Novel highly efficient prime editor and applications thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a gene leading editing system, which comprises a carrier, the carrier contains a gene coding pegRNA and a gene coding fusion protein, the pegRNA comprises sgRNA, reverse transcription template RTT, primary binding site PBS and RNA stem loop structure, and the fusion protein is a fusion protein of Cas9 nickase or a variant thereof and reverse transcriptase. The application develops sPEs by adding an RNA neck ring structure to the 3' end of the pegRNA, develops tPEs by allowing the 3' end of the pegRNA to bind to Cas9, so as to improve the stability of the Cas9 / pegRNA complex, and further improve the editing efficiency of different genomic sites in different cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to novel and efficient prime editors and their applications. The novel and efficient prime editors developed, namely sPEs and tPEs, are platform technologies for gene therapy and can be used for gene therapy of diseases such as site-directed mutation, insertion, and screening of genomic DNA fragments. Background Technology

[0002] Most human diseases are closely related to genes, so targeted modifications based on genes have long been a goal of life sciences. Despite rapid progress in gene editing technology, most of the more than 75,000 known human genetic variations associated with diseases still present challenges in correcting them.

[0003] Engineered nucleases such as ZFPs, TALENs, or CRISPR-Cas can effectively utilize DNA double-strand breaks (DSBs)-mediated DNA editing to perform insertions and deletions at specific genomic sites. However, the generation of DSBs can lead to many undesirable outcomes, such as non-single-segmentation editing, gene sequence translocation, and p53 activation.

[0004] Within cells, DNA double-strand breaks (DSBs) frequently occur in chromatin. These are essential for meiotic recombination and the development of the vertebrate immune system. However, erroneous repair of DSBs can lead to mutations and promote genomic instability, while failure to repair DSBs results in cell death. Generally, two main repair mechanisms are activated by DSBs: homology-directed repair (HDR) and non-homologous end joining (NHEJ). As the primary pathway for DNA double-strand break repair, HDR uses homologous sequences as templates to correct gene sequences and can only occur during the G2 / S phase. Conversely, NHEJ introduces insertions and deletions at genomic sites near DSBs without requiring homologous sequences. However, due to the inherent limitations of homologous recombination, the efficiency of HDR-mediated gene correction has remained low (typically less than 5%). This significantly restricts the transition of CRISPR / Cas gene editing tools from research to practical applications. The base editor (BE) that emerged to address this need, cytosine and adenosine base editors can convert C·G to T·A and A·T to G·C. Although this method can greatly improve the efficiency of gene site editing, the mutation mode is singular and requires DNA double-strand breaks. For applications in precision medicine, these methods have certain limitations.

[0005] Prime editing was invented in late 2019 by David Liu's lab at MIT. This technology is considered a third-generation gene-editing technology based on CRISPR, capable of precise site mutations, insertions, and deletions of small DNA fragments at any site in the genome. Currently, the main problem with prime editing is that its efficiency varies greatly across different cells and at different sites. Therefore, optimizing the efficiency of prime editing is urgently needed. Summary of the Invention

[0006] Prime editing allows for site-directed mutation, insertion, and screening of genomic DNA fragments without double-strand breaks. The stability and misfolding of pegRNA in prime editors affect the efficiency of prime editing. Compared to sgRNA protected by the Cas9 protein, the 3' extension of pegRNA may be exposed in the cell and more susceptible to degradation by exonucleases. We developed sPEs by adding an RNA neck loop structure to the 3' end of pegRNA and tPEs by binding the 3' end of pegRNA to Cas9, thereby improving the stability of the Cas9 / pegRNA complex and reducing the degradation aptitude of the 3' end. sPEs and tPEs significantly improve the efficiency of prime editing.

[0007] One objective of this invention is to provide a gene-guided editing system comprising a vector containing a gene encoding pegRNA and a gene encoding a fusion protein, wherein the pegRNA comprises sgRNA, a reverse transcription template RTT, a major binding site PBS, and an RNA stem-loop structure; and the fusion protein is a fusion protein of Cas9 nickase or a variant thereof with reverse transcriptase.

[0008] The RNA stem-loop structure is selected from at least one of the following:

[0009] 1) The sequences are MS2, PP7, ComBS or Csy4BS as shown in SEQ ID NO.1 to 4 respectively;

[0010] 2) An RNA sequence obtained by adding one or more nucleotides to the 5′ end and / or 3′ end of 1);

[0011] 3) RNA sequences that share more than 85% identity with RNA sequences of 1) or 2).

[0012] The RNA stem-loop structure is attached to the 3' end of the primary binding site PBS;

[0013] The pegRNA is an RNA molecule, consisting of sgRNA, reverse transcription template RTT, major binding site PBS, and RNA stem-loop structure from the 5' end to the 3' end.

[0014] The fusion protein also contains an RNA-binding protein RBP; the RBP is a protein that specifically binds to RNA stem-loop structures; the RBP may be one or more.

[0015] The fusion protein also contains the RNA-binding protein RBP; the fusion protein comprises, from the N-terminus to the C-terminus, RBP, Cas9 or a variant thereof, and reverse transcriptase.

[0016] The carrier may be one or more;

[0017] The gene encoding pegRNA or the gene encoding fusion protein may be one or more;

[0018] The gene encoding pegRNA or the gene encoding fusion protein is located on the same vector or different vectors.

[0019] The RBP is selected from any one of tdMCP, tdPCP, COM, and Csy4H29A;

[0020] The Cas9 cleavage enzyme is H840A;

[0021] The reverse transcriptase is MMLV;

[0022] The system also contains screening marker proteins.

[0023] Another object of the present invention is to provide a pegRNA, wherein the pegRNA is the pegRNA described in any of the preceding claims.

[0024] Another object of the present invention is to provide a fusion protein, said fusion protein being the fusion protein described in any of the preceding claims.

[0025] Another object of the present invention is to provide a nucleic acid that encodes a pegRNA and / or a fusion protein as described above.

[0026] Another object of the present invention is to provide a construct containing the nucleic acid as described above.

[0027] Another object of the present invention is a host cell comprising the constructs described above or having exogenous nucleic acids as described above integrated into its genome.

[0028] The use of the systems, pegRNAs, fusion proteins, nucleic acids, constructs, or host cells described above in at least one of the following:

[0029] 1) Used for editing the genome sequence of organisms or biological cells; preferably, for base substitution;

[0030] 2) Products used for editing the genome sequence of organisms or biological cells;

[0031] 3) Used to improve the efficiency of editing genome sequences of organisms or biological cells;

[0032] 4) Products used to improve the efficiency of editing genome sequences of organisms or biological cells.

[0033] Specifically, it is used for DNA editing or RNA editing; preferably, it is used for DNA editing; more preferably, it is used for editing SRD5A3, DYRK1A, HDAC1, BCL11A, GFAP, RUNX1, JAK2, SRD5A1, DMD, and EED sites; even more preferably, it is used for editing SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, RUNX1_+5G·C to T·A, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A.

[0034] Another object of the present invention is to provide a method for gene editing, comprising introducing the system, pegRNA, fusion protein, nucleic acid, construct or host cell as described above into the organism to be edited to achieve gene editing. Attached Figure Description

[0035] Figure 1 .sPE-MS2 and tPE-MS2 improved PE efficiency at different sites in U2OS cells.

[0036] Figure 2 .sPE-MS2 and tPE-MS2 improved PE efficiency at different sites in 293FT cells.

[0037] Figure 3 .sPE-MS2 and tPE-MS2 improved PE efficiency at different sites in HeLa cells.

[0038] Figure 4 Editing efficiency of different aptamers of .sPEs and tPEs

[0039] Figure 5(a) Prime Editing (PE) System. The Prime Editing (PE) complex consists of the C-terminus of Streptococcus pyogenes Cas9-H840A (sky blue) and reverse transcriptase MMLV (yellow) to form the Cas9-H840A-MMLV fusion protein, and pegRNA containing the prime binding site (PBS) (blue-green) and the reverse transcription template (RTT, red-purple) at the 3' end of the sgRNA. (b) sPE System. Based on the PE system, an RNA stem-loop ligand (orange) is added to the 3' end of the pegRNA. (c) tPE System. Based on the PE system, an RNA-binding protein (RBP, blue) is fused to the N-terminus of Cas9, which can bind to the 3' end RNA stem-loop structure.

[0040] Figure 6 Construct pPB-PE-ALL-RNA for PE and pPB-sPE-ALL-RNA for sPE and tPE plasmids. Detailed Implementation

[0041] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0042] This invention develops sPEs by adding an RNA neck loop structure to the 3' end of pegRNA and tPEs by binding the 3' end of pegRNA to Cas9, thereby reducing the degradation ability of the 3' end and further improving the editing efficiency of different genomic sites in different cells.

[0043] This invention uses the editing efficiency of sPE and tPE at different gene loci (SRD5A3, DYRK1A, HDAC1, BCL11A, GFAP, RUNX1, JAK2, SRD5A1, DMD, and EED) in U2OS cells (Example 1), 293FT cells (Example 2), and HeLa cells (Example 3) as examples to illustrate that sPE and tPE improve editing efficiency compared to PE in different cell types. To demonstrate that this method is not limited to a single aptamer (MS2), this invention also tested the editing efficiency of sPE and tPE in different aptamers (com, boxB, pp7, and csy4) (Example 4).

[0044] Example 1:

[0045] In U2OS cells, this invention tested 10 gene loci, including SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, RUNX1_+5G·C to T·A, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A. The editing efficiency using the sPE-MS2 or tPE-MS2 system was on average 2.5 or 3.1 times higher than that of the PE system. sPE-MS2 or tPE-MS2 can improve the editing of PE-edited sites with very low C·G to A·T, DYRK1A_+1C·G to G·C, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, and DMD_+1T·A to C·G by an average of 3.4 or 3.9 times.

[0046] Figure 1 .sPE-MS2 and tPE-MS2 improved the PE efficiency at different sites in U2OS cells. (Left) Editing efficiency of PE, sPE-MS2, and tPEb-MS2-mediated point mutations SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, and RUNX1_+5G·C to T·A in U2OS cells. (Right) Editing efficiency of PE, sPE-MS2, and tPE-MS2-mediated point mutations JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A in U2OS cells.

[0047] Example 2:

[0048] In 293FT cells, this invention tested 10 gene loci including SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, RUNX1_+5G·C to T·A, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A. The editing efficiency using the sPE-MS2 or tPE-MS2 system was on average 1.8 or 1.9 times higher than that of the PE system. sPE-MS2 or tPE-MS2 can improve the editing of PE-edited sites with very low C·G to A·T, DYRK1A_+1C·G to G·C, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, and DMD_+1T·A to C·G by an average of 2.5 or 2.6 times.

[0049] Figure 2 .sPE-MS2 and tPE-MS2 improved the PE efficiency at different sites in 293FT cells. (Left) Editing efficiency of PE, sPE-MS2, and tPEb-MS2-mediated point mutations SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, and RUNX1_+5G·C to T·A in 293FT cells. (Right) Editing efficiency of PE, sPE-MS2, and tPE-MS2-mediated point mutations JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A in 293FT cells.

[0050] Example 3:

[0051] In HeLa cells, this invention tested 10 gene loci, including SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, RUNX1_+5G·C to T·A, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A. The editing efficiency using the sPE-MS2 or tPE-MS2 system was on average 3.7 or 2.7 times higher than that of the PE system. sPE-MS2 or tPE-MS2 can improve the editing efficiency of low-level PE sites such as SRD5A3_+2 C·G to A·T, DYRK1A_+1C·G to G·C, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, and DMD_+1T·A to C·G by an average of 6.2 or 4.9 times.

[0052] Figure 3 .sPE-MS2 and tPE-MS2 improved PE efficiency at different sites in HeLa cells.

