to the situation in which such diseases are caused by long repeat sequences in the human body.

[0281] (4) Currently, there is no similar commercial system. The systems currently used generally only focus on the characterization of their effects on long repeat sequences, while ignoring the characterization of short repeat sequences.

[0282] (5) The system of the present invention can also be used in cells and model animals (zebrafish, mice).

[0283] (6) The system of the present invention can be used to screen for drugs for treating diseases associated with long RNA repeat sequences, such as artificial RNA degrading enzymes having the specific structure of Formula IV or IV' of the present invention.

[0284] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0285] Unless otherwise specified, the materials and reagents in the examples are commercially available products.

[0286] Example 1. Construction of a reporter plasmid containing CTG repeat sequences.

[0287] First, DNA fragments expressing mCherry and EGFP were obtained by PCR amplification, and pcDNA5-FRT / TO plasmids (purchased from Thermo Fisher) were linearized by PCR. The mCherry fragment and the linearized vector pcDNA5-FRT / TO were cyclized by homologous recombination, and the reagent used was Gibson Assembly Master Mix (purchased from New England Biolabs). The cyclized product was transformed into DH5a competent medium (purchased from Beijing Quanshijin Biology) and cultured according to the instructions. The obtained plasmids were confirmed to be pcDNA5-FRT / TO-mCherry and pcDNA5-FRT / TO-EGFP by Sanger sequencing.

[0288] The synthesized (CTG) 14 The DNA fragment (purchased from Genewiz) was inserted into the pre-constructed pcDNA5-FRT / TO-mCherry by PCR, specifically at the C-terminus of the mCherry gene, thereby obtaining a plasmid that can express mCherry-(CTG) 14 Plasmid pcDNA5-FRT / TO-mCherry-(CTG) 14 .

[0289] The restriction site of restriction enzyme SapI was inserted into the pre-constructed pcDNA5-FRT / TO-EGFP by PCR, specifically at the C-terminus of the EGFP gene, thereby obtaining pcDNA5-FRT / TO-EGFP-IN.

[0290] The gene synthesized (CTG) 50The DNA fragment was digested with EarI and ligated into the pcDNA5-FRT / TO-EGFP-IN vector digested with SapI to obtain pcDNA5-FRT / TO-EGFP-(CTG) 50 (Recorded as 50CUG). Then repeat the restriction enzyme cutting and ligation process to obtain pcDNA5-FRT / TO-EGFP-(CTG) 100 (denoted as 100CUG), pcDNA5-FRT / TO-EGFP-(CTG) 150 (denoted as 150CUG) and pcDNA5-FRT / TO-EGFP-(CTG) 200 (denoted as 200CUG). During the construction process, since the CTG repeat sequence will condense during the culture of E. coli, pcDNA5-FRT / TO-EGFP-(CTG) was also obtained. 130 (denoted as 130CUG) plasmid.

[0291] Place pcDNA5-FRT / TO-mCherry-(CTG) 14 Plasmid PCR was used to introduce NotI and KpnI single restriction sites to obtain vectors. DNA fragments with EGFP and CUG of different lengths were obtained by using NotI and KpnI to digest plasmids such as 50CUG, 100CUG, 130CUG, 150CUG, and 200CUG. After homologous recombination, vectors with two different CUG repeat sequence lengths were obtained that could express the same intensity at the same time. The vectors expressed two different fluorescent proteins for the indication of RNA expression amount ( Figure 1 A).

[0292] Example 2. Construction of a reporter plasmid containing CCTG repeat sequences

[0293] The synthesized (CCTG)8 DNA fragment was inserted into the pre-constructed pcDNA5-FRT / TO-mCherry by PCR, specifically at the C-terminus of the mCherry gene, thereby obtaining pcDNA5-FRT / TO-mCherry-(CCTG)8.

[0294] The restriction site of restriction enzyme BbsI was inserted into the pre-constructed pcDNA5-FRT / TO-EGFP by PCR, specifically at the C-terminus of the EGFP gene, thereby obtaining pcDNA5-FRT / TO-EGFP-IN.

[0295] Gene synthesis (CCTG) 50The DNA fragment was digested with BbsI and Esp3I and ligated into the pcDNA5-FRT / TO-EGFP-IN vector digested with BbsI to obtain pcDNA5-FRT / TO-EGFP-(CCTG) 50 (Recorded as 50CCUG). Then repeat the restriction digestion and ligation process to obtain pcDNA5-FRT / TO-EGFP-(CCTG) 100 (denoted as 100CCUG), pcDNA5-FRT / TO-EGFP-(CCTG) 150 (denoted as 150CCUG) and pcDNA5-FRT / TO-EGFP-(CCTG) 200 (denoted as 200CCUG). Similarly, the CCTG repeat sequence undergoes condensation during the culture of E. coli, and pcDNA5-FRT / TO-EGFP-(CCTG) was obtained. 170 (denoted as 170CCUG) plasmid.

[0296] The pcDNA5-FRT / TO-mCherry-(CCTG)8 plasmid was introduced into the NotI and KpnI single restriction sites by PCR to obtain a vector. The plasmids of 50CCUG, 100CCUG, 150CCUG, 200CCUG, etc. were digested with NotI and KpnI to obtain DNA fragments with EGFP and CCUG of different lengths. After homologous recombination reaction, a vector with two different CCUG repeat sequence lengths was obtained that could express at the same intensity. The vector expressed two different fluorescent proteins for the indication of RNA expression amount ( Figure 1 B).

[0297] Example 3. Construction of a reporter plasmid containing CAG repeat sequences

[0298] The synthesized (CAG) 23 The DNA fragment was inserted into the pre-constructed pcDNA5-FRT / TO-mCherry by PCR, specifically at the C-terminus of the mCherry gene, thus obtaining pcDNA5-FRT / TO-mCherry-

[0299] (CAG) 23 .

[0300] The restriction site of restriction enzyme BbsI was inserted into the pre-constructed pcDNA5-FRT / TO-EGFP by PCR, specifically at the C-terminus of the EGFP gene, thereby obtaining pcDNA5-FRT / TO-EGFP-IN.

[0301] The synthetically produced (CAG) 50The DNA fragment was digested with BbsI and Esp3I and ligated into the pcDNA5-FRT / TO-EGFP-IN vector digested with BbsI to obtain pcDNA5-FRT / TO-EGFP-(CAG) 50 (Note: 50CAG). Then repeat the restriction enzyme cutting and ligation process to obtain pcDNA5-FRT / TO-EGFP-(CAG) 100 (denoted as 100CAG), pcDNA5-FRT / TO-EGFP-(CAG) 150 (denoted as 150CAG) and pcDNA5-FRT / TO-EGFP-(CAG) 200 (denoted as 200CAG).

[0302] Place pcDNA5-FRT / TO-mCherry-(CAG) 23 Plasmid PCR was used to introduce NotI and KpnI single restriction sites to obtain a vector. DNA fragments with EGFP and CAG of different lengths were obtained by using NotI and KpnI to digest plasmids such as 50CAG, 100CAG, 150CAG, and 200CAG. After homologous recombination, a vector with two different CAG repeat sequence lengths was obtained that could express the same intensity at the same time. The vector produced two different fluorescent proteins for the indication of RNA expression amount ( Figure 1 C).

[0303] Example 4. Construction of a reporter plasmid containing TGGAA repeat sequences

[0304] The synthesized (TGGAA)3 DNA fragment was inserted into the pre-constructed pcDNA5-FRT / TO-mCherry by PCR, specifically at the C-terminus of the mCherry gene, thereby obtaining pcDNA5-FRT / TO-mCherry-(TGGAA)3.

[0305] The restriction site of restriction enzyme BbsI was inserted into the pre-constructed pcDNA5-FRT / TO-EGFP by PCR, specifically at the C-terminus of the EGFP gene, thereby obtaining pcDNA5-FRT / TO-EGFP-IN.

[0306] The gene synthesized (TGGAA) 50 The DNA fragment was digested with BbsI and Esp3I and ligated into the pcDNA5-FRT / TO-EGFP-IN vector digested with BbsI to obtain pcDNA5-FRT / TO-EGFP-(TGGAA) 50(Recorded as 50TGGAA). Then repeat the restriction enzyme cutting and ligation process to obtain pcDNA5-FRT / TO-EGFP-(TGGAA) 100 (denoted as 100TGGAA).

[0307] The pcDNA5-FRT / TO-mCherry-(TGGAA)3 plasmid was PCR introduced into the NotI and KpnI single restriction sites to obtain a vector. The plasmids 50TGGAA, 100TGGAA, etc. were digested with NotI and KpnI to obtain DNA fragments with EGFP and TGGAA of different lengths. After homologous recombination, a vector that can express two different TGGAA repeat sequence lengths at the same intensity was obtained. The vector expressed two different fluorescent proteins for the indication of RNA expression amount ( Figure 1 D).

[0308] Example 5. Construction of a cell line that can stably express a reporter plasmid

[0309] The plasmids containing different CTG / CCTG / CAG / TGGAA repeat sequence lengths obtained above were co-transfected with plasmid pOG44 (purchased from Thermo Fisher) at a ratio of 1:9 into Flp-In T-Rex 293 cells (purchased from ThermoFisher). 48 hours after transfection, the cells were washed and plated into fresh culture medium (<25% cell density) and cultured overnight at 37°C under 5% carbon dioxide. Subsequently, the cells were cultured every 3 days with fresh culture medium containing 100 μg / mL Hygromycin (purchased from Thermo Fisher) until resistant single cell colonies appeared. 3-4 individual colonies were selected for further use. The correctness of the inserted sequence was verified by Sanger sequencing.

