WO2022083425A1 - 一种目标rna单碱基编辑的系统和方法 - Google Patents

一种目标rna单碱基编辑的系统和方法 Download PDF

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WO2022083425A1
WO2022083425A1 PCT/CN2021/121066 CN2021121066W WO2022083425A1 WO 2022083425 A1 WO2022083425 A1 WO 2022083425A1 CN 2021121066 W CN2021121066 W CN 2021121066W WO 2022083425 A1 WO2022083425 A1 WO 2022083425A1
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nucleic acid
base
editing
sulfur
base editor
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French (fr)
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贺新义
于昊
刘光
邓子新
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上海交通大学
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Definitions

  • the invention belongs to the field of biotechnology, and relates to a system and method for single-base editing of target RNA, in particular to an RNA base editor based on sulfur-modified nucleic acid and sulfur-modified nucleic acid recognition protein and its application.
  • RNA Ribonucleic Acid
  • Ribonucleic Acid is a genetic information carrier that exists in biological cells and some viruses and viroids. It plays a vital role in the process of life. Its main function is to realize the expression of genetic information on proteins. A bridge in the transformation of genetic information to phenotype. DNA is the carrier of genetic information, and RNA, as an intermediate product transcribed from DNA, is responsible for directing the production of downstream proteins.
  • a major advantage of RNA editing is its reversibility, whereas changes at the DNA level, in contrast, are permanent. Therefore, for disease-related genetic mutations, only short-lived modification of RNA mutations avoids irreversible modifications to the genome and enables correction of mutant proteins.
  • RNA mainly include precise editing of RNA using adenosine deaminase or cytidine deaminase.
  • ADAR proteins can convert adenosine (A) to inosine (I), an A ⁇ I conversion.
  • A adenosine
  • I inosine
  • Three types of ADAR proteins have been identified in mammals: ADAR1 (isoforms p110 and p150), ADAR2 and ADAR3, all of which are double-stranded RNA (dsRNA) substrates.
  • the ADAR protein or its catalytic domain is fused to a ⁇ N peptide, a SNAP tag, or a Cas protein (dCas13b), and a guide RNA (gRNA) is designed to fuse this
  • gRNA guide RNA
  • Zhang Feng's team published a new CRISPR system for RNA editing, REPAIR, in the journal Science.
  • the researchers combined the efficient RNA-targeting Cas13b protein with the ADAR2 enzyme.
  • the REPAIR editing system was directed to a specific RNA position, specifically modifying the adenine base (A) on the RNA to inosine (I), which is structurally similar to the guanine base (G).
  • Zhang Feng's team developed a new RNA single-base editor by improving ADAR2 so that the improved ADAR2 can convert cytosine C to uracil U.
  • Cas protein-based base editors and guide elements in one vector (eg, viral vectors such as AAV).
  • the purpose of the present invention is to provide a novel base editor and a method of using the same.
  • a first aspect of the present invention provides a base editor, the base editor comprising a targeting element and an editing effect element, wherein the targeting element and the editing effect element form a fusion protein, wherein the The targeting element comprises a sulfur-modified nucleic acid recognition protein, and the editing effector element is an RNA editing effector protein.
  • the base editor can bind to a predetermined nucleic acid region under the guidance of a guide element, and perform base editing on nucleotides in or adjacent to the predetermined nucleic acid region.
  • the guide element and the nucleic acid sequence to be base edited form a double-stranded complementary structure in a predetermined nucleic acid region.
  • the guide element is a single-stranded sulfur-modified nucleic acid.
  • the guide element comprises sulfur-modified gDNA or gRNA.
  • the base editor is a single-stranded nucleic acid base editor.
  • the single-stranded nucleic acid is single-stranded DNA, single-stranded RNA, or a combination thereof.
  • the sulfur-modified nucleic acid recognition protein is a sulfur-binding domain protein (SBD), which specifically recognizes sulfur-modified nucleic acid.
  • SBD sulfur-binding domain protein
  • the sulfur-modified nucleic acid refers to the modified as or its corresponding isomer.
  • the sulfur-binding domain protein binds RNA/RNA double-stranded or RNA/DNA hybrid double-stranded.
  • the sulfur binding domain protein includes: SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, SBDtcu, or SBD homologous protein.
  • the sulfur binding domain protein is derived from a species selected from the group consisting of MMoMcrA, ScoMcrA, SprMcrA, HgaMcrA, EcoMcrA; preferably, derived from MMoMcrA.
  • the SBD homologous protein can bind to sulfur-modified oligonucleotides, and has one or more of the following conditions:
  • oligonucleotides including deoxyribonucleic acid DNA or ribonucleic acid RNA
  • C3 contains a P-L-W motif or an A-L-W motif (eg: P75-L79-W85 in SBDmmo; P79-L83-W89 in SBDspr; A73-L77-W83 in SBDtcu).
  • RNA editing effector protein is selected from the group consisting of ADAR2DD, ADAR2DD(E488Q), ADAR2DD(E488Q/T375G), APOBEC, AID or CDA1.
  • the targeting element and/or the editing effect element are each independently the corresponding element of the base editor prepared in the Examples.
  • the targeting element and the editing effect element are directly connected by chemical bonds (eg, peptide bonds) or connected by a linker.
  • the joints include flexible joints and non-flexible joints.
  • the fusion protein contains one or more SBDs.
  • the multiple SBDs may be the same or different.
  • the multiple SBDs may be located on the same side (eg, left or right) of the RNA editing effector protein, or may be located on both sides of the editing effector protein.
  • the structure of the fusion protein is shown in the following formula I or I' or I":
  • A is the targeting element SBD
  • B is the editing effector element RNA editing effector protein
  • L is each independently none or a linker peptide
  • Each "-" is independently a chemical bond.
  • the chemical bond includes a peptide bond or a covalent bond.
  • sequence of the fusion protein is shown in SEQ ID No:4, or the 1-565th position or the 2-565th position in SEQ ID No:4.
  • sequence length of the gDNA or gRNA is greater than 15nt and less than 100nt, which contains a mismatch mutation (C) for the base to be edited (A), and is about 9-20nt away from the site to be edited of at least one sulfur-modified base.
  • sequence length of the gDNA or gRNA is 15-25 nt.
  • the sulfur-modified base may be one base.
  • the sulfur-modified bases may be N consecutive bases (1 ⁇ N ⁇ 10).
  • the sulfur-modified base is preferably C or G.
  • the sulfur-modified base can be in the 5'-end direction or the 3'-end direction of the editing site.
  • the guide element may also carry other chemical modifications, such as fluorine modification, methylation modification, methoxy modification, locked nucleic acid (LNA) and the like.
  • reaction system for base editing, the reaction system comprising:
  • the nucleic acid to be base edited is single-stranded DNA, single-stranded RNA, or a combination thereof.
  • reaction system further includes (c) nucleic acid to be base edited.
  • the detection system further comprises: (d) a buffer.
  • the concentration of the nucleic acid to be base edited in the reaction system is 0.0001-1000 nM, preferably 0.01-500 nM, more preferably 0.1-100 nM.
  • the concentration of the nucleic acid to be base edited in the reaction system is 1 to 1 ⁇ 10 8 copies/microliter, preferably 2 to 1 ⁇ 10 5 copies/microliter, More preferably 5 to 1 x 103 copies/microliter.
  • the molar ratio of the guide element to the nucleic acid to be base edited is 1:10 to 10 5 :1, preferably 1:1 to 1000:1 , more preferably 2:1 to 100:1.
  • the guide element is a sulfur-modified gDNA or gRNA.
  • the length of the gDNA or gRNA is 15-100 nt, preferably 16-50 nt, more preferably 18-40 nt.
  • the nucleic acid to be base-edited comprises a nucleic acid to be base-edited from the group consisting of plants, animals, insects, microorganisms, viruses, or a combination thereof.
  • the nucleic acid to be base edited is artificially synthesized or naturally occurring mRNA.
  • the nucleic acid to be base edited includes wild-type or mutant mRNA.
  • the SBD is selected from the following group: the amino acid sequence identity of any one of SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, SBDtcu is ⁇ 20% or its combination; Said SBD is SBDmmo.
  • nucleic acid encoding the base editor according to the first aspect of the present invention.
  • the nucleic acid is a linear sequence.
  • the nucleic acid has the structure of formula II of 5'-3' (5' to 3'):
  • P1 is the first promoter sequence
  • X1 is the coding sequence of sulfur binding domain protein (SBD);
  • L1 is the coding sequence without or linked sequence
  • X2 is the coding sequence of the RNA editing effector protein
  • X3 is the polyA sequence
  • each "-" is a key independently.
  • the nucleic acid construct contains one or more SBD coding sequences.
  • the multiple SBD coding sequences may be the same or different.