[0053] (Left) Editing efficiency of PE, sPE-MS2, and tPEb-MS2-mediated point mutations SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, and RUNX1_+5G·C to T·A in HeLa cells. (Right) Editing efficiency of PE, sPE-MS2, and tPE-MS2-mediated point mutations JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A in HeLa cells.

[0054] Example 4:

[0055] To ensure that the efficiency enhancement of PE by sPE or tPE is not limited to MS2 stem-loop mutations, we tested three additional RNA stem-loop aptamers: Com binding site (ComBS), Csy4 binding site (Csy4BS), and PP7. RNA aptamers ComBS, Csy4BS, or PP7 were labeled to the 3' end of pegRNA to generate sPE-Com, sPE-Cys4, or sPE-PP7, respectively. In 293FT cells, all sPE-Com, sPE-Cys4, and sPE-PP7 systems enhanced the efficiency of G-to-T transversion mutations at the RUNX1 site. The tPE-Com and tPE-Cys4 systems also enhanced the efficiency of point mutations at the RUNX1 site.

[0056] Figure 4 Editing efficiency of different aptamers of .sPEs and tPEs

[0057] The efficiency of sPEs and tPEs in RUNX1_+5G·C to T·A transversion point mutation editing in HEK293FT cells was assessed using PE3. ComBS, Csy4BS, PP7, or MS2 RNA aptamers were attached to the 3' ends of the pegRNAs of sPE-Com (Com), sPE-Csy4 (Csy4), sPE-PP7 (tdPCP), and sPE-MS2 (tdMCP), respectively. The N-terminus of Cas9H840A-MMLV was fused with the homologous RNA-binding proteins Com of tPE-Com, Csy4 of tPE-Csy4, tPE-PP7 (tdPCP), and tPE-MS2 (tdMCP).

[0058] Specifically, the experimental method includes the following steps:

[0059] 1.1 Experimental Materials

[0060] 1.1.1 Reagents and Plasmids

[0061] Primers were synthesized by Suzhou Genewise Biotech Co., Ltd.; restriction endonucleases, DNA ligases, and high-fidelity DNA polymerases were used. Purchased from NEB; Plasmid recombination kit Clone And glue recovery kit Purchased from Vazyme; pCMV-BE3 from AddGene website; transfection reagent 2000, Purchased from Thermo Fisher; QuickExtract TM Genomic DNA extraction reagents were purchased from Illumina.

[0062] 1.1.2 Cell lines

[0063] Human fetal kidney cells HEK293FT (Thermo Fisher Scientific), human osteosarcoma cells U2OS, and human cervical cancer cells HeLa were cultured in adherent DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gbico) and 1% penicillin-dextrose antibody.

[0064] 1.2 Experimental Methods

[0065] 1.2.1 Constructing expression plasmids

[0066] The pegRNA expression plasmids pPB-PE-ALL-RNA and pPB-sPE-ALL-RNA, used for the modification of sPE and tPE, are generated through two steps. Figure 6 First, pPE-pegRNA, psPE-pegRNA, and pPE3-sgRNA were constructed. For pPE-pegRNA and psPE-pegRNA, oligonucleotides were synthesized (Supplementary Table 1), and 5'ACCG and 3'AAAA were added to the upstream and downstream primers, followed by annealing (Table 1.2.1.3), and then cloned into the pLH-AAAA backbone plasmid (Supplementary sequence S1). For pPE3-sgRNA, oligonucleotides were synthesized, and 5'ACCG and 3'AAAC were added to the upstream and downstream primers, followed by annealing, and then cloned into the pLH-sgRNA3 backbone vector (Supplementary sequence S1). Finally, the relevant expression components were cloned into a vector pDONOR5.1 (Supplementary sequence S1) using the Golden Gate cloning method (Table 1.2.1.5), generating pPB-PE-ALL-RNA and pPB-sPE-ALL-RNA plasmids.

[0067] The RBP-SpCas9-MMLV expression plasmid was generated based on pCMV-PE2(#addgene 132775), where PE2 refers to SpCas9H840A-MMLV( Figure 6b). First, T2A-GFP was inserted into the C-terminus of pCMV-PE2 to form the pCMV-PE2-T2A-GFP plasmid. Second, RNA-binding proteins of the stem-loop aptamer, including tdMCP, Com, Csy4H29A, and tdPCP (supplementary sequence S2), were synthesized and cloned into pCMV-PE2-T2A-GFP using the ClonExpress II one-step cloning kit (Vazyme, C112-01) (Table 1.2.1.4), resulting in pCMV-tdMCP-PE2-T2A-GFP, pCMV-Com-PE2-T2A-GFP, pCMV-Csy4H29A-PE2-T2A-GFP, and pCMV-tdPCP-PE2-T2A-GFP plasmids. All PCR amplifications involved are detailed in Table 1.2.1.1, and the PCR procedures are detailed in Table 1.2.1.2.

[0068] In step 1.2.1, the PCR system used to amplify various fragments in the plasmid construction experiment is as follows:

[0069] Table 1.2.1.1 PCR amplification system

[0070]

[0071] Table 1.2.1.2 PCR Procedure

[0072]

[0073]

[0074] Table 1.2.1.3 Annealing Procedure

[0075]

[0076] Table 1.2.1.4 Homologous recombination reactions

[0077]

[0078] Table 1.2.1.5 sgRNA Golden Gate Assembly Reaction

[0079]

[0080] 1.2.2 Cell Culture, Transfection, and Genome Extraction

[0081] HEK293FT (Thermo Fisher Scientific), HeLa (ATCC, CCL-2), and U2OS (ATCC, HTB-96) cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum, Thermo Fisher Scientific) with high glucose. All cells were cultured in a humidified incubator at 37°C and 5% CO2. Before transfection, cells were seeded in 12-well plates at approximately 60% confluency and transfected using Lipofectamine 2000 (Thermo Fisher Scientific). To measure gene editing efficiency, 50 μL of opti-DMEM, 1.5 μg of Primeeditor 2 (PE2, dCas9-MMLV) or RBP-PE2, and 500 ng of PE-all-RNA, sPE-all-RNA, or tPE-all-RNA expression plasmids were added to 1.5 mL centrifuge tubes. Vortex to mix thoroughly. In a separate 1.5 mL centrifuge tube, add 50 μL of opti-DMEM and 4 μL of Lipofectamine 2000 transfection reagent and incubate for 5 min. Combine the two solutions, mix well, and incubate for 15 min to co-transfect HEK293FT, U2OS, or HeLa cells. 72 hours after transfection, collect Prime editor (GFP+) and PE-all-RNA, sPE-all-RNA, and tPE-all-RNA double-positive cells using flow cytometry (BD FACS Aria).

[0082] 1.2.3 Genomic DNA extraction and PCR amplification

[0083] The genomic DNA of the cells was extracted using QuickExtract DNA Extraction Solution (Lucigen) according to the reagent instructions. The extracted genomic DNA was then amplified by PCR using the following primers (Supplementary Table 2).

[0084] 1.2.4 Deep Sequencing

[0085] Genomic loci of interest were amplified from genomic DNA using locus-specific primers containing Truseq adapters. Equal volumes of PCR products were pooled and recovered using a gel extraction kit. The purified library was purified using a gel. Deep sequencing of the purified library was performed using a 150bp paired-end Illumina MiniSeq analyzer.

[0086] Figure 6Construct pPB-PE-ALL-RNA for PE and pPB-sPE-ALL-RNA for sPE and tPE plasmids.

[0087] (a) pPB-PE-ALL-RNA was assembled from pPE-pegRNA, pPE3-sgRNA, and pDONOR5.1 via a golden gate. pPE-pegRNA consisted of sgRNA (blue), RTT (gold), and PBS (magenta). pPE3-sgRNA contained sgRNAnick (orange). pDONOR5.1 consisted of the CcdB gene (green), puromycin (yellow), P2A (purple), and TagBFP2 (cyan). pPB-sPE-ALL-RNA was recombined from pPE-pegRNA, pPE3-sgRNA, and pDONOR5.1. pPE-pegRNA contained sgRNA (blue), RTT (gold), PBS (magenta), and a stem-loop RNA aptamer (dark green). (b) Construction of pCMV-RBP-PE2-T2A-sfGFP. T2A-sfGFP is inserted into the C-terminus of pCMV-PE2 to generate pCMV-PE2-T2A-sfGFP, and then the RNA-binding protein including the stem-loop aptamer is cloned into pCMV-PE2-T2A-sfGFP.

[0088] MS2 (SEQ ID NO.1)

[0089] Acatgaggatcacccatgt

[0090] PP7 (SEQ ID NO.2)

[0091] Ggagcagacgatatggcgtcgctcc

[0092] ComBS (Com protein binding site) (SEQ ID NO.3)

[0093] CTGAATGCCTGCGAGCATC

[0094] Csy4BS (Csy4 protein binding site) (SEQ ID NO.4)

[0095] actgccgtataggcagctaagaaa

[0096] tdMCP (an RNA-binding protein that tandemly binds to the MS2 outer shell protein and the MS2 RNA neck loop structure) (SEQ ID NO.5)

[0097] ASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQ SSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYAMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSN SRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYA

[0098] tdPCP (a tandem PP7 capsid protein that binds to the PP7 RNA neck loop structure) (SEQ ID NO.6)

[0099] SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAY RVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGRADPLASKTIVLSVGEATRTLTEIQSTADRQIFEEKV GPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHD VTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR

[0100] COM (Com RNA-binding protein, an RNA-binding protein that binds to the ComBS RNA neck loop structure) (SEQ ID NO.7)

[0101] GSMKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKR EKITHSDETVRYGSTSGHKLNGGGGGMDAKSLTAWS

[0102] Csy4H29A (Csy4 binding protein H29A mutant, an RNA-binding protein that binds to the Csy4BS RNA neck loop structure) (SEQ ID NO.8)

[0103] MDHYLDIRLRPDPEFPPAQLMSVLFGKLAQALVAQGGDRIGVSFPDLDESR SRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQAKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQST GQHFRLFIRHGPLQVTAEEGGFTCYGLSKGGFVPWF

[0104] Supplementary Table 1

[0105] pPE-pegRNA

[0106] JAK2-F:

[0107] ataattgaagacATaccgACCTGTGACTCATGAAACACgtttgagagctagaaatagcaagttcaaataaggc(SEQ ID NO.9)

[0108] JAK2-R:

[0109] ataattgaagacGCaaaaCTGTGACTCATGAAATACAGGAAGAATGTgcaccgact cggtgcc(SEQID NO.10)

[0110] HDAC1-F:

[0111] ataattgaagacATaccgGCACCATGCAAAGAAGTCCGgtttgagagctagaaatagcaagttcaaataaggc(SEQ ID NO.11)

[0112] HDAC1-R:

[0113] ataattgaagacGCaaaaTGCAAAGAAGTGCGAGGCATCTGGCTgcaccgactcgg tgcc(SEQ IDNO.12)

[0114] EED-F:

[0115] ataattgaagacATaccgCCGGTTGTTGCAATCTTACGgtttgagagctagaaatagcaagttcaaataaggc(SEQ ID NO.13)

[0116] EED-R:

[0117] ataattgaagacGCaaaaGTTGTTGCAATCTTTCGTGGATGCTGATgcaccgactc ggtgcc(SEQID NO.14)

[0118] BCL11A-F:

[0119] ATAAACTTCTGCACgtttgagagctagaaatagcaagttcaaataggc(SEQ ID NO.15)

[0120] BCL11A-R:

[0121] ataattgaagacGCaaaaCACAGATAAACTTCTGAACTGGAGGGGCCgcaccgac tcggtgcc(SEQID NO.16)

[0122] SRD5A3-F:

[0123] aatattgagacATaccgCCTTACTCAATCTCTGTTCCgtttgagagctagaaatagcaagttcaaataggc(SEQ ID NO.17)

[0124] SRD5A3-R:

[0125] ataattgaagacGCaaaaTTACTCAATCTCTGTTACTGGGAGCACCTTTTCgcac cgactcggtgcc(SEQ ID NO.18)