[0310] The cell line carrying the stably expressed reporter plasmid can be induced to express by adding doxycycline (purchased from Sigma-Aldrich) or tetracycline (Tetracycline) at a final concentration of 1 μg / mL, and the expression of the reporter system at the cellular level can be detected by fluorescence microscopy, flow cytometry or other detection methods. Figure 2 The results of induced expression of the dual reporter gene system with CUG repeat sequences in Flp-In T-REX 293 cells. Figure 2 A shows that when no inducer was added, both fluorescent reporter genes were not expressed; however, after induction with 1 μg / mL of doxycycline ( Figure 2 B), Both fluorescent proteins showed high expression. Figure 2 C is the fluorescence microscopy result shown. Figure 2 The results showed that stably transfected cell lines carrying reporter genes with RNA repeat sequences of different lengths can stably express these two different reporter genes and the RNA repeat sequences they carry.

[0311] Example 6. Comparison of existing methods for regulating RNA repetitive sequences at the cellular level

[0312] The plasmid of the Cas13d system that can express the CUG repeat sequence targeting (purchased from addgene), the PIN-RCas9 system plasmid (purchased from addgene), the ASRE (CUG) plasmid (self-owned), the shRNA (CUG) plasmid (purchased from addgene), the antisense nucleotide (ASO) (purchased from Jima Gene), and the control plasmid empty vector pcDNA3.1 (purchased from Thermo Fisher) and the 150CUG plasmid described in the present invention were transfected with Lipofectamine 3000 (purchased from Thermo Fisher) at a ratio of 1:10 and transfected into HEK 293T cells (purchased from ATCC). For specific steps, refer to the instruction manual of Lipofectamine 3000. After 48 hours of transfection, the adherent cells were digested with trypsin, the cells were collected by centrifugation at 800g, and then the cells were washed with PBS solution (see the experimental design and process). Figure 3 ).

[0313] The cells were then divided into three parts. The first part of the cells was used to detect the fluorescence levels of EGFP and mCherry by flow cytometry (see the results). Figure 4 ). Figure 4 It is shown that, using the screening and comparison system provided by the present invention, it can be found that among the currently available regulatory methods, only the PIN-RCas9 system achieves a significant reduction in the long RNA repeat sequence EGFP-130CUG, which is manifested in a decrease in the level of the EGFP protein expressed.

[0314] The second part of the cells was centrifuged again, lysed with RIPA solution (purchased from Thermo Fisher), added with 2x Laemmli Sample Buffer (purchased from Biorad), heated to 99°C for 10 min, and then used for protein immunoblotting identification.

[0315] The third part of the cells was lysed with TRIzol Reagent (purchased from Thermo Fisher), and the total RNA of the cells was extracted according to the instructions. The extracted total RNA was reverse transcribed using TAKARA's PrimeScript RT Reagent Kit with gDNA Eraser or other companies' reverse transcription kits. Subsequently, 2X Universal SYBR GreenFast qPCR Mix reagent (purchased from Wuhan ABclonal) was used for quantitative PCR detection to compare the clearance effect and off-target effect of the regulatory method used (see the test results). Figure 5 ), Figure 5 It was shown that among all the methods involved in the comparison, only shCUG and PIN-RCas9 (CUG) achieved the elimination of target long RNA repeat sequences, but the selectivity of shCUG was poor.

[0316] The primer sequences used for quantitative PCR were: EGFP-F1: 5'-AGTGCTTCAGCCGCTACCC-3'; EGFP-R1: 5'-GTTGTACTCCAGCTTGTGCC-3'; mCherry-F1: 5'-CTTCAAGTGGGAGCGCGTG-3'; mCherry-R1: 5'-CAGCCTCTGCTTGATCTCGCC-3'; GAPDH-F: 5'-TTGCCATCAATGACCCCTTCA-3'; GAPDH-R: 5'-CGCCCCACTTGATTTTGGA-3'.

[0317] Example 7. Construction and detection of new artificial RNase

[0318] The plasmid expressing ASRE (CUG) was linearized by PCR linearization, and the primer sequences used were PUF-R: 5'-CTGAGAGCCGTTGCCGGTGTCGACCCCTAAGTCAACACCGTTC-3'; CDNA-F: 5'-GCTAGCGTTTAAACTTAAGCTTGGTACC-3'. The structure of the artificial RNase part of ASRE (CUG) selected was NLS-PUF (CUG)-Linker-PIN.

[0319] The full-length DNA or active partial DNA fragments of the following RNases or RNA helicases were amplified from the cDNA of HEK 293T by PCR amplification: including but not limited to ANG (UNIPROT: P03950), RNASEL (UNIPROT: Q05823), RRP44 (UNIPROT: Q9Y2L1), RNASE1 (UNIPROT: P07998), RNASET2 (UNIPROT: O00584), SUPV3L1 (UNIPROT: Q8IYB8), PNPT1 (UNIPROT: Q8TCS8), REXO2 (UNIPROT: Q9Y3B8), MRPL44 (UNIPROT: Q9H9J2), LACTB2 (UNIPROT: Q53H82), TSEN15 (UNIPROT: Q8WW01), and ZC3H12A (UNIPROT: Q5D1E8). The amplified fragments all carry DNA bases (>20 bp) that can complement the linearized ASRE (CUG) plasmid. The obtained DNA fragments were circularly connected with the linearized ASRE (CUG) plasmid vector using Gibson Assembly Master Mix (purchased from New England Biolabs), thereby replacing the original artificial RNA enzymes PUF-ANG, PUF-ANG-S, PUF-RNASEL, PUF-SUPV3L1, PUF-SUPV3L1 FL, PUF-RNASET2, PUF-RRP44, PUF-RNASE1, PUF-PNPT1, PUF-REXO2, PUF-MRPL44, PUF-LACTB2, PUF-TSEN15 and PUF-ZC3H12A. Similarly, PCR and homologous recombination methods can also be used to construct ANG-PUF, ANG-S-PUF, SUPV3L1-PUF, SUPV3L1 FL-PUF, RNASET2-PUF, RNASE1-PUF, PNPT1-PUF, REXO2-PUF and ZC3H12A-PUF (the structures of the new artificial RNA enzyme elements are shown in Figure 6 A, Figure 6 A is only a schematic diagram).

[0320] 1 μg of the new artificial RNA enzyme plasmid and 0.1 μg of the reporter plasmid pcDNA5-EGFP-CTG130-mCherry-CTG14 were co-transfected into HEK 293T cells (cell density was about 80%) according to the instructions of Lipofectamine 3000 (purchased from Thermo Fisher), and the transfected cells were collected 48 hours after transfection. The resulting cells were lysed with TRIzol Reagent (purchased from ThermoFisher), and the total RNA of the cells was extracted according to the instructions. The extracted total RNA was reverse transcribed using PrimeScript RT Reagent Kitwith gDNA Eraser (purchased from TAKARA) or reverse transcription kits from other companies. Subsequently, quantitative PCR detection was performed using 2X Universal SYBR Green Fast qPCR Mix reagent (purchased from Wuhan Abotek ABclonal) (see the test results). Figure 6 B) From Figure 6 From the detection results of B, we can see that PUF-RNASE1, PUF-SUPV3L1 (ARC-S), PUF-SUPV3L1 FL, PUF-RNASET2, PUF-PNPT1, PUF-REXO2, RNASE1-PUF, PNPT1-PUF and REXO2-PUF, SUPV3L1-PUF, SUPV3L1 FL-PUF, RNASET2-PUF (ARC-T) (ARC, Artificial RNA Cleaver, artificial RNA enzyme) showed good selectivity for two different lengths of CTG repeat sequences, and had a good effect on reducing long CUG repeat sequences.

[0321] Subsequently, we used Lipofectamine 3000 (purchased from Thermo Fisher) to transfect ARC-S and ARC-T with outstanding test results and the 130CUG plasmid described in the present invention at a ratio of 1:10 into HEK 293T cells (purchased from ATCC). For specific steps, refer to the instructions of Lipofectamine 3000. After 48 hours of transfection, the adherent cells were digested with trypsin, centrifuged at 800g to collect the cells, and then washed with PBS solution (see the experimental design and process for details). Figure 3 ).

[0322] The cells were then used to detect the fluorescence levels of EGFP and mCherry using flow cytometry (see Figure 7 ), Figure 7It is shown that, using the screening and comparison system provided by the present invention, it can be found that the new artificial RNA enzymes ARC-S and ARC-T constructed by us have achieved a significant reduction in the long RNA repeat sequence EGFP-130CUG, which is manifested as a decrease in the level of EGFP protein expressed by it, but have no obvious effect on mCherry-14CUG containing a short repeat sequence.

[0323] Example 9

[0324] According to the results of fluorescent RNA imaging reported in the literature, the DNA sequence with fluorescent RNA fragment (4Pepper, obtained from East China University of Science and Technology) was connected into plasmid DT12 (purchased from Addgene, Cat#80416) and DT960 (purchased from Addgene, Cat#80412) by PCR restriction ligation method. The insertion position was before DMPK exon12 and after CMV promoter. Thus, plasmid DT12-4Pepper and plasmid DT960-4Pepper ( Figure 8 A).

[0325] DT12-4Pepper and DT960-4Pepper were co-transfected with NLS-PUF-BFP-CUG plasmid (constructed according to the construction method of Example 1) using Lipofectamine 3000 (purchased from Thermo Fisher) and transfected into HEK293T cells (purchased from ATCC). The steps were referred to the instructions of Lipofectamine 3000. After 48 hours of transfection, the cells were imaged using a fluorescent confocal microscope, and the number of RNA aggregates in the cell nucleus was counted and detected ( Figure 8 B) (n=50). Figure 8 The statistical results of B show that DT960-4Pepper can form more RNA aggregates in the cell nucleus (consistent with reports in the literature), while DT12-4Pepper can only form a small amount or no RNA aggregates. At the same time, the distribution of fluorescent RNA labeled with CUG repeat sequences and the distribution of fluorescent protein NLS-PUF-BFP targeting CUG repeat sequences were quantitatively counted ( Figure 8 C), and found that the distribution of the two is positively correlated ( Figure 8 D).