  • the multiple SBD coding sequences may be located on the same side (eg, left or right) of the RNA editing effector protein coding sequence, or may be located on both sides of the editing effector protein coding sequence.
  • a vector is provided, and the vector contains the nucleic acid according to the third aspect of the present invention.
  • the vector includes an expression vector.
  • the vectors include plasmids and viral vectors.
  • a genetically engineered cell is provided, the cell contains the vector of the fourth aspect, or the nucleic acid of the third aspect is integrated into the genome.
  • the cells are transformed or transfected with the vector or nucleic acid.
  • the cells are also transfected with gDNA or gRNA or an expression vector thereof.
  • the cells include eukaryotic cells and prokaryotic cells.
  • the cells include bacteria, yeast, mammalian cells, and plant cells.
  • a kit comprising:
  • the base editor can bind to a predetermined nucleic acid region under the guidance of the guide element, and perform base editing on nucleotides in or adjacent to the predetermined nucleic acid region.
  • the guide element includes gDNA, gRNA or a combination thereof.
  • a method for base editing comprising the steps of:
  • the base editor according to the first aspect of the present invention and the nucleic acid to be base edited form a "base editor-nucleic acid to be base edited-guide element" complex, whereby, the base editor is made to perform base editing in the predetermined nucleic acid region or adjacent nucleotides.
  • the nucleotides in or adjacent to the predetermined nucleic acid region refer to any one or more nucleotides within ⁇ 30nt (preferably ⁇ 20nt, more preferably ⁇ 10nt) of the binding site .
  • the nucleic acid to be base edited is selected from the group consisting of single-stranded DNA, single-stranded RNA, or a combination thereof
  • the nucleic acid to be base edited is mRNA.
  • the nucleic acid to be base edited is an isolated, intracellular, or target nucleic acid of a cell-free system.
  • the mRNA is isolated mRNA, intracellular mRNA, or cell-free mRNA.
  • the base editing includes single base editing and multiple base editing.
  • the method includes an in vivo method and an in vitro method.
  • the method is a non-diagnostic and non-therapeutic method.
  • the method includes:
  • the base editor according to the first aspect of the present invention which is used to prepare a reagent or kit for base editing mRNA.
  • the base editing includes in vivo or in vitro base editing.
  • the kit further comprises: a guide element.
  • a gene therapy method comprising the steps of: applying the base editor or its coding sequence according to the first aspect of the present invention to a subject in need; and specifically binding to the base editor to be edited nucleic acid guide element.
  • the guide element binds to a predetermined region of a pathogenic gene.
  • Figure 1 shows an amino acid sequence alignment of SBD homologous proteins. Red triangles indicate the P-L-W or A-L-W motifs.
  • Figure 2 shows the chemical structure of sulfur-modified DNA.
  • Figure 3 shows the binding activity of sulfur-modified DNA binding domains to sulfur-modified nucleic acids.
  • Figure 4 shows a schematic flow chart of RNA single base editing using sulfur-modified nucleic acid and sulfur-modified nucleic acid recognition protein.
  • Figure 5 shows the gDNA-mediated in vitro editing efficiency of GFP mRNA by SBD Mmo -ADAR2 fusion protein (plate).
  • Figure 6 shows the gDNA-mediated in vitro editing efficiency of green fluorescent protein mRNA by SBD Mmo -ADAR2 fusion protein (plate).
  • Figure 7 shows the gRNA-mediated in vitro editing efficiency of GFP mRNA by SBD Mmo -ADAR2 fusion protein (plate).
  • Figure 8 shows the in vitro editing efficiency (sequencing) of SBD Mmo -ADAR2 fusion protein mediated by gRNA to green fluorescent protein mRNA.
  • Figure 9 shows the in vivo editing efficiency of green fluorescent protein mRNA by SBD Mmo and ADAR2 fusion protein (fluorescence microscopy imaging).
  • the left and right views are darkfield and brightfield imaging, respectively, with a brightfield exposure time of 5ms and a darkfield exposure time of 800ms.
  • the base editor of the present invention is based on a sulfur-modified DNA recognition domain, and under the guidance of a sulfur-modified nucleic acid (such as gDNA or gRNA) as a guide element, it can perform efficient and specific processing on single-stranded nucleic acid (such as single-stranded RNA or DNA). single base editing.
  • a sulfur-modified nucleic acid such as gDNA or gRNA
  • single base editing single base editing.
  • the experimental results show that the base editor of the present invention can successfully perform in vitro and in vivo single-base directed editing of the reporter gene RNA. On this basis, the invention was completed.
  • sulfur modification may also be referred to as "phosphorylation modification” and refers to modifications made to the guide elements of the single base editors described herein; in particular, to the DNA or RNA phosphate backbone A modification that occurs when the upper non-bridging oxygen atom is replaced by a sulfur atom.
  • sulfur-modified nucleic acid recognition protein As used herein, the terms "sulfur-modified nucleic acid recognition protein,” “sulfur-binding domain protein,” and “SBD” are used interchangeably and refer to proteins capable of specifically recognizing sulfur-modified nucleic acids.
  • nucleic acid As used herein, the terms "sulfur-modified nucleic acid”, or “sulfur-modified nucleic acid” and both refer to nucleic acid modified with sulfur groups; wherein the nucleic acid can be either RNA or DNA.
  • sulfur-modified DNA and "S-gDNA” are used interchangeably; the terms “sulfur-modified RNA” and “S-gRNA” are used interchangeably.
  • gRNA or "gDNA” refers to a single-stranded nucleic acid sequence that can complementarily pair with bases preceding and following an RNA target editing site, comprising a base mismatch with the target editing site, and Sulfur modification at one or more sites near the 3' end guides the SBD to the RNA target editing site.
  • insertion/deletion refers to the insertion or deletion of nucleotide bases within a nucleic acid. Such insertions or deletions can result in frameshift mutations within the coding region of the gene.
  • base editor of the present invention and “fusion protein of the present invention” are used interchangeably to refer to the base editor described in the first aspect of the present invention, ie comprising at least one of said targets A fusion protein of a targeting element and at least one of the editing effector elements, wherein the targeting element comprises a sulfur-modified nucleic acid recognition protein, and the editing effector element is an RNA editing effector protein.
  • the base editors of the present invention are capable of modifying specific nucleotide bases without producing a significant proportion of insertions/deletions.
  • any of the base editors provided herein are capable of producing a greater proportion of intended modifications (eg, point mutations or deaminations) relative to insertions/deletions.
  • any of the base editors of the present invention are capable of efficiently generating intended mutations, such as point mutations, in nucleic acids (eg, nucleic acids within a genome) without generating large numbers of unintended mutations, such as unintended point mutations.
  • the base editor mainly includes adenine deaminase, and other types of base editors are also within the protection scope of the present invention as long as they have the functions of the base editor of the present invention.
  • SBD Sulfur binding domain protein
  • SBD Sulfur-binding domain protein
  • SBD is the recognition domain of restriction endonucleases.
  • the fusion protein is constructed, and the SBD is used as the targeting domain to bring the base editor to the RNA to be edited.
  • the SBD can be located at the N-terminus or the C-terminus of the fusion protein (in the embodiment, the SBD is located at the N-terminus of the fusion protein). in the 5' direction of the editing site. Multiple SBD domains can also be present in the fusion protein, such as in the form of SBD-ADAR-SBD, to improve editing efficiency.
  • the gDNA or gRNA used have both the 5' and 3' ends of the editing site, respectively. Sulfur-modified bases.
  • SBD and editor can also have a variety of arrangements, such as SBD-SBD-ADAR, ADAR-SBD-SBD and so on.
  • representative said sulfur binding domain proteins include: SBDsco, SBDspr, SBDmmo, SBDhga, SBDeco, SBDtcu, or SBD homologous proteins.
  • the base editor of the present invention further comprises an editing effect element.
  • the editing effector elements that can be used in the present invention are not particularly limited.
  • the editing effector elements applicable to the present invention include wild-type and mutant editing effector elements, as long as the editing effector element can catalyze the base at a specific site, thereby, for example, by deamination reaction or other similar A reaction that converts one base to another, such as converting A to I.
  • representative editing effector elements include, but are not limited to, the following RNA editing effector proteins: ADAR2DD, ADAR2DD(E488Q), ADAR2DD(E488Q/T375G), APOBEC, AID or CDA1.
  • ADAR2DD, ADAR2DD(E488Q), and ADAR2DD(E488Q/T375G) are the wild type of adenosine deaminase ADAR2 (Adenosine to inosine acting on RNA enzyme 2) and mutants with improved editing effect, respectively.
  • a preferred editing response element is the ADAR2 mutant used in the RESCUE system.
  • the sulfur-modified nucleic acid refers to the modified as or its corresponding isomer.