[0126] SRD5A1-F:

[0127] ataattgaagacATaccgTCCATTCAGATCATATCCTAgtttgagagctagaaatagcaagttcaaataaggc(SEQ ID NO.19)

[0128] SRD5A1-R:

[0129] ataattgaagacGCaaaaCATTCAGATCATATCATAAGGAATCTCAGAAAgcaccg actcggtgcc(SEQ ID NO.20)

[0130] DYRK1A-F:

[0131] atattgagacATaccgagcacacaatcattctagtttgagagctagaatagcaagttcaataggc (SEQ ID NO.21)

[0132] DYRK1A-R:atattgaagacGCaaaaaaggacattgtaggatgcaccgactcggtgcc (SEQID NO.22)

[0133] DMD-F:

[0134] atattgagacATaccgCTACCTCTGTGATACTCTTCgttttgagagcttaggcaagttcaataggc(SEQ ID NO.23)

[0135] DMD-R:

[0136] atattgagacGCaaaaACCTCTGTGATACTCCTCAGGTGCACCTgcaccgactc ggtgcc(SEQID NO.24)

[0137] RUNX1-F:

[0138] atattgagacATaccggcattttcaggaagcgagtttgagagctagaatagcaagttcaataggc(SEQ ID NO.25)

[0139] RUNX1-R:

[0140] atattgaagacGCaaaaattttcaggaagcgattgcttcagacagcaccgactcggtgcc (SEQID NO.26)

[0141] GFAP-F:

[0142] atattgaagacATaccgagcctgtccatataaagggttttagagctagaatagcaagttaaata ggctagtc(SEQ ID NO.27)

[0143] GFAP-R:

[0144] atattgagacGCaaaaTGTGTCCATATAATGGAGGAGTTGGAAgcaccgactcgg tgcc(SEQ IDNO.28)

[0145] psPE-pegRNA

[0146] JAK2 -MS2-F:

[0147] atattgagacATaccgACCTGTGACTCATGAAAACACgttttgagagctagaatagcaagttcaataggc (SEQ ID NO.29)

[0148] JAK2 -MS2-R:

[0149] atattgaagacGCaaaaaacatgggtgatcctcatgtCTGTGACTCATGAATACAGGAATGTgcaccgactcggtgcc(SEQ ID NO.30)

[0150] HDAC1 -MS2-F:

[0151] atattgagacATaccgGCACCATGCAAAGAGTCCGgttttgagagctagaatagcaagttcaataggc (SEQ ID NO.31)

[0152] HDAC1 -MS2-R:

[0153] atattgaagacGCaaaaaacatgggtgatcctcatgtTGCAAAGAAGTGCGAGGCATCTGGCTgcaccgactcggtgcc(SEQ ID NO.32)

[0154] EED -MS2-F:

[0155] atattgagacATaccgCCGGTTGTTGCAATCTTACGgttttgagagctagaatagcaagttcaataggc(SEQ ID NO.33)

[0156] EED -MS2-R:

[0157] ataattgaagacGCaaaaacatgggtgatcctcatgtGTTGTTGCAATCTTTCGTGGATGCTGATgcaccgactcggtgcc(SEQ ID NO.34)

[0158] BCL11A -MS2-F:

[0159] aatattgagacATaccgtcacagATAAACTTCTGCACgtttgagagctagaaatagcaagttcaaataggc(SEQ ID NO.35)

[0160] BCL11A -MS2-R:

[0161] ataattgaagacGCaaaaacatgggtgatcctcatgtCACAGATAAACTTCTGAACTGGAGGGGCCgcaccgactcggtgcc(SEQ ID NO.36)

[0162] SRD5A3 -MS2-F:

[0163] aatattgagacATaccgCCTTACTCAATCTCTGTTCCgtttgagagctagaaatagcaagttcaaataggc(SEQ ID NO.37)

[0164] SRD5A3 -MS2-R:

[0165] TACTCAATCTCTGTTACTGGGAGCACCTTTTCgcaccgactcggtgcc(SEQ ID NO.38)

[0166] SRD5A1 -MS2-F:

[0167] ataattgaagacATaccgTCCATTCAGATCATATCCTAgtttgagagctagaaatagcaagttcaaataaggc(SEQ ID NO.39)

[0168] SRD5A1 -MS2-R:

[0169] atattgaagacGCaaaaaacatgggtgatcctcatgtCATTCAGATCATCATAAGGAATCTCAGAAAgcaccgactcggtgcc(SEQ ID NO.40)

[0170] DYRK1A -MS2-F:

[0171] atattgagacATaccgagcacacaatcattctagtttgagagctagaatagcaagttcaataggc(SEQ ID NO.41)

[0172] DYRK1A -MS2-R:

[0173] atattgaagacGCaaaaaacatgggtgatcctcatgtcaggacattgtaggatggatgcaccgactcggtgcc(SEQ ID NO.42)

[0174] DMD -MS2-F:

[0175] atattgagacATaccgCTACCTCTGTGATACTCTTCgttttgagagcttaggcaagttcaataggc(SEQ ID NO.43)

[0176] DMD -MS2-R:

[0177] atattgaagacGCaaaaaacatgggtgatcctcatgtACCTCTGTGATACTCCTCAGGTGCACCTgcaccgactcggtgcc(SEQ ID NO.44)

[0178] RUNX1 -MS2-F:

[0179] atattgagacATaccggcattttcaggaagcgagtttgagagctagaatagcaagttcaataggc(SEQ ID NO.45)

[0180] RUNX1 -MS2-R:atattgaagacGCaaaaaacatgggtgatctcatgtattttcaggaagcgattgcttcagacag caccgactcggtgcc(SEQ ID NO.46)

[0181] GFAP -MS2-F:

[0182] atattgaagacATaccgagcctgtccatataaagggttttagagctagaatagcaagttaaata ggctagtc(SEQ ID NO.47)

[0183] GFAP -MS2-R:

[0184] atattgaagacGCaaaaaacatgggtgatcctcatgtTGTGCCATAATGGAGGAGTTGGAAgcaccgactcggtgcc(SEQ ID NO.48)

[0185] RUNX1 -com-F:

[0186] atattgagacATaccggcattttcaggaagcgagtttgagagctagaatagcaagttcaataggc(SEQ ID NO.49)

[0187] RUNX1 -com-R:

[0188] atattgaagacGCaaaaGATGCTCGCAGGCATTCAGatttcaggaagcgattgcttcagacagcaccgactcggtgcc(SEQ ID NO.50)

[0189] RUNX1 -pp7-F:

[0190] atattgagacATaccggcattttcaggaagcgagtttgagagctagaatagcaagttcaataggc(SEQ ID NO.51)

[0191] RUNX1 -pp7-R:

[0192] ataattgaagacGCaaaaggagcgacgccatatcgtctgctccattttcaggaggagcgattgcttcagacagcaccgactcggtgcc(SEQ ID NO.52);

[0193] RUNX1 -csy4-F:

[0194] atattgaagacATaccggcattttcaggaggagcgagttttgagagctagaaatagcaagttcaaataggc(SEQ ID NO.53)

[0195] RUNX1 -csy4-R:

[0196] ataattgaagacGCaaaaCTGCCTATACGGCAGTGAACatttcaggaggagcgattgcttcagacagcaccgactcggtgcc(SEQ ID NO.54)

[0197] pPE3-sgRNA

[0198] JAK2-F:accgTGGGGTTTGATCGTTTTCTT(SEQ ID NO.55)JAK2-R:aaacAAGAAAACGATCAAACCCCA(SEQ ID NO.56)

[0199] HDAC1-F:accgATACTTTAGCAGTTCCAGG(SEQ ID NO.57)HDAC1-R:aaacTCCTGGAACTGCTAAAGTAT(SEQ ID NO.58)

[0200] EED-F:accgTGATTAATTTATAAAAGTTT(SEQ ID NO.59)EED-R:aaacAAACTTTTATAAATTAATCA(SEQ ID NO.60)

[0201] BCL11A-F:accgCTGCACTCATCCCAGGCGTG(SEQ ID NO.61)BCL11A- R:aaacCACGCCTGGGATGAGTGCAG(SEQ ID NO.62)

[0202] SRD5A3-F: accgCCAGTAACAGAGATTGAGTA(SEQ ID NO.63) SRD5A3-R: aaacTACTCAATCTCTGTTACTGG(SEQ ID NO.64)

[0203] SRD5A1-F: accgTCCTTATGATATGATCTGAA(SEQ ID NO.65) SRD5A1-R: aaacTTCAGATCATATCATAAGGA(SEQ ID NO.66)

[0204] DYRK1A-F: accgAAGAGTGAGGAGAAGGCAGG(SEQ ID NO.67) DYRK1A-R: aaacCCTGCCTTCTCCTCACTCTT(SEQ ID NO.68)

[0205] DMD-F: accgGTCAATGCTCTCCTTTTCAC(SEQ ID NO.69) DMD-R: aaacGTGAAAAGGAGAGCATTGAC(SEQ ID NO.70)

[0206] RUNX1-F: accgCATGTACTCACCTCTCATGA(SEQ ID NO.71) RUNX1-R: aaacTCATGAGAGGTGAGTACATG(SEQ ID NO.72)

[0207] Supplementary Table 2

[0208] Gene

[0209] JAK2-F: ctacacgacgctcttccgatctgTGGCGGCATGATTTTGT(SEQ ID NO.73)

[0210] JAK2-R: agacgtgtgctcttccgatctccaagcattttagataag(SEQ ID NO.74)

[0211] HDAC1-F: ctacacgacgctcttccgatcttcatgcccagCAAGTGCTGTGAAAC(SEQ IDNO.75) HDAC1-R: agacgtgtgctcttccgatctgacactcaccgagcacatcctagcc(SEQ ID NO.76)

[0212] EED-F:ctacacgacgctcttccgatctagttggagagagtatgttttctatacttgagatga(SEQID NO.77)EED-R:agacgtgtgctcttccgatctaactagatacaggattctcttgggtgatagtg(SEQ IDNO.78)

[0213] BCL11A-F:ctacacgacgctcttccgatcttattttaatggaattgaaatgtgatatcctgtggtttatgatg (SEQ ID NO.79)BCL11A-R:agacgtgtgctcttccgatctccttcagtacttaaaaagtaagggcaatttccagaaa(SEQ ID NO.80)

[0214] SRD5A3-F:ctacacgacgctcttccgatcttgcctcgcttgttttccttttgcagAT(SEQ IDNO.81)

[0215] SRD5A3-R:agacgtgtgctcttccgatctgcccttctgaggaggtgccg(SEQ ID NO.82)

[0216] SRD5A1-F:ctacacgacgctcttccgatctgaaattttacggtttattagccataatcatcttgcaa(SEQ ID NO.83)SRD5A1-R: agacgtgtgctcttccgatctgatgacttacactgaaaacttgtagctaataactactacg(SEQ ID NO.84)

[0217] DYRK1A-F:ctacacgacgctcttccgatctcttgtcacacacaatgaaactttgctgttcact(SEQID NO.85)DYRK1A-R:agacgtgtgctcttccgatctagtagctaacatttttcagtacttaactatatgccagctat(SEQ ID NO.86)

[0218] DMD-F:

[0219] ctacacgacgctcttccgatctAAGCATTAAATCTTAAGACTACAAGACATTACTTGA AGG(SEQID NO.87)DMD-R: agacgtgtgctcttccgatctGACAGAGAAGGGTGTAAAAGCTTCTAGC(SEQ IDNO.88)

[0220] RUNX1-F:ctacacgacgctcttccgatcttggttttcgctccgaagGTAAAAGAA(SEQ IDNO.89)RUNX1-R:agacgtgtgctcttccgatctatatgcctcagtttgaattcctctcacaaacaag(SEQ IDNO.90)