[0326] Example 10. Clearance of RNA repetitive sequence nuclear aggregation by different systems

[0327] The existing different systems for RNA repeat sequences (plasmids were all labeled with BFP for imaging detection) were co-transfected with DT960-4Pepper using Lipofectamine 3000 (purchased from Thermo Fisher) and transfected into HEK 293T cells (purchased from ATCC). The steps refer to the instructions of Lipofectamine 3000. After 48 hours of transfection, the cells were imaged with a fluorescent confocal microscope to detect the number of RNA aggregates in the cell nucleus. In the imaging results, the dye SYTO 9 was used to distinguish the cytoplasm from the nucleus (FITC channel, green), the blue fluorescent protein BFP labeled with different system plasmids was used to characterize the cells with successful transfection system, and the TRITC channel was used to select cells successfully transfected with DT960-4Ppepper (orange). Cells expressing BFP and DT960-4Ppepper at the same time were used to count the number of RNA aggregates in the cell nucleus.

[0328] Imaging results showed that the PIN-RCas9 system had the best clearance ability in the non-PUF protein-related regulatory system ( Fig. 9 A) (n=50). In the PUF protein-related regulatory system, the ARC-T constructed in this application has the best clearance ability ( Fig. 9 B) (n = 50). In all system comparisons, the PIN-RCas9 system has a similar clearance effect to ARC-T ( Fig. 9 C).

[0329] Example 11. Construction of a mouse strain that can stably express a reporter plasmid

[0330] A mouse strain (denoted as mouse-CUG-R160G14) that can stably express the reporter plasmid was constructed by a contract research organization (CRO) (purchased from Saiye Bio). Specifically, a mouse strain that can simultaneously express EGFP-(CUG) 14 with mCherry-(CUG) 160 The DNA fragment of the RNA sequence was inserted into the intron 1 position of the ROSA26 gene (NCBI Reference Sequence: NR_027008.1) in the mouse genome (the construction strategy is shown in Fig.10 ).

[0331] A mouse strain (denoted as mouse-CAG-G155R23) that can stably express the reporter plasmid was constructed by a contract research organization (CRO) (purchased from Saiye Bio). Specifically, a mouse strain that can simultaneously express EGFP-(CAG) 155 with mCherry-(CAG) 23The DNA fragment of the RNA sequence was inserted into the intron 1 position of the ROSA26 gene (NCBI Reference Sequence: NR_027008.1) in the mouse genome (the construction strategy is shown in Fig.11 ).

[0332] The transgenic mouse mouse-CUG-R160G14 obtained can simultaneously express EGFP-(CUG) 14 with mCherry-(CUG) 160 RNA sequences, therefore, carry two different fluorescent ( Fig.12 ). This feature can be used to quickly screen and determine the effects of drugs specific to CUG repeat sequences.

[0333] Example 12. Comparison of existing methods for regulating RNA repetitive sequences at the in vivo level

[0334] AAV virus particles expressing the Cas13d system targeting CUG repeat sequences (purchased from Heyuan Biotechnology), AAV virus particles of the PIN-RCas9 system plasmid (purchased from Heyuan Biotechnology), AAV virus particles of PUF-PIN (purchased from Heyuan Biotechnology), AAV virus particles of ARC-S (purchased from Heyuan Biotechnology), and AAV virus particles of ARC-T (purchased from Heyuan Biotechnology) were added at 10 11 vg were injected into the thigh skeletal muscle (right leg) of mouse-CUG-R160G14 mice (purchased from Saiye Biotechnology), and the respective control AAV virus particles (purchased from Heyuan Biotechnology) were injected into the left leg ( Fig.13 A).

[0335] Two months after the injection, the injected leg muscles were removed and total RNA was extracted using TRIzol Reagent (purchased from ThermoFisher). The specific steps were performed according to the instructions. The extracted total RNA was reverse transcribed using TAKARA's PrimeScript RTReagent Kit with gDNA Eraser or other companies' reverse transcription kits. Subsequently, 2X Universal SYBR Green Fast qPCR Mix reagent (purchased from Wuhan Aibotek ABclonal) was used for quantitative PCR detection to compare the clearance effect of the regulatory method used and the off-target effect ( Fig.13 B and Fig.13 C) From Fig.13As can be seen in B, ARC-T virus particles have the best clearance effect on mCherry-160CUG with long RNA repeat sequences, followed by Cas13d-CUG, ARC-S, PUF-PIN-CUG and PIN-RCas9-CUG. Fig.13 As can be seen in C, Cas13d-CUG virus particles have the greatest off-target clearance effect (about 50%) on EGFP-14CUG with short RNA repeat sequences, while the other four have comparable and lower off-target effects (about 20%), and ARC-T has the lowest off-target rate.

[0336] The primer sequences used for quantitative PCR were: EGFP-F2: 5'-CCCGACAACCACTACCTGAG-3'; EGFP-R2: 5'-TGCATCTTACACCAAACTCATCTG-3'; mCherry-F2: 5'-AAGACTATGGGCTGGGAGGC-3'; mCherry-R2: 5'-CTCGTTGTGGGAGGTGATGT-3'; mGAPDH-F: 5'-GTCAAGGCCGAGAATGGGAA-3'; mGAPDH-R: 5'-CTCGTGGTTCACACCCATCA-3'.

[0337] Sequence information

[0338] SEQ ID NO: 1 PUF protein fragment

[0339] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALS THPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0340] CMV promoter SEQ ID NO: 2

[0341] GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

[0342] SEQ ID NO:3 CAG promoter

[0343] GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0344] SEQ ID NO:4 PGK promoter

[0345] GGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCG

[0346] SEQ ID NO:5 EF-1α promoter

[0347]

[0348] SEQ ID NO:6 SV40 promoter

[0349] GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA

[0350] SEQ ID NO:7 chicken beta-actin promoter

[0351] TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0352] SEQ ID NO:8 HSV-TK promoter

[0353] AAATGAGTCTTCGGACCTCGCGGGGGCCGCTTAAGCGGTGGTTAGGGTTTGTCTGACGCGGGGGGAGGGGGAAGGAACGAAACACTCTCATTCGGAGGCGGCTCGGGGTTTGGTCTTGGTGGCCACGGGCACGCAGAAGAGCGCCGCGATCCTCTTAAGCACCCCCCCGCCCTCCGTGGAGGCGGGGGTTTGGTCGGCGGGTGGTAACTGGCGGGCCGCTGACTCGGGCGGGTCGCGCGCCCCAGAGTGTGACCTTTTCGGTCTGCTCGCAGACCCCCGGGCGGCGCCGCCGCGGCGGCGACGGGCTCGCTGGGTCCTAGGCTCCATGGGGACCGTATACGTGGACAGGCTCTGGAGCATCCGCACGACTGCGGTGATATTACCGGAGACCTTCTGCGGGACGAGCCGGGTCACGCGGCTGACGCGGAGCGTCCGTTGGGCGACAAACACCAGGACGGGGCACAGGTACACTATCTTGTCACCCGGAGGCGCGAGGGACTGCAGGAGCTTCAGGGAGTGGCGCAGCTGCTTCATCCCCGTGGCCCGTTGCTCGCGTTTGCTGGCGGTGTCCCCGGAAGAAATATATTTGCATGTCTTTAGTTCTATGATGACACAAACCCCGCCCAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGCAGTCGGGGCGGCGCGGTCCCAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAA

[0354] SEQ ID NO:9 Ubc promoter

[0355]

[0356] SEQ ID NO:10 TRE promoter

[0357] GAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCC

[0358] SEQ ID NO:11 UAS promoter

[0359] CGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGACTCGAGCGGAGTACTGTCCTCCGATCGGAGTACTGTCCTCCGCGAATTCCGGAGTACTGTCCTCCG

[0360] SEQ ID NO:12 Ad promoter

[0361] GGGGGGCTATAAAAGGGGGTGGGGGCGTTCGTCCTCACTCT

[0362] SEQ ID NO:13 Ac5 promoter

[0363]

[0364] SEQ ID NO:14 Polyhedrin promoter

[0365] ATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAAT

[0366] SEQ ID NO:15 p10 promoter

[0367] GACCTTTAATTCAACCCAACACAATATATTATAGTTAAATAAGAATTATTATCAAATCATTTGTATATTAATTAAAATACTATACTGTAAATTACATTTTATTTACAATC

[0368] SEQ ID NO:16 CaMKIIa promoter

[0369]

[0370] SEQ ID NO:17 IL-2 promoter

[0371] CATTTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCAAGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGC

[0372] SEQ ID NO:18 RSV promoter

[0373] AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAAC

[0374] SEQ ID NO:19 Gal1 promoter

[0375] CGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAG

[0376] SEQ ID NO:20 TEF1 promoter

[0377] AATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAG

[0378] SEQ ID NO:21 TDH3 promoter

[0379] TCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA

[0380] SEQ ID NO:22 ADH1 promoter

[0381] ATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACT

[0382] SEQ ID NO:23 T7 promoter TAATACGACTCACTATAGG

[0383] SEQ ID NO:24 T3 promoter AATTAACCCTCACTAAAGG

[0384] SEQ ID NO:25 SP6 promoter ATTTAGGTGACACTATAGA

[0385] SEQ ID NO:26 araBAD promoter

[0386] AAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCAT

[0387] SEQ ID NO:27 trp promoter

[0388] GTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGT

[0389] SEQ ID NO:28 tac promoter TTGACAATTAATCATCGGCTCGTATAATG

[0390] SEQ ID NO:29 lac promoter TTTACACTTTATGCTTCCGGCTCGTATGTTG

[0391] SEQ ID NO:30 pL promoter

[0392] TCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATATAAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACATCAGCAGGACGCACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAAGGGCAGCATTCAAAGCAGAAGGCTTTGGGGTGTGTGATACGAAACGAAGCATTGG

