Heterodimer type gene editor as well as preparation method and application thereof

Abstract

The invention provides a heterodimer type gene editor as well as a preparation method and application thereof. Specifically, a Cas9 nicking enzyme (nCas9) element and a reverse transcriptase (RT) element (such as an RT element of an engineered Moroy mouse leukemia virus (M-MLV)) are respectively utilized, and a split PE (split PE, sPE) system (also called as a heterodimer gene editor) is constructed in a Cipe-coil (CC) peptide mediation mode. Compared with a non-split PE working system, the gene editor disclosed by the invention can be conveniently packaged into AAV and other virus vectors due to a unique split structure, so that the virus vectors can be efficiently delivered into cells, and wide application of the gene editor in gene editing, gene treatment and clinic is promoted.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a heterodimeric gene editor, its preparation method, and its application. Background Technology

[0002] Since 2013, next-generation gene editing technologies, represented by CRISPR / Cas9, have entered various experiments in the field of biology, changing traditional gene manipulation methods.

[0003] Prime Editing (PE) is a fusion protein of Cas9 nickase (nCas9) and engineered Moroni mouse leukemia virus (M-MLV) reverse transcriptase (RT). It also requires a 3′ extended sgRNA containing a reverse transcription template (RTT) and a primer binding site (PBS). It can perform precise arbitrary base substitutions, small fragment insertions and deletions of four nucleotides at the target site without double-strand DNA breaks (DSB) or exogenous donor DNA templates.

[0004] Despite their versatility and precision in genome editing, current PE (penetrating organelle) systems are large, hindering their efficient delivery in vivo. Adeno-associated virus (AAV), with its low immunogenicity, broad infectivity, and ease of production, is one of the most suitable vectors for delivering PE systems to target tissues for in vivo gene therapy. However, the large size of PE (greater than 6.3 kb) exceeds the packaging capacity of AAV.

[0005] Several splitting strategies have been reported, including those using integrins, MS2, SunTag, and direct splitting PE (sPE) systems. In the integrin-mediated splitting PE system, nCas9 is split into two parts, and the coding sequence of nCas9 is assembled by integrin-mediated trans-splicing. However, the selection of splitting sites is very difficult and affects editing efficiency. The existing MS2-PE and SunTag-PE systems are relatively complex because MS2 requires specially processed sgRNA, while SunTag requires multiple GCN4 repeat sequences. In addition, the editing efficiency of MS2-PE and SunTag-PE systems cannot be compared with that of unsplit PE systems [3]. In the direct splitting PE system, nCas9 is not connected to RT, however, considering the lack of affinity modules, RT may not bind to the target genomic site. Theoretically, the flexibility of PE may be further improved by creating protein-protein affinity module fusions.

[0006] Therefore, there is an urgent need in this field to develop split gene editing systems that can maintain comparable or better efficiency in targeting gene mutations. Summary of the Invention

[0007] The purpose of this invention is to provide a heterodimeric gene editor that can maintain comparable or better efficiency in targeting gene mutations.

[0008] In a first aspect of the present invention, a heterodimeric gene editor complex is provided, the complex comprising:

[0009] (a) A first fusion protein comprising a fused Cas9 nuclease and a first coiled-helical peptide element; and

[0010] (b) A second fusion protein comprising a reverse transcriptase and a second coiled-coil peptide element fused together;

[0011] The first fusion protein and the second fusion protein form a heterodimeric gene editor through the first coiled helical peptide element and the second coiled helical peptide element.

[0012] In another preferred embodiment, a dimerized pairing structure is formed through the interaction of the first coiled-coil peptide element and the second coiled-coil peptide element, thereby enabling the first fusion protein and the second fusion protein to combine and form a heterodimer, i.e., a heterodimer-type gene editor.

[0013] In another preferred embodiment, the heterodimeric gene editor has the function of precise genome editing.

[0014] In another preferred embodiment, the first fusion protein has a structure of Formula I from the N-terminus to the C-terminus or from the C-terminus to the N-terminus:

[0015] Y1-X1-L1-Z1(I)

[0016] in,

[0017] Y1 is either absent or a signal peptide;

[0018] X1 is a Cas9 nuclease element;

[0019] L1 is either absent or linked to a peptide element;

[0020] Z1 is the first coiled helical peptide element;

[0021] "-" indicates a peptide bond or peptide linker.

[0022] In another preferred embodiment, the second fusion protein has a structure of formula II from the N-terminus to the C-terminus or from the C-terminus to the N-terminus:

[0023] Y2-Z2-L2-X2(II)

[0024] in,

[0025] Y2 is either absent or a signal peptide;

[0026] Z2 is the second coiled helical peptide element;

[0027] L2 is either absent or linked to a peptide element;

[0028] X2 is a reverse transcriptase peptide element;

[0029] "-" indicates a peptide bond or peptide linker.