  • the present invention also provides a method, a corresponding kit, a reaction system and an application for base editing using the base editor of the present invention.
  • the base editing method of the present invention comprises the steps of: in the presence of a guide element, the base editor of claim 1 and the nucleic acid to be base edited form a "base editor-to be base edited".
  • a nucleic acid-guide element" complex thereby allowing the base editor to perform base editing within a predetermined nucleic acid region or adjacent nucleotides.
  • the base editing method can be an in vitro or in vivo method.
  • the base editing methods of the present invention can be used for gene therapy, eg, the treatment of certain diseases in mammals (eg, humans).
  • the method of the present invention can effectively edit intracellular mRNA without changing the genomic DNA.
  • the present invention proposes a novel base editor, which modifies gDNA or gRNA with sulfur to target the editing site, and uses SBD as the targeting domain to bring the editor to the RNA to be edited.
  • the sulfur-modified DNA or S-modified RNA used in the present invention has a longer half-life in the cell body, is more stable, and is not easily degraded.
  • the SBD used in the present invention is smaller than Cas13 (SBD is only 16kDa), and the minimum S-gRNA or S-gDNA can be 20nt, which makes the editing composed of sulfur-modified nucleic acid and SBD.
  • the volume of the system is much smaller than the editing system relying on Cas protein, which is more convenient for packaging and delivery into the cell.
  • the method of the present invention can effectively edit intracellular mRNA without changing the genomic DNA.
  • ADAR2DD E488Q/T375G
  • pESC-URA yeast expression vector
  • SalI and BamHI restriction sites are added, and the synthetic sequence is as follows (SEQ ID No: 1):
  • the bold part is the vector sequence
  • the lowercase sequence is the SalI and BamHI restriction sites
  • the italic part is the ADAR2DD (E488Q/T375G) sequence
  • the underlined part is the histidine tag sequence.
  • the SBDmmo-ADAR2 fusion protein expression vector was constructed from the above-mentioned plasmid, and the amplification primer sequence was as follows:
  • SBDmmo-F 5'-CCGctcgagATGATTAGCCCGGAAACCCT-3' (SEQ ID No: 2)
  • SBDmmo-R 5'-CGCggatccGCTTCCACCTCCTCCACGGAAATGCGGATCGCGGT-3' (SEQ ID No::3)
  • Bacterial liquid lysis centrifugally collect the bacterial liquid, discard the supernatant, resuspend the bacteria in 50 mL of protein purification Buffer A (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole), and use a pre-cooled cell disruptor to break the bacteria. The cell lysate was centrifuged at 10,000 rpm for 1 h at 4°C, and the supernatant was taken, filtered with a 0.22 ⁇ M filter, and placed on ice.
  • Buffer A 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole
  • Ni column purification Equilibrate the Ni affinity chromatography column (GE) with Buffer A (20mM Tris-HCl pH 8.0, 300mM NaCl, 50mM imidazole,), load the cell lysate onto the Ni affinity chromatography column, use Buffer A After A was washed for 5 column volumes, the concentration of Buffer B (20mM Tris-HCl pH 8.0, 300mM NaCl, 500mM imidazole) was linearly increased, and the SBDmmo-ADAR2 fusion protein was eluted in the concentration range of about 35%-65%. All procedures were performed on an AKTA purification system (GE).
  • Buffer A 20mM Tris-HCl pH 8.0, 300mM NaCl, 50mM imidazole
  • Heparin column purification Equilibrate Heparin affinity chromatography column (GE) with Buffer A (20mM Tris-HCl pH 8.0, 50mM NaCl, 1mM DTT), dilute the protein obtained by Ni column purification and load the sample, and wash 5 cells with Buffer A After the column volume, the concentration of Buffer B (20 mM Tris-HCl pH 8.0, 1000 mM NaCl, 1 mM DTT) was linearly increased, and the SBDmmo-ADAR2 fusion protein was eluted in the concentration range of about 30%-60%. All procedures were performed on an AKTA purification system (GE).
  • SBDmmo-ADAR2 fusion protein The sequence of the obtained SBDmmo-ADAR2 fusion protein is shown in SEQ ID NO: 4.
  • the GFP gene was constructed on the pET28a E. coli protein expression vector, and the GFP DNA sequence was point mutated using mutation primers to mutate one of the G to A.
  • the sequence of the mutation primers is as follows:
  • GFP-Mut-F 5'-TGTTCCATAGCCAACACTTGTCACT-3' (SEQ ID No: 5)
  • GFP-Mut-R 5'-GTGTTGGCTATGGAACAGGCAGCTT-3' (SEQ ID No: 6)
  • GFP After mutation, a stop codon is introduced inside GFP, so that GFP cannot be fully expressed in the expression host, and the host does not emit green fluorescence.
  • RNA transcription kit Use the RNA transcription kit to obtain the mutated GFP mutant RNA substrate, add DNaseI to remove the residual DNA in the system, and verify with PCR to ensure that there is no DNA residue.
  • a series of single-stranded sulfur-modified gDNAs are designed at the GFP mutation site.
  • the gDNA as a whole is complementary to the GFP sequence, but a C-A mismatch is formed at the mutation site.
  • the sulfur modification site in the gDNA is located at 3 of the mutation site. ' end, and designed a gDNA-0 without sulfur modification as a negative control, the sequence is as follows:
  • gDNA-0 5'-ACAAGTGTTGGCCATGGAACAGGCAGCTTGCCGGTAGTGC-3' (SEQ ID No: 7)
  • gDNA-9 5'-ACAAGTGTTGGCCATGGAACAG * GCAGCTTGCCGGTAGTGC-3' (SEQ ID No: 8)
  • gDNA-13 5'-ACAAGTGTTGGCCATGGAACAGGCAG * CTTGCCGGTAGTGC-3' (SEQ ID No: 9)
  • gDNA-17 5'-ACAAGTGTTGGCCATGGAACAGGCAGCTTG * CCGGTAGTGC-3' (SEQ ID No: 10)
  • gDNA-20 5'-ACAAGTGTTGGCCATGGAACAGGCAGCTTGCCG * GTAGTGC-3' (SEQ ID No: 11)
  • gDNA-7S 5'-ACAAGTGTTGGCCATGGAACAGGCAG * C * T * T * G * C * C * GGTAGTGC-3' (SEQ ID No: 12)
  • the substrate mRNA and gDNA were annealed in a PCR machine, and the final concentration of 100nM substrate RNA, 200nM gDNA, and 100mM NaCl, 1mM DTT, 20mM Tris-HCl pH8.0 were added to the system.
  • RNA after the editing reaction was reverse transcribed, and pfu polymerase was used to amplify the cDNA after reverse transcription.
  • the primer sequences used were as follows:
  • cDNA-F AGGAGATATACATATGAGTAAAGGAGAAGA (SEQ ID No: 13)
  • cDNA-R TAGCAGCCGGATCTCAGTGGTGGTGGTGGT (SEQ ID No: 14)
  • the amplified fragment was cloned into Pet28a vector and expressed in E. coli BL21 host. If RNA editing occurs, the GFP protein can be translated and expressed normally, causing the host cell to fluoresce.
  • the GFP (green fluorescent protein) mutant RNA substrate is the same as in Example 2.
  • gRNA-13 5'-ACAAGUGUUGGCCAUGGAACAGGCAG*CUUGCCGGUAGUGC-3' (SEQ ID No: 15)
  • gRNA-7S 5'-ACAAGUGUUGGCCAUGGAACAGGCAG*C*U*U*G*C*C*GGUAGUGC-3' (SEQ ID No: 16)
  • gRNA-0 5'-ACAAGUGUUGGCCAUGGAACAGGCAGCUUGCCGGUAGUGC-3' (SEQ ID No: 17)
  • the substrate mRNA and gRNA were annealed in a PCR machine, and the final concentration of 100nM substrate RNA, 200nM gRNA, and 100mM NaCl, 1mM DTT, 20mM Tris-HCl pH8.0 were added to the system.
  • RNA after the editing reaction was reverse transcribed, and pfu polymerase was used to amplify the cDNA after reverse transcription.
  • the primers used were cDNA-F (SEQ ID No: 13) and cDNA-R (SEQ ID No: 14).
  • the amplified fragment was cloned into Pet28a vector and expressed in E. coli BL21 host. If RNA editing occurs, the GFP protein can be translated and expressed normally, causing the host cell to fluoresce.