[0393] SEQ ID NO:31 Nucleic acid sequence encoding β-galactosidase LacZ

[0394]

[0395] SEQ ID NO:32β-galactosidase LacZ protein sequence

[0396]

[0397] The nucleic acid sequence encoding mCherry with SEQ ID NO:33

[0398] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAG

[0399] The mCherry protein sequence with SEQ ID NO:34

[0400] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK*

[0401] SEQ ID NO:35 encoding the GFP nucleic acid sequence

[0402] ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAG

[0403] SEQ ID NO:36 GFP protein sequence

[0404] MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK*

[0405] Nucleic acid sequence encoding EGFP with SEQ ID NO:37

[0406] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0407] SEQ ID NO:38 EGFP protein sequence

[0408] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0409] The nucleic acid sequence encoding dTomato of SEQ ID NO:39

[0410] ATGGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGTAA

[0411] SEQ ID NO: 40 dTomato protein sequence

[0412] MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLYGMDELYK*

[0413] The nucleic acid sequence of BFP encoded by SEQ ID NO:41

[0414] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAGCCACGGCGTCCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTTCAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAGCCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0415] SEQ ID NO: 42 BFP protein sequence

[0416] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGICANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0417] SEQ ID NO:43 code YFP nucleic acid sequence

[0418] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGCGCCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0419] SEQ ID NO:44 YFP protein sequence

[0420] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMRQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

[0421] The nucleic acid sequence encoding ZsGreen of SEQ ID NO:45

[0422] ATGGCTCAGTCAAAGCACGGTCTAACAAAAGAAATGACAATGAAATACCGTATGGAAGGGTGCGTCGATGGACATAAATTTGTGATCACGGGAGAGGGCATTGGATATCCGTTCAAAGGGAAACAGGCTATTAATCTGTGTGTGGTCGAAGGTGGACCATTGCCATTTGCCGAAGACATATTGTCAGCTGCCTTTATGTACGGAAACAGGGTTTTCACTGAATATCCTCAAGACATAGCTGACTATTTCAAGAACTCGTGTCCTGCTGGTTATACATGGGACAGGTCTTTTCTCTTTGAGGATGGAGCAGTTTGCATATGTAATGCAGATATAACAGTGAGTGTTGAAGAAAACTGCATGTATCATGAGTCCAAATTTTATGGAGTGAATTTTCCTGCTGATGGACCTGTGATGAAAAAGATGACAGATAACTGGGAGCCATCCTGCGAGAAGATCATACCAGTACCTAAGCAGGGGATATTGAAAGGGGATGTCTCCATGTACCTCCTTCTGAAGGATGGTGGGCGTTTACGGTGCCAATTCGACACAGTTTACAAAGCAAAGTCTGTGCCAAGAAAGATGCCGGACTGGCACTTCATCCAGCATAAGCTCACCCGTGAAGACCGCAGCGATGCTAAGAATCAGAAATGGCATCTGACAGAACATGCTATTGCATCCGGATCTGCATTGCCCTGA

[0423] SEQ ID NO:46 ZsGreen protein sequence

[0424] MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIADYFKNSCPAGYTWDRSFLFEDGAVCICNADITVSVE ENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEHAIASGSALP*

[0425] SEQ ID NO:47 Nucleic acid sequence encoding firefly luciferase (FLuc)

[0426]

[0427] SEQ ID NO:48 Firefly luciferase (FLuc) protein sequence

[0428] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV*

[0429] SEQ ID NO:49 Nucleic acid sequence encoding Renilla Luciferase (RLuc)

[0430] ATGACCAGCAAAGTGTATGATCCGGAACAGCGCAAACGCATGATTACCGGCCCGCAGTGGTGGGCGCGCTGCAAACAGATGAACGTGCTGGATAGCTTTATTAACTATTATGATAGCGAAAAACATGCGGAAAACGCGGTGATTTTTCTGCATGGCAACGCGGCGAGCAGCTATCTGTGGCGGCATGTGGTGCCGCATATTGAACCAGTGGCGCGCTGCATTATTCCGGATCTGATTGGCATGGGCAAAAGCGGCAAAAGCGGCAACGGCAGCTATCGCCTGCTGGATCATTATAAATATCTGACCGCGTGGTTTGAACTGCTGAACCTGCCGAAAAAAATTATTTTTGTGGGCCATGATTGGGGCGCGTGCCTGGCGTTTCATTATAGCTATGAACATCAGGATAAAATTAAAGCGATTGTGCACGCGGAAAGCGTGGTGGATGTGATTGAAAGCTGGGATGAATGGCCGGATATTGAAGAAGATATTGCGCTGATTAAAAGCGAAGAAGGCGAAAAAATGGTGCTGGAAAACAACTTTTTTGTGGAAACCATGCTGCCGAGCAAAATTATGCGCAAACTGGAACCGGAAGAATTTGCGGCGTATCTGGAACCGTTTAAAGAAAAAGGCGAAGTGCGCCGCCCGACCCTGAGCTGGCCGCGCGAAATTCCGCTGGTGAAAGGCGGCAAACCGGATGTGGTCCAGATTGTGCGCAACTATAACGCGTATCTGCGCGCGAGCGATGATCTGCCGAAAATGTTTATTGAAAGCGATCCGGGCTTTTTTAGCAACGCGATTGTGGAAGGCGCGAAAAAATTTCCGAACACCGAATTTGTGAAAGTGAAGGGCCTGCATTTTAGCCAGGAAGATGCGCCGGATGAAATGGGCAAATATATTAAGAGCTTTGTGGAACGCGTGCTGAAAAACGAACAGGGCTAG

[0431] SEQ ID NO:50 Renilla Luciferase (RLuc) protein sequence

[0432] MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEW PDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQG*

[0433] SEQ ID NO:51 Nucleic acid sequence encoding Gaussia Luciferase (GLuc)

[0434] ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAA

[0435] SEQ ID NO:52 Gaussia Luciferase (GLuc) protein sequence

[0436] MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD*

[0437] SEQ ID NO:53 Nucleic acid sequence encoding Nano - Glo Luciferase (NLuc)

[0438] ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAA

[0439] SEQ ID NO:54 Nano - Glo Luciferase (NLuc) protein sequence

[0440] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILA*

[0441] SEQ ID NO:55 Cypridina Luciferase (CLuc) nucleic acid sequence encoding

[0442]

[0443] SEQ ID NO: 56 Cypridina Luciferase (CLuc) protein sequence of Cypridina luciferin

[0444] MKTLILAVALVYCATVHCQDCPYEPDPPNTVPTSCEAKEGECIDSSCGTCTRDILSDGLCENKPGKTCCRMCQYVIECRVEAAGWFRTFYGKRFQFQEPGTYVLGQGTKGGDWKVSITLENLDGTKGAVLTKTRLEVAGDIIDIAQATENPITVNGGADPIIANPYTIGEVTIAVVEMPGFNITVIEFFKLIVIDILGGRSVRIAPDTANKGMISGLCGDLKMMEDTDFTSDPEQLAIQPKINQEFDGCPLYGNPDDVAYCKGLLEPYKDSCRNPINFYYYTISCAFARCMGGDERASHVLLDYRETCAAPETRGTCVLSGHTFYDTFDKARYQFQGPCKEILMAADCFWNTWDVKVSHRNVDSYTEVEKVRIRKQSTVVELIVDGKQILVGGEAVSVPYSSQNTSIYWQDGDILTTAILPEALVVKFNFKQLLVVHIRDPFDGKTCGICGNYNQDFSDDSFDAEGACDLTPNPPGCTEEQKPEAERLCNSLFAGQSDLDQKCNVCHKPDRVERCMYEYCLRGQQGFCDHAWEFKKECYIKHGDTLEVPDECK*

[0445] SEQ ID NO: 57 Nucleic acid sequence of CaMV poly(A) signal

[0446] TTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC

[0447] SEQ ID NO: 58 Nucleic acid sequence of EF-1α poly(A) signal

[0448] ATTATCCCTAATACCTGCCACCCCACTCTTAATCAGTGGTGGAAGAACGGTCTCAGAACTGTTTGTTTCAATTGGCCATTTAAGTTTAGTAGTAAAAGACTGGTTAATGATAACAATGCATCGTAAAACCTTCAGAAGGAAAGGAGAATGTTTTGTGGACCACTTTGGTTTTCTTTTTTGCGTGTGGCAGTTTTAAGTTATTAGTTTTTAAAATCAGTACTTTTTAATGGAAACAACTTGACCAAAAATTTGTCACAGAATTTTGAGACCCATTAAAAAAGTTAAATGAGAAACCTGTGTGTTCCTTTGGTCAACACCGAGACATTTAGGTGAAAGACATCTAATTCTGGTTTTACGAATCTGGAAACTTCTTGAAAATGTAATTCTTGAGTTAACACTTCTGGGTGGAGAATAGGGTTGTTTTCCCCCCACATAATTGGAAGGGGAAGGAATATCATTTAAAGCTATGGGAGGGTTTCTTTGATTACAACACTGGAGAGAAATGCAGCATGTTGCTGATTGCCTGTCACTAAAACAGGCCAAAAACTGAGTCCTTGGGTTGCATAGAAAGCT