[0030] In another preferred embodiment, the Cas9 nuclease is selected from the group consisting of Cas9, nCas9, or combinations thereof.

[0031] In another preferred embodiment, the Cas9 nuclease is an nCas9 nuclease, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0032] In another preferred embodiment, the Cas9 nuclease is selected from Streptococcus pyogenes.

[0033] In another preferred embodiment, the first coiled helical peptide and the second coiled helical peptide form a coiled helical peptide pair, wherein the first coiled helical peptide is selected from P3 or N5; the second coiled peptide is selected from P4 or N6, and the peptide pair is selected from the P3-P4 peptide pair or the N5-N6 peptide pair.

[0034] In another preferred embodiment, the first coiled helical peptide is selected from P3; the second coiled peptide is selected from P4, and the peptide pair is a P3-P4 peptide pair.

[0035] In another preferred embodiment, the amino acid sequence of the reverse transcriptase is shown in SEQ ID NO: 2.

[0036] In a second aspect of the invention, a polynucleotide encoding the heterodimeric gene editor complex described in the first aspect of the invention is provided, the polynucleotide comprising:

[0037] (i) a first polynucleotide encoding the first fusion protein; and

[0038] (ii) A second polynucleotide, which encodes the second fusion protein.

[0039] In another preferred embodiment, the first polynucleotide and the second polynucleotide are located on the same nucleic acid chain.

[0040] In another preferred embodiment, the first polynucleotide and the second polynucleotide are located on different nucleic acid chains.

[0041] In another preferred embodiment, the length of the first polynucleotide is ≤5kb, more preferably ≤4.5kb, and even more preferably about 4.1kb.

[0042] In another preferred embodiment, the length of the second polynucleotide is ≤2.5kb, more preferably ≤2.3kb, and even more preferably about 2.1kb.

[0043] In a third aspect of the invention, a carrier is provided, comprising a first carrier and a second carrier, wherein the first carrier contains the first fusion protein and the second carrier contains the second fusion protein, wherein the first fusion protein and the second fusion protein are as defined in the first aspect of the invention.

[0044] In another preferred embodiment, the vector is selected from the group consisting of plasmids and viral vectors.

[0045] In another preferred embodiment, the vector is selected from adenovirus, adeno-associated virus, lentivirus, or a combination thereof.

[0046] In another preferred embodiment, the vector is adeno-associated virus.

[0047] In a fourth aspect of the invention, a genetically engineered cell is provided, the cell containing the polynucleotide described in the second aspect of the invention or the vector described in the third aspect of the invention.

[0048] In another preferred embodiment, the cells are selected from the group consisting of prokaryotic cells, eukaryotic cells, or combinations thereof.

[0049] In another preferred embodiment, the cells are selected from the group consisting of Escherichia coli and mammalian cells.

[0050] In another preferred embodiment, the mammal is a somatic cell.

[0051] In a fifth aspect of the invention, a composition is provided comprising the complex described in the first aspect of the invention, or the carrier described in the third aspect of the invention.

[0052] In another preferred embodiment, the composition includes a pharmaceutical composition or a laboratory formulation composition.

[0053] In a sixth aspect of the invention, the use of the composition described in the fifth aspect of the invention in the editing of a predetermined target gene is provided.

[0054] In another preferred embodiment, the gene editing includes in vivo gene editing and in vitro gene editing.

[0055] In another preferred embodiment, the gene editing includes both therapeutic and non-therapeutic gene editing.

[0056] In another preferred embodiment, the gene editing is non-diagnostic and non-therapeutic in vitro gene editing.

[0057] In another preferred embodiment, the gene editing is performed on mammalian cells (such as somatic cells of humans or non-human primates).

[0058] In a seventh aspect of the present invention, a gene editing method is provided, comprising the steps of:

[0059] (1) Provide the target nucleic acid sequence to be edited and the heterodimeric gene editor complex described in the first aspect of this invention; and

[0060] (2) In the presence of the heterodimeric gene editor complex, gene editing is performed on the provided nucleic acid target sequence to be edited.

[0061] In another preferred embodiment, the target sequence is from an in vitro or from a living organism.

[0062] In an eighth aspect of the present invention, a kit is provided, the kit comprising:

[0063] (a) a first container, and a first carrier located within the first container; and

[0064] (b) a second container, and a second carrier located within the second container; the first carrier containing the

[0065] The first fusion protein and the second carrier contain the second fusion protein, wherein the first fusion protein and the second fusion protein are as defined in the first aspect of the present invention.

[0066] In a ninth aspect of the invention, the use of the heterodimeric gene editor complex described in the first aspect of the invention or the vector described in the third aspect of the invention in the preparation of reagents and / or drugs for gene editing is provided.

