Selective polyadenylation site editing tool and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a selective polyadenylation site editing tool and application thereof. The PABPN1 is modified according to the structural domain characteristics of the PABPN1: an effect structural domain CCD structural domain and a phase separation key structural domain EP structural domain are reserved, and an RRM structural domain and other structural domains are removed, so that a minimum unit structural domain with a high-specificity and high-efficiency APA regulation and control function is obtained. According to the present invention, the truncated body protein PABPN1-EP / CCD loses the original RNA binding ability and cannot singly provide the APA regulation function, the PABPN1-EP / CCD is combined with the dPspCas13b protein, the dPspCas13b protein is combined with the crRNA, and the EP / CCD is guided to the target precursor RNA so as to specifically inhibit the target poly (A) site of the target gene;

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a selective polyadenylation site editing tool and its application. Background Art

[0002] The CRISPR / Cas system is an innate immune mechanism derived from bacteria and archaea that recognizes and cleaves foreign genetic material (such as phage DNA), thereby protecting the host from invasion. CRISPR / Cas systems are primarily divided into two categories: Class 1 (Types I, III, and IV) rely on multiprotein complexes to function, while Class 2 (Types II, V, and VI) require only a single Cas protein to complete gene editing. Type II systems have become a core tool in the field of gene editing due to the high efficiency and ease of use of the Cas9 protein. By artificially designing and in vitro transcribing RNA, sgRNA can be synthesized, which guides the Cas protein to specifically cleave the target DNA sequence. By modifying Cas proteins, the CRISPR / Cas system can perform a variety of operations, including cleavage, modification, silencing, knockout, and expression regulation. It has become a vital tool for gene editing and research, with broad application prospects. The CRISPR / Cas9 system edits genes by specifically recognizing target DNA, primarily for gene expression regulation. Studies have shown that the Cas13 protein can target mRNA for degradation.

[0003] During gene transcription in eukaryotic cells, precursor mRNA undergoes a series of processing steps, such as capping, cleavage, and tailing, ultimately becoming mature mRNA. Alternative polyadenylation (APA) often occurs at the 3' end of the precursor mRNA, resulting in mRNAs with varying 3' UTR lengths and even altering protein-coding sequences. Studies have found that APA is closely linked to tumorigenesis and progression, embryonic development, and immune responses. Therefore, a tool that can specifically intervene in APA is needed for basic APA research and targeted drug development.

[0004] However, currently, tools for specifically intervening against APA are limited and have drawbacks. Using CRISPR / Cas9 technology, sgRNAs are designed near the poly(A) site to induce site-directed mutagenesis in the target DNA, thereby altering the APA events in the target mRNA transcript (Wang et al., 2018)(Austin et al., 2024). However, this approach has significant drawbacks. CRISPR directly edits genomic DNA, resulting in low efficiency and potential harmful effects. Naveed et al. (Naveed et al., 2021) used antisense oligonucleotides (ASOs) that bind to sequences surrounding the poly(A) site. These ASOs physically block recognition of nascent RNA by 3' end processing factors, thereby preventing tailing at the cleaved PAS. Although ASOs effectively interfere with cleavage and polyadenylation and have even entered clinical trials (Havens and Hastings, 2016; MacLeod and Crooke, 2017), their cost is prohibitive for large-scale studies. Tian et al. (Tian et al., 2022) developed a CRISPR-iPAS approach to regulate poly(A) site utilization. They recruited a 3xEGFP-fused dPguCas13b protein to core regulatory elements near the poly(A) site, using steric hindrance to prevent CPA factor recruitment and effectively inhibit poly(A) site utilization. However, this approach requires precise gRNA targeting of poly(A) site elements. The high sequence similarity of poly(A) site elements increases the difficulty of gRNA design and affects gRNA specificity. Shin et al. (Shin et al., 2022) developed a CRISPR-pas approach to regulate APA by recruiting dCas9 to sequences downstream of the PAS. Steric hindrance by dCas9 leads to disengagement of the Pol II transcription complex, enhancing proximal PAS utilization. However, this approach also requires a high distance between the target sequence and the PAS site, which limits gRNA design.

[0005] Kelly et al. designed a fusion protein of dCas13b and the 3' processing factor NUDT21 to regulate APA (Kelly et al., 2019). However, NUDT21 itself binds to RNA, reducing its specificity. Therefore, designing an APA regulatory tool that achieves high specificity and target sequence flexibility has become a challenge in this field. Summary of the Invention

[0006] In order to solve the above-mentioned problems existing in the art, the present invention has modified PABPN1 based on its domain characteristics: retaining the effector domain CCD domain and the phase separation key domain EP domain, while removing the RRM domain and other domains to obtain the minimum unit domain with high specificity and high efficiency APA regulatory function.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a PABPN1 truncated protein consisting of the EP domain and the CCD domain of PABPN1.

[0009] Nuclear poly(A)-binding protein 1 (PABPN1) is a 3'-end processing factor that plays an important role in polyadenylation by inhibiting the use of proximal poly(A) sites. The PABPN1 protein consists of five main domains: an N-terminal domain consisting of 10 alanine repeats (Ala10); a coiled-coil region that is important for its interaction with poly(A) polymerase (CCD domain) (Kerwitz et al., 2003); an RNA-binding domain that binds to poly(A) RNA (RRM domain) (Kühn et al., 2003); and an arginine-rich C-terminal domain that contains a nuclear localization signal and facilitates oligomerization. Furthermore, the applicant team first identified a Pro / Glu-rich domain (EP) that is important for phase separation and normal function of PABPN1 (Hu et al., 2024).