[0221] GFAP-F:ctacacgacgctcttccgatcttactggggaaagcagtgcaggagca(SEQ ID NO.91)GFAP-R:agacgtgtgctcttccgatctaccaccgcttcacagctgtgc(SEQ ID NO.92)

[0222] Supplementary Table 3

[0223] Gene

[0224] JAK2:gTGGCGGCATGATTTTGTGCACGGATGGATAAAAGTACCTGTGAC TCATGAAACACAGGAAGAATGTCTTGGGATGGCAGTGTTAGATATGATGAGAATAGCCAAAGAAAACGATCAAACCCCACTGGCCATCTATAACTCTAT CAGgtaattttcttttgcaaatccttacacataagtgtgagtagagattttatataattcgtatatattttctgtgtttacccatgccttttgattttgtaatactagttaagtactccttatctaaaatgcttgg(SEQ IDNO.93)

[0225] HDAC1:tcatgcccagCAAGTGCTGTGAAACTTAATAAGCAGCAGACGGACAT CGCTGTGAATTGGGCTGGGGGCCTGCACCATGCAAAGAAGTCCGAGGCATCTGGCTTCTGTTACGTCAATGATATCGTCTTGGCCATCCTGGAACTG CTAAAgtatgcctgcctggccttgtctcttggaagagcaccttaggccaggttcccatttccctcttcccctgggcttgcctccctagtttgcttttcctaccgatgtgctggctaggatgtgctcggtgagtgtc(SEQ IDNO.94)

[0226] EED:agttggagagagtatgttttctatacttgagatgaaatttactgtatttttattttcattagGTTACCTT GTATGAATGTCATTCACAAGGAGAAATCCGGTTGTTGCAATCTTACGTGG ATGCTGATgtatcctttcctgggtttttaatatctttagtcaaaggaatttattttcaataagtgcaccaaaacttttataaattaatcatttccctaaagttattgttctttcttaaccgatttcttcactatcacccaagagaatcctgt atctagtt(SEQ IDNO.95)

[0227] BCL11A:tattttaatggaattgaaatgtgatatcctgtggtttatgatgcacgttgtttgtagctgtagtgcttg attttgggtttctttcacagATAAACTTCTGCACTGGAGGGGCCTCTCCTCCCCTCGTTCTGCACATGGAGCTCTAATCCCCACGCCTGGGATGAGTGCAGAATAT GCCCCGCAGGGTATTTgtaagttgagccttatttcttctacaaatgtccatgtgtatagagatgagaatttctggaaattgcccttactttttaagtactgaagg(SEQ IDNO.96)

[0228] SRD5A3:tgcctcgcttgttttccttttgcagATATTTTTCCCACTTTTATATCATCTCAGT GCTGTGGAATGGCTTCCTGCTTTGGTGCCTTACTCAATCTCTGTTCCTGGGAGCACCTTTTCCAAGCTGGCTTCATGGTTTGCTCAGAATTCTCGGGG CGGCACAGTTCCAGGgtaaggactccctgggcttatgacaacgctgcatcccgtttctgttgttcctgtctgcaagggagcagtttttggcattttgtctcctctctcggcacctcctcagaagggc(SEQ IDNO.97)

[0229] SRD5A1:

[0230] gaaattttacggtttattagccataatcatcttgcaatttttttcctttagGTTTTGGCTTGTGGTTAACGGGCATGTTGATAAACATCCATTCAGATCATATCCTAAGGAATCTCAGAAAACCAGGAGATACTGGATACAAAATACCAAGGGgtacgtacagaaagtgaaga atttctgtgaaagttgcttgccatggttcctggctattttagtgttgccagctctaagaagtagtagcgtagtagttattagctacaagttttcagtgtaagtcatc(SEQ ID NO.98)

[0231] DYRK1A:

[0232] cttgtcacacacaatgaaactttgctgttcactgtcagttataacttacatgaggtgacccatttccattcaag ggttttagaagcacatcaaggacattctaaggatgattgacttacacaatgatctctgaacatgcctcctgccttctcctcactcttgagtatttgcttaggggagcagaaaaaatactggtagcattactgtaacattttaattct gctttatttaaaaaagacatagctggcatatagttaagtactgaaaaatgttagctact(SEQ ID NO.99)

[0233] DMD:

[0234] AAGCATTAAATCTTAAGACTACAAGACATTACTTGAAGGTCAATGCTCTC CTTTTCACAGGCTACTCAAAAAGAGATTGAGAAACAGAAGGTGCACCTGAAGAGTATCACAGAGGTAGGAGAGGCCTTGAAAACAGTTTTGGGCAAG AAGGAGACGTTGGTGGAAGATAAACTCAGTCTTCTGAATAGTAACTGGATAGCTGTCACCTCCCGAGCAGAAGAGTGGTTAAATCTTTTGTTGGTAAG AGAAAAGGCTAGAAGCTTTTACACCCTTCTCTGTC(SEQ IDNO.100)

[0235] RUNX1:

[0236] tggttttcgctccgaagGTAAAAGAAATCATTGAGTCCCCCGCCTTCAGAAGAGG GTGCATTTTCAGGAGGAAGCGATGGCTTCAGACAGCATATTTGAGTCATTTCCTTCGTACCCACAGTGCTTCATGAGAGgtgagtacatgctggtcttgtaatatctac ttttgctcagctttgcctgtaatgaaatggcagcttgtttcacctcggtgcagagatgcctcggtgcctgccagttccctgtcttgtttgtgagaggaattcaaactgaggcatat(SEQ ID NO.101)

[0237] GFAP:

[0238] tactggggaaagcagtgcaggagcagcggggcccctgtgtttcattcatggctgggctttgtgactgtggg cagcgagctcacctattctgagcctgtgtccatataaaggaggagttggaagcggagaaggttgatgtccatgagggagattggattctggggtgaagaaagtgagggaaagagcaggcaggtctgggcgcaaagc acaggtgactgcctgccaccagcttgtgacccccatcaagttactttgacttgcacagctgtgaagcggtggt(SEQ ID NO.102)

[0239] Supplementary sequence 1

[0240] pDONOR 5.1

[0241] acggatcgggagatctaccggtacgcgttgacgtgtcttcggatccggcttactaaaagccagataacag tatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatat gatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctga cgagaacaggggctggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgttt gtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatg accaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggctcccttatacacagcca gtctgcaggtcgacgaagacccactcgcggccgcttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcac attcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgc gggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcc tccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatccgccaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtac gcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatc gagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttc gccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgac caccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtca ccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgccgctagcggtgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggtagtggaacgcgtatggtgtctaagggcgaagagctgattaaggagaacatgcacatgaagctgtacatggagg gcaccgtggacaaccatcacttcaagtgcacatccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagcttc ctctacggcagcaagaccttcatcaaccacacccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgaagatgggggcgtgctgaccgctacccaggacac cagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggctgggaggccttcaccgagacgctgtaccccgctgacggcggc ctggaaggcagaaacgacatggccctgaagctcgtgggcgggagccatctgatcgcaaacgccaag accacatatagatccaagaaacccgctaagaacctcaagatgcctggcatctactatgtggactacagactggaaagaatcaaggaggccaacaacgaaacctacgtcgagcagcacgaggtggcagtggccag atactgcgacctccctagcaaactggggcacaagcttaatccaaagaaaaagcggaaagtgtgagtcgacctcgagggggggcccggtacctttaagaccaatgacttacaaggcagctgtagatcttagccacatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttg tggtttgtccaaactcatcaatgtatcttatcatgtctggaattcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcaca aaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggg aagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaaccc ggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgc gctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgatacc gcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagt tcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtt agctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctg agaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatag cagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttga atgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc(SEQ ID NO.103)

[0242] pLH-AAAA

[0243] ttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgg gaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggtt cgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcg cgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttg accgaatcaccgacctctctccccagggggatccaccggatcaagcttgccagctggggcgccctctggtaaggttgggaagccctgcagatctgatcccggggggcaatgagatatgaaaaagcctgaactcaccg cgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccg atggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagatctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtg atttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgc acgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatgg agcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgc agggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagca ctcgtccgagggcaaaggaatagagtagatgccgaccgggatctatcgataccgtcgacctcgagggggggcccggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaagg ggggactggaagggctaattcactcccaacgaagaaaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgg ctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtga ggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcccc ctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctga atggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggc tttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaat tttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaa aggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaa ctggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttta aagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaag agaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaacc ggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtt atctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatt taaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccactt caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaac ggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgcca gcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaa tgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcac taaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagta aggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtgga aaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgcca aaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacat atagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatata atacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtggga ataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtg ccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaa agaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctata gtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtg aacggatctcgacggtatcgatcacgagactagcctcgagcggccgcccccttcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaac acaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccggtgtcttcgaggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtatt acagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaag ggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattg acacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgcc ggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaaccctgcaggacgaagaccctttttttgaattctcgacctcgagacaaat ggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttc gggtttattacagggacagcagagatccactttggccgcggctcgaggggg(SEQ ID NO.104)

[0244] pLH-sgRNA3

[0245] ttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgg gaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggtt cgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcg cgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccc tgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatccaccggatcaagcttgccagctggggcgccctctgg taaggttgggaagccctgcagatctgatcccggggggcaatgagatatgaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggaggg cgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagatctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgt ccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcg aggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtat atgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcag aagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaatagagtagatgccgaccgggatctatcgataccgtcgacctcgagggg gggcccggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagaaaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttag tcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaa tttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgcccc atggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactgg ccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctga atggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttg gagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaat tttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaa aggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaa ctggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacac tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggagg cggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtt atctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatt taaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcg ataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtt tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattc tgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatgg taacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagta aggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtgga aaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgcca aaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacat atagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtggga ataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtg ccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaa agaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggc tgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatcacgagactagcctcgagcggccgcccccttcaccgagggcctatttc ccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatg ttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccggtgtcttcgaggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaa gcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtgaaat gcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtg aactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaa cctgatgttctggggaatataaaccctgcaggacgaagacccgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattctcgacctcgaga caaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaa attttcgggtttattacagggacagcagagatccactttggccgcggctcgaggggg(SEQ ID NO.105)

[0246] Supplementary sequence 2

[0247] tdMCP coding gene

[0248] gcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaac ttcgctaacgggatcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgcctaaaggcgcctggcgttcg tacttaaatatggaactaaccattccaattttcgccacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctacgccatgg ccagcaacttcacccagttcgtgctggtggacaacggcggcaccggcgacgtgaccgtggcccccagc aacttcgccaacggcatcgccgagtggatcagcagcaacagcagaagccaggcctacaaggtgacctgcagcgtgagacagagcagcgcccagaacagaaagtacaccatcaaggtggaggtgcccaagggc gcctggagaagctacctgaacatggagctgaccatccccatcttcgccaccaacagcgactgcgagctgatcgtgaaggccatgcagggcctgctgaaggacggcaaccccatccccagcgccatcgccgccaac agcggcatctacgcc(SEQ ID NO.106)

[0249] COM coding gene

[0250] ggtagtatgaaatcaattcgctgtaaaaactgcaacaaactgttatttaaggcggatagttttgatcacattg aaatcaggtgtccgcgttgcaaacgtcacatcataatgctgaatgcctgcgagcatcccacggagaaacattgtgggaaaagagaaaaaatcacgcattctgacgaaaccgtgcgttatggctccactagtgggcacaagcttaatggcggtggcggagggatggatgctaagtcactaactgcctggtcc(SEQ ID NO.107)