  • SBD-ADAR2-F cgggatcccATGATTAGCCCGGAAACC (SEQ ID No: 18)
  • SBD-ADAR2-R gctctagaTTAGTGGTGGTGGTGGTGGTGCTCGAGCGTGAGTG (SEQ ID No: 19)
  • GFP-F cgggatccc atgagtaaaggagaag (SEQ ID No: 20)
  • GFP-R gctctagatcagtggtggtggtggtggtggtgctcgagtttgtata (SEQ ID No: 21)
  • HEK293T cells Fresh HEK293T cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin and 1% streptomycin. The SBDmmo-ADAR2 fusion protein expression vector and the green fluorescent protein mutant expression vector were transfected into HEK293T cells. Lipofectamine transfection reagent was used, and the cells were cultured overnight and passaged.
  • the gRNAs used in the in vivo editing experiments were the same as in Example 2.
  • Transfection gRNAs were transfected into HEK293T cells expressing the SBDmmo-ADAR2 fusion and GFP mutant and imaged with a fluorescence microscope. The results are shown in Figure 9.
  • sulfur modification has been deeply utilized for its anti-nuclease activity; and this modification exists in nature as a natural modification, and has functions such as being recognized by proteins and participating in restriction modification systems.
  • the present invention establishes a new type of positioning module based on sulfur-modified nucleic acid and its specific binding protein, that is, the sulfur-modified ssRNA is used as the gRNA to match the complementary sequence of the mRNA target region to generate the sulfur-modified dsRNA; Combine with the target area to achieve the purpose of positioning. Furthermore, a novel RNA editing system is established by fusing this module with the RNA editing module.
  • the SBD domain has only about 170 amino acids, which is smaller than CRISPR-Cas13, so compared with the REPAIR system and the RESCUE system, the base editor of the present invention is easier to be introduced into cells.
  • RNA editors including C->U editors from directed evolution, with a wider editing scope.
  • the SBD-based RNA editing system of the present invention can theoretically use gRNAs of any length. By using longer gRNAs, a more stable dsRNA structure may be obtained, and a lower off-target rate and higher efficiency may be obtained. Broad application prospects.

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Abstract

本发明提供了一种目标RNA单碱基编辑的系统和方法。具体地，本发明提供了一种碱基编辑器,包括靶向元件和编辑效应元件，其中所述靶向元件与所述编辑效应元件形成一融合蛋白,其中所述靶向元件包含硫修饰核酸识别蛋白，所述编辑效应元件为RNA编辑效应蛋白。本发明首次提出了由硫修饰核酸介导的碱基编辑的概念，将硫修饰识别蛋白与碱基编辑相结合，构建了基于硫修饰核酸及硫修饰核酸识别蛋白的单碱基编辑系统，从而实现了RNA编辑的精准调控。本发明的碱基编辑器具有小型化、靶向性好、碱基编辑效率高等优点。

Description

一种目标RNA单碱基编辑的系统和方法 技术领域
本发明属于生物技术领域，涉及一种目标RNA单碱基编辑的系统和方法，尤其涉及一种基于硫修饰核酸及硫修饰核酸识别蛋白的RNA碱基编辑器及其应用。
背景技术
核糖核酸(Ribonucleic Acid，RNA)，是存在于生物细胞以及部分病毒、类病毒中的遗传信息载体，在生命过程中发挥至关重要的作用，主要功能是实现遗传信息在蛋白质上的表达，是遗传信息向表型转化过程中的桥梁。DNA是遗传信息的载体，RNA作为DNA转录出来的中间产物，负责指导下游蛋白质的生产。RNA编辑的一个主要优点是其可逆性，而DNA水平的变化则相反，是一种永久性的改变。因此，针对与疾病相关的基因突变，仅短暂对RNA突变进行修改，既避免了对基因组的不可逆修改，又实现了突变蛋白质的纠正。
目前能够对RNA进行编辑的方法主要包括有使用腺苷脱氨酶或胞苷脱氨酶对RNA进行精确编辑。例如，ADAR蛋白可以将腺苷(A)转换为肌苷(I)，即A→I转换。三种类型的ADAR蛋白已在哺乳动物中鉴定出：ADAR1(异构体p110和p150)、ADAR2和ADAR3，它们的底物都是双链RNA(dsRNA)。为了实现靶向RNA编辑，ADAR蛋白或者它的催化结构域与一种λN肽、一种SNAP标签或一种Cas蛋白(dCas13b)融合在一起，而且向导RNA(gRNA)经设计后将这种融合的ADAR蛋白招募到特定位点上，并进行RNA的精确编辑。
2017年，张锋团队就在《科学》杂志发表了一项针对RNA编辑的全新CRISPR系统——REPAIR，在这款RNA编辑器中，研究人员将高效靶向RNA的Cas13b蛋白与ADAR2酶结合在一起，将REPAIR编辑系统引导至特定的RNA位置，特异性地将RNA上的腺嘌呤碱基(A)修改为与鸟嘌呤碱基(G)结构类似的肌苷(I)。2019年，张锋团队研究通过改进了ADAR2，使得改进后的ADAR2能够将胞嘧啶C转化为尿嘧啶U，开发出了一种全新的RNA单碱基编辑器。
然而，由于Cas蛋白的结构和分子量大，因此难以在一个载体(例如病毒载体，例如AAV)中容纳基于Cas蛋白的碱基编辑器和引导元件。
因此，本领域迫切需要开发新的结构更为紧凑，并可高效地进行碱基编辑的编辑器和相应的编辑方法。
发明内容
本发明的目的在于提供一种新型的碱基编辑器及其使用方法。
本发明的第一方面，提供了一种碱基编辑器,所述碱基编辑器包括靶向元件和编辑效应元件，其中所述靶向元件与所述编辑效应元件形成一融合蛋白,其中所述靶向元件包含硫修饰核酸识别蛋白，所述编辑效应元件为RNA编辑效应蛋白。
在另一优选例中，所述的碱基编辑器在向导元件的引导下，可结合于预定的核酸区域，并对预定的核酸区域内或邻近的核苷酸进行碱基编辑。
在另一优选例中，所述的向导元件与待碱基编辑的核酸序列，在预定的核酸区域内形成双链互补结构。
在另一优选例中，所述的向导元件为单链的硫修饰核酸。
在另一优选例中，所述的向导元件包含硫修饰的gDNA或gRNA。
在另一优选例中，所述的碱基编辑器为单链核酸的碱基编辑器。
在另一优选例中，所述的单链核酸为单链DNA、单链RNA、或其组合。
在另一优选例中，所述硫修饰核酸识别蛋白为硫结合结构域蛋白(SBD)，其特异性识别被硫修饰的核酸。
在另一优选例中，所述的硫修饰的核酸指核酸中磷酸基团中的一个OH(O或O -)被SH(或S -)所取代和或一个＝0被＝S替换。