[0067] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0068] Figure 1 The diagrams show the unresolved PE2 and CC-PE structures. Figure A shows the structure of the unresolved PE2, which is formed by fusing nCas9 and RT based on a linker under the guidance of pegRNA; Figure B shows the coiled coil peptide-mediated PE (CC-PE) structure of the present invention, which is formed by fusing nCas9 and RT based on a coiled coil peptide under the guidance of pegRNA.

[0069] Figure 2A schematic diagram of CC-PE is shown. Wherein, CMV: promoter; NLS: nuclear localization signal; pA: PolyA sequence; PE2: unresolved PE; sPE-P3P4 contains the C-terminal fusion fragment of the detached peptide P3 with nCas9 and the N-terminal fusion fragment of peptide P4 with RT; sPE-N5N6 contains the C-terminal fusion fragment of the detached peptide N5 with nCas9 and the N-terminal fusion fragment of peptide N6 with RT; sPE-ctrl: directly detached PE (sPE) without affinity modules (P3-P4 pair or N5-N6 pair).

[0070] Figure 3 This image shows the results of Sanger sequencing detection of CC-PE's ability to detect GAAT base deletion at the human B2M locus. In the upper image, within the DNA double strand at the human B2M locus, red base sequences represent the spacer sequence of gRNA; green base sequences represent the PAM recognition region; blue background base sequences represent the pegRNA PBS sequence; and gray background base sequences represent the pegRNA RT sequence. The purple arrows in the lower image indicate the positions of the deleted bases. The red, green, blue, and black peaks represent thymine, adenine, cytosine, and guanine, respectively.

[0071] Figure 4 The results of deep sequencing detection of CC-PE's ability to detect GAAT base deletions at the human B2M locus are shown. Figure A illustrates the spacer and PAM sequences at the B2M locus, with the spacer and PAM sequences (sPE-P3P4, sPE-N5N6, sPE-ctrl, and PE2) at the B2M locus without CAAT deletions as a reference, and the spacer and PAM sequences (sPE-P3P4, sPE-N5N6, sPE-ctrl, and PE2) at the B2M locus with CAAT deletions, from top to bottom. Figure B shows the results of deep sequencing detection of GAAT base deletions at the human B2M locus, where "precise editing" indicates precise editing efficiency, "Indels" indicates insertion and deletion efficiency, and "Untreated" represents the untreated control group.

[0072] Figure 5 The results of deep sequencing detection of CC-PE's ability to detect a 1-T base deletion at the VEFGA locus are shown in the figure.

[0073] Figure 6The results of deep sequencing detection of CC-PE substitution of a single T base for A at the HEK3 locus are shown. Figure A represents a schematic diagram of the spacer and PAM sequences at the HEK3 locus, where, from top to bottom, the spacer and PAM sequences of the HEK3 locus without substitution of a single T base for A are shown as a reference, and the spacer and PAM sequences of the HEK3 locus with substitution of a single T base for A are shown as sPE-P3P4, sPE-N5N6, sPE-ctrl, and PE2. Figure B represents the results of deep sequencing detection of substitution of a single T base for A at the HEK3 locus, where Precise editing indicates precise editing efficiency, Indels indicate insertion and deletion efficiency, and Untreated represents the untreated control group.

[0074] Figure 7 The results of deep sequencing detection of CC-PE for inserting a single C base at the VEGFA locus are shown. Figure A shows a schematic diagram of the spacer and PAM sequences at the VEGFA locus, where, from top to bottom, the spacer and PAM sequences of the VEGFA locus without a single C base insertion are shown as a reference, and the spacer and PAM sequences of the VEGFA locus with a single C base insertion are sPE-P3P4, sPE-N5N6, sPE-ctrl, and PE2. Figure B shows the results of deep sequencing detection of a single C base insertion at the VEGFA locus, where Precise editing indicates precise editing efficiency, Indels indicate insertion and deletion efficiency, and Untreated represents the untreated control group.

[0075] Figure 8 The image shows the results of deep sequencing detection of CC-PE insertion of a single A base into the HEK3 locus. Precise editing indicates the precision editing efficiency, Indels indicate insertion and deletion efficiency, and Untreated represents the untreated control group.

[0076] Figure 9The results of Sanger sequencing detection of CC-PE's ability to insert GT bases into the human DMD locus are shown. In Figure A, the upper part represents the DNA double strand at the human DMD locus; red base sequences represent the spacer sequence of gRNA; green base sequences represent the PAM recognition region; blue background base sequences represent the pegRNA PBS sequence; and gray background base sequences represent the pegRNA RT sequence. The purple arrows in the lower part indicate the insertion positions; red, green, blue, and black peaks represent thymine, adenine, cytosine, and guanine, respectively. Figure B shows the results of deep sequencing detection of CC-PE inserting one AC base into the DMD locus. Precise editing indicates the precision editing efficiency, Indels indicate insertion and deletion efficiency, and Untreated represents the untreated control group.