[0010] In some embodiments of the present invention, the amino acid sequence of the EP domain is shown in SEQ ID NO: 1.

[0011] In some embodiments of the present invention, the amino acid sequence of the CCD domain is shown in SEQ ID NO: 2.

[0012] In some embodiments of the present invention, the EP domain and the CCD domain may be connected via a connecting peptide, or directly connected.

[0013] In some embodiments of the present invention, the EP domain is located at the N-terminus; the CCD domain is located at the C-terminus; further, the amino acid sequence of the PABPN1 truncated protein is shown in SEQ ID NO: 3.

[0014] The second aspect of the present invention provides a fusion protein consisting of the PABPN1 truncated protein of the first aspect of the present invention and the dPspCas13b protein.

[0015] In some embodiments of the present invention, the amino acid sequence of the dPspCas13b protein is shown in SEQ ID NO: 4.

[0016] In some embodiments of the present invention, the connection order of the dPspCas13b protein and the PABPN1 truncated protein is not limited, and the dPspCas13b protein can be located at the N-terminus or C-terminus of the PABPN1 truncated protein.

[0017] In some embodiments of the present invention, the dPspCas13b protein and the PABPN1 truncated protein can be connected by a connecting peptide, an NLS signal sequence can be inserted, or they can be directly connected.

[0018] The third aspect of the present invention provides a polyadenylation site editing tool, comprising a PABPN1 truncated protein, a dPspCas13b protein, a nuclear import signal (peptide), a screening protein, and a tag protein.

[0019] In some embodiments of the present invention, the structure of the polyadenylation site editing tool is:

[0020] Nuclear import signal-A-Nuclear import signal-B-Tag protein-Screening protein; or

[0021] Nuclear import signal-BA-nuclear import signal-tag protein-screening protein.

[0022] A and B are dPspCas13b protein and PABPN1 truncated protein, respectively.

[0023] In some embodiments of the present invention, the nuclear localization signal (NLS) is selected from at least one of nucleoplasmin NLS and SV40 NLS.

[0024] In some embodiments of the present invention, the screening protein is selected from at least one of BSD protein, PAC protein, Hygro protein, and Neo protein.

[0025] In some embodiments of the present invention, a self-cleaving polypeptide 2A may be present between the screening protein and the tag protein.

[0026] In some embodiments of the present invention, the self-cleaving polypeptide 2A is P2A.

[0027] In some embodiments of the present invention, the tag protein includes but is not limited to HA, Flag, GFP, GST, His and other conventional tag proteins in the art.

[0028] In some embodiments of the present invention, the components of the polyadenylation site editing tool can be connected by a connecting peptide. The connecting peptide is conventionally selected in the art, including but not limited to GGGGS n 、GSS n .

[0029] In some embodiments of the present invention, the sequence of the connecting peptide is GGGGS (SEQ ID NO: 6).

[0030] In some embodiments of the present invention, a polyadenylation site editing tool dPspCas13b-PABPN1-EP / CCD is provided, the amino acid sequence of which is shown in SEQ ID NO: 11.

[0031] In some embodiments of the present invention, a polyadenylation site editing tool PABPN1-EP / CCD-dPspCas13b is provided, the amino acid sequence of which is shown in SEQ ID NO: 12.

[0032] In some embodiments of the present invention, the polyadenylation site editing tool actually performs editing on combinations of PABPN1 truncated proteins and dPspCas13b proteins in different sequences.

[0033] The purpose of the NLS nuclear import signal (peptide) is to help the editing tool enter the cell nucleus. In non-nuclear scenarios, the nuclear import signal can also be omitted.

[0034] The purpose of the tag protein, screening protein, and polypeptide between the screening protein and the tag protein is to facilitate the screening, expression, and purification of the editing tool and is unrelated to polyadenylation site editing. The HA tag, P2A cleavage peptide, and BSD screening protein used in the examples of the present invention are merely one option for the polyadenylation site editing screening, expression, and purification process of the present invention. Routine modifications to these components by those skilled in the art fall within the same technical concept as the present invention and should be included in the scope of protection of the present invention.

[0035] In the above aspects of the present invention, simple replacements, additions, and deletions to the above amino acid sequences with a homology of more than 95% all belong to the same technical concept as the present invention. Other modifications obtained by technicians in this field without creative work all fall within the scope of protection of the present invention.

[0036] In some embodiments of the present invention, the polyadenylation site editing tool may further include a multiple cloning site sequence on the vector, which provides an enzyme cutting site to facilitate the insertion of the target gene.

[0037] The fourth aspect of the present invention provides a nucleic acid molecule, which can encode the PABPN1 truncated protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, and / or the polyadenylation site editing tool described in the third aspect of the present invention.

[0038] The fifth aspect of the present invention provides a vector comprising the nucleic acid molecule described in the fourth aspect of the present invention.

[0039] The sixth aspect of the present invention provides the use of the PABPN1 truncated protein described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polyadenylation site editing tool described in the third aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, and / or the vector described in the fifth aspect of the present invention in selective polyadenylation regulation.

[0040] In some embodiments of the present invention, the selective polyadenylation regulation is the inhibition of the poly(A) site of the target gene.

[0041] In some embodiments of the present invention, the applications include the development of molecular biological reagents, the development of disease target drugs, and the development of agricultural resources.

[0042] The seventh aspect of the present invention provides a method for inhibiting the poly(A) site of the target gene precursor mRNA, using the polyadenylation site editing tool described in the third aspect of the present invention or the vector of the fifth aspect of the present invention to treat the sample.