在另一优选例中，所述的硫修饰的核酸指核酸中的
Figure PCTCN2021121066-appb-000001
被修饰为
Figure PCTCN2021121066-appb-000002
或其对应异构体。
在另一优选例中，所述硫结合结构域蛋白(SBD)结合RNA/RNA双链或RNA/DNA杂合双链。
在另一优选例中，所述硫结合结构域蛋白(SBD)包括：SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu、或SBD同源蛋白。
在另一优选例中，硫结合结构域蛋白(SBD)来源于选自下组的物种：MMoMcrA、ScoMcrA、SprMcrA、HgaMcrA、EcoMcrA；优选地，来源于MMoMcrA。
在另一优选例中，所述SBD同源蛋白可结合于硫修饰寡核苷酸，并且具有一个或多个以下条件：
C1)具有硫修饰寡核苷酸(包括脱氧核糖核酸DNA或核糖核酸RNA在内的核苷酸)结合活性的蛋白结构域；
C2)与SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu中任意一个蛋白的氨基酸序列的同一性≥20％(较佳地≥50％，更佳地≥70％，最佳地≥80％ 或≥90％)；
C3)包含P-L-W基序或A-L-W基序(例如：SBDmmo中的P75-L79-W85；SBDspr中的P79-L83-W89；SBDtcu中的A73-L77-W83)。
在另一优选例中，所述RNA编辑效应蛋白选自下组：ADAR2DD、ADAR2DD(E488Q)、ADAR2DD(E488Q/T375G)、APOBEC、AID或CDA1。
在另一优选例中，所述的靶向元件和/或所述编辑效应元件各自独立地为实施例中所制备的碱基编辑器的对应元件。
在另一优选例中，所述的靶向元件和所述编辑效应元件通过化学键(如肽键)直接相连或通过接头连接。
在另一优选例中，所述的接头包括柔性接头和非柔性接头。
在另一优选例中,所述融合蛋白含有一个或多个SBD。
在另一优选例中,所述多个SBD可以相同也可以不同。
在另一优选例中,所述多个SBD可位于RNA编辑效应蛋白的同一侧(例如左侧或右侧)，也可以位于编辑效应蛋白的两侧。
在另一优选例中，所述融合蛋白的结构如下式I或I'或I”所示：
A-L-B(I)
B-L-A(I')
A-L-B-L-A(I”)
式中，
A为靶向元件SBD；
B为编辑效应元件RNA编辑效应蛋白；
L各自独立地为无或连接肽；
各“-”独立地为化学键。
在另一优选例中，所述的化学键包括肽键、或共价键。
在另一优选例中，所述的融合蛋白的序列如SEQ ID No:4所示，或SEQ ID No:4中第1-565位或第2-565位所示。
在另一优选例中，所述gDNA或gRNA的序列长度大于15nt，小于100nt,其中含有一针对待编辑碱基(A)的错配突变(C)，以及距离待编辑位点约9-20nt的至少一个硫修饰碱基。
在另一优选例中，所述gDNA或gRNA的序列长度为15-25nt。
在另一优选例中，所述硫修饰碱基可以为一个碱基。
在另一优选例中，所述硫修饰碱基可以为N个连续的碱基(1≤N≤10)。
在另一优选例中，所述硫修饰碱基优选地为C或G。
在另一优选例中，所述硫修饰碱基可在编辑位点的5'端方向或3'端方向。
在另一优选例中，所述的向导元件除了硫修饰，还可以带有其他化学修饰， 比如氟代修饰、甲基化修饰、甲氧基修饰、锁核酸(LNA)等等。
在本发明的第二方面，提供了一种用于碱基编辑的反应体系，所述的反应体系包括：
(a)本发明第一方面所述的碱基编辑器；和
(b)向导元件，所述向导元件引导SBD融合蛋白特异性结合于待碱基编辑的核酸序列的预定区域；
其中，所述的待碱基编辑的核酸为单链DNA、单链RNA、或其组合。
在另一优选例中，所述的反应体系还包括(c)待碱基编辑的核酸。
在另一优选例中，所述的检测体系还包括：(d)缓冲液。
在另一优选例中，所述的待碱基编辑的核酸在所述反应体系(尤其是体外反应系统)中的浓度为0.0001-1000nM，较佳地0.01-500nM，更佳地0.1-100nM。
在另一优选例中，所述的待碱基编辑的核酸在所述反应体系中的浓度为1至1×10 8拷贝/微升，较佳地2至1×10 5拷贝/微升，更佳地5至1×10 3拷贝/微升。
在另一优选例中，所述的反应体系中，所述向导元件与所述待碱基编辑的核酸的摩尔比为1:10至10 5:1，较佳地1:1至1000:1，更佳地2:1至100：1。
在另一优选例中，所述的向导元件为硫修饰的gDNA或gRNA。
在另一优选例中，所述的gDNA或gRNA的长度为15-100nt，较佳地16-50nt，更佳地18-40nt。
在另一优选例中，所述待碱基编辑的核酸包括来源于选自下组的待碱基编辑的核酸：植物、动物、昆虫、微生物、病毒、或其组合。
在另一优选例中，所述的待碱基编辑的核酸是人工合成或天然存在的mRNA。
在另一优选例中，所述的待碱基编辑的核酸包括野生型或突变型的mRNA。
在另一优选例中，所述的SBD选自下组：与SBDsco，SBDspr，SBDmmo，SBDhga，SBDeco，SBDtcu中任意一个蛋白的氨基酸序列的同一性≥20％或其组合；更佳的，所述SBD为SBDmmo。
在本发明的第三方面，提供了一种核酸，所述的核酸编码本发明第一方面所述的碱基编辑器。
在另一优选例中，所述的核酸为线性序列。
在另一优选例中，所述核酸具有5'-3'(5'至3')的式II结构：
P1-X1-L1-X2-X3(II)
P1-X2-L1-X1-X3(II')；
式中，P1为第一启动子序列；
X1为硫结合结构域蛋白(SBD)的编码序列；
L1为无或连接序列的编码序列；
X2为RNA编辑效应蛋白的编码序列；
X3为polyA序列；
并且，各“-”独立地为键。
在另一优选例中,所述核酸构建物含有一个或多个SBD编码序列。
在另一优选例中,所述多个SBD编码序列可以相同也可以不同。
在另一优选例中,所述多个SBD编码序列可位于RNA编辑效应蛋白编码序列的同一侧(例如左侧或右侧)，也可以位于编辑效应蛋白编码序列的两侧。
在本发明第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸。
在另一优选例中，所述的载体包括表达载体。
在另一优选例中，所述的载体包括质粒、病毒载体。
在本发明的第五方面，提供了一种基因工程细胞，所述细胞含有第四方面所述的载体，或者基因组中整合有第三方面所述的核酸。
在另一优选例中，所述的细胞被所述的载体或核酸所转化或转染。
在另一优选例中，所述的细胞还被gDNA或gRNA或其表达载体所转染。
在另一优选例中，所述的细胞包括真核细胞、原核细胞。
在另一优选例中，所述的细胞包括细菌、酵母、哺乳动物细胞、植物细胞。
在本发明的第六方面，提供了一种试剂盒，所述试剂盒包含：
(a1)第一容器，以及位于所述第一容器中的本发明第一方面所述的碱基编辑器、或其编码序列、或含所述编码序列的载体；和
(b1)第二容器，以及位于所述第二容器中的向导元件，
其中，所述的碱基编辑器在向导元件的引导下，可结合于预定的核酸区域，并对预定的核酸区域内或邻近的核苷酸进行碱基编辑。
在另一优选例中，所述的向导元件包括gDNA、gRNA或其组合。
在本发明的第七方面，提供了一种碱基编辑的方法，包括步骤：
(a)在向导元件的存在下，使得本发明第一方面所述的碱基编辑器与待碱基编辑的核酸形成“碱基编辑器-待碱基编辑的核酸-向导元件”复合物，从而使得所述碱基编辑器在预定的核酸区域内或邻近的核苷酸进行碱基编辑。
在另一优选例中，所述的预定的核酸区域内或邻近的核苷酸指与结合位点±30nt(较佳地±20nt，更佳地±10nt)的任何一个或多个核苷酸。
在另一优选例中，所述的待碱基编辑的核酸选自下组：单链DNA、单链RNA、或其组合
在另一优选例中，所述的待碱基编辑的核酸为mRNA。
在另一优选例中，所述的待碱基编辑的核酸为分离的、胞内的、或无细胞体系的靶核酸。
在另一优选例中，所述的mRNA为分离的mRNA、胞内的mRNA、或无细胞的mRNA。
在另一优选例中，所述的碱基编辑包括单碱基编辑、多碱基编辑。
在另一优选例中，所述的方法包括体内方法、体外方法。
在另一优选例中，所述的方法为非诊断和非治疗的方法。
在另一优选例中，所述方法包括：
(i)对靶mRNA与gRNA或gDNA进行退火反应，从而形成第一反应溶液；
(ii)在第一反应溶液中添加所述的碱基编辑器。
在本发明的第八方面，提供了本发明第一方面所述的碱基编辑器的用途，它用于制备对mRNA进行碱基编辑的试剂或试剂盒。
在另一优选例中，所述的碱基编辑包括体内或体外的碱基编辑。
在另一优选例中，所述的试剂盒还包括：向导元件。
在本发明的第八方面，提供了一种基因治疗方法，包括步骤：给需要的对象使用本发明第一方面所述的碱基编辑器或其编码序列；和特异性结合于待碱基编辑的核酸的向导元件。
在另一优选例中，所述的向导元件结合于致病基因的预定区域。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了SBD同源蛋白的氨基酸序列比对。红色三角标注的是P-L-W或A-L-W基序。
图2显示了硫修饰DNA的化学结构。
图3显示了硫修饰DNA结合结构域对硫修饰核酸的结合活性。