[0077] Figure 10 The graphs show the unexpected insertion and deletion efficiency of CC-PE at the HEK3 gene site (where one T base is replaced with an A). Figure A represents the total insertion and deletion frequency of CC-PE at the HEK3 gene site (where one T base is replaced with an A), where Unmodified: total insertion and deletion frequency; Undesired indel: unexpected insertion and deletion frequency. Figures B and CF represent the specific unexpected insertion and deletion frequencies of CC-PE at the HEK3 gene site (where one T base is replaced with an A), respectively.

[0078] Figure 11 The graphs show the unexpected insertion and deletion efficiencies of CC-PE at the B2M gene (1 GAAT base deletion) site. Figure A represents the total insertion and deletion frequency of CC-PE at the B2M gene (1 GAAT base deletion) site, where Unmodified: total insertion and deletion frequency; Undesired indel: unexpected insertion and deletion frequency. Figures B and CF represent the specific unexpected insertion and deletion frequencies of CC-PE at the B2M gene (1 GAAT base deletion) site, respectively.

[0079] Figure 12 The graphs show the unexpected insertion and deletion efficiencies of CC-PE at the VEGFA gene (with a 1-C base insertion) site. Figure A represents the total insertion and deletion frequency of CC-PE at the VEGFA gene (with a 1-C base insertion) site, where Unmodified represents the total insertion and deletion frequency, and Undesired indel represents the unexpected insertion and deletion frequency. Figures B and CF represent the specific unexpected insertion and deletion frequencies of CC-PE at the VEGFA gene (with a 1-C base insertion) site, respectively.

[0080] Figure 13 The results of deep sequencing detection of CC-PE base insertion at the mouse mPcsk9 locus are shown. Figure A shows the result of deep sequencing detection of CC-PE inserting 2 TGA bases at the mouse mPcsk9 locus; Figure B shows the result of deep sequencing detection of CC-PE inserting 1 T base at the mouse mPcsk9 locus; EpigRNA: pegRNA modified with the RNA structural group evopreQ1.

[0081] Figure 14 The diagram illustrates the use of CC-PE for in vivo genome editing via dual AAVs. Figure A shows the structure of sPE-P3P4, where ITR is the terminal repeat sequence of the viral vector, EFS is the EF1a short promoter, P2A is the cleavage peptide, mCherry is red fluorescent protein, and U6 is the U6 promoter. Figure B shows the structure of sPE-ctrl, where ITR is the terminal repeat sequence of the viral vector, EFS is the EF1a short promoter, P2A is the cleavage peptide, mCherry is red fluorescent protein, and U6 is the U6 promoter. Figure C shows the flowchart of intraperitoneal injection of dual AAV8 sPE-P3P4 or sPE-ctrl in mice and subsequent experiments.

[0082] Figure 15 The image shows the results of Sanger sequencing detection of TGA base insertion at the mouse Pcsk9 locus by CC-PE. In the upper image, within the double-stranded DNA at the mouse Pcsk9 locus, red base sequences represent the spacer sequence of the gRNA; green base sequences represent the PAM recognition region; blue background base sequences represent the pegRNA PBS sequence; and gray background base sequences represent the pegRNA RT sequence. In the lower image, purple arrows indicate the insertion sites; red, green, blue, and black peaks represent thymine, adenine, cytosine, and guanine, respectively.

[0083] Figure 16 The graph shows the results of precise editing frequency and unexpected insertion / deletion frequency of AAV8 sPE-P3P4 in mice.

[0084] Figure A shows the in vivo precise insertion frequency of AAV8 sPE-P3P4, from top to bottom representing 12 liver samples from 3 sPE-P3P4 mice (numbered 1-3), 3 sPE-ctrl mice (numbered 1-3), and 2 untreated mice (numbered 1 and 2). Figure B shows the average precise insertion frequency after summing all samples in Figure A. Figure C shows the in vivo unintended insertion and deletion frequency of AAV8 sPE-P3P4, from top to bottom representing 12 liver samples from 3 sPE-P3P4 mice (numbered 1-3), 3 sPE-ctrl mice (numbered 1-3), and 2 untreated mice (numbered 1 and 2).