[0043] In some embodiments of the invention, the method comprises the steps of:

[0044] (1) Using crRNA targeting the target pre-mRNA to recognize the target pre-mRNA;

[0045] (2) using polyadenylation site editing tools to inhibit the target poly(A) site of the target pre-mRNA to achieve APA editing;

[0046] (3) verify the APA events and 3'UTR length of the mature mRNA transcribed after treatment;

[0047] (4) Use polyadenylation site editing tools and corresponding crRNA to edit the APA of the target gene precursor mRNA, thereby regulating the target gene mRNA.

[0048] In some embodiments of the present invention, the 5' end of the crRNA is a specific sequence that targets the target precursor mRNA through complementary pairing with the template; and the 3' end of the crRNA contains a repetitive sequence.

[0049] In some embodiments of the present invention, the 5' end specific sequence of the crRNA can be complementary to the upstream sequence of the poly (A) site of the target mRNA, and the crRNA can bind to the region 0-200bp upstream of the poly (A) site; the 3' end of the crRNA can form an mRNA secondary structure similar to a hairpin structure, and can be recognized and bound by dPspCas13b, the polyadenylation site editing tool of the third aspect of the present invention, other active Cas13b, or other Cas13 protease variants.

[0050] In some embodiments of the present invention, in step (1) of the method, the 5' end of the crRNA is a specific sequence that can complementarily pair with the template; the 3' end of the crRNA is a dPspCas13b / Cas13b recognition sequence.

[0051] In some embodiments of the present invention, in step (2) of the method, the polyadenylation site editing tool comprises a fusion combination of multiple Cas13b protease variants and PABPN1-EP / CCD protein variants.

[0052] In some embodiments of the present invention, in step (2) of the method, the polyadenylation site editing tool and a single or multiple crRNAs are added to the reaction system together, the polyadenylation site editing tool binds to the crRNA, the crRNA recognizes the target mRNA, and the polyadenylation site editing tool inhibits the target poly (A) site of the target precursor mRNA to achieve APA editing.

[0053] In some embodiments of the present invention, in step (3) of the method, the 3'UTR verification includes verification of the efficiency of the polyadenylation site editing tool / crRNA system and verification of various biological functions of the processed mRNA.

[0054] In some embodiments of the present invention, in step (4) of the method, the polyadenylation site editing tool / crRNA system is used to perform targeted regulation of specific mRNA, including expression regulation, stability regulation, translation efficiency regulation, regulation of nuclear entry and exit ability, etc.

[0055] In some embodiments of the present invention, the method is performed in living cells, living animals, or in vitro; when the method is used in living animals, the method is for purposes other than disease diagnosis and treatment.

[0056] The eighth aspect of the present invention provides a polyadenylation site editing system, comprising the polyadenylation site editing tool of the third aspect of the present invention and crRNA designed for the target gene.

[0057] In some embodiments of the present invention, the present invention takes the three genes WAPL, SMAD4, and COL1A2 as examples and designs different crRNAs for verification.

[0058] Those skilled in the art can design different crRNAs for other target genes and perform APA editing of other target genes.

[0059] The design of the above-mentioned crRNA is a conventional technical means in the field, and the specific crRNA used in the examples of the present invention does not constitute a limitation of the present invention.

[0060] The beneficial effects of the present invention are:

[0061] The present invention modifies PABPN1 based on its domain characteristics: retaining the effector domain CCD domain and the phase separation key domain EP domain, while removing the RRM domain and other domains to obtain a minimum unit domain with high specificity and high efficiency APA regulation function. This truncated protein PABPN1-EP / CCD loses its original ability to bind RNA and cannot perform APA regulation function alone. By combining PABPN1-EP / CCD with the dPspCas13b protein, the dPspCas13b protein binds to the crRNA and then guides the EP / CCD to the target precursor RNA, thereby achieving specific inhibition of the target poly(A) site of the target gene.

[0062] Quantitative PCR results showed that the dPspCas13b-PABPN1-EP / CCD fusion protein can effectively inhibit the use of the proximal target poly(A) site of the target gene in the cell, transcribing mRNA with a longer 3'UTR. The present invention integrates dPspCas13b and the modified PABPN1-EP / CCD to achieve efficient and specific inhibition of the gene target poly(A) site, providing a selective polyadenylation site editing tool with high specificity and flexibility in crRNA design for regulating the expression and function of the target gene. This method can be used to study the biological functions of selective polyadenylation and provide new tools for the treatment of tumors and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0064] Figure 1 It is a schematic diagram of the principle of the present invention.

[0065] Figure 2 Schematic diagram of dPspCas13b-PABPN1-EP / CCD fusion protein and its variants.

[0066] Figure 3 Schematic diagram of the dual-luciferase reporter system.

[0067] Figure 4 The dPspCas13b-PABPN1-EP / CCD fusion protein and its variant PABPN1-EP / CCD-dPspCas13b fusion protein inhibit the use of the target poly(A) site of the target pre-mRNA to achieve APA editing.

[0068] Figure 5 Schematic diagram of crRNA targeting the endogenous WAPL site.

[0069] Figure 6 Quantitative qPCR showed that the dPspCas13b-PABPN1-EP / CCD fusion protein bound to the crRNA targeting WAPL inhibited the efficiency of target poly(A) site usage in its pre-mRNA.

[0070] Figure 7 Schematic diagram of crRNA targeting the SMAD4 site.

[0071] Figure 8 Schematic diagram of crRNA targeting the COL1A2 site.