[0251] Csy4H29A coding gene

[0252] atggaccactacctcgacattcgcttgcgaccggacccggaatttcccccggcgcaactcatgagcgtgc tcttcggcaagctcgcccaggccctggtggcacagggcggggacaggatcggcgtgagcttccccgacctcgacgaaagccgctcccggctgggcgagcgcctgcgcattcatgcctcggcggacgaccttcgtgcc ctgctcgcccggccctggctggaagggttgcgggaccatctgcaattcggagaaccggcagtcgtgcctcaccccacaccgtaccgtcaggtcagtcgggttcaggcgaaaagcaatccggaacgcctgcggcggc ggctcatgcgccggcacgatctgagtgaggaggaggctcggaaacgcattcccgatacggtcgcgagagccttggacctgcccttcgtcacgctacgcagccagagcaccggacagcacttccgtctcttcatccgcc acgggccgttgcaggtgacggcagaggaaggaggattcacctgttacgggttgagcaaaggaggtttcgttccctggttc(SEQ ID NO.108)

[0253] tdPCP coding gene

[0254] tccaaaactatcgttctgagcgtaggtgaagcgaccagaacgttaactgaaattcaatctacagcagaca ggcaaatatttgaagagaaagtcggtccattagtagggaggttaagattgacagctagcttaagacaaaatggagctaaaactgcatacagggtcaatttaaaattggatcaagcagatgtcgttgatagcggtctacct aaggttagatacacgcaagtgtggtcacacgatgtcactattgtcgcgaattctaccgaagctagtagaaaatcattgtatgatttaacaaaatccttggtggctacctctcaggtggaagatcttgttgtgaatttagttccatt aggtagggcggacccgctagcctccaaaaccatcgttctttcggtcggcgaggctactcgcactctgactgagatccagtccaccgcagaccgtcagatcttcgaagagaaggtcgggcctctggtgggtcggctgcgc ctcacggcttcgctccgtcaaaacggagccaagaccgcgtatcgcgtcaacctaaaactggatcaggcggacgtcgttgattccggacttccgaaagtgcgctacactcaggtatggtcgcacgacgtgacaatcgttg cgaatagcaccgaggcctcgcgcaaatcgttgtacgatttgaccaagtccctcgtcgcgacctcgcaggtcgaagatcttgtcgtcaaccttgtgccgctgggccgt(SEQ IDNO.109) Sequence Listing <110> ShanghaiTech University <120> Novel and Efficient Prime Editors and Their Applications <160> 109 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 acatgaggat cacccatgt 19 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 ggagcagacg atatggcgtc gctcc 25 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <400> 3 ctgaatgcct gcgagcatc 19 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 actgccgtat aggcagctaa gaaa 24 <210> 5 <211> 235 <212> PRT <213> Artificial Sequence <400> 5 Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu Trp 20 25 30 Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45 Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50 55 60 Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro 65 70 75 80 Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln 85 90 95 Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn 100 105 110 Ser Gly Ile Tyr Ala Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val 115 120 125 Asp Asn Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala 130 135 140 Asn Gly Ile Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr 145 150 155 160 Lys Val Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr 165 170 175 Thr Ile Lys Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn 180 185 190 Met Glu Leu Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu 195 200 205 Ile Val Lys Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro 210 215 220 Ser Ala Ile Ala Ala Asn Ser Gly Ile Tyr Ala 225 230 235 <210> 6 <211> 247 <212> PRT <213> Artificial Sequence <400> 6 Ser Lys Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu Thr 1 5 10 15 Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val Gly 20 25 30 Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn Gly 35 40 45 Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln Ala Asp Val 50 55 60 Val Asp Ser Gly Leu Pro Lys Val Arg Tyr Thr Gln Val Trp Ser His 65 70 75 80 Asp Val Thr Ile Val Ala Asn Ser Thr Glu Ala Ser Arg Lys Ser Leu 85 90 95 Tyr Asp Leu Thr Lys Ser Leu Val Ala Thr Ser Gln Val Glu Asp Leu 100 105 110 Val Val Asn Leu Val Pro Leu Gly Arg Ala Asp Pro Leu Ala Ser Lys 115 120 125 Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu Thr Glu Ile 130 135 140 Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val Gly Pro Leu 145 150 155 160 Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn Gly Ala Lys 165 170 175 Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln Ala Asp Val Val Asp 180 185 190 Ser Gly Leu Pro Lys Val Arg Tyr Thr Gln Val Trp Ser His Asp Val 195 200 205 Thr Ile Val Ala Asn Ser Thr Glu Ala Ser Arg Lys Ser Leu Tyr Asp 210 215 220 Leu Thr Lys Ser Leu Val Ala Thr Ser Gln Val Glu Asp Leu Val Val 225 230 235 240 Asn Leu Val Pro Leu Gly Arg 245 <210> 7 <211> 88 <212> PRT <213> Artificial Sequence <400> 7 Gly Ser Met Lys Ser Ile Arg Cys Lys Asn Cys Asn Lys Leu Leu Phe 1 5 10 15 Lys Ala Asp Ser Phe Asp His Ile Glu Ile Arg Cys Pro Arg Cys Lys 20 25 30 Arg His Ile Ile Met Leu Asn Ala Cys Glu His Pro Thr Glu Lys His 35 40 45 Cys Gly Lys Arg Glu Lys Ile Thr His Ser Asp Glu Thr Val Arg Tyr 50 55 60 Gly Ser Thr Ser Gly His Lys Leu Asn Gly Gly Gly Gly Gly Met Asp 65 70 75 80 Ala Lys Ser Leu Thr Ala Trp Ser 85 <210> 8 <211> 187 <212> PRT <213> Artificial Sequence <400> 8 Met Asp His Tyr Leu Asp Ile Arg Leu Arg Pro Asp Pro Glu Phe Pro 1 5 10 15 Pro Ala Gln Leu Met Ser Val Leu Phe Gly Lys Leu Ala Gln Ala Leu 20 25 30 Val Ala Gln Gly Gly Asp Arg Ile Gly Val Ser Phe Pro Asp Leu Asp 35 40 45 Glu Ser Arg Ser Arg Leu Gly Glu Arg Leu Arg Ile His Ala Ser Ala 50 55 60 Asp Asp Leu Arg Ala Leu Leu Ala Arg Pro Trp Leu Glu Gly Leu Arg 65 70 75 80 Asp His Leu Gln Phe Gly Glu Pro Ala Val Val Pro His Pro Thr Pro 85 90 95 Tyr Arg Gln Val Ser Arg Val Gln Ala Lys Ser Asn Pro Glu Arg Leu 100 105 110 Arg Arg Arg Leu Met Arg Arg His Asp Leu Ser Glu Glu Glu Ala Arg 115 120 125 Lys Arg Ile Pro Asp Thr Val Ala Arg Ala Leu Asp Leu Pro Phe Val 130 135 140 Thr Leu Arg Ser Gln Ser Thr Gly Gln His Phe Arg Leu Phe Ile Arg 145 150 155 160 His Gly Pro Leu Gln Val Thr Ala Glu Glu Gly Gly Phe Thr Cys Tyr 165 170 175 Gly Leu Ser Lys Gly Gly Phe Val Pro Trp Phe 180 185 <210> 9 <211> 73 <212> DNA <213> Artificial Sequence <400> 9 ataattgaag acataccgac ctgtgactca tgaaacacgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 10 <211> 63 <212> DNA <213> Artificial Sequence <400> 10 ataattgaag acgcaaaact gtgactcatg aaatacagga agaatgtgca ccgactcggt 60 gcc 63 <210> 11 <211> 73 <212> DNA <213> Artificial Sequence <400> 11 ataattgaag acataccggc accatgcaaa gaagtccggt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <400> 12 ataattgaag acgcaaaatg caaagaagtg cgaggcatct ggctgcaccg actcggtgcc 60 <210> 13 <211> 73 <212> DNA <213> Artificial Sequence <400> 13 ataattgaag acataccgcc ggttgttgca atcttacggt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 14 <211> 62 <212> DNA <213> Artificial Sequence <400> 14 ataattgaag acgcaaaagt tgttgcaatc tttcgtggat gctgatgcac cgactcggtg 60 cc 62 <210> 15 <211> 73 <212> DNA <213> Artificial Sequence <400> 15 ataattgaag acataccgtc acagataaac ttctgcacgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 16 <211> 63 <212> DNA <213> Artificial Sequence <400> 16 ataattgaag acgcaaaaca cagataaact tctgaactgg aggggccgca ccgactcggt 60 gcc 63 <210> 17 <211> 73<00个0769><212> DNA <213> Artificial Sequence <400> 17 ataattgaag acataccgcc ttactcaatc tctgttccgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 18 <211> 67 It should be noted that the tag seems to be incorrect in the original. It's presented as <00个0769> here. You may want to double-check the accuracy of the original text for a more precise translation. <212> DNA <213> Artificial Sequence <400> 18 father acgcaaaatt actcaatctc tgttactggg agcacctttt cgcaccgact cggtgcc 67 <210> 19 <211> 73 <212> DNA <213> Artificial Sequence <400> 19 father acataccgtc cattcagatc father ttgagagcta gaaatagcaa gttcaata ggc <210> 20 <211> 66 <212> DNA <213> Artificial Sequence <400> 20 60. ataattgaag acgcaaaaca ttcagatcat atcataagga atctcagaaa gcaccgactc ggtgcc 66 <210> 21 <211> 73 <212> DNA <213> Artificial Sequence <400> 21 father acataccgag cacatcaagg acattctagt ttgagagcta gaaatagcaa gttcaata ggc <210> 22 <211> 55 <212> DNA <213> Artificial Sequence <400> 22 ataattgaag acgcaaaaca aggacattgt aaggatgatg caccgactcg gtgcc 55 <210> 23 <211> 73 <212> DNA <213> Artificial Sequence <400> 23 ataattgaag acataccgct acctctgtga tactcttcgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 24 <211> 62​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ataattgaag acgcaaaaat tttcaggagg aagcgattgc ttcagacagc accgactcgg 60 tgcc 64 <210> 27 <211> 78 <212> DNA <213> Artificial Sequence <400> 27 ataattgaag acataccgag cctgtgtcca tataaagggt tttagagcta gaaatagcaa 60​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ataattgaag acgcaaaaac atgggtgatc ctcatgtctg tgactcatga aatacaggaa 60 gaatgtgcac cgactcggtg cc 82 <210> 31 <211> 73 <212> DNA <213> Artificial Sequence <400> 31 ataattgaag acataccggc accatgcaaa gaagtccggt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 32 <211> 79 <212> DNA <213> Artificial Sequence <400> 32 / ataattgaag acgcaaaaac atgggtgatc ctcatgttgc aaagaagtgc gaggcatctg 60 gctgcaccga ctcggtgcc 79 <21S> 33 <211> 73 <212> DNA <213> Artificial Sequence <400> 33 ataattgaag acataccgcc ggttgttgca atcttacggt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 34 <211> 81 <212> DNA <213> Artificial Sequence <400> 34 ataattgaag acgcaaaaac atgggtgatc ctcatgtgtt gttgcaatct ttcgtggatg 60 ctgatgcacc gactcggtgc c 81 <210> 35 <211> 73 <212> DNA <213> Artificial Sequence <400> 35 ataattgaag acataccgtc acagataaac ttctgcacgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 36 <211> 82 <212> DNA <213> Artificial Sequence <400> 36 ataattgaag acgcaaaaac atgggtgatc ctcatgtcac agataaactt ctgaactgga 60 ggggccgcac cgactcggtg cc 82 <210> 37 <211> 73 <212> DNA <213> Artificial Sequence <400> 37 ataattgaag acataccgcc ttactcaatc tctgttccgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 38 <211> 86 <212> DNA <213> Artificial Sequence <400> 38 ataattgaag acgcaaaaac atgggtgatc ctcatgttta ctcaatctct gttactggga 60 gcaccttttc gcaccgactc ggtgcc 86 <210> 39 <211> 73 <212> DNA <213> Artificial Sequence <400> 39 ataattgaag acataccgtc cattcagatc atatcctagt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 40 <211> 85 <212> DNA <213> Artificial Sequence <400> 40 ataattgaag acgcaaaaac atgggtgatc ctcatgtcat tcagatcata tcataaggaa 60 tctcagaaag caccgactcg gtgcc 85 <210> 41 <211> 73 <212> DNA <213> Artificial Sequence <400> 41 [[ID=?]]ataattgaag acataccgag cacatcaagg acattctagt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 42 <211> 74 <212> DNA <213> Artificial Sequence <400> 42 ataattgaag acgcaaaaac atgggtgatc ctcatgtcaa ggacattgta aggatgatgc 60 accgactcgg tgcc 74 <210> 43 <211> 73 <212> DNA <213> Artificial Sequence <400> 43 ataattgaag acataccgct acctctgtga tactcttcgt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 44 <211> 81 <212> DNA <213> Artificial Sequence <400> 44 ataattgaag acgcaaaaac atgggtgatc ctcatgtacc tctgtgatac tcctcaggtg 60 cacctgcacc gactcggtgc c 81 <210> 45 <211> 73 <212> DNA <213> Artificial Sequence <400> 45 ataattgaag acataccggc attttcagga ggaagcgagt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 46 <211> 83 <212> DNA <213> Artificial Sequence <400> 46 ataattgaag acgcaaaaac atgggtgatc ctcatgtatt ttcaggagga agcgattgct 60 tcagacagca ccgactcggt gcc 83 <210> 47 <211> 78 <212> DNA <213> Artificial Sequence <400> 47 ataattgaag acataccgag cctgtgtcca tataaagggt tttagagcta gaaatagcaa 60 gttaaaataa ggctagtc 78 <210> 48 <211> 80 <212> DNA <213> Artificial Sequence <400> 用英语翻译“用英语翻译”这句话是“Translate this sentence in English”。48 ataattgaag acgcaaaaac atgggtgatc ctcatgttgt gtccatataa tggaggagtt 60 ggaagcaccg actcggtgcc 80 <210> 49 [[ID=用英语翻译“用英语翻译”这句话是“Translate this sentence in English”。34]]<211> 73 <212> DNA <213> Artificial Sequence <400> 49 ataattgaag acataccggc attttcagga ggaagcgagt ttgagagcta gaaatagcaa 60 用英语翻译“用英语翻译”这句话是“Translate this sentence in English”。 gttcaaataa ggc 73 <210> 50 <211> 83 <212> DNA <2用英语翻译“用英语翻译”这句话是“Translate this sentence in English”。13> Artificial Sequence[[ID=用英语翻译“用英语翻译”这句话是“Translate this sentence in English”。53]] <400> 50 需要注意的是,原文中存在一些重复且表述不太清晰的内容,我按照要求进行了完整翻译。你可以检查一下是否符合你的预期,若有疑问可随时告诉我。ataattgaag acgcaaaaga tgctcgcagg cattcagatt ttcaggagga agcgattgct 60 tcagacagca ccgactcggt gcc 83 <210> 51 <211> 73 <212> DNA <213> Artificial Sequence <400> 51 ataattgaag acataccggc attttcagga ggaagcgagt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 52 <211> 89 <212> DNA <213> Artificial Sequence <400> 52 ataattgaag acgcaaaagg agcgacgcca tatcgtctgc tccattttca ggaggaagcg 60 attgcttcag acagcaccga ctcggtgcc 89 <210> 53 <211> 73 <212> DNA <213> Artificial Sequence <400> 53 ataattgaag acataccggc attttcagga ggaagcgagt ttgagagcta gaaatagcaa 60 gttcaaataa ggc 73 <210> 54 <211> 84 <212> DNA <213> Artificial Sequence <400> 54 ataattgaag acgcaaaact gcctatacgg cagtgaacat tttcaggagg aagcgattgc 60 ttcagacagc accgactcgg tgcc 84 <210> 55 <211> twenty four <212> DNA <213> Artificial Sequence <400> 55 accgtggggt ttgatcgttt tctt 24 <210> 56 <211> twenty four <212> DNA <213> Artificial Sequence <400> 56 aaacaagaaa acgatcaaac ccca 24 <210> 57 <211> twenty three <212> DNA <213> Artificial Sequence <400> 57 accgatactt tagcagttcc agg 23 <210> 58 <211> twenty four <212> DNA <213> Artificial Sequence <400> 58 aaactcctgg aactgctaaa gtat 24 <210> 59 <211> twenty four <212> DNA <213> Artificial Sequence <400> 59 accgtgatta atttataaaa gttt 24 <210> 60 <211> twenty four <212> DNA <213> Artificial Sequence <400> 60 aaacaaactt ttataaatta atca 24 <210> 61 <211> twenty four <212> DNA <213> Artificial Sequence <400> 61 accgctgcac tcatcccagg cgtg 24 <210> 62 <211> twenty four <212> DNA <213> Artificial Sequence <400> 62 aaaccacgcc tgggatgagt gcag 24 <210> 63 <211> twenty four <212> DNA <213> Artificial Sequence <400> 63 accgccagta acagagattg agta 24 <210> 64 <211> twenty four <212> DNA <213> Artificial Sequence <400> 64 aaactactca atctctgtta ctgg 24 <210> 65 <211> twenty four <212> DNA <213> Artificial Sequence <400> 65 accgtcctta tgatatgatc tgaa 24 <210> 66 <211> twenty four <212> DNA <213> Artificial Sequence <400> 66 aaacttcaga tcatatcata agga 24 <210> 67 <211> twenty four <212> DNA <213> Artificial Sequence <400> 67 accgaagagt gaggagaagg cagg 24 <210> 68 <211> twenty four <212> DNA <213> Artificial Sequence <400> 68 aaaccctgcc ttctcctcac tctt 24 <210> 69 <211> twenty four <212> DNA <213> Artificial Sequence <400> 69 accggtcaat gctctccttt tcac 24 <210> 70 <211> twenty four <212> DNA <213> Artificial Sequence <400> 70 aaacgtgaaa aggagagcat tgac 24 <210> 71 <211> twenty four <212> DNA <213> Artificial Sequence <400> 71 accgcatgta ctcacctctc atga 24 <210> 72 <211> twenty four <212> DNA <213> Artificial Sequence <400> 72 aaactcatga gaggtgagta catg 24 <210> 73 <211> 40 <212> DNA <213> Artificial Sequence <400> 73 ctacacgacg ctcttccgat ctgtggcggc atgattttgt 40 <210> 74 <211> 39 <212> DNA <213> Artificial Sequence <400> 74 agacgtgtgc tcttccgatc tccaagcattttagataag 39 <210> 75 <211> 47 <212> DNA <213> Artificial Sequence <400> 75 ctacacgacg