图4显示了利用硫修饰核酸及硫修饰核酸识别蛋白进行RNA单碱基编辑的 流程示意图。
图5显示了SBD Mmo-ADAR2融合蛋白在gDNA介导下，对绿色荧光蛋白mRNA的体外编辑效率(平板)。
图6显示了SBD Mmo-ADAR2融合蛋白在gDNA介导下，对绿色荧光蛋白mRNA的体外编辑效率(平板)。
图7显示了SBD Mmo-ADAR2融合蛋白在gRNA介导下，对绿色荧光蛋白mRNA的体外编辑效率(平板)。
图8显示了SBD Mmo-ADAR2融合蛋白在gRNA介导下，对绿色荧光蛋白mRNA的体外编辑效率(测序)。
图9显示了SBD Mmo与ADAR2融合蛋白对绿色荧光蛋白mRNA的细胞体内编辑效率(荧光显微镜成像)。A)正对照，在293T细胞中转染GFP蛋白表达质粒；B)负对照，在293T细胞中转染GFP突变体蛋白表达质粒，并转染gRNA-7S；C)实验组，在293T细胞中共转染GFP突变体蛋白表达质粒及SBDMmo-ADAR2融合蛋白表达质粒，并并转染gRNA-7S。左右视图分别是暗场和明场成像，明场曝光时间为5ms，暗场曝光时间为800ms。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次开发了一种结构新颖，体积小巧的碱基编辑器。本发明的碱基编辑器基于硫修饰DNA识别结构域，在硫修饰核酸(如gDNA或gRNA)作为向导元件的引导下，可对单链核酸(如单链的RNA或DNA)进行高效而特异的单碱基编辑。实验结果表明，本发明的碱基编辑器可成功地对报告基因RNA进行体外及体内的单碱基定向编辑。在此基础上,完成了此发明。
术语
如本文所用，术语“硫修饰”也可称为“磷硫酰化修饰”，是指对本文所述的单碱基编辑器的向导元件进行的修饰；具体地，是指DNA或RNA磷酸骨架上非桥联氧原子被硫原子取代而发生的修饰。
如本文所用，术语“硫修饰核酸识别蛋白”，“硫结合结构域蛋白”以及“SBD”可互换使用，均指能够特异性识别带有硫修饰核酸的蛋白。
如本文所用，术语“硫修饰核酸”，或“被硫修饰的核酸”以及均表示核酸带有硫基团修饰；其中，所述核酸可以是RNA，也可以是DNA。
如本文所用，术语“硫修饰DNA”与“S-gDNA”可互换使用；术语“硫修饰RNA”与“S-gRNA”可互换使用。
如本文所用，术语“gRNA”或“gDNA”是指可与RNA靶编辑位点前后的 的碱基互补配对的一段单链核酸序列，其包含一与靶编辑位点错配的碱基，以及靠近3'端的一个或多个位点的硫修饰，引导SBD到达RNA靶编辑位点。
如本文所用，“插入/缺失”指核酸内的核苷酸碱基的插入或缺失。此类插入或缺失可以导致基因编码区内的移码突变。
碱基编辑器
如本文所用，术语“本发明的碱基编辑器”、“本发明的融合蛋白”可互换使用，指本发明第一方面中所述的碱基编辑器,即包含了至少一个所述靶向元件和至少一个所述编辑效应元件的融合蛋白,其中所述靶向元件包含硫修饰核酸识别蛋白，而所述编辑效应元件为RNA编辑效应蛋白。
本发明的碱基编辑器能够修饰特定核苷酸碱基而不产生显著比例的插入/缺失。
在一些实施方案中，期望产生有效修饰(例如突变或脱氨基化)核酸内的特定核苷酸，而不在核酸中产生大量插入或缺失(即插入/缺失)的碱基编辑器。在某些实施方案中，本文提供的任何碱基编辑器能够产生相对于插入/缺失更大比例的意图修饰(例如，点突变或脱氨基化)。
本发明的任何碱基编辑器能够有效地在核酸(例如基因组内的核酸)中产生意图的突变，如点突变，而不产生大量的非意图突变，诸如非意图点突变。
在本发明中，碱基编辑器主要包括腺嘌呤脱氨酶，其他类型的碱基编辑器只要具备本发明的碱基编辑器的功能也在本发明的保护范围内。
硫结合结构域蛋白(SBD)
硫结合结构域蛋白简称SBD,是限制性内切酶的识别结构域。在本发明中，进行了融合蛋白的构建，SBD作为靶向结构域，将碱基编辑器带到待编辑的RNA处。
SBD可以位于融合蛋白的N端或C端(实施例中SBD位于融合蛋白的N端)，若SBD位于融合蛋白C端，即编辑器位于融合蛋白N端时，gDNA或gRNA的硫修饰碱基位于编辑位点的5'方向。融合蛋白中也可以存在多个SBD结构域，比如SBD-ADAR-SBD的形式，以提高编辑效率，这种情况下使用的gDNA或gRNA在编辑位点的5'端和3'端都分别有硫修饰碱基。此外，SBD和编辑器还可以有多种排列组合方式，如SBD-SBD-ADAR,ADAR-SBD-SBD等等。
在本发明中，代表性的所述硫结合结构域蛋白(SBD)包括：SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu、或SBD同源蛋白。
一些代表性的SBD同源蛋白的氨基酸序列比对结果见图1。
RNA编辑效应蛋白
本发明的碱基编辑器，还包含一编辑效应元件。应理解，可用于本发明的编辑效应元件没有特别限制。应理解，可适用于本发明的编辑效应元件包括野生型和突变型的编辑效应元件，只要该编辑效应元件能够对将特定位点的碱基进行催化，从而通过例如脱氨反应或其他类似的反应，将一种碱基转化为另一种碱基，例如将A转变为I。
在本发明中，代表性的编辑效应元件包括(但并不限于)以下RNA编辑效应蛋白：ADAR2DD、ADAR2DD(E488Q)、ADAR2DD(E488Q/T375G)、APOBEC、AID或CDA1。其中ADAR2DD、ADAR2DD(E488Q)、ADAR2DD(E488Q/T375G)分别为腺苷脱氨酶ADAR2(Adenosine to inosine acting on RNA enzyme 2)的野生型以及改良后编辑效应提高的突变体。
在本发明中，一种优选的编辑效应元件是在RESCUE系统中使用的ADAR2突变体。
硫修饰的核酸
在本发明中，所述的硫修饰的核酸指核酸中磷酸基团中的一个OH(O或O -)被SH(或S -)所取代和或一个＝0被＝S替换。
在另一优选例中，所述的硫修饰的核酸指核酸中的
Figure PCTCN2021121066-appb-000003
被修饰为
Figure PCTCN2021121066-appb-000004
或其对应异构体。
一种代表性的硫修饰的核酸的结构如图2所示。
硫修饰DNA结合结构域对某些硫修饰核酸的结合活性如图3所示。
碱基编辑方法、试剂盒、反应体系和应用
本发明还提供了一种采用本发明碱基编辑器进行碱基编辑的方法、相应的试剂盒、反应体系和应用。
典型地，本发明的碱基编辑方法包括步骤：在向导元件的存在下，使得权利要求1所述的碱基编辑器与待碱基编辑的核酸形成“碱基编辑器-待碱基编辑的核酸-向导元件”复合物，从而使得所述碱基编辑器在预定的核酸区域内或邻近的核苷酸进行碱基编辑。
一种代表性的利用硫修饰核酸及硫修饰核酸识别蛋白进行RNA单碱基编辑 的流程如图4所示。
在本发明中，所述的碱基编辑方法可以是体外或体内方法。
本发明的碱基编辑方法可用于基因治疗，例如治疗哺乳动物(如人)的某些疾病。本发明方法可以在不改变基因组DNA的情况下，对胞内的mRNA进行有效的编辑。
本发明的主要优点在于：
(1)本发明提出了一种新型的碱基编辑器，以硫修饰gDNA或gRNA靶向编辑位点，通过SBD作为靶向结构域，将编辑器带到待编辑的RNA处。
(2)与现有技术相比，本发明所用硫修饰DNA或S修饰RNA在细胞体内的半衰期更长，更稳定，不易降解。
(3)与现有技术相比，本发明所用SBD相较于Cas13更小(SBD仅有16kDa)，S-gRNA或S-gDNA最小可为20nt，这使得由硫修饰核酸和SBD组成的编辑系统体积上要远远小于依赖于Cas蛋白的编辑系统，更利于包装和递送至胞内。
(4)本发明方法可以在不改变基因组DNA的情况下，对胞内的mRNA进行有效的编辑。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条例，例如J.Sambrook等人，分子克隆实验指南(第四版)(科学出版社有限责任公司，2017)中所述的条件，或按照产品制造商提供的产品说明书中所述条件。实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。
实施例1
SBDmmo-ADAR2融合蛋白纯化
1.通过基因合成，将ADAR2DD(E488Q/T375G)合成到酵母表达载体pESC-URA，并添加SalI和BamHI酶切位点，合成序列如下(SEQ ID No：1)：
Figure PCTCN2021121066-appb-000005
Figure PCTCN2021121066-appb-000006
其中，粗体部分为载体序列，小写字母序列为SalI及BamHI酶切位点，斜体部分为ADAR2DD(E488Q/T375G)序列，下划线部分为组氨酸标签序列。
以上述质粒出发构建SBDmmo-ADAR2融合蛋白表达载体，扩增引物序列如下：
SBDmmo-F：5'-CCGctcgagATGATTAGCCCGGAAACCCT-3'(SEQ ID No：2)
SBDmmo-R：5'-CGCggatccGCTTCCACCTCCTCCACGGAAATGCGGATCGCGGT-3'(SEQ ID No：:3)