[0072] Figure 9 Quantitative qPCR showed that the dPspCas13b-PABPN1-EP / CCD fusion protein bound to crRNA targeting SMAD4 and COL1A2 and inhibited the target poly(A) site usage efficiency of their pre-mRNA. DETAILED DESCRIPTION

[0073] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0074] Example 1 Construction of fusion protein dPspCas13b-PABPN1-EP / CCD

[0075] This example analyzes the domain characteristics of PABPN1 and modifies it based on its domain characteristics: retains the effector domain CCD domain and the phase separation key domain EP domain, and removes the RRM domain and other domains to obtain the minimum unit domain with high specificity and high efficiency APA regulatory function.

[0076] The truncated protein includes the CCD structure and EP domain of PABPN1, and from the N-terminus to the C-terminus are the EP structure and the CCD domain respectively.

[0077] in:

[0078] The amino acid sequence of the PABPN1-EP fragment is:

[0079] ESEELEPEELLLEPEPEPEPEEEEPPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGD GAIEDPE(SEQ ID NO: 1);

[0080] The amino acid sequence of the PABPN1-CCD fragment is:

[0081] LEAIKARVREMEEEAEKLKELQNEVEKQ (SEQ ID NO: 2).

[0082] The amino acid sequence of the truncated protein PABPN1-EP / CCD is:

[0083] ESEELEPEELLLEPEPEPEPEEEEPPRAPPGAPGPGPGSGAPGSQEEEEEPGLVEGDPGD GAIEDPELEAIKARVREMEEEAEKLKELQNEVEKQ (SEQ ID NO: 3).

[0084] At this point, the truncated protein PABPN1-EP / CCD loses its original ability to bind RNA and cannot play the APA regulatory function alone. Furthermore, in order to achieve APA editing, this embodiment fuses the truncated protein with dPspCas13b, and the dPspCas13b protein binds to crRNA and then guides EP / CCD to the target precursor RNA, thereby achieving specific inhibition of the target poly (A) site of the target gene.

[0085] The amino acid sequence of the dPspCas13b protein is as follows:

[0086] NIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNNENNLWFHPVMSHL

[0087] YNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKR

[0088] AFGVLKMYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTE

[0089] DLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQ

[0090] YINIFLSRLPIFSSYNAQSEERIIIRSFGINSIKLPKDRIHSEKSNKSVADMMLNEVKRCPDEL

[0091] FTTLSAEKQSRFRIISDDHNEVLMKRSSDRVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLK

[0092] ADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPAN

[0093] YPYIVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHM

[0094] FLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKD

[0095] VDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGGKRGFKQISTGKLADFLAKDIVLFQ

[0096] PSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKV

[0097] FARSIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSE

[0098] DLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQWNRN

[0099] YRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNAS

[0100] SEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPD

[0101] AEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKE

[0102] DIMEEFNKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQ

[0103] SDILRKIRNAFDANNYPDKGVVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 4).

[0104] In this example, two fusion proteins were designed, including dPspCas13b-PABPN1-EP / CCD and PABPN1-EP / CCD-dPspCas13b.

[0105] dPspCas13b-PABPN1-EP / CCD, from N-terminus to C-terminus, contains: nucleoplasmin NLS (nuclear import signal) peptide fragment, dPspCas13b fragment, SV40 NLS (nuclear import signal) peptide fragment, linker peptide fragment, PABPN1-EP / CCD fragment, linker peptide fragment, 3×HA fragment, P2A fragment, and BSD fragment.

[0106] PABPN1-EP / CCD-dPspCas13b, from N-terminus to C-terminus, contains: nucleoplasmin NLS (nuclear import signal) peptide fragment, PABPN1-EP / CCD fragment, linker fragment, dPspCas13b fragment, SV40 NLS (nuclear import signal) peptide fragment, linker peptide fragment, 3×HA fragment, P2A fragment, and BSD fragment.

[0107] Among them, the amino acid sequence of the nucleoplasmin NLS (nuclear import signal) peptide fragment is:

[0108] KRPAATKKAGQAKKKK (SEQ ID NO: 5);

[0109] The amino acid sequence of the linker fragment is GGGGS (SEQ ID NO: 6);

[0110] The amino acid sequence of the SV40 NLS (nuclear import signal) peptide fragment is: PKKKRKV (SEQ ID NO: 7);

[0111] The amino acid sequence of the 3×HA fragment is: YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 8); the amino acid sequence of the P2A fragment is: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 9)

[0112] The amino acid sequence of the BSD fragment is:

[0113] MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVNVYHFTGGPCAELVV LGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKAIVKDSDGQPTAVGIRELL PSGYVWEG (SEQ ID NO: 10).

[0114] Specifically, the amino acid sequence of dPspCas13b-PABPN1-EP / CCD is:

[0115]

[0116] The amino acid sequence of PABPN1-EP / CCD-dPspCas13b is:

[0117]

[0118] The structural diagrams of the two fusion proteins are shown in Figure 2. Figure 2 shown.

[0119] Example 2dPspCas13b-PABPN1-EP / CCD fusion protein or its variant PABPN1-EP / CCD-dPspCas13b can inhibit the use of the WAPL proximal poly (A) site

[0120] 1 Experimental Materials and Methods

[0121] 1.1 Main reagents and instruments

[0122] HEK293T cells were maintained in our laboratory or obtained from other routine laboratory laboratories and cell banks. Fetal bovine serum was purchased from Gbico, jetPRIME DNA / siRNA transfection reagent from Polyplus, Dual Luciferase Reporter Assay Kit from Vazyme, and luminescence detector from Glomax.