[0449] SEQ ID NO:59 bGH poly(A) signal nucleic acid sequence

[0450] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATC

[0451] SEQ ID NO:60 hGH poly(A) signal nucleic acid sequence

[0452] GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTT

[0453] SEQ ID NO:61 SV40 poly(A) signal nucleic acid sequence

[0454] AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTA

[0455] SEQ ID NO:62 human β - globin poly(A) signal nucleic acid sequence

[0456] GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTT AAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTA

[0457] SEQ ID NO:63 Nucleic acid sequence encoding the nuclear localization signal peptide sequence CCAAAAAAGAAGAGAAAGGTA

[0458] SEQ ID NO:64 Nuclear localization signal peptide protein sequence PKKKRKV

[0459] SEQ ID NO:65 Nucleic acid sequence encoding mitochondrial localization signal peptide sequence

[0460] ATGCTGTTTAATCTGAGGATCCTGTTAAACAATGCAGCTTTTAGAAATGGTCACAACTTCATGGTTCGAAATTTTCGGTGTGGACAACCACTACAAAATAAAGTGCAG

[0461] SEQ ID NO:66 Mitochondrial localization signal peptide protein sequence

[0462] MLFNLRILLNNAAFRNGHNFMVRNFRCGQPLQNKVQ

[0463] SEQ ID NO:67 Nucleic acid sequence encoding a nuclear export signal peptide sequence

[0464] CTGCCCCCCCTGGAGCGGCTGACCCTG

[0465] SEQ ID NO:68 Nuclear export signal peptide protein sequence LPPLERLTL

[0466] The nucleic acid sequence of SEQ ID NO:69 encoding ANG protein

[0467] ATGGTGATGGGCCTGGGCGTTTTGTTGTTGGTCTTCGTGCTGGGTCTGGGTCTGACCCCACCGACCCTGGCTCAGGATAACTCCAGGTACACACACTTCCTGACCCAGCACTATGATGCCAAACCACAGGGCCGGGATGACAGATACTGTGAAAGCATCATGAGGAGACGGGGCCTGACCTCACCCTGCAAAGACATCAACACATTTATTCATGGCAACAAGCGCAGCATCAAGGCCATCTGTGAAAACAAGAATGGAAACCCTCACAGAGAAAACCTAAGAATAAGCAAGTCTTCTTTCCAGGTCACCACTTGCAAGCTACATGGAGGTTCCCCCTGGCCTCCATGCCAGTACCGAGCCACAGCGGGGTTCAGAAACGTTGTTGTTGCTTGTGAAAATGGCTTACCTGTCCACTTGGATCAGTCAATTTTCCGTCGTCCG

[0468] SEQ ID NO:70 ANG protein sequence

[0469] MVMGLGVLLLVFVLGLGLTPPTLAQDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRP

[0470] The nucleic acid sequence of SEQ ID NO:71 encoding ANG-S protein

[0471] CAGGATAACTCCAGGTACACACACTTCCTGACCCAGCACTATGATGCCAAACCACAGGGCCGGGATGACAGATACTGTGAAAGCATCATGAGGAGACGGGGCCTGACCTCACCCTGCAAAGACATCAACACATTTATTCATGGCAACAAGCGCAGCATCAAGGCCATCTGTGAAAACAAGAATGGAAACCCTCACAGAGAAAACCTAAGAATAAGCAAGTCTTCTTTCCAGGTCACCACTTGCAAGCTACATGGAGGTTCCCCCTGGCCTCCATGCCAGTACCGAGCCACAGCGGGGTTCAGAAACGTTGTTGTTGCTTGTGAAAATGGCTTACCTGTCCACTTGGATCAGTCAATTTTCCGTCGTCCG

[0472] SEQ ID NO:72 ANG-S protein sequence

[0473] QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRP

[0474] SEQ ID NO:73 Nucleic acid sequence encoding RNASEL protein

[0475]

[0476] SEQ ID NO:74 RNASEL protein sequence

[0477] IDEKYKIADTSEGGIYLGFYEKQEVAVKTFCEGSPRAQREVSCLQSSRENSHLVTFYGSESHRGHLFVCVTLCEQTLEACLDVHRGEDVENEEDEFARNVLSSIFKAVQELHLSCGYTHQDLQPQNILIDSKKAAHLADFDKSIKWAGDPQEVKRDLEDLGRLVLYVVKKGSISFEDLKAQSNEEVVQLSPDEETKDLIHRLFHPGEHVRDCLSDLLGHPFFWTWESRYRTLRNVGNESDIKTRKSESEILRLLQPGPSEHSKSFDKWTTKINECVMKKMNKFYEKRGNFYQNTVGDLLKFIRNLGEHIDEEKHKKMKLKIGDPSLYFQKTFPDLVIYVYTKLQNTEYRKHFPQTHSPN

[0478] SEQ ID NO:75 Nucleic acid sequence encoding the RRP44 protein

[0479] CACTACTTGCTGCCCGACACTAATGTGTTACTGCACCAGATTGATGTTCTTGAGGACCCTGCCATCAGGAATGTAATTGTGCTACAAACAGTTCTTCAAGAAGTGAGAAATCGCAGTGCCCCCGTATATAAACGCATCCGAGATGTGACTAATAACCAAGAGAAGCATTTCTATACTTTCACTAATGAGCACCATAGAGAAACCTATGTAGAACAAGAACAGGGAGAAAATGCTAATGACAGGAATGATAGAGCGATTCGAGTAGCAGCAAAATGGTACAATGAACATTTGAAAAAAATGTCAGCAGACAACCAGCTGCAAGTTATCTTCATAACAAATGACAGGAGAAACAAAGAG

[0480] SEQ ID NO:76 RRP44 protein sequence

[0481] HYLLPDTNVLLHQIDVLEDPAIRNVIVLQTVLQEVRNRSAPVYKRIRDVTNNQEKHFYTFTNEHHRETYVEQEQGENANDRNDRAIRVAAKWYNEHLKKMSADNQLQVIFITNDRRNKE

[0482] Nucleic acid sequence encoding RNASE1 protein of SEQ ID NO:77

[0483] AAGGAATCCCGGGCCAAGAAATTCCAGCGGCAGCATATGGACTCAGACAGTTCCCCCAGCAGCAGCTCCACCTACTGTAACCAAATGATGAGGCGCCGGAATATGACACAGGGGCGGTGCAAACCAGTGAACACCTTTGTGCACGAGCCCCTGGTAGATGTCCAGAATGTCTGTTTCCAGGAAAAGGTCACCTGCAAGAACGGGCAGGGCAACTGCTACAAGAGCAACTCCAGCATGCACATCACAGACTGCCGCCTGACAAACGGCTCCAGGTACCCCAACTGTGCATACCGGACCAGCCCGAAGGAGAGACACATCATTGTGGCCTGTGAAGGGAGCCCATATGTGCCAGTCCACTTTGATGCTTCTGTGGAGGACTCTACC

[0484] SEQ ID NO:78 RNASE1 protein sequence

[0485] KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST

[0486] Nucleic acid sequence encoding PNPT1 protein of SEQ ID NO:79

[0487]

[0488] SEQ ID NO:80 PNPT1 protein sequence

[0489] AVAVDLGNRKLEISSGKLARFADGSAVVQSGDTAVMVTAVSKTKPSPSQFMPLVVDYRQKAAAAGRIPTNYLRREIGTSDKEILTSRIIDRSIRPLFPAGYFYDTQVLCNLLAVDGVNEPDVLAINGASVALSLSDIPWNGPVGAVRIGIIDGEYVVNPTRKEMSSSTLNLVVAGAPKSQIVMLEASAENILQQDFCHAIKVGVKYTQQIIQGIQQLVKETGVTKRTPQKLFTPSPEIVKYTHKLAMERLYAVFTDYEHDKVSRDEAVNKIRLDTEEQLKEKFPEADPYEIIESFNVVAKEVFRSIVLNEYKRCDGRDLTSLRNVSCEVDMFKTLHGSALFQRGQTQVLCTVTFDSLESGIKSDQVITAINGIKDKNFMLHYEFPPYATNEIGKVTGLNRRELGHGALAEKALYPVIPRDFPFTIRVTSEVLESNGSSSMASACGGSLALMDSGVPISSAVAGVAIGLVTKTDPEKGEIEDYRLLTDILGIEDYNGDMDFKIAGTNKGITALQADIKLPGIPIKIVMEAIQQASVAKKEILQIMNKTISKPRASRKENG

[0490] SEQ ID NO:81 Nucleic acid sequence encoding REXO2 protein

[0491] ATGGTCTGGGTGGACCTGGAGATGACAGGATTGGACATTGAGAAGGACCAGATTATTGAGATGGCCTGTCTGATAACTGACTCTGATCTCAACATTTTGGCTGAAGGTCCTAACCTGATTATAAAACAACCAGATGAGTTGCTGGACAGCATGTCAGATTGGTGTAAGGAGCATCACGGGAAGTCTGGCCTTACCAAGGCAGTGAAGGAGAGTACAATTACATTGCAGCAGGCAGAGTATGAATTTCTGTCCTTTGTACGACAGCAGACTCCTCCAGGGCTCTGTCCACTTGCAGGAAATTCAGTTCATGAAGATAAGAAGTTTCTTGACAAATACATGCCCCAGTTCATGAAACATCTTCATTATAGAATAATTGATGTGAGCACTGTTAAAGAACTGTGCAGACGCTGGTATCCAGAAGAATATGAATTTGCACCAAAGAAGGCTGCTTCTCATAGGGCACTTGATGACATTAGTGAAAGCATCAAAGAGCTT

[0492] SEQ ID NO:82 REXO2 protein sequence

[0493] MVWVDLEMTGLDIEKDQIIEMACLITDSDLNILAEGPNLIIKQPDELLDSMSDWCKEHHGKSGLTKAVKESTITLQQAEYEFLSFVRQQTPPGLCPLAGNSVHEDKKFLDKYMPQFMKHLHYRIIDVSTVKELCRRWYPEEYEFAPKKAASHRALDDISESIKEL