2.在酿酒酵母(Saccharomyces cerevisiae)宿主BY4741中进行SBDmmo-ADAR2融合蛋白的异源表达。挑取携带表达载体的单克隆至U -液体培养基中(含2％葡萄糖)，30℃，280rpm过夜生长，作为种子液。将种子液离心处理去上清，接种于U -液体培养基(含2％raffinose)中，30℃，280rpm，诱导蛋白表达。
3.SBDmmo-ADAR2融合蛋白的纯化。
菌液裂解：离心收集菌液，弃上清，将菌体重悬于50mL蛋白纯化Buffer A(20mM Tris-HCl pH 8.0，300mM NaCl,20mM imidazole)中，使用预冷的细胞破碎仪破碎菌体，细胞裂解液4℃，10000rpm离心1h，取上清，用0.22μM滤膜过滤后冰上放置。
Ni柱纯化：用Buffer A(20mM Tris-HCl pH 8.0，300mM NaCl,50mM imidazole,)平衡Ni亲和层析柱(GE)，将细胞裂解液上样至Ni亲和层析柱上，用 Buffer A冲洗5个柱体积后，线性提高Buffer B(20mM Tris-HCl pH 8.0，300mM NaCl,500mM imidazole)浓度，SBDmmo-ADAR2融合蛋白大约在35％-65％浓度区间内被洗脱下来。全部过程在AKTA纯化系统(GE)上进行。
肝素柱纯化：用Buffer A(20mM Tris-HCl pH 8.0，50mM NaCl,1mM DTT)平衡Heparin亲和层析柱(GE)，将Ni柱纯化得到的蛋白稀释后上样，用Buffer A冲洗5个柱体积后，线性提高Buffer B(20mM Tris-HCl pH 8.0，1000mM NaCl,1mM DTT)浓度，SBDmmo-ADAR2融合蛋白大约在30％-60％浓度区间内被洗脱下来。全部过程在AKTA纯化系统(GE)上进行。
得到的SBDmmo-ADAR2融合蛋白的序列如SEQ ID NO：4所示。
Figure PCTCN2021121066-appb-000007
实施例2
利用SBDmmo-ADAR2融合蛋白与gDNA体外进行绿色荧光蛋白的RNA单碱基编辑
1.GFP(绿色荧光蛋白)突变RNA底物的制备
将GFP基因构建在pET28a大肠杆菌蛋白表达载体上，使用突变引物对GFP DNA序列进行点突变，使其中一个G突变为A，突变引物序列如下：
GFP-Mut-F：5'-TGTTCCATAGCCAACACTTGTCACT-3'(SEQ ID No：5)
GFP-Mut-R：5'-GTGTTGGCTATGGAACAGGCAGCTT-3'(SEQ ID No：6)
突变后在GFP内部引入一个终止密码子，使得GFP无法在表达宿主中完整表达，宿主不发出绿色荧光。
使用RNA转录试剂盒，获得突变后的GFP突变RNA底物，并添加DNaseI去除体系中的残留DNA，用PCR验证，保证无DNA残留。
2.gDNA(guid DNA)准备
在GFP突变位点处设计一系列单链硫修饰gDNA，gDNA整体上与GFP序列 互补配对，但在突变位点处形成一个C-A的错配，gDNA中的硫修饰位点位于突变位点的3'端，并设计了一条不带硫修饰的gDNA-0作为阴性对照，序列如下：
gDNA-0：5'-ACAAGTGTTGGCCATGGAACAGGCAGCTTGCCGGTAGTGC-3'(SEQ ID No：7)
gDNA-9：5'-ACAAGTGTTGGCCATGGAACAG *GCAGCTTGCCGGTAGTGC-3'(SEQ ID No：8)
gDNA-13：5'-ACAAGTGTTGGCCATGGAACAGGCAG *CTTGCCGGTAGTGC-3'(SEQ ID No：9)
gDNA-17：5'-ACAAGTGTTGGCCATGGAACAGGCAGCTTG *CCGGTAGTGC-3'(SEQ ID No：10)
gDNA-20：5'-ACAAGTGTTGGCCATGGAACAGGCAGCTTGCCG *GTAGTGC-3'(SEQ ID No：11)
gDNA-7S：5'-ACAAGTGTTGGCCATGGAACAGGCAG *C *T *T *G *C *C *GGTAGTGC-3'(SEQ ID No：12)
备注：其中，红色加粗的C代表与突变位点A形成错配的C，*代表硫修饰位点
3.在gDNA介导下，SBDmmo-ADAR2融合蛋白对GFP突变RNA的编辑反应
首先将底物mRNA与gDNA在PCR仪中进行退火反应，体系中添加终浓度为100nM的底物RNA，200nM的gDNA，并添加100mM NaCl、1mM DTT、20mM Tris-HCl pH8.0。
退火结束后，在体系中添加800nM的SBDmmo-ADAR2融合蛋白，37℃反应1h。
4.编辑效率检测
将编辑反应后的RNA进行反转录，反转录后用pfu聚合酶进行cDNA的扩增，使用的引物序列如下：
cDNA-F：AGGAGATATACATATGAGTAAAGGAGAAGA(SEQ ID No：13)
cDNA-R：TAGCAGCCGGATCTCAGTGGTGGTGGTGGT(SEQ ID No：14)
将扩增得到的片段克隆至Pet28a载体上，并在大肠杆菌BL21宿主中进行表 达。若发生RNA编辑，则GFP蛋白可以正常翻译并表达，使得宿主细胞发荧光。
5.结果
如图5所示。当无SBDmmo-ADAR2融合蛋白添加时，发光克隆在总克隆数中占比极低；当无gDNA添加时，编辑效率为0％；当gDNA为未硫修饰的gDNA-0时，编辑效率低；当gDNA选取为gDNA-13和gDNA-7S时，分别有37.64％和48.55％的编辑效率。此外，图6也显示了以gDNA-13和gDNA-7S为gDNA进行RNA编辑后，样本进行测序时在待编辑位点出现了明显的双峰，突变频率与图5中结果相接近。
实施例3
利用SBDmmo-ADAR2融合蛋白与硫修饰gRNA体外进行绿色荧光蛋白的RNA单碱基编辑
1.GFP(绿色荧光蛋白)突变RNA底物与实施例2中一致。
2.gRNA(guid RNA)准备
在GFP突变位点处设计单链硫修饰gRNA，参考实施例1中的结果，设计Grna-13及gRNA-7S两条单链硫修饰gRNA，gRNA整体上与GFP序列互补配对，但在突变位点处形成一个C-A的错配，gRNA中的硫修饰位点位于突变位点的3'端，并设计了一条不带硫修饰的gRNA-0作为阴性对照，序列如下：
gRNA-13:5'-ACAAGUGUUGGCCAUGGAACAGGCAG*CUUGCCGGUAGUGC-3'(SEQ ID No：15)
gRNA-7S:5'-ACAAGUGUUGGCCAUGGAACAGGCAG*C*U*U*G*C*C*GGUAGUGC-3'(SEQ ID No：16)
gRNA-0：5'-ACAAGUGUUGGCCAUGGAACAGGCAGCUUGCCGGUAGUGC-3'(SEQ ID No：17)
将gRNA溶解于Rnase free水中，终浓度为10pmol/μl，并置于-80℃冰箱中保存。
3.在gRNA介导下，SBDmmo-ADAR2融合蛋白对GFP突变RNA的编辑反应。
首先将底物mRNA与gRNA在PCR仪中进行退火反应，体系中添加终浓度为100nM的底物RNA，200nM的gRNA，并添加100mM NaCl、1mM DTT、20mM Tris-HCl pH8.0。
退火结束后，在体系中添加800nM的SBDmmo-ADAR2融合蛋白，37℃反 应1h。
4.编辑效率检测
将编辑反应后的RNA进行反转录，反转录后用pfu聚合酶进行cDNA的扩增，使用引物为cDNA-F(SEQ ID No：13)、cDNA-R(SEQ ID No：14)。将扩增得到的片段克隆至Pet28a载体上，并在大肠杆菌BL21宿主中进行表达。若发生RNA编辑，则GFP蛋白可以正常翻译并表达，使得宿主细胞发荧光。
结果如图7所示。结果表明，当无SBDmmo-ADAR2融合蛋白添加时，发光克隆在总克隆数中占比极低；当gDNA为未硫修饰的gDNA-0时，编辑效率低；当gDNA选取为gDNA-13和gDNA-7S时，分别有48.2％和69.2％的编辑效率。此外，图8也显示了以gDNA-13和gDNA-7S为gDNA进行RNA编辑后，样本进行测序时在待编辑位点出现了明显的双峰，突变频率与图7中结果相接近。
实施例4
利用SBDmmo-ADAR2融合蛋白与硫修饰gRNA体内进行绿色荧光蛋白的RNA单碱基编辑
1.SBDmmo-ADAR2融合蛋白表达载体、报告基因(绿色荧光蛋白突变体)表达载体的构建。选取pcDNA3.1质粒作为出发质粒，将SBDmmo-ADAR2基因或绿色荧光蛋白突变基因插入到BamH1/Xba1位点，并加入Kozak序列，使用的引物序列如下：
SBD-ADAR2-F：cgggatcccATGATTAGCCCGGAAACC(SEQ ID No：18)
SBD-ADAR2-R：gctctagaTTAGTGGTGGTGGTGGTGGTGCTCGAGCGTGAGTG(SEQ ID No：19)
GFP-F：cgggatccc atgagtaaaggagaag(SEQ ID No：20)
GFP-R：gctctaga tcagtggtggtggtggtggtgctcgagtttgtata(SEQ ID No：21)
2.宿主细胞的构建。将新鲜的HEK293T细胞用DMEM培养基培养，并补充10％FBS、1％青霉素及1％链霉素。将SBDmmo-ADAR2融合蛋白表达载体和绿色荧光蛋白突变体表达载体转染至HEK293T细胞中，使用Lipofectamine转染试剂，过夜培养并进行细胞传代。