[0123] 1.2 Dual luciferase reporter system

[0124] The proximal poly (A) site signal of the WAPL gene and 100bp upstream and downstream were cloned into the psiCHECK-2 plasmid. An element that can be accurately recognized and targeted by crRNA was also designed at the 5' end of this element. The dPspCas13b-PABPN1-EP / CCD fusion protein or its variant can recognize and target this site through crRNA. The reporter system contains a Renilla luciferase reporter gene (hRluc) and a Firefly luciferase reporter gene (hluc) at both ends, which are mediated by IRES to enable independent translation. When the proximal poly (A) site of WAPL is used, the transcribed mRNA will be cut after the poly (A) site, and the Firefly luciferase reporter gene at the 3' end will not be translated, and hRluc / hluc will become larger, otherwise it will become smaller. Therefore, the value of hRluc / hluc can reflect the usage of the poly (A) site. The structural diagram of the dual luciferase reporter system is shown in the figure. Figure 3 shown.

[0125] The sequence of the dual-luciferase reporter system is as follows:

[0126] ATGGCTTCCAAGGTGTACGACCCCGAGCAACGCAAACGCATGATCACTGGGCCTCA

[0127] GTGGTGGGCTCGCTGCAAGCAAATGAACGTGCTGGACTCCTTCATCAACTACTATGATTC

[0128] CGAGAAGCACGCCGAGAACGCCGTGATTTTTCTGCATGGTAACGCTGCCTCCAGCTACC

[0129] TGTGGAGGCACGTCGTGCCTCACATCGAGCCCGTGGCTAGATGCATCATCCCTGATCTGA

[0130] TCGGAATGGGTAAGTCCGGCAAGAGCGGGAATGGCTCATATCGCCTCCTGGATCACTAC

[0131] AAGTACCTCACCGCTTGGTTCGAGCTGCTGAACCTTCCAAAGAAAATCATCTTTGTGGG

[0132] CCACGACTGGGGGGCTTGTCTGGCCTTTCACTACTCCTACGAGCACCAAGACAAGATCA

[0133] AGGCCATCGTCCATGCTGAGAGTGTCGTGGACGTGATCGAGTCCTGGGACGAGTGGCCT

[0134] GACATCGAGGAGGATATCGCCCTGATCAAGAGCGAAGAGGGCGAGAAAATGGTGCTTG

[0135] AGAATAACTTCTTCGTCGAGACCATGCTCCCAAGCAAGATCATGCGGAAACTGGAGCCT

[0136] GAGGAGTTCGCTGCCTACCTGGAGCCATTCAAGGAGAAGGGCGAGGTTAGACGGCCTA

[0137] CCCTCTCCTGGCCTCGCGAGATCCCTCTCGTTAAGGGAGGCAAGCCCGACGTCGTCCAG

[0138] ATTGTCCGCAACTACAACGCCTACCTTCGGGCCAGCGACGATCTGCCTAAGATGTTCATC

[0139] GAGTCCGACCCTGGGTTCTTTTCCAACGCTATTGTCGAGGGAGCTAAGAAGTTCCCTAA

[0140] CACCGAGTTCGTGAAGGTGAAGGGCCTCCACTTCAGCCAGGAGGACGCTCCAGATGAA

[0141] ATGGGTAAGTACATCAAGAGCTTCGTGGAGCGCGTGCTGAAGAACGAGCAGTAATTCTA

[0142] GGCGATCGCTCGAGctgctttcaaagaattaccatactgaggtagcgaatgttctgaggacattctagacaacagcttagttcctttttc

[0143] aggctcatttgcttttgcttttttgttgaatgattccaatcgtaaataaagcttttaataattttgtgaattttttggttgttgttccctgaactactgtctatattt

[0144] aaaattagatggaatccaaagatacacgggattaatagtataGAATTCGCCCCTCTCCCTCCCCCCCCCCTAACGT