[0494] SEQ ID NO:83 Nucleic acid sequence encoding MRPL44 protein

[0495] GATCTTCTCAAAACTGCATTTGTTAATAGCTGCTATATTAAAAGTGAGGAGGCCAAACGCCAACAACTTGGGATAGAGAAAGAAGCTGTTCTTCTGAATCTTAAAAGTAATCAAGAACTATCCGAACAAGGGACATCTTTTTCACAGACTTGCCTTACACAGTTTCTTGAAGACGAGTACCCAGACATGCCCACTGAAGGCATAAAAAATCTTGTTGACTTTCTCACTGGTGAGGAAGTCGTGTGTCACGTGGCTAGAAACTTGGCTGTGGAGCAGTTAACACTGAGTGAAGAATTCCCAGTGCCCCCAGCTGTGTTACAGCAGACTTTCTTTGCAGTTATTGGAGCCCTGTTACAGAGCAGTGGACCTGAGAGGACTGCACTTTTCATCAGGGACTTCTTAATTACTCAAATGACTGGAAAAGAGCTC

[0496] SEQ ID NO:84 MRPL44 protein sequence

[0497] DLLKTAFVNSCYIKSEEAKRQQLGIEKEAVLLNLKSNQELSEQGTSFSQTCLTQFLEDEYPDMPTEGIKNLVDFLTGEEVVCHVARNLAVEQLTLSEEFPVPPAVLQQTFFAVIGALLQSSGPERTALFIRDFLITQMTGKEL

[0498] SEQ ID NO:85 Nucleic acid sequence encoding LACTB2 protein

[0499] ATGGCTGCTGTACTGCAGCGCGTCGAGCGGCTGTCCAATCGAGTCGTGCGTGTGTTGGGCTGTAACCCGGGTCCCATGACCCTCCAAGGCACCAACACCTACCTAGTGGGGACCGGCCCCAGGAGAATCCTCATTGACACTGGAGAACCAGCAATTCCAGAATACATCAGCTGTTTAAAGCAGGCTCTAACTGAATTTAACACAGCAATCCAGGAAATTGTAGTGACTCACTGGCACCGAGATCATTCTGGAGGCATAGGAGATATTTGTAAAAGCATCAATAATGACACTACCTATTGCATTAAAAAACTCCCACGGAATCCTCAGAGAGAAGAAATTATAGGAAATGGAGAGCAACAATATGTTTATCTGAAAGATGGAGATGTGATTAAGACTGAGGGAGCCACTCTAAGAGTTCTATATACCCCTGGCCACACTGATGATCACATGGCTCTACTCTTAGAAGAGGAAAATGCTATCTTTTCTGGAGATTGCATCCTAGGGGAAGGAACAACGGTATTTGAAGACCTCTATGATTATATGAACTCTTTAAAAGAGTTATTGAAAATCAAAGCTGATATTATATATCCAGGACATGGCCCAGTAATTCATAATGCTGAAGCTAAAATTCAACAATACATTTCTCACAGAAATATTCGAGAGCAGCAAATTCTTACATTATTTCGTGAGAACTTTGAGAAATCATTTACAGTAATGGAGCTTGTAAAAATTATTTACAAGAATACTCCTGAGAATTTACATGAAATGGCTAAACATAATCTCTTACTTCATTTGAAAAAACTAGAAAAAGAAGGAAAAATATTTAGCAACACAGATCCTGACAAGAAATGGAAAGCTCATCTT

[0500] SEQ ID NO:86 LACTB2 protein sequence

[0501] MAAVLQRVERLSNRVVRVLGCNPGPMTLQGTNTYLVGTGPRRILIDTGEPAIPEYISCLKQALTEFNTAIQEIVVTHWHRDHSGGIGDICKSINNDTTYCIKKLPRNPQREEIIGNGEQQYVYLKDGDVIKTEGATLRVLYTPGHTDDHMALLLEEENAIFSGDCILGEGTTVFEDLYDYMNSLKELLKIKADIIYPGHGPVIHNAEAKIQQYISHRNIREQQILTLFRENFEKSFTVMELVKIIYKNTPENLHEMAKHNLLLHLKKLEKEGKIFSNTDPDKKWKAHL

[0502] The nucleic acid sequence encoding the TSEN15 protein of SEQ ID NO:87

[0503] ATGGAGGAGCGCGGCGATTCCGAGCCGACCCCCGGCTGCAGCGGCCTGGGTCCGGGCGGTGTTCGCGGCTTTGGCGACGGCGGTGGAGCTCCTTCGTGGGCCCCTGAGGACGCCTGGATGGGCACTCACCCTAAGTATCTAGAAATGATGGAATTAGATATAGGAGATGCCACCCAAGTTTATGTAGCGTTCTTGGTTTACCTGGACCTCATGGAAAGCAAAAGCTGGCATGAAGTAAACTGTGTAGGATTACCAGAACTCCAGCTCATCTGCCTTGTTGGTACTGAGATAGAAGGGGAGGGGTTACAGACTGTGGTGCCTACCCCCATCACTGCTTCCCTCAGCCATAACAGGATAAGGGAGATCTTGAAGGCATCTCGAAAGTTGCAAGGTGATCCAGATTTGCCGATGTCTTTTACTTTGGCCATAGTGGAGTCTGATTCTACAATAGTCTATTATAAACTTACTGATGGATTTATGCTGCCAGACCCTCAGAATATTTCTCTTAGAAGA

[0504] The TSEN15 protein sequence of SEQ ID NO:88

[0505] MEERGDSEPTPGCSGLGPGGVRGFGDGGGAPSWAPEDAWMGTHPKYLEMMELDIGDATQVYVAFLVYLDLMESKSWHEVNCVGLPELQLICLVGTEIEGEGLQTVVPTPITASLSHNRIREILKASRKLQGDPDLPMSFTLAIVESDSTIVYYKLTDGFMLPDPQNISLRR

[0506] The nucleic acid sequence encoding the ZC3H12A protein of SEQ ID NO:89

[0507] GGTGGTGGCACCCCTAAGGCTCCCAACCTGGAGCCTCCACTCCCAGAAGAGGAAAAGGAGGGCAGCGACCTGAGACCAGTGGTCATCGATGGGAGCAACGTGGCCATGAGCCATGGGAACAAGGAGGTCTTCTCCTGCCGGGGCATCCTGCTGGCAGTGAACTGGTTTCTGGAGCGGGGCCACACAGACATCACAGTGTTTGTGCCATCCTGGAGGAAGGAGCAGCCTCGGCCCGACGTGCCCATCACAGACCAGCACATCCTGCGGGAACTGGAGAAGAAGAAGATCCTGGTGTTCACACCATCACGACGCGTGGGTGGCAAGCGGGTGGTGTGCTATGACGACAGATTCATTGTGAAGCTGGCCTACGAGTCTGACGGGATCGTGGTTTCCAACGACACATACCGTGACCTCCAAGGCGAGCGGCAGGAGTGGAAGCGCTTCATCGAGGAGCGGCTGCTCATGTACTCCTTCGTCAATGACAAGTTTATGCCCCCTGATGACCCACTGGGCCGGCACGGGCCCAGCCTGGACAACTTCCTGCGTAAGAAGCCACTC

[0508] The ZC3H12A protein sequence of SEQ ID NO:90

[0509] GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPL

[0510] SEQ ID NO:91 XTEN connected peptide sequence SGSETPGTSESATPES

[0511] SEQ ID NO:92 Connecting peptide protein sequence GGGGS

[0512] SEQ ID NO:93(CTG)14 nucleic acid sequence

[0513] CTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTG

[0514] SEQ ID NO:94mCherry-(CTG)14 nucleic acid sequence

[0515] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACTATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAGGTTGCTAGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTCGAG

[0516] SEQ ID NO:95 PUF-ANG protein sequence

[0517] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQMVMGLGVLLLVFVLGLGLTPPTLAQDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRP

[0518] SEQ ID NO:96 PUF-ANG-S protein sequence

[0519] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQQDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRP

[0520] SEQ ID NO:97 PUF-RNASEL protein sequence

[0521] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQIDEKYKIADTSEGGIYLGFYEKQEVAVKTFCEGSPRAQREVSCLQSSRENSHLVTFYGSESHRGHLFVCVTLCEQTLEACLDVHRGEDVENEEDEFARNVLSSIFKAVQELHLSCGYTHQDLQPQNILIDSKKAAHLADFDKSIKWAGDPQEVKRDLEDLGRLVLYVVKKGSISFEDLKAQSNEEVVQLSPDEETKDLIHRLFHPGEHVRDCLSDLLGHPFFWTWESRYRTLRNVGNESDIKTRKSESEILRLLQPGPSEHSKSFDKWTTKINECVMKKMNKFYEKRGNFYQNTVGDLLKFIRNLGEHIDEEKHKKMKLKIGDPSLYFQKTFPDLVIYVYTKLQNTEYRKHFPQTHSPN

[0522] SEQ ID NO:98 PUF-RRP44 protein sequence

[0523] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQHYLLPDTNVLLHQIDVLEDPAIRNVIVLQTVLQEVRNRSAPVYKRIRDVTNNQEKHFYTFTNEHHRETYVEQEQGENANDRNDRAIRVAAKWYNEHLKKMSADNQLQVIFITNDRRNKE