[0085] Figure 17 The results of the effects of CC-PE on mouse hepatocyte damage are shown in the figure. Figure A shows the effect of AAV8 sPE-P3P4 on total cholesterol levels in mice; Figure B shows the effect of AAV8 sPE-P3P4 on low-density lipoprotein cholesterol levels in mice; Figures C and D show the effects of AAV8 sPE-P3P4 on alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice, respectively. Detailed Implementation

[0086] Through extensive and in-depth research and screening, the inventors have developed for the first time a structurally unique heterodimeric gene editor (hereinafter referred to as "the gene editor of this invention"). The inventors utilized Cas9 nickase (nCas9) elements and reverse transcriptase (RT) elements (such as the RT elements of engineered Moroni mouse leukemia virus (M-MLV)) to construct a split PE (sPE) system (also known as a heterodimeric gene editor) via a coiled-coil (CC) peptide-mediated approach. Compared to the unsplit PE working system, the gene editor of this invention, due to its unique split structure, can be easily packaged into viral vectors such as AAV, thereby enabling efficient delivery into cells and promoting its widespread application in gene editing, gene therapy, and clinical settings. Based on this, the inventors completed this invention.

[0087] definition

[0088] As used herein, the terms "heterodimeric gene editor complex of the present invention", "gene editor of the present invention", "lead gene editor of the present invention", "gene editor complex of the present invention", "complex of the present invention", and "heterodimer of the present invention" are used interchangeably to refer to the heterodimeric gene editor complex described in the first aspect of the present invention.

[0089] As used herein, the term CRISPR / Cas system refers to more than one gRNA that directs a catalytically active Cas9 or a variant thereof, or an inactivated nicking enzyme Cas9 or a variant thereof, to a desired genomic site where, upon recognition by an appropriate PAM, a DSB form is formed via specific Cas9 action. The nicking enzyme nCas9 in this invention is fused to a heterodimerization system at its N-terminus.

[0090] As described in this article, genome editing involves any modification to genomic and / or non-genomic DNA due to the CRISPR / Cas system and its ability to cause DSBs. This allows for single-base editing, knock-in, or knock-out of cells and / or organisms.

[0091] As used herein, the term "heterodimerization system" refers to a pair of peptides or peptide domains that are linked together in a coiled helix. As used herein, the term "heterodimerization" refers to a protein domain or peptide pair that is linked to other domains of a different type via non-covalent interactions, such as, for example, a coiled helix.

[0092] As used in this article, the term "organism" refers to any living organism.

[0093] As used herein, the terms "reverse transcriptase" and "RT" refer to multifunctional enzymes that typically possess three enzymatic activities: RNA-dependent and DNA-dependent DNA polymerization activity and RNase H activity, which catalyzes the cleavage of RNA in RNA-DNA hybrids. Reverse transcriptase can generate DNA from an RNA template.

[0094] As used herein, the term "peptide pair forming a coiled helix" refers to any peptide pair, heterodimerized peptide, or homodimerized peptide that expresses a coiled helix structural motif, which is a combination of hydrophobic and electrostatic interactions between amino acid residues in a heptameric α-helix that leads to heterodimerization or homodimerization.

[0095] As used herein, the term "cell" refers to a eukaryotic or prokaryotic cell, cellular organism, or multicellular organism (cell line) cultured as a single-celled entity used as a recipient of nucleic acids, and includes daughter cells of the original cell that have been genetically modified by containing nucleic acids. The term primarily refers to cells of higher-developing eukaryotic organisms, preferably vertebrates, and preferably mammals. The invention also relies on invertebrate cells, preferably plant cells. The term "cell" also refers to human cell lines and plant cells. Naturally, as a result of natural, random, or planned mutations, the offspring of a cell are not necessarily identical to the parent in morphology and its DNA complement. A "genetically modified host cell" (also called a "recombinant host cell") is a host cell in which nucleic acids have been introduced. Eukaryotic genetically modified host cells are formed by introducing suitable nucleic acids or recombinant nucleic acids into suitable eukaryotic host cells. The invention described below includes host cells and organisms containing (transiently or stably) nucleic acids according to the invention, carrying the operon record according to the invention. Suitable host cells are known in the art and include eukaryotic cells. Proteins are known to be expressed in the cells of the following organisms: humans, rodents, cattle, pigs, poultry, rabbits, etc. Host cells can include primary cell lines or cultured cell lines of immortalized origin.

[0096] As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length, and is not limited to single-stranded, double-stranded, or longer-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers having a thiophosphate polymer backbone made of purine and pyrimidine bases or other naturally, chemically or biochemically modified, synthetic or derived nucleotide bases.

[0097] As used herein, the term “recombination” refers to a specific nucleic acid (DNA or RNA) as the product of various combinations of cloning, restriction, and / or ligation that produce constructs having structural coding or non-coding sequences different from those of endogenous nucleic acids in the natural host system.

[0098] In some embodiments, the Cas9 nuclease of the present invention is a Cas9 CRISPR nuclease. In some embodiments, the Cas9 nuclease is a nicking enzyme, for example, including nCas9 nuclease with an amino acid sequence as shown in SEQ ID NO:1.