[0145] TACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCAC

[0146] CATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAG

[0147] CATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGA

[0148] AGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGC

[0149] AGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTAT

[0150] AAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTG

[0151] GAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGA

[0152] AGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTA

[0153] GTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGA

[0154] AAAACACGATGATAATATGGCCACAACCATGGCCGATGCTAAGAACATTAAGAAGGGCC

[0155] CTGCTCCCTTCTACCCTCTGGAGGATGGCACCGCTGGCGAGCAGCTGCACAAGGCCATG

[0156] AAGAGGTATGCCCTGGTGCCTGGCACCATTGCCTTCACCGATGCCCACATTGAGGTGGA

[0157] CATCACCTATGCCGAGTACTTCGAGATGTCTGTGCGCCTGGCCGAGGCCATGAAGAGGT

[0158] ACGGCCTGAACACCAACCACCGCATCGTGGTGTGCTCTGAGAACTCTCTGCAGTTCTTC

[0159] ATGCCAGTGCTGGGCGCCCTGTTCATCGGAGTGGCCGTGGCCCCTGCTAACGACATTTA

[0160] CAACGAGCGCGAGCTGCTGAACAGCATGGGCATTTCTCAGCCTACCGTGGTGTTCGTGT

[0161] CTAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCTATCATCCAGAA

[0162] GATCATCATCATGGACTCTAAGACCGACTACCAGGGCTTCCAGAGCATGTACACATTCGT

[0163] GACATCTCATCTGCCTCCTGGCTTCAACGAGTACGACTTCGTGCCAGAGTCTTTCGACA

[0164] GGGACAAAACCATTGCCCTGATCATGAACAGCTCTGGGTCTACCGGCCTGCCTAAGGGC

[0165] GTGGCCCTGCCTCATCGCACCGCCTGTGTGCGCTTCTCTCACGCCCGCGACCCTATTTTC

[0166] GGCAACCAGATCATCCCCGACACCGCTATTCTGAGCGTGGTGCCATTCCACCACGGCTT

[0167] CGGCATGTTCACCACCCTGGGCTACCTGATTTGCGGCTTTCGGGTGGTGCTGATGTACCG

[0168] CTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAAATTCAGTCTGCCCTGC

[0169] TGGTGCCAACCCTGTTCAGCTTCTTCGCTAAGAGCACCCTGATCGACAAGTACGACCTG

[0170] TCTAACCTGCACGAGATTGCCTCTGGCGGCGCCCCACTGTCTAAGGAGGTGGGCGAAGC

[0171] CGTGGCCAAGCGCTTTCATCTGCCAGGCATCCGCCAGGGCTACGGCCTGACCGAGACAA

[0172] CCAGCGCCATTCTGATTACCCCAGAGGGCGACGACAAGCCTGGCGCCGTGGGCAAGGT

[0173] GGTGCCATTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGAGTG

[0174] AACCAGCGCGGCGAGCTGTGTGTGCGCGGCCCTATGATTATGTCCGGCTACGTGAATAA

[0175] CCCTGAGGCCACAAACGCCCTGATCGACAAGGACGGCTGGCTGCACTCTGGCGACATT

[0176] GCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGCCTGAAGTCTCTGATCAA

[0177] GTACAAGGGCTACCAGGTGGCCCCAGCCGAGCTGGAGTCTATCCTGCTGCAGCACCCTA

[0178] ACATTTTCGACGCCGGAGTGGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCTGC

[0179] CGCCGTCGTCGTGCTGGAACACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTAT

[0180] GTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGCGGAGTGGTGTTCGTGGAC

[0181] GAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGCAAGATCCGCGAGATCCTGA

[0182] TCAAGGCTAAGAAAGGCGGCAAGATCGCCGTGTAA(SEQ ID NO:13)。

[0183] 1.3 Cell resuscitation and culture

[0184] Remove HEK293T cells from a liquid nitrogen tank and rapidly thaw in a 37°C water bath. Transfer the cell suspension to a 1.5 mL centrifuge tube in a clean bench and centrifuge at 290 g for 5 minutes. Discard the supernatant and add 5 mL of DMEM medium containing 10% FBS. Incubate in a 5% CO2, 37°C atmosphere.

[0185] 1.4 Cell treatment and transfection experiments

[0186] Adherent HEK293T cells were digested and resuspended, plated into 48-well plates, and 5x104 cells were added to each well. The culture plates were pre-cultured in the incubator for 24 hours. When the cell density reached 50-60% confluence, transfection was performed using jetPRIME DNA / siRNA transfection reagent. The main steps were as follows: 500ng of dPspCas13b-PABPN1-EP / CCD / PABPN1-EP / CCD-dPspCas13b overexpression plasmid, 500ng of crRNA overexpression plasmid, and 300ng of dual-luciferase reporter system overexpression plasmid were diluted with 100μL jetPRIME buffer, 2.6μL jetPRIME was added and vortexed to mix, incubated at room temperature for 10 minutes, and then added to each well of the culture plate seeded with cells. Four replicate wells were cultured in each group. After culturing the cells in a CO2 incubator at 37°C for 4-6 hours, the culture medium containing the transfection reagent was removed and replaced with fresh cell culture medium containing 10% FBS and cultured for a further 48 hours.

[0187] The crRNA overexpression plasmid is U6-BsmBI-crRNA plasmid, and the sequence is as follows:

[0188] gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaat tatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccggagacgggataccgtctctgttgtggaaggtccagttttgaggggctattacaactttttt(SEQ ID NO: 14).

[0189] According to the target site sequence, upstream and downstream primers with specific structure and complementary base pairing are designed respectively, and after annealing, they are connected to the U6-BsmBI-crRNA vector to construct target-specific crRNA.

[0190] Primer structure: linker crRNA; the upstream primer linker is acaccg, the downstream primer linker is acaaca, and the crRNA sequence is as follows:

[0191] target-crRNA: tggtaattctttgaaagcag (SEQ ID NO: 15).

[0192] 1.5 Dual luciferase reporter system to test the use of proximal poly(A) sites

[0193] The Dual Luciferase Reporter Assay Kit (Vazyme) was used to detect the fluorescence signal of the dual-luciferase reporter system. The main steps were as follows: After 48 hours of culture, cells were lysed with 50 μL of 1× Cell Lysis Buffer on a shaker at room temperature for 5 minutes. The lysate was aspirated into a 1.5 ml centrifuge tube, 20 μL of Luciferase Substrate equilibrated to room temperature was added, mixed rapidly, and the activity of the Firefly luciferase reporter gene (hluc) was immediately measured in a luminometer. To this reaction solution, 20 μL of freshly prepared Renilla substrate was added, mixed rapidly, and the activity of the Renilla luciferase reporter gene (hRluc) was immediately measured in a luminometer. The results were statistically analyzed, and the hRluc / hluc ratio of each group was normalized to the control group's hRluc / hluc ratio of 1. The resulting values ​​were used to compare the treated and control groups.