ctcttccgat cttcatgccc agcaagtgct gtgaaac 47 <210> 76 <211> 46 <212> DNA <213> Artificial Sequence <400> 76 agacgtgtgc tcttccgatc tgacactcac cgagcacatc ctagcc 46 <210> 77 <211> 57 <212> DNA <213> Artificial Sequence <400> 77 ctacacgacg ctcttccgat ctagttggag agagtatgtt ttctatactt gagatga 57 <210> 78 <211> 53 <212> DNA <213> Artificial Sequence <400> 78 agacgtgtgc tcttccgatc taactagata caggattctc ttgggtgata gtg 53 <210> 79 <211> 65 <212> DNA <213> Artificial Sequence <400> 79 ctacacgacg ctcttccgat cttattttaa tggaattgaa atgtgatatc ctgtggttta 60 tgatg 65 <210> 80 <211> 58 <212> DNA <213> Artificial Sequence <400> 80 agacgtgtgc tcttccgatc tccttcagta cttaaaaagt aagggcaatt tccagaaa 58 <210> 81 <211> 49 <212> DNA <213> Artificial Sequence <400> 81 ctacacgacg ctcttccgat cttgcctcgc ttgttttcct tttgcagat 49 <210> 82 <211> 41 <212> DNA <213> Artificial Sequence <400> 82 agacgtgtgc tcttccgatc tgcccttctg aggaggtgcc g 41 <210> 83 <211> 59 <212> DNA <213> Artificial Sequence <400> 83 ctacacgacg ctcttccgat ctgaaatttt acggtttatt agccataatc atcttgcaa 59 <210> 84 <211> 61 <212> DNA <213> Artificial Sequence <400> 84 agacgtgtgc tcttccgatc tgatgactta cactgaaaac ttgtagctaa taactactac 60 g 61 <210> 85 <211> 55 <212> DNA <213> Artificial Sequence <400> 85 ctacacgacg ctcttccgat ctcttgtcac acacaatgaa actttgctgt tcact 55 <210> 86 <211> 62 <212> DNA <213> Artificial Sequence <400> 86 agacgtgtgc tcttccgatc tagtagctaa catttttcag tacttaacta tatgccagct at 62 <210> 87 <211> 61 <212> DNA <213> Artificial Sequence <400> 87 ctacacgacg ctcttccgat ctaagcatta aatcttaaga ctacaagaca ttacttgaag g 61 <210> 88 <211> 49 <212> DNA <213> Artificial Sequence <400> 88 agacgtgtgc tcttccgatc tgacagagaa gggtgtaaaa gcttctagc <210> 89 <211> 48 <212> DNA <213> Artificial Sequence <400> 89 ctacacgacg ctcttccgat cttggttttc gctccgaagg taaaga <210> 90 <211> 55 <212> DNA <213> Artificial Sequence <400> 90 agcgtgtgc tcttccgatc fathergcctc agtttgatt cctctcacaa acaag <210> 91 <211> 47 <212> DNA <213> Artificial Sequence <400> 91 ctacacgacg ctcttccgat cttactgggg aaagcagtgc aggagca 47 <210> 92 <211> 42 <212> DNA <213> Artificial Sequence <400> 92 agacgtgtgc tcttccgatc taccaccgct tcacagctgt gc 42 <210> 93 <211> 280 <212> DNA <213> Artificial Sequence <400> 93 gtggcggcat gattttgtgc acggatggat aaaagtacct gtgactcatg aaacacagga 60 agaatgtctt gggatggcag tgttagatat gatgagaata gccaaagaaa acgatcaaac 120 cccactggcc atctataact ctatcaggta attttctttt gcaaatcctt acacataagt 180 gtgagtagag attttatata attcgtatat attttctgtg tttacccatg ccttttgatt 240 ttgtaatact agttaagtac tccttatcta aaatgcttgg 280 <210> 94 <211> 280 <212> DNA <213> Artificial Sequence <400> 94 tcatgcccag caagtgctgt gaaacttaat aagcagcaga cggacatcgc tgtgaattgg 60 gctgggggcc tgcaccatgc aaagaagtcc gaggcatctg gcttctgtta cgtcaatgat 120 atcgtcttgg ccatcctgga actgctaaag tatgcctgcc tggccttgtc tcttggaaga 180 gcaccttagg ccaggttccc atttccctct tcccctgggc ttgcctccct agtttgcttt 240 tcctaccgat gtgctggcta ggatgtgctc ggtgagtgtc 280 <210> 95 <211> 280 <212> DNA <213> Artificial Sequence <400> 95 agttggagag agtatgtttt ctatacttga gatgaaattt actgtatttt tattttcatt 60 aggttacctt gtatgaatgt cattcacaag gagaaatccg gttgttgcaa tcttacgtgg 120 atgctgatgt atcctttcct gggtttttaa tatctttagt caaaggaatt tattttcaat 180 aagtgcacca aaacttttat aaattaatca tttccctaaa gttattgttc tttcttaacc 240 gatttcttca ctatcaccca agagaatcct gtatctagtt 280<e <210> 96 <211> 280 <212> DNA <213> Artificial Sequence <400> 96 tattttaatg gaattgaaat gtgatatcct gtggtttatg atgcacgttg tttgtagctg 60 tagtgcttga ttttgggttt ctttcacaga taaacttctg cactggaggg gcctctcctc 120 ccctcgttct gcacatggag ctctaatccc cacgcctggg atgagtgcag aatatgcccc 180 gcagggtatt tgtaagttga gccttatttc ttctacaaat gtccatgtgt atagagatga 240 gaatttctgg aaattgccct tactttttaa gtactgaagg 280 <210> 97 <211> 280 <212> DNA <213> Artificial Sequence <400> 97 tgcctcgctt gttttccttt tgcagatatt tttcccactt ttatatcatc tcagtgctgt 60 ggaatggctt cctgctttgg tgccttactc aatctctgtt cctgggagca ccttttccaa 120 gctggcttca tggtttgctc agaattctcg gggcggcaca gttccagggt aaggactccc 180 tgggcttatg acaacgctgc atcccgtttc tgttgttcct gtctgcaagg gagcagtttt 240 tggcattttg tctcctctct cggcacctcc tcagaagggc 280 <210> 98 <211> 280 <212> DNA <213> Artificial Sequence <400> 98 gaaattttac ggtttattag ccataatcat cttgcaattt ttttccttta ggttttggct 60 tgtggttaac gggcatgttg ataaacatcc attcagatca tatcctaagg aatctcagaa 120 aaccaggaga tactggatac aaaataccaa ggggtacgta cagaaagtga agaatttctg 180 tgaaagttgc ttgccatggt tcctggctat tttagtgttg ccagctctaa gaagtagtag 240 cgtagtagtt attagctaca agttttcagt gtaagtcatc 280 <210> 99 <211> 280<​​​​​​​​​​​​​​​​​​​ <212> DNA <213> Artificial Sequence <400> 100 aagcattaaa tcttaagact acaagacatt acttgaaggt caatgctctc cttttcacag 60 gctactcaaa aagagattga gaaacagaag gtgcacctga agagtatcac agaggtagga 120 gaggccttga aaacagtttt gggcaagaag gagacgttgg tggaagataa actcagtctt 180 ctgaatagta actggatagc tgtcacctcc cgagcagaag agtggttaaa tcttttgttg 240 gtaagagaaa aggctagaag cttttacacc cttctctgtc 280 <210> 101 <211> 280 <212> DNA <213> Artificial Sequence <400> 101 tggttttcgc tccgaaggta aaagaaatca ttgagtcccc cgccttcaga agagggtgca 60 ttttcaggag gaagcgatgg cttcagacag catatttgag tcatttcctt cgtacccaca 120 gtgcttcatg agaggtgagt acatgctggt cttgtaatat ctacttttgc tcagctttgc 180 ctgtaatgaa atggcagctt gtttcacctc ggtgcagaga tgcctcggtg cctgccagtt 240 ccctgtcttg tttgtgagag gaattcaaac tgaggcatat 280 <210> 102 <211> 280 <212> DNA <213> Artificial Sequence <400> 102 tactggggaa agcagtgcag gagcagcggg gcccctgtgt ttcattcatg gctgggcttt 60 gtgactgtgg gcagcgagct cacctattct gagcctgtgt ccatataaag gaggagttgg 120 aagcggagaa ggttgatgtc catgagggag attggattct ggggtgaaga aagtgaggga 180 aagagcaggc aggtctgggc gcaaagcaca ggtgactgcc tgccaccagc ttgtgacccc 240 catcaagtta ctttgacttg cacagctgtg aagcggtggt 280 <210> 103 <211> 4757 <212> DNA <213> Artificial Sequence <400> 103 acggatcggg agatctaccg gtacgcgttg acgtgtcttc ggatccggct tactaaaagc 60 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat 120 gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt 180 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa 240 gcacaacat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc 300 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga 360 acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt 420 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc 480 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat 540 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt 600 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac 660 ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga 720 cgaagaccca ctcgcggccg cttggggttg cgccttttcc aaggcagccc tgggtttgcg 780 cagggaacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct 840 cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc 900 cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg 960 tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc 1020 cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca 1080 gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta 1140 gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg 1200 tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccgcca 1260 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg 1320 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg 1380 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg 1440 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg 1500 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg 1560 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg 1620 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg 1680 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc 1740 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg 1800 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg 1860 ctagcggtgc tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg 1920 gacctggtag tggaacgcgt atggtgtcta agggcgaaga gctgattaag gagaacatgc 1980 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg 2040 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc 2100 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca 2160 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat 2220 gggagagagt caccacatac gaagatgggg gcgtgctgac cgctacccag gacaccagcc 2280 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg 2340 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg 2400 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc 2460 tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc 2520 ctggcatcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgaaacct 2580 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc 2640 acaagcttaa tccaaagaaa aagcggaaag tgtgagtcga cctcgagggg gggcccggta 2700 cctttaagac caatgactta caaggcagct gtagatctta gccacatgtc atcttattat 2760 tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc 2820 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 2880 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggaa 2940 ttcacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3000 cgtttttcca tagctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 3060 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 3120 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 3180 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 3240 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 3300 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 3360 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 3420 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 3480 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 3540 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 3600 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 3660 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 3720 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 3780 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 3840 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 3900 gagaaccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 3960 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 4020 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 4080 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 4140 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 4200 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 4260 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 4320 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 4380 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 4440 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 4500 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 4560 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 4620 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 4680 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 4740 aagtgccacc tgacgtc 4757 <210> 104 <211> 8191 <212> DNA <213> Artificial Sequence <400> 104 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 60 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 120 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 180 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 240 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 300 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 360 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 420 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 480 gaccgaatca ccgacctctc tccccagggg gatccaccgg atcaagcttg ccagctgggg 540 cgccctctgg taaggttggg aagccctgca gatctgatcc cggggggcaa tgagatatga 600 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg 660 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 720 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt 780 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 840 aatttagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag 900 atctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga 960 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 1020 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 1080 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga 1140 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 1200 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 1260 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 1320 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc 1380 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 1440 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 1500 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 1560 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat 1620 agaggtagg ccgaccggga tctatcgata ccgtcgacct cgagggggg cccggtacct 1680 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg 1740 ggactggaag ggctaattca ctcccaacga agaaaagatc tgctttttgc ttgtactggg 1800 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 1860 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag rich acttgtttat tgcagcttat aatggttaca aataagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atccccgcccc 2220. taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccaga gtagtgagga ggctttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 2640 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 2700 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 2760 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 2820 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 2880 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 2940 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 3000 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 3060 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 3120 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 3180 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 3240 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 3300 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 3360 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 3420 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 3480 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 3540 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 3600 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 3660 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 3720 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 3780 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 3840 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3900 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3960 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 4020 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4320. ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4440 ttggactcaa gacgatagtt accggataag gcgcagcggt cggggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcgga caggagagcg cacgaggggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4740 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4800 tggccttttg ctcacatgtt ctttcctgcg ttcccctg attctgtgga taaccgtatt 4860 accgccttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 4920 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4980 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 5040 gcaattaatg tgagttagct cactcattag gcaccccagg cttacactt tatgcttccg 5100 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 5160 catgattacg ccaagcgcgc aattaacct cactaaaggg aaaaagct ggagctgcaa 5220 gcttaatgta gtcttatgca atactcttgt agctttgcaa catggtaacg atgagttagc 5280 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 5340 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg 5400 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc 5460 tctggttaga ccagatctga gcctgggagc tctctggcta actagggac ccactgctta 5520 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgccgtctg tgtgtgact 5580 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 5640 cccgaacagg gacttgaaag cgaaagggaa accagagg ctctcgac gcaggactcg 5700 gcttgctgaa gcgcgcacgg caaggcga ggggcggcga ctggtgagta cgccaaaaat 5760 tttgactagc ggaggctaga aggagaga tggtgcgag agcgtcagta ttaagcgggg 5820 gagaattaga tcgcgatggg aaaaaatttcg gttaaggcca gggggaaga aaaatataa 5880 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 5940 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 6000 aggatcagaa gaacttagat cattatataa tacagtagca accctctat gtgtgcatca 6060 aggtagag aaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaasca 6120 aagtaagacc accgcacagc aagcggccgc tgatctcag acctggagga ggagatatga 6180 gggacaattg gagaagtgaa tatataat aataagtagt aaaaattgaa ccattaggag 6240 tagcaccac caggcaag agaagtgg tgcagagaga aaaaagagca gtgggaatag 6300 gagctttgtt ccttgggttc tgggagcag caggaagcac tatggcgca gcgtcaatga 6360 cgctgacggt acaggccaga cattattgt ctggtatagt gcagcagcag aacaatttgc 6420 tgaggctat tgaggcgca cagcatgt tgcactcac agtctggggc atcagcagc 6480 tccaggcaag aatcctggct gtggaagat acctaagga tcacagctc ctgggattt 6540 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 6600 aaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaatta 6660 acaattacac aagcttaata cactccttaa ttgagaatc gcaaaccag cahaaaga 6720 atgacaaga attattgga ttagataat gggcaagtttt gtggaatttgg tttacataa 6780 caattggct gtgtatata aattattca taatgagt aggaggcttg gtaggtttta 6840 gatagtttt tgctgtactt tctatagtga atagagttag gcagggat tcaccattat 6900 cgtttcagac ccaccccca accccgaggg gacccgacag gcccgaagga atagagaag 6960 aaggtggaga gagagacaga gatacca ttcgattagt gaacggatct cgacggtatc 7020 gatcacgaga ctagcctcga gcggccgcccc ccttcaccga gggcctattt cccatgattc 7080 cttcatattt gcatatacga tacaggctg ttagagagat attggaattt aatttgactg 7140 taacacaaa gattagta aaatacgt gacgtagaa gtaataattt cttgggtagt 7200 ttgcagtttt aaaattatgt ttaaatgg actacatat gcttaccgta acttgaaagt 7260 atttcgattt cttggcttta tatatcttgt ggaaggacg aacaccggt gtcttcgagg 7320 cttactaaaa gccagataac agtatgcgta ttgcgcgct gatttttgcg gtataagaat 7380 atatactgat atgtataccc gaagtatgtc aaaagaggt gtgctatgaa gcagcgtatt 7440 acagtgacag ttgacagcga cagctatcag tgctcagg catatatgat gtcaatct 7500 ccggtctgt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga 7560 aagcggaaaa tcaggaaggg atggctgagg tcgccggtt tattgaatg aacggctctt 7620 ttgctgacga gaacagggac tggtgaaatg cagtttaagg tttacaccta taaaagagag 7680 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg 7740 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac 7800 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg 7860 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa 7920 aacgccatta acctgatgtt ctggggaata taaaccctgc aggacgaaga cccttttttt 7980 gaattctcga cctcgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg 8040 attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact 8100 aaagaattac aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc 8160 agagatccac tttggccgcg gctcgagggg g 8191 <210> 105 <211> 8267 <212> DNA <213> Artificial Sequence <400> 105 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 60 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 120 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 180 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 240 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 300 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 360 cgggaaggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 420 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 480 gaccgaatca ccgacctctc tccccagggg gatccaccgg atcaagcttg ccagctgggg 540 cgccctctgg taaggttggg aagccctgca gatctgatcc cggggggcaa tgagatatga 600 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg 660 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 720 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt 780 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 840 aatttagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag 900 atctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga 960 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 1020 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 1080 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga 1140 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 1200 aaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 1260 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 1320 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc 1380 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 1440 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 1500 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 1560 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat 1620 agaggtagg ccgaccggga tctatcgata ccgtcgacct cgagggggg cccggtacct 1680 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg 1740 ggactggaag ggctaattca ctcccaacga agaaaagatc tgctttttgc ttgtactggg 1800 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 1860 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 1920 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 1980 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag 2040 agtgagagga acttgtttat tgcagcttat aatggttaca aataaaagcaa tagcatcaca 2100 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 2160 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc 2220 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccaga gtagtgagga ggctttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 2640 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 2700 aaatcggggg ctccctttag ggttccgatt taggcttta cggcacctcg accccaaaaa 2760 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagcgg tttttcgccc 2820 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aaaaaaatt taacgcgaat ttaaaaaa tattaacgct 3000 tacaatttag gtggcacttt tcggggaat gtgcgcggaa cccctatttg tttattttc 3060 taaatacatt caatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 3120 tattgaaaaa ggaagagtat gagtattca cattccgtg tcgcccttat tccctttttt 3180 gcggcatttt gccttcctgt tttgctcac ccagaaacgc tggtgaagt aaagatgct 3240 gagatcagt tggtgcacg agtgggttac atcgactgg atctcacag cggtaagatc 3300 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttta agttctgcta 3360 tgtggcgcgg tattacccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 3420 tattctcaga atgacttggt tgagtactca ccagtcacag aaagcatct tacggatggc 3480 atgacagtaa gagaattatg cagtgctgcc ataccatga gtgatacac tgcggccaac 3540 ttactctga caacgatcgg aggaccgaag gagctaccg cttttgca caacatgggg 3600 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaacgac 3660 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg tagccctcc 3900 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 4320. ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 4440 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 4500 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 4560 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4620 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4680 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4740 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4800 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4860 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 4920 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4980 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 5040 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 5100 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 5160 catgattacg ccaagcgcgc aattaacct cactaaaggg aaaaagct ggagctgcaa 5220 gcttaatgta gtcttatgca atactcttgt agctttgcaa catggtaacg atgagttagc 5280 aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 5340 acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg 5400 aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc 5460 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 5520 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 5580 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 5640 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 5700 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 5760 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 5820 gagaattaga tcgcgatggg aaaaaatttcg gttaaggcca gggggaaga aaaatataa 5880 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 5940 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 6000 aggatcagaa gaacttagat cattatataa tacagtagca accctctat gtgtgcatca 6060 aggtagag aaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaasca 6120 aagtaagacc accgcacagc aagcggccgc tgatctcag acctggagga ggagatatga 6180 gggacaattg gagaagtgaa tatataat aataagtagt aaaaattgaa ccattaggag 6240 tagcaccac caggcaag agaagtgg tgcagagaga aaaaagagca gtgggaatag 6300 gagctttgtt ccttgggttc tgggagcag caggaagcac tatggcgca gcgtcaatga 6360 cgctgacggt acaggccaga cattattgt ctggtatagt gcagcagcag aacaatttgc 6420 tgaggctat tgaggcgca cagcatgt tgcactcac agtctggggc atcagcagc 6480 tccaggcaag aatcctggct gtggaagat acctaagga tcacagctc ctgggattt 6540 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 6600 aaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaatta 6660 acaattacac aagcttaata cactccttaa ttgagaatc gcaaaccag cahaaaga 6720 atgacaaga attattgga ttagataat gggcaagtttt gtggaatttgg tttacataa 6780 caattggct gtgtatata aattattca taatgagt aggaggcttg gtaggtttta 6840 gatagtttt tgctgtactt tctatagtga atagagttag gcagggat tcaccattat 6900 cgtttcagac ccaccccca accccgaggg gacccgacag gcccgaagga atagagaag 6960 aaggtggaga gagagacaga gatacca ttcgattagt gaacggatct cgacggtatc 7020 gatcacgaga ctagcctcga gcggccgcccc ccttcaccga gggcctattt cccatgattc 7080 cttcatattt gcatatacga tacaggctg ttagagagat attggaattt aatttgactg 7140 taacacaaa gattagta aaatacgt gacgtagaa gtaataattt cttgggtagt 7200 ttgcagtttt aaaattatgt ttaaatgg actacatat gcttaccgta acttgaaagt 7260 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggt gtcttcgagg 7320 cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat 7380 atatactgat atgtataccc gaagtatgtc aaaaagaggt gtgctatgaa gcagcgtatt 7440 acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct 7500 ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga 7560 aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt 7620 ttgctgacga gaacagggac tggtgaaatg cagtttaagg tttacaccta taaaagagag 7680 agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg 7740 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac 7800 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg 7860 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa 7920 aacgccatta acctgatgtt ctggggaata taaaccctgc aggacgaaga cccgttttag 7980 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg 8040 agtcggtgct ttttttgaat tctcgacctc gagacaaatg gcagtattca tccacaattt 8100 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 8160 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt 8220 ttattacagg gacagcagag atccactttg gccgcggctc gaggggg 8267 <210> 106 <211> 705 <212> DNA <213> Artificial Sequence <400> 106 gcttctaact ttactcagtt cgttctcgtc gacaatggcg gaactggcga cgtgactgtc 60 gccccaagca acttcgctaa cgggatcgct gaatggatca gctctaactc gcgttcacag 120 gcttacaaag taacctgtag cgttcgtcag agctctgcgc agaatcgcaa atacaccatc 180 aaagtcgagg tgcctaaagg cgcctggcgt tcgtacttaa atatggaact aaccattcca 240 attttcgcca cgaattccga ctgcgagctt attgttaagg caatgcaagg tctcctaaaa 300 gatggaaacc cgattccctc agcaatcgca gcaaactccg gcatctacgc catggccagc 360 aacttcaccc agttcgtgct ggtggacaac ggcggcaccg gcgacgtgac cgtggccccc 420 agcaacttcg ccaacggcat cgccgagtgg atcagcagca acagcagaag ccaggcctac 480 aaggtgacct gcagcgtgag acagagcagc gcccagaaca gaaagtacac catcaaggtg 540 gaggtgccca agggcgcctg gagaagctac ctgaacatgg agctgaccat ccccatcttc 600 gccaccaaca gcgactgcga gctgatcgtg aaggccatgc agggcctgct gaaggacggc 660 aaccccatcc ccagcgccat cgccgccaac agcggcatct acgcc 705 <210> 107 <211> 264 <212> DNA <213> Artificial Sequence(人工序列) <400> 107 ggtagtatga aatcaattcg ctgtaaaaac tgcaacaaac tgttatttaa ggcggatagt 60 tttgatcaca ttgaaatcag gtgtccgcgt tgcaaacgtc acatcataat gctgaatgcc 120 tgcgagcatc ccacggagaa acattgtggg aaaagagaaa aaatcacgca ttctgacgaa 180 accgtgcgtt atggctccac tagtgggcac aagcttaatg gcggtggcgg agggatggat 240 gctaagtcac taactgcctg gtcc 264 <210> 108 <211> 561 <212> DNA <213> Artificial Sequence <400> 108 atggaccact acctcgacat tcgcttgcga ccggacccgg aatttccccc ggcgcaactc atgagcgtgc tcttcggcaa gctcgcccag gccctggtgg cacagggcgg ggacaggatc 120 ggcgtgagct tccccgacct cgacgaaagc cgctcccggc tggcgagcg cctgcgcatt 180 catgcctcgg cggacgacct tcgtgccctg ctcgcccggc cctggctgga agggttgcgg 240 gaccatctgc aattcggaga accggcagtc gtgcctcacc ccacaccgta ccgtcaggtc 300 agtcgggttc aggcgaaag caatccgga cgcctgcggc ggcggctcat gcgccggcac 360 gatctgagtg aggaggagc tcggaaacgc attcccgata cggtcgcgag agccttggac 420 ctgcccttcg tcacgctacg cagccagagc accggacagc acttccgtct cttcatccgc 480 cacgggccgt tgcaggtgac ggcagagga ggaggattca cctgttacgg gttgagcaaa ggaggtttcg ttccctggtt c 561 <210> 109 <211> 741 <212> DNA <213> Artificial Sequence <400> 109 60. tccaaaacta tcgttctgag cgtaggtga gcgaccaga cgttaactga aattcaatct acagcagaca ggcaaatatt tgaagagaaa gtcggtccat tagtagggag gttaagattg acagctagct tagcaaaa tggagctaaa actgcataca gggtcaattt aaaattggat caagcagatg tcgttgatag cggtctacct aaggttagat acacgcaagt gtggtcacac gatgtcacta ttgtcgcga ttctaccga gctagtaga aatcattgta tgatttaaca aaatccttgg tggctacctc tcaggtgga gatcttgttg tgatttagt tccattaggt 360 agggcggacc cgctagcctc caaaaccatc gttctttcgg tcggcgaggc tactcgcact 420 480. ctgactgaga tccagtccac cgcagaccgt cagatcttcg aagagaaggt cggggcctctg gtgggtcggc tgcgcctcac ggcttcgctc cgtcaaaacg gagccaagac cgcgtatcgc 540 gtcaacctaa aactggatca ggcggacgtc gttgattccg gacttccgaa agtgcgctac actcaggtat ggtcgcacga cgtgacaatc gttgcgaata gcaccgaggc ctcgcgcaaa 660 tcgttgtacg atttgaccaa gtccctcgtc gcgacctcgc aggtcgaaga tcttgtcgtc 720 aaccttgtgc cgctgggccg t 741