[0099] In some embodiments, the CRISPR nuclease in any of the embodiments described herein includes at least one (e.g., two, three, four, five, six or more) nuclear localization signals (NLS).

[0100] PE system

[0101] The PE system is a CRISPR-Cas9-based prime editing (PE) system, consisting of two core components: pegRNA (Prime Editing Guide RNA) and a fusion protein. The pegRNA is based on sgRNA with an RNA sequence added to its 3' end. This sequence has a dual role: one end serves as a primer binding site (PBS) for reverse transcription, and the other end serves as a reverse transcription template (RTT) that can bind to reverse transcriptase (RT), carrying a designed target point mutation or insertion / deletion mutation. The fusion protein is a novel protein obtained by fusing nCas9 (H840A mutant, which only cleaves the target DNA strand containing PAM) with reverse transcriptase.

[0102] The basic principle of the PE system is as follows: First, guided by pegRNA, the Cas9 H840 cleavage enzyme cleaves the target DNA strand containing PAM. The broken target DNA strand is complementary to and binds to the 3' end PBS sequence of the pegRNA. Then, the reverse transcriptase performs its function, initiating a reverse transcription reaction along the RT template sequence. After the reaction, a dynamic equilibrium of 5'- and 3'-flap structures is formed at the cleavage site of the DNA strand. The DNA strand with the 3'-flap structure carries the target mutation, while the DNA strand with the 5'-flap structure has no mutation. The intracellular 5'-flap structure is easily recognized and excised by structure-specific endonucleases. After DNA ligation and repair, precise gene editing is achieved at the target site.

[0103] coiled helical peptide

[0104] In direct PE splitting systems, nCas9 is not linked to RT; however, given the lack of an affinity module, RT may not bind to the target genomic site. Theoretically, the flexibility of PE could be further enhanced by creating protein-protein affinity module fusions. As an alternative to subunit oligomerization, coiled-coil (CC) peptides can achieve tight and specific dimerization pairing based on electrostatic and hydrophobic interactions. Taking advantage of this structural simplicity, we use CC peptides to non-covalently recruit RT to nCas9, further termed the coiled-coil peptide-mediated PE system (CC-PE). The first coiled-coil peptide in the peptide pair coiled-coil-induced heterodimerization system can be at least one peptide or peptide domain from a peptide pair forming a coiled-coil of a selected peptide pair (e.g., P3-P4, N5-N6, P3S-P4S, etc.), genetically fused to a Cas protein or a variant thereof; for example, P3, N5, or P3S can be linked to a Cas9 protein or a variant thereof (e.g., Cas9, Cas9 nickase, Cpf1, etc.).

[0105] The second coiled-coil peptide in the peptide pair coiled-coil induced heterodimerization system can be at least one peptide or peptide domain from a peptide pair that forms a coiled-coil of a selected peptide pair (e.g., P3-P4, N5-N6, P3S-P4S, etc.), whose gene is fused to a reverse transcriptase.

[0106] Reverse transcriptase

[0107] In various embodiments, the compositions disclosed herein comprise a polymerase (e.g., a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase) or a variant thereof, which may be provided as a fusion with a CRISPR nuclease. The polymerase may be a wild-type polymerase, a functional fragment, a variant, a truncated variant, etc. The polymerase may comprise a wild-type polymerase derived from eukaryotic, prokaryotic, or viral organisms, and / or the polymerase may be modified through genetic engineering, mutagenesis, or directed evolution-based processes.

[0108] Any Cas9 nuclease-RT fusion polypeptide encoding a nucleic acid, a vector containing such encoded nucleic acid, and a method for preparing such encoded nucleic acid are also within the scope of this disclosure. In some embodiments, the polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase polypeptide is obtained from any naturally occurring organism or virus, or from any wild-type reverse transcriptase obtained from commercial or non-commercial sources. The reverse transcriptase polypeptide may also be a variant reverse transcriptase polypeptide.

[0109] Reverse transcriptase peptides can be obtained from many different sources. For example, the gene can be obtained from eukaryotic cells infected with retroviruses or from plasmids containing part or all of the retroviral genome. Additionally, RNA containing the reverse transcriptase gene can be obtained from retroviruses. In some embodiments, the reverse transcriptase is expressed as a separate component or otherwise provided, i.e., not as a fusion protein with the Cas9 nuclease peptide.