[0194] 2 Results

[0195] In this example, the crRNA targeting site (ctgctttcaaagaattacca, SEQ ID NO: 16) and the proximal PAS of WAPL were cloned into the psiCHECK-2 plasmid to construct a dual luciferase reporter system ( Figure 3). dPspCas13b-PABPN1-EP / CCD fusion protein or its variant PABPN1-EP / CCD-dPspCas13b was combined with crRNA that can target the target position in the dual luciferase reporter system and transfected into HEK293T cells together with the dual luciferase reporter system. The experiment found that hRluc / hluc decreased, indicating that dPspCas13b-PABPN1-EP / CCD fusion protein or its variant PABPN1-EP / CCD-dPspCas13b can inhibit the use of proximal PAS in WAPL ( Figure 4 ).

[0196] Example 3d PspCas13b-PABPN1-EP / CCD fusion protein achieves inhibition of the target poly(A) site of WAPL pre-mRNA

[0197] 1 Experimental Materials and Methods

[0198] 1.1 Main reagents and instruments

[0199] The SW620-dPspCas13b-PABPN1-EP / CCD stably expressing cell line (SW620 cells stably expressing dPspCas13b-PABPN1-EP / CCD) was maintained in our laboratory. Fetal bovine serum was purchased from Gbico, jetPRIME DNA / siRNA transfection reagent was purchased from Polyplus, the high-temperature resistant fully premixed first-strand cDNA synthesis kit was purchased from TransGen Biotech, and the SYBR Green Pro Taq HS premixed qPCR kit was purchased from Accurium Biotech (AG). The LightCycler 480II fluorescence quantitative PCR instrument was purchased from Roche.

[0200] 1.2 Cell recovery and culture

[0201] Remove the SW620-dPspCas13b-PABPN1-EP / CCD stable expression cell line from a liquid nitrogen tank and rapidly thaw in a 37°C water bath. Transfer the cell suspension to a 1.5 mL centrifuge tube in a clean bench and centrifuge at low speed at 290 g for 5 minutes. Discard the supernatant and add 5 mL of 1640 medium supplemented with 10% FBS. Incubate in a 5% CO2, 37°C atmosphere.

[0202] 1.3 Cell treatment and transfection experiments

[0203] Adherent SW620-dPspCas13b-PABPN1-EP / CCD cells were digested and resuspended, then plated into 12-well plates at 2x105 cells per well. The plates were pre-incubated in an incubator for 24 hours. When the cells reached 50-60% confluence, transfection was performed using jetPRIME DNA / siRNA transfection reagent. The main steps were: dilute 1.5 μg of crRNA overexpression plasmid in 150 μL jetPRIME buffer, add 3 μL jetPRIME, and vortex to mix. Incubate at room temperature for 10 minutes before adding the cells to the wells of the plate seeded with cells. After incubating the cells in a 37°C CO2 incubator for 4-6 hours, the medium containing the transfection reagent was removed and replaced with fresh cell culture medium containing 10% FBS, which was then cultured for an additional 48 hours.

[0204] The crRNA overexpression plasmid was U6-BsmBI-crRNA plasmid, the same as in Example 2.

[0205] The crRNA was designed for the WAPL gene, and the specific sequence is as follows:

[0206] WAPL-crRNA1:cacagcagttgcactaacagca (SEQ ID NO: 17);

[0207] WAPL-crRNA2: acaacagtggctcaacatacaca (SEQ ID NO: 18);

[0208] WAPL-crRNA3: tgcacctggctggcatgacaaca (SEQ ID NO: 19);

[0209] WAPL-crRNA4: tcctcagaacattcgctacctca (SEQ ID NO: 20).

[0210] 1.4 Total RNA extraction from cells and RT-qPCR detection of target mRNA APA editing

[0211] Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and the extracted RNA was reverse transcribed using a high-temperature premixed first-strand cDNA synthesis kit (TransGen Biotech). The reverse transcription reaction was performed at 50°C for 5 minutes to obtain cDNA, which was then analyzed by quantitative real-time PCR using a SYBR Green Pro Taq HS premixed qPCR kit (AG). After the completion of the quantitative real-time PCR reaction, the amplification curve and melting curve of the quantitative real-time PCR were confirmed, and the results were statistically analyzed using the △△Ct method. Where △Ct = mRNA proximal PAS Ct value - mRNA distal PAS Ct value, △△Ct = experimental group △Ct - control group △Ct, gene relative expression = 2 -△△Ct .

[0212] 2 Results

[0213] In this example, the crRNA targeting position is located 70-150 bp upstream of the proximal poly(A) site of the endogenous WAPL ( Figure 5 RT-qPCR analysis showed that crRNA combined with dPspCas13b-PABPN1-EP / CCD targeting upstream of WAPL significantly reduced the ratio of proximal PAS usage to distal PAS usage ( Figure 6 ), indicating that the dPspCas13b-PABPN1-EP / CCD / crRNA system can significantly inhibit single-gene APA.

[0214] Example 4d PspCas13b-PABPN1-EP / CCD fusion protein enables multiple crRNAs to inhibit the target poly(A) site of the target gene pre-mRNA, achieving APA editing

[0215] 1 Experimental Materials and Methods

[0216] 1.1 Main reagents and instruments

[0217] The SW620-dPspCas13b-PABPN1-EP / CCD stably expressing cell line was maintained in our laboratory. Fetal bovine serum was purchased from Gbico, jetPRIME DNA / siRNA transfection reagent from Polyplus, a high-temperature premixed first-strand cDNA synthesis kit from TransGen Biotech, and a SYBR Green Pro Taq HS premixed qPCR kit from Accurate Biotech (AG). The LightCycler 480II fluorescence quantitative PCR instrument was purchased from Roche.