Claims

1. A gene-guided editing system comprising a vector, said vector containing a gene encoding pegRNA and a gene encoding a fusion protein, characterized in that, The pegRNA comprises sgRNA, reverse transcription template RTT, major binding site PBS, and RNA stem-loop structure; the fusion protein is a fusion protein of Cas9 nickase, reverse transcriptase, and RBP; The RBP is a protein that specifically binds to the stem-loop structure of RNA; The pegRNA is an RNA molecule, consisting of sgRNA, reverse transcription template RTT, major binding site PBS, and RNA stem-loop structure from the 5' end to the 3' end. The fusion protein comprises RBP, Cas9 nickase, and reverse transcriptase from the N-terminus to the C-terminus.

2. The system as described in claim 1, characterized in that, The RNA stem-loop structure is selected from MS2, PP7, ComBS or Csy4BS, as shown in SEQ ID NO. 1 to 4, respectively.

3. The system according to claim 1, characterized in that, Choose from at least one of the following: 1) The carrier may be one or more; 2) The gene encoding pegRNA or the gene encoding fusion protein are located on the same vector or different vectors.

4. The system according to claim 3, characterized in that, Choose from any of the following: 1) The RBP is selected from any one of tdMCP, tdPCP, COM, and Csy4H29A; 2) The Cas9 cleavage enzyme is H840A; 3) The reverse transcriptase is MMLV.

5. A nucleic acid, characterized in that, The nucleic acid encodes the pegRNA and the fusion protein as described in any one of claims 1 to 4.

6. A construct comprising the nucleic acid as described in claim 5.

7. A host cell, characterized in that, The host cell comprises the construct of claim 6 or the genome in which exogenous nucleic acids of claim 5 are integrated.

8. The following applications of the system according to any one of claims 1-4, the nucleic acid according to claim 5, the construct according to claim 6, or the host cell according to claim 7: 1) Editing the genome sequence of organisms or biological cells for non-therapeutic purposes; 2) Products used for editing the genome sequence of organisms or biological cells; 3) Used for non-therapeutic purposes to improve the efficiency of genome sequence editing in organisms or biological cells; 4) Products used to improve the efficiency of editing genome sequences of organisms or biological cells.

9. The application according to claim 8, characterized in that, Used for base substitution.

10. The application according to claim 8, characterized in that, Used for DNA editing.

11. The application according to claim 10, characterized in that, Editing of SRD5A3, DYRK1A, HDAC1, BCL11A, GFAP, RUNX1, JAK2, SRD5A1, DMD, and EED sites.

12. The application according to claim 10, characterized in that, Editing for SRD5A3_+2C·G to A·T, DYRK1A_+1C·G to G·C, HDAC1_+1C·G to G·C, BCL11A_+1C·G to A·T, GFAP_+1A·T to T·A, RUNX1_+5G·C to T·A, JAK2_+1C·G to T·A, SRD5A1_+1C·G to A·T, DMD_+1T·A to C·G, and EED_+1A·T to T·A.

13. A method for non-therapeutic gene editing, comprising introducing the system of any one of claims 1-4, the nucleic acid of claim 5, the construct of claim 6, or the host cell of claim 7 into the organism to be edited to achieve gene editing.

Images