[0524] SEQ ID NO:99 PUF-RNASE1 protein sequence

[0525] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST

[0526] SEQ ID NO:100 PUF-PNPT1 protein sequence

[0527] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQAVAVDLGNRKLEISSGKLARFADGSAVVQSGDTAVMVTAVSKTKPSPSQFMPLVVDYRQKAAAAGRIPTNYLRREIGTSDKEILTSRIIDRSIRPLFPAGYFYDTQVLCNLLAVDGVNEPDVLAINGASVALSLSDIPWNGPVGAVRIGIIDGEYVVNPTRKEMSSSTLNLVVAGAPKSQIVMLEASAENILQQDFCHAIKVGVKYTQQIIQGIQQLVKETGVTKRTPQKLFTPSPEIVKYTHKLAMERLYAVFTDYEHDKVSRDEAVNKIRLDTEEQLKEKFPEADPYEIIESFNVVAKEVFRSIVLNEYKRCDGRDLTSLRNVSCEVDMFKTLHGSALFQRGQTQVLCTVTFDSLESGIKSDQVITAINGIKDKNFMLHYEFPPYATNEIGKVTGLNRRELGHGALAEKALYPVIPRDFPFTIRVTSEVLESNGSSSMASACGGSLALMDSGVPISSAVAGVAIGLVTKTDPEKGEIEDYRLLTDILGIEDYNGDMDFKIAGTNKGITALQADIKLPGIPIKIVMEAIQQASVAKKEILQIMNKTISKPRASRKENG

[0528] SEQ ID NO:101 PUF-REXO2 protein sequence

[0529] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQMVWVDLEMTGLDIEKDQIIEMACLITDSDLNILAEGPNLIIKQPDELLDSMSDWCKEHHGKSGLTKAVKESTITLQQAEYEFLSFVRQQTPPGLCPLAGNSVHEDKKFLDKYMPQFMKHLHYRIIDVSTVKELCRRWYPEEYEFAPKKAASHRALDDISESIKEL

[0530] SEQ ID NO:102 PUF-MRPL44 protein sequence

[0531] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQDLLKTAFVNSCYIKSEEAKRQQLGIEKEAVLLNLKSNQELSEQGTSFSQTCLTQFLEDEYPDMPTEGIKNLVDFLTGEEVVCHVARNLAVEQLTLSEEFPVPPAVLQQTFFAVIGALLQSSGPERTALFIRDFLITQMTGKEL

[0532] SEQ ID NO:103 PUF-LACTB2 protein sequence

[0533] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQMAAVLQRVERLSNRVVRVLGCNPGPMTLQGTNTYLVGTGPRRILIDTGEPAIPEYISCLKQALTEFNTAIQEIVVTHWHRDHSGGIGDICKSINNDTTYCIKKLPRNPQREEIIGNGEQQYVYLKDGDVIKTEGATLRVLYTPGHTDDHMALLLEEENAIFSGDCILGEGTTVFEDLYDYMNSLKELLKIKADIIYPGHGPVIHNAEAKIQQYISHRNIREQQILTLFRENFEKSFTVMELVKIIYKNTPENLHEMAKHNLLLHLKKLEKEGKIFSNTDPDKKWKAHL

[0534] SEQ ID NO:104 PUF-TSEN15 protein sequence

[0535] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQMEERGDSEPTPGCSGLGPGGVRGFGDGGGAPSWAPEDAWMGTHPKYLEMMELDIGDATQVYVAFLVYLDLMESKSWHEVNCVGLPELQLICLVGTEIEGEGLQTVVPTPITASLSHNRIREILKASRKLQGDPDLPMSFTLAIVESDSTIVYYKLTDGFMLPDPQNISLRR

[0536] SEQ ID NO:105 PUF-ZC3H12A protein sequence

[0537] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVDTGNGSQGGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPL

[0538] SEQ ID NO:106 ANG-PUF protein sequence

[0539] MVMGLGVLLLVFVLGLGLTPPTLAQDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRPVDTGNGSQGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0540] SEQ ID NO:107 ANG-S-PUF protein sequence

[0541] QDNSRYTHFLTQHYDAKPQGRDDRYCESIMRRRGLTSPCKDINTFIHGNKRSIKAICENKNGNPHRENLRISKSSFQVTTCKLHGGSPWPPCQYRATAGFRNVVVACENGLPVHLDQSIFRRPVDTGNGSQGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0542] SEQ ID NO:108 RNASE1-PUF protein sequence

[0543] KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTVDTGNGSQGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0544] SEQ ID NO:109 PNPT1-PUF protein sequence

[0545] AVAVDLGNRKLEISSGKLARFADGSAVVQSGDTAVMVTAVSKTKPSPSQFMPLVVDYRQKAAAAGRIPTNYLRREIGTSDKEILTSRIIDRSIRPLFPAGYFYDTQVLCNLLAVDGVNEPDVLAINGASVALSLSDIPWNGPVGAVRIGIIDGEYVVNPTRKEMSSSTLNLVVAGAPKSQIVMLEASAENILQQDFCHAIKVGVKYTQQIIQGIQQLVKETGVTKRTPQKLFTPSPEIVKYTHKLAMERLYAVFTDYEHDKVSRDEAVNKIRLDTEEQLKEKFPEADPYEIIESFNVVAKEVFRSIVLNEYKRCDGRDLTSLRNVSCEVDMFKTLHGSALFQRGQTQVLCTVTFDSLESGIKSDQVITAINGIKDKNFMLHYEFPPYATNEIGKVTGLNRRELGHGALAEKALYPVIPRDFPFTIRVTSEVLESNGSSSMASACGGSLALMDSGVPISSAVAGVAIGLVTKTDPEKGEIEDYRLLTDILGIEDYNGDMDFKIAGTNKGITALQADIKLPGIPIKIVMEAIQQASVAKKEILQIMNKTISKPRASRKENGVDTGNGSQGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0546] SEQ ID NO:110 REXO2-PUF protein sequence

[0547] MVWVDLEMTGLDIEKDQIIEMACLITDSDLNILAEGPNLIIKQPDELLDSMSDWCKEHHGKSGLTKAVKESTITLQQAEYEFLSFVRQQTPPGLCPLAGNSVHEDKKFLDKYMPQFMKHLHYRIIDVSTVKELCRRWYPEEYEFAPKKAASHRALDDISESIKELVDTGNGSQGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0548] SEQ ID NO:111 ZC3H12A - PUF protein sequence

[0549] GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFLERGHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDDRFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGPSLDNFLRKKPLVDTGNGSQGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0550] The nucleic acid sequence encoding the RNASET2 protein of SEQ ID NO: 112

[0551] ATGCGCCCTGCAGCCCTGCGCGGGGCCCTGCTGGGCTGCCTCTGCCTGGCGTTGCTTTGCCTGGGCGGTGCGGACAAGCGCCTGCGTGACAACCATGAGTGGAAAAAACTAATTATGGTTCAGCACTGGCCTGAGACAGTATGCGAGAAAATTCAAAACGACTGTAGAGACCCTCCGGATTACTGGACAATACATGGACTATGGCCCGATAAAAGTGAAGGATGTAATAGATCGTGGCCCTTCAATTTAGAAGAGATTAAGGATCTTTTGCCAGAAATGAGGGCATACTGGCCTGACGTAATTCACTCGTTTCCCAATCGCAGCCGCTTCTGGAAGCATGAGTGGGAAAAGCATGGGACCTGCGCCGCCCAGGTGGATGCGCTCAACTCCCAGAAGAAGTACTTTGGCAGAAGCCTGGAACTCTACAGGGAGCTGGACCTCAACAGTGTGCTTCTAAAATTGGGGATAAAACCATCCATCAATTACTACCAAGTTGCAGATTTTAAAGATGCCCTTGCCAGAGTATATGGAGTGATACCCAAAATCCAGTGCCTTCCACCAAGCCAGGATGAGGAAGTACAGACAATTGGTCAGATAGAACTGTGCCTCACTAAGCAAGACCAGCAGCTGCAAAACTGCACCGAGCCGGGGGAGCAGCCGTCCCCCAAGCAGGAAGTCTGGCTGGCAAATGGGGCCGCCGAGAGCCGGGGTCTGAGAGTCTGTGAAGATGGCCCAGTCTTCTATCCCCCACCTAAAAAGACCAAGCAT

[0552] SEQ ID NO:113 RNASET2 protein sequence

[0553] MRPAALRGALLGCLCLALLCLGGADKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPFNLEEIKDLLPEMRAYWPDVIHSFPNRSRFWKHEWEKHGTCAAQVDALNSQKKYFGRSLELYRELDLNSVLLKLGIKPSINYYQVADFKDALARVYGVIPKIQCLPPSQDEEVQTIGQIELCLTKQDQQLQNCTEPGEQPSPKQEVWLANGAAESRGLRVCEDGPVFYPPPKKTKH

[0554] The nucleic acid sequence encoding the SUPV3L1 protein of SEQ ID NO:114

[0555]

[0556] SEQ ID NO:115 SUPV3L1 protein sequence

[0557] MSFSRALLWARLPAGRQAGHRAAICSALRPHFGPFPGVLGQVSVLATASSSASGGSKIPNTSLFVPLTVKPQGPSADGDVGAELTRPLDKNEVKKVLDKFYKRKEIQKLGADYGLDARLFHQAFISFRNYIMQSHSLDVDIHIVLNDICFGAAHADDLFPFFLRHAKQIFPVLDCKDDLRKISDLRIPPNWYPDARAMQRKIIFHSGPTNSGKTYHAIQKYFSAKSGVYCGPLKLLAHEIFEKSNAAGVPCDLVTGEERVTVQPNGKQASHVSCTVEMCSVTTPYEVAVIDEIQMIRDPARGWAWTRALLGLCAEEVHLCGEPAAIDLVMELMYTTGEEVEVRDYKRLTPISVLDHALESLDNLRPGDCIVCFSKNDIYSVSRQIEIRGLESAVIYGSLPPGTKLAQAKKFNDPNDPCKILVATDAIGMGLNLSIRRIIFYSLIKPSINEKGERELEPITTSQALQIAGRAGRFSSRFKEGEVTTMNHEDLSLLKEILKRPVDPIRAAGLHPTAEQIEMFAYHLPDATLSNLIDIFVDFSQVDGQYFVCNMDDFKFSAELIQHIPLSLRVRYVFCTAPINKKQPFVCSSLLQFARQYSRNEPLTFAWLRRYIKWPLLPPKNIKDLMDLEAVHDVLDLYLWLSYRFMDMFPDASLIRDLQKELDGIIQDGVHNITKLIKMSETHKLLNLEGFPSGSQSRLSGTLKSQARRTRGTKALGSKATEPPSPDAGELSLASRLVQQGLLTPDMLKQLEKEWMTQQTEHNKEKTESGTHPKGTRRKKKEPDSD