[0218] 1.2 Cell recovery and culture

[0219] Remove the SW620-dPspCas13b-PABPN1-EP / CCD stable expression cell line from a liquid nitrogen tank and rapidly thaw in a 37°C water bath. Transfer the cell suspension to a 1.5 mL centrifuge tube in a clean bench and centrifuge at low speed at 290 g for 5 minutes. Discard the supernatant and add 5 mL of 1640 medium supplemented with 10% FBS. Incubate in a 5% CO2, 37°C atmosphere.

[0220] 1.3 Cell treatment and transfection experiments

[0221] The adherent SW620-dPspCas13b-PABPN1-EP / CCD cells were digested and resuspended, plated into 12-well plates, 2x105 cells were added to each well, and the culture plates were pre-cultured in the incubator for 24 hours. When the cell density reached 50-60% confluence, transfection was performed using jetPRIME DNA / siRNA transfection reagent. The main steps are: dilute 1.5μg of crRNA overexpression plasmid (three crRNAs targeting the same gene) with 150μL jetPRIME buffer, add 3μL jetPRIME and vortex to mix, incubate at room temperature for 10 minutes, and then add to each well of the culture plate seeded with cells. After the cells were cultured in a CO2 incubator at 37°C for 4-6 hours, the culture medium containing the transfection reagent was removed and replaced with fresh cell culture medium containing 10% FBS and continued to be cultured for 48 hours.

[0222] The crRNA overexpression plasmid was U6-BsmBI-crRNA plasmid, the same as in Example 2.

[0223] crRNA was designed for SMAD4 and COL1A2 genes, and the specific sequences are as follows:

[0224] SMAD4-crRNA1: attgatggcaaagggcttagaat (SEQ ID NO: 21);

[0225] SMAD4-crRNA2: caaagtcactgccaattgatatg (SEQ ID NO: 22);

[0226] SMAD4-crRNA3:ttctacttaaattctctatacaa (SEQ ID NO: 23);

[0227] COL1A2-crRNA1: aatccttctttttctcttttgcc (SEQ ID NO: 24);

[0228] COL1A2-crRNA2: atgaaactgtattgcacaatgct (SEQ ID NO: 25);

[0229] COL1A2-crRNA3: tttgggggagcgggggaaggagt (SEQ ID NO: 26).

[0230] 1.4 Total RNA extraction from cells and RT-qPCR detection of target mRNA APA editing

[0231] Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and the extracted RNA was reverse transcribed using a high-temperature resistant fully premixed first-strand cDNA synthesis kit (TransGen Biotech). The reverse transcription reaction was carried out at 50°C for 5 minutes to obtain cDNA, which was then analyzed by quantitative real-time PCR using a SYBR Green Pro Taq HS premixed qPCR kit (AG). After the completion of the quantitative real-time PCR reaction, the amplification curve and melting curve of the quantitative real-time PCR were confirmed, and the results were statistically analyzed using the △△Ct method. Where △Ct = mRNA proximal PAS Ct value - mRNA distal PAS Ct value, △△Ct = experimental group △Ct - control group △Ct, and gene relative expression = 2 -△△Ct .

[0232] 2 Results

[0233] In this embodiment, the crRNA targeting SMAD4 is located 60-120 bp upstream of the proximal poly(A) site of endogenous SMAD4 ( Figure 7 ), the crRNA targeting COL1A2 is located 60-130 bp upstream of the proximal poly(A) site of endogenous COL1A2 ( Figure 8RT-qPCR analysis showed that crRNA combined with dPspCas13b-PABPN1-EP / CCD targeting to the upstream of the corresponding gene significantly reduced the ratio of proximal PAS usage to distal PAS usage ( Figure 9 ), indicating that the simultaneous application of multiple crRNAs with the dPspCas13b-PABPN1-EP / CCD fusion protein can significantly inhibit single-gene APA.

[0234] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A truncated PABPN1 protein, characterized in that: It is composed of the EP domain and CCD domain of PABPN1; The amino acid sequence of the EP domain is shown in SEQ ID NO: 1; The amino acid sequence of the CCD domain is shown in SEQ ID NO:

2.

2. The PABPN1 truncated protein according to claim 1, characterized in that: The amino acid sequence of the truncated protein is shown in SEQ ID NO:

3.

3. A fusion protein, characterized in that: Comprising the PABPN1 truncated protein and dPspCas13b protein according to claim 1; The amino acid sequence of the dPspCas13b protein is shown in SEQ ID NO:

4.

4. A polyadenylation site editing tool, characterized in that: The polyadenylation site editing tool includes the PABPN1 truncated protein according to claim 1, as well as the dPspCas13b protein, a nuclear import signal, a screening protein, and a tag protein.

5. The polyadenylation site editing tool according to claim 4, wherein: The nuclear import signal is selected from at least one of nucleoplasmin NLS and SV40 NLS.

6. The polyadenylation site editing tool according to claim 4, wherein: The screening protein is selected from at least one of BSD protein, PAC protein, Hygro protein and Neo protein.

7. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the PABPN1 truncated protein according to claim 1 or 2, the fusion protein according to claim 3, and / or the polyadenylation site editing tool according to any one of claims 4 to 6. A vector comprising the nucleic acid molecule according to claim 7 .

9. Use of the PABPN1 truncated protein according to claim 1 or 2, the fusion protein according to claim 3, the polyadenylation site editing tool according to any one of claims 4 to 6, the nucleic acid molecule according to claim 7, and / or the vector according to claim 8 in selective polyadenylation regulation; The selective polyadenylation regulation is to inhibit the poly (A) site of the target gene.

10. A method for inhibiting the poly(A) site of a target gene pre-mRNA, comprising treating a sample with the polyadenylation site editing tool according to any one of claims 4 to 6 or the vector according to claim 8.

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