[0110] Those skilled in the art will recognize that reverse transcriptases are known in the art, including but not limited to Moroni murine leukosis virus (MMLV) reverse transcriptase, human immunodeficiency virus (HIV) reverse transcriptase, and avian sarcoma-leukosis virus (ASLV) reverse transcriptase, including but not limited to Rouss sarcoma virus (RSV) reverse transcriptase, avian myeloblastoma virus (AMV) reverse transcriptase, avian erythroblastovirus (AEV) helper virus MCAV reverse transcriptase, avian myelomavirus MC29 helper virus MCAV reverse transcriptase, avian reticuloendotheliosis virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus UR2AV reverse transcriptase, avian sarcoma virus Y73 helper virus YAV reverse transcriptase, Rouss-associated virus (RAV) reverse transcriptase, and myeloblastosis-associated virus (MAV) reverse transcriptase, which can be used in the compositions described herein.

[0111] The heterodimeric gene editor of the present invention

[0112] The present invention provides a heterodimeric gene editor comprising a first fusion protein and a second fusion protein (as described in the first aspect of the present invention).

[0113] Preferably, the gene editor of the present invention utilizes Cas9 nickase (nCas9) and engineered Moroni mouse leukemia virus (M-MLV) reverse transcriptase (RT) to develop a coiled-coil peptide-mediated splitting PE (sPE) system. This system enables precise arbitrary base substitutions, small fragment insertions, and deletions of four nucleotides at the target site, achieving a targeting gene mutation efficiency comparable to that of the unsplit PE working system. This makes it possible to package it into AAV for delivery, promoting its widespread application in gene editing, gene therapy, and clinical settings.

[0114] There are no particular limitations on the various elements used in the heterodimeric gene editor of this invention; elements known in the art or derived similar elements can be used. Those skilled in the art can obtain the corresponding elements using conventional methods, such as PCR, fully artificial chemical synthesis, and enzyme digestion, and then link them together using well-known DNA ligation techniques to form the construct assembly of this invention. Inserting the heterodimeric gene editor (first nucleic acid element and second nucleic acid element) of this invention into a foreign vector constitutes the vector assembly of this invention (first vector and second vector).

[0115] With the development of the PE system, the difference between the second-generation PE system (PE2) and the third-generation PE system (PE3) is only that there are no additional gaps mediated by sgRNA on the non-edited strand, thereby minimizing unwanted insertions and deletions (indels). Almost all experiments in this invention were conducted using the PE2 system.

[0116] Specifically, the construction process and application of the heterodimeric gene editor of the present invention are as follows:

[0117] Based on the human B2M gene, VEGFA gene, HEK3 gene, and DMD gene, different pegRNA sequences were designed as shown in Table 2. In addition, primer sequences for different genes used in high-throughput sequencing and Sanger sequencing were designed in this invention (as shown in Table 3).

[0118] To improve the existing unsplit PE2 system (nCas9-RT fusion) Figure 1 A) The flexibility of pegRNA allows nCas9 and RT to fuse with coiled-coil peptide pairs (P3-P4 or N5-N6 pairs) that form coiled dimers, respectively, enabling nCas9 and RT to polymerize at the target site, forming a coiled-coil peptide-mediated PE system (CC-PE). Figure 1 B).

[0119] In this invention, a preferred process for constructing a CC-PE is as follows:

[0120] Referring to the unsplit PE2 system (reference), in which nCas9 and RT are connected via a linker; firstly, this invention fuses P3 or N5 with the C-end of nCas9, naming them nCas9-P3 or nCas9-N5 respectively; and fuses P4 or N6 with the N-end of RT, naming them P4-RT or N6-RT respectively.

[0121] Subsequently, P4-RT will be recruited by nCas9-P3 into nCas9-P3 / P4-RT, and N6-RT will be recruited by nCas9-N5 into nCas9-N5 / N6-RT; nCas9-P3 / P4-RT and nCas9-N5 / N6-RT will be named sPE-P3P4 and sPE-N5N6 respectively;

[0122] The direct split PE (sPE) system without affinity modules (P3-P4 pairs or N5-N6 pairs) is used as a counterpart to sPE-P3P4 and sPE-N5N6 and is named sPE-ctrl.

[0123] To test the effectiveness of the CC-PE system in mammalian cells, plasmids expressing nCas9, RT, and pegRNA were constructed based on the different genes and corresponding pegRNAs described in Table 2. These plasmids were co-transfected into HEK293T cells to delete the 4-GAAT base fragment at the human B2M locus. Cells were collected and analyzed three days after treatment. Sanger sequencing showed that the 4-bp (GAAT base) deletion signal generated by sPE-N5N6 was higher than that of sPE-ctrl, and comparable to the efficiency of the un-dissected PE2.

[0124] To test the precise editing capabilities of the CC-PE system, targeted amplicon deep sequencing was used to detect precise base deletion, substitution, and insertion capabilities. The results showed that the CC-PE system's precise editing capabilities are similar to or even stronger than those of the unsplit PE2 system.