[0558] SEQ ID NO:116 PUF-RNASET2 protein sequence

[0559] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVEEHRRSTDKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPFNLEEIKDLLPEMRAYWPDVIHSFPNRSRFWKHEWEKHGTCAAQVDALNSQKKYFGRSLELYRELDLNSVLLKLGIKPSINYYQVADFKDALARVYGVIPKIQCLPPSQDEEVQTIGQIELCLTKQDQQLQNCTEPGEQPSPKQEVWLANGAAESRGLRVCEDGPVFYPPPKKTKH

[0560] SEQ ID NO:117 RNASET2 - PUF protein sequence

[0561] DKRLRDNHEWKKLIMVQHWPETVCEKIQNDCRDPPDYWTIHGLWPDKSEGCNRSWPFNLEEIKDLLPEMRAYWPDVIHSFPNRSRFWKHEWEKHGTCAAQVDALNSQKKYFGRSLELYRELDLNSVLLKLGIKPSINYYQVADFKDALARVYGVIPKIQCLPPSQDEEVQTIGQIELCLTKQDQQLQNCTEPGEQPSPKQEVWLANGAAESRGLRVCEDGPVFYPPPKKTKHVEEHRRSTGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0562] SEQ ID NO:118 PUF - SUPV3L1 protein sequence

[0563] GRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQKLALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYVIRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLGVEEHRRSTVLDHALESLDNLRPGDCIVCFSKNDIYSVSRQIEIRGLESAVIYGSLPPGTKLAQAKKFNDPNDPCKILVATDAIGMGLNLSIRRIIFYSLIKPSINEKGERELEPITTSQALQIAGRAGRFSSRFKEGEVTTMNHEDLSLLKEILKRPVDPIRAAGLHPTAEQIE

[0564] SEQ ID NO:119 SUPV3L1 - PUF protein sequence

[0565] VLDHALESLDNLRPGDCIVCFSKNDIYSVSRQIEIRGLESAVIYGSLPPGTKLAQAKKFNDPNDPCKILVATDAIGMGLNLSIRRIIFYSLIKPSINEKGERELEPITTSQALQIAGRAGRFSSRFKEGE VTTMNHEDLSLLKEILKRPVDPIRAAGLHPTAEQIEVEEHRRSTGRSRLLEDFRNNRYPNLQLREIAGHIMEFSQDQHGSRFIELKLERATPAERQLVFNEILQAAYQLMVDVFGNYVIQKFFEFGSLEQK LALAERIRGHVLSLALQMYGSYVIRKALEFIPSDQQNEMVRELDGHVLKCVKDQNGSYVVEKCIECVQPQSLQFIIDAFKGQVFALSTHPYGNRVIQRILEHCLPDQTLPILEELHQHTEQLVQDQYGSYV IRHVLEHGRPEDKSKIVAEIRGNVLVLSQHKFASNVVEKCVTHASRTERAVLIDEVCTMNDGPHSALYTMMKDQYANYVVQKMIDVAEPGQRKIVMHKIRPHIATLRKYTYGKHILAKLEKYYMKNGVDLG

[0566] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A nucleic acid construct set, characterized in that: The nucleic acid construct group includes: a first nucleic acid construct and a second nucleic acid construct; Furthermore, the first nucleic acid construct contains a first expression cassette, the first expression cassette expresses a long RNA repeat sequence, and the first expression cassette comprises a repeat sequence unit of 3-6 nucleotides, the repeat sequence is repeated 41-2000 times, preferably 45-1500 times, more preferably 50-1000 times; The second nucleic acid construct contains a second expression cassette, which expresses a short RNA repeat sequence, and the second expression cassette contains a repeat sequence unit of 3-6 nucleotides corresponding to the first expression cassette, and the repeat sequence is repeated 2-40 times, preferably 5-40 times.

2. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid construct set as claimed in claim 1.

3. A genetically engineered cell, characterized in that: The genetically engineered cell contains the nucleic acid construct set according to claim 1, or contains the recombinant vector according to claim 2.

4. A kit, characterized in that: The kit comprises: a container and the nucleic acid construct set according to claim 1 located in the container.

5. A method for screening drugs for treating diseases associated with long RNA repeat sequences, characterized in that: Includes steps: (a) introducing the nucleic acid construct set according to claim 1 and the drug to be screened into cells together, (b) judging whether the drug to be tested is a drug for treating a disease associated with a long RNA repeat sequence by the intensity change of the first detectable marker and the second detectable marker in the nucleic acid construct group.

6. A system for screening drugs for treating diseases associated with long RNA repeat sequences, characterized in that: The system comprises: (a) The nucleic acid construct set of claim 1; (b) drugs to be screened; Among them, when the expression level or intensity of the first detectable marker in the nucleic acid construct group is significantly reduced, and the expression level or intensity of the second detectable marker changes very little or remains basically unchanged, it indicates that the drug to be screened is a candidate or potential drug that can be used to treat long RNA repeat sequence-related diseases.

7. A screening composition for screening drugs for treating diseases associated with long RNA repeat sequences, characterized in that: The screening composition comprises: (a) drugs to be screened; (b) the nucleic acid construct set according to claim 1; Among them, when the expression amount or intensity of the first detectable marker in the nucleic acid construct group is significantly reduced, and the expression amount or intensity of the second detection marker changes slightly or remains basically unchanged, it indicates that the drug to be screened is a candidate or potential drug that can be used to treat long RNA repeat sequence-related diseases.

8. Use of the system of claim 6 or the screening composition of claim 7, characterized in that: Used to screen drugs for treating diseases associated with long RNA repeat sequences.

9. A composition, characterized in that include: (a) A drug for treating a disease associated with long RNA repeat sequences obtained by the method of claim 5 or the system of claim 6, wherein the drug for treating a disease associated with long RNA repeat sequences comprises an artificial RNA degrading enzyme, and preferably, the RNA degrading enzyme has a structure shown in Formula IV or IV' from the NC end of the protein: D1-A-L1-B-D2(IV); D1-B-L1-A-D2(IV') In the formula, each "-" is independently a connecting peptide or a peptide bond; A is the PUF protein fragment; B is the full length or active fragment of an RNase or RNA helicase; L1 is none or a linker peptide; D1 is no or localized peptide; D2 is a peptide without or positioned, and the full length or active fragment of the RNA enzyme or RNA helicase is selected from the following group: PIN (from SMG6, UNIPROT: Q86US8), ANG (UNIPROT: P03950), RNASEL (UNIPROT: Q05823), RRP44 (UNIPROT: Q9Y2L1), RNASE1 (UNIPROT: P07998), RNASET2 (UNIPROT: O00584) 、SUPV3L1(UNIPROT:Q8IYB8)、PNPT1(UNIPROT:Q8TCS8)、REXO2(UNIPROT:Q9Y3B8)、MRPL44(UNIPROT:Q9H9J2)、LACTB2(UNIPROT:Q53H82)、TSEN15(UNIPROT:Q8WW01)、ZC3H12A(UNIPROT:Q5D1E8), or their active regions and sequence combinations.

10. A method for preparing an animal model for screening drugs for treating diseases associated with long RNA repeat sequences, characterized in that: The method comprises the following steps: (a) providing cells of a non-human mammal, and introducing the nucleic acid construct set according to claim 1 into the cells, thereby obtaining cells that simultaneously express long RNA repeat sequences and short RNA repeat sequences; (b) Using the cells obtained in step (a) that simultaneously express long RNA repeat sequences and short RNA repeat sequences, an animal model is prepared for screening drugs for treating diseases associated with long RNA repeat sequences.

11. Use of a non-human mammal model prepared by the method according to claim 10, characterized in that: This model is used to screen or identify substances (therapeutic agents) for treating long RNA repeat sequence-related diseases.

12. A method for screening or identifying potential therapeutic agents for treating long RNA repeat sequence-related diseases, characterized in that: The following steps are involved: (a) in a test group, administering the test drug to the non-human mammal model prepared by the method of claim 9 in the presence of the test drug, and analyzing the expression amount or intensity of the first detectable marker and the second detectable marker in the animal model of the test group respectively; and in a control group to which the test drug is not administered and other conditions are the same, analyzing the expression amount or intensity of the first detectable marker and the second detectable marker of the animal model of the control group; (b) comparing the expression levels or intensities of the first detectable marker and the second detectable marker in the test group and the control group animal models, wherein, if the expression level or intensity of the first detectable marker in the animal model administered with the test drug is significantly reduced and the expression level or intensity of the second detectable marker remains essentially unchanged compared with the control group, it indicates that the test compound can be used as a potential therapeutic agent for treating diseases associated with long RNA repeat sequences.

13. A use of the genetically engineered cell according to claim 3, characterized in that: Used to prepare biological preparations for constructing animal models for screening drugs for treating diseases associated with long RNA repeat sequences in non-human mammals.

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