3.体内编辑及检测
体内编辑实验使用的gRNA与实施例2中一致。
转染gRNA转染至表达有SBDmmo-ADAR2融合和绿色荧光蛋白突变体的HEK293T细胞中，用荧光显微镜进行成像。结果如图9所示。
讨论
硫修饰作为一种化学修饰，其抗核酸酶活性等已经被深入利用；而这种修饰作为一种天然修饰存在于自然界中，具有能够被蛋白识别、参与限制修饰系统等功能。
本发明基于硫修饰核酸及其特异性结合蛋白建立一种新型的定位模块，即以硫修饰的ssRNA作为gRNA，与mRNA目标区域互补序列进行匹配，产生含有硫修饰的dsRNA；然后以SBD结构域与目标区域结合，实现定位的目的。进而通过将此模块与RNA编辑模块融合建立新型RNA编辑系统。
与目前的RNA编辑系统相比，SBD结构域仅有约170个氨基酸，比CRISPR-Cas13更为小巧，因此与比REPAIR系统和RESCUE系统相比，本发明的碱基编辑器更易被导入细胞。
与通过招募内源性的ADAR的RESTORE系统和LEAPER系统相比，SBD可以融合不同的RNA编辑器，包括定向进化而来的C->U编辑器，具有更广泛的编辑范围。
本发明的基于SBD的RNA编辑系统，理论上可以使用任意长度的gRNA，通过使用较长的gRNA，可能获得更加稳定的dsRNA结构，也可能获得较低的脱靶率和较高的效率，因此具有广阔的应用前景。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种碱基编辑器,其特征在于,所述碱基编辑器包括靶向元件和编辑效应元件，其中所述靶向元件与所述编辑效应元件形成一融合蛋白，其中所述靶向元件包含硫修饰核酸识别蛋白，所述编辑效应元件为RNA编辑效应蛋白。
  2. 如权利要求1所述的碱基编辑器，其特征在于，所述的碱基编辑器在向导元件的引导下，可结合于预定的核酸区域，并对预定的核酸区域内或邻近的核苷酸进行碱基编辑，所述的向导元件为单链的硫修饰核酸。
  3. 如权利要求2所述的碱基编辑器，其特征在于，所述的硫修饰核酸指核酸中磷酸基团中的一个或多个OH(O或O -)被SH(或S -)所取代和或一个或多个＝O被＝S替换。
  4. 如权利要求2所述的碱基编辑器，其特征在于，所述的向导元件包含硫修饰的gDNA或gRNA，所述gDNA或gRNA的序列长度大于15nt，小于100nt,其中含有一针对待编辑碱基(A)的错配突变(C)或者针对待编辑碱基(C)的错配突变(A)，以及在一侧或两侧距离待编辑位点约9-20nt的至少一个或多个硫修饰碱基。
  5. 如权利要求1所述的碱基编辑器，其特征在于，所述的碱基编辑器为单链核酸的碱基编辑器；较佳地，所述的单链核酸为单链DNA、单链RNA、或其组合。
  6. 如权利要求1所述的碱基编辑器，其特征在于，所述的硫修饰核酸识别蛋白为硫结合结构域蛋白(SBD)，所述SBD包括：SBDsco、SBDspr、SBDmmo、SBDhga、SBDeco、SBDtcu、或SBD同源蛋白。
  7. 如权利要求1所述的碱基编辑器，其特征在于，所述RNA编辑效应蛋白选自下组：ADAR2DD、ADAR2DD(E488Q)、ADAR2DD(E488Q/T375G)、APOBEC、AID或CDA1或其他具有RNA编辑功能的效应器。8.如权利要求1所述的碱基编辑器，其特征在于，所述融合蛋白的结构如下式I或I'或I”所示：
    A-L-B(I)
    B-L-A(I')
    A-L-B-L-A(I”)
    式中，
    A为靶向元件SBD；
    B为编辑效应元件RNA编辑效应蛋白；
    L各自独立地为无或连接肽；
    各“-”独立地为化学键。
  8. 如权利要求8所述的碱基编辑器，其特征在于，所述的融合蛋白的序列如 SEQ ID No:4所示，或SEQ ID No:4中第1-565位或第2-565位所示。
  9. 一种用于碱基编辑的反应体系，其特征在于，所述的反应体系包括：
    (a)如权利要求1所述的碱基编辑器；和
    (b)向导元件，所述向导元件引导SBD融合蛋白特异性结合于待碱基编辑的核酸序列的预定区域；
    其中，所述的待碱基编辑的核酸为单链DNA、单链RNA、或其组合。
  10. 一种核酸，其特征在于，所述的核酸编码权利要求1所述的碱基编辑器。
  11. 一种载体，其特征在于，所述的载体含有如权利要求11所述的核酸。
  12. 一种基因工程细胞，其特征在于，所述细胞含有权利要求12所述的载体，或者基因组中整合有权利要求11所述的核酸。
  13. 一种试剂盒，其特征在于，所述试剂盒包含：
    (a1)第一容器，以及位于所述第一容器中的权利要求1所述的碱基编辑器、或其编码序列、或含所述编码序列的载体；和
    (b1)第二容器，以及位于所述第二容器中的向导元件，
    其中，所述的碱基编辑器在向导元件的引导下，可结合于预定的核酸区域，并对预定的核酸区域内或邻近的核苷酸进行碱基编辑。
  14. 一种碱基编辑的方法，其特征在于，包括步骤：
    (a)在向导元件的存在下，使得权利要求1所述的碱基编辑器与待碱基编辑的核酸形成“碱基编辑器-待碱基编辑的核酸-向导元件”复合物，从而使得所述碱基编辑器在预定的核酸区域内或邻近的核苷酸进行碱基编辑。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180119141A1 (en) * 2016-10-28 2018-05-03 Massachusetts Institute Of Technology Crispr/cas global regulator screening platform
CN110511286A (zh) * 2019-08-29 2019-11-29 上海科技大学 一种rna碱基编辑分子
CN110527697A (zh) * 2018-05-23 2019-12-03 中国科学院上海生命科学研究院 基于CRISPR-Cas13a的RNA定点编辑技术
CN110869498A (zh) * 2017-05-10 2020-03-06 加利福尼亚大学董事会 经由核递送crispr/cas9导向编辑细胞rna
CN111629786A (zh) * 2017-10-06 2020-09-04 俄勒冈健康与科学大学 用于编辑rna的组合物和方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3234134B1 (en) * 2014-12-17 2020-05-27 ProQR Therapeutics II B.V. Targeted rna editing
US11390865B2 (en) * 2015-07-14 2022-07-19 Fukuoka University Method for introducing site-directed RNA mutation, target editing guide RNA used in the method and target RNA-target editing guide RNA complex
CA3035293A1 (en) * 2016-09-01 2018-03-08 Proqr Therapeutics Ii B.V. Chemically modified single-stranded rna-editing oligonucleotides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180119141A1 (en) * 2016-10-28 2018-05-03 Massachusetts Institute Of Technology Crispr/cas global regulator screening platform
CN110869498A (zh) * 2017-05-10 2020-03-06 加利福尼亚大学董事会 经由核递送crispr/cas9导向编辑细胞rna
CN111629786A (zh) * 2017-10-06 2020-09-04 俄勒冈健康与科学大学 用于编辑rna的组合物和方法
CN110527697A (zh) * 2018-05-23 2019-12-03 中国科学院上海生命科学研究院 基于CRISPR-Cas13a的RNA定点编辑技术
CN110511286A (zh) * 2019-08-29 2019-11-29 上海科技大学 一种rna碱基编辑分子

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAO YU, ET AL.: "DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses", vol. 15, no. 48, 4 September 2020 (2020-09-04), XP055922693 *

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