[0125] To assess whether the CC-PE system leads to excessive undesired indels, the inventors analyzed and compared the total insertion / deletion frequencies of CC-PE, PE2, and sPE-ctrl at three gene loci: HEK3 (+1 T to A), B2M (+1 GAAT deletion), and VEGFA (+1 C insertion). The results indicate that CC-PE does not generate excessive undesirable insertion / deletion byproducts, thus endowing it with potential for gene therapy applications.

[0126] The inventors also used dual AAV to verify the flexible delivery of CC-PE in vivo and its precise editing effect. To maximize the efficiency of in vivo lead editing, the inventors used the RNA structural group evopreQ1 to modify the 3′ end of pegRNA (EpegRNA). This method has been reported to improve the efficiency of lead editing by enhancing the stability of pegRNA and preventing its degradation.

[0127] Because mouse prorotein convertase subtilisin / kexin type 9 (Pcsk9) has the potential to treat familial hypercholesterolemia, it was selected as a target gene for in vivo validation. The inventors chose to encapsulate sPE-P3P4 and epigRNA inserted into TGA into AAV8, which has high affinity for hepatocytes and specifically targets the liver, in order to disrupt the Pcsk9 gene in vivo.

[0128] In a preferred embodiment, two AAV vectors were constructed: the first vector contained nCas9 (H840A) driven by the EF-1a short promoter (EFS), with the C-terminus fused to express the coiled-coil peptide P3; the second vector contained EFS-driven RT (N-terminus fused to express the coiled-coil peptide P4), a Pcsk9-EpegRNA directed by the U6 promoter, and the corresponding nicking sgRNA (ngRNA) expression cassette. An AAV vector that directly disassembled the PE system was also constructed as a control. All of the above vectors were within the AAV 4.8kb packaging capacity and were packaged into AAV8.

[0129] like Figure 14 The flowchart shown in C describes the intraperitoneal injection of either dual AAV8 sPE-P3P4 or sPE-ctrl (5 × 10⁻⁶ each). 11 The viral genome was injected into 2-week-old mice (n=3 per group); four weeks after injection, total plasma and liver were collected from mice in the AAV8-injected group and the untreated control group; 12 samples were collected from the liver of each mouse injected with AAV8 (3 mice per group) or the control mice not injected with AAV8 (2 mice per group), and genomic DNA was extracted and analyzed. The results showed that sPE-P3P4 had a higher in vivo plasmid editing efficiency than sPE-ctrl; sPE-P3P4 and sPE-ctrl successfully inserted the TGA stop codon into the Pcsk9 gene in vivo; and the average precise insertion efficiency of sPE-P3P4 was significantly higher than that of sPE-ctrl.

[0130] Compared to the untreated control group, mice injected with dual AAV8sPE-P3P4 showed significantly lower levels of total cholesterol and low-density lipoprotein cholesterol, which is completely consistent with the function of Pcsk9. Importantly, mice injected with dual AAV8sPE-P3P4 did not show elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating no significant hepatocellular damage. In summary, these data strongly demonstrate that CC-PE can effectively perform precise in vivo editing.

[0131] The main advantages of this invention include:

[0132] (1) This invention utilizes the existing protein structure information of nCas9 and its disassembly method to develop a coiled-helical peptide-mediated disassembly PE (sPE) system, which has a similar gene mutation targeting efficiency as the undisassembled PE working system, making it possible to package it into AAV for delivery and promoting its widespread application in gene editing, gene therapy and clinical practice.

[0133] (2) The split gene editing system (CC-PE) of the present invention, guided by pegRNA, fuses nCas9 and RT with coiled-coil peptide pairs (P3-P4 or N5-N6) that form a coiled dimer. The length of each pair is less than the adeno-associated virus (AAV) packaging limit of 4.7 kb, which can be packaged and delivered with AAV, further expanding the application scope of lead editing.

[0134] (3) The split gene editing system (CC-PE) of the present invention is more flexible in splitting PE, and it has similar or even higher precision editing efficiency than the unsplit PE2 system (such as the gene editor of the present invention based on P3-P4 coiled helical peptide pairs). Therefore, the present invention provides a simple, flexible and efficient splitting method, and is expected to be widely used in gene editing, gene therapy and clinical applications.

[0135] (4) The split gene editing system (CC-PE) of the present invention is more accurate, and its frequency of unintended insertions and deletions is as low as that of the PE2 system compared with the PE3 system, and it produces very few byproducts.

[0136] (5) The split gene editing system (CC-PE) of the present invention has better versatility. Its coiled helical peptide, composed of only 28 amino acids, has simple modularity and can be split into different effectors, including adenine base editor, cytosine base editor, dibase editor, transcription activator / inhibitor and HDR stimulator.

[0137] (6) The split gene editing system (CC-PE) of the present invention can effectively perform precise in vivo editing. In practical applications, when AAV-encapsulated CC-PE is administered to mice to achieve precise editing of the Pcsk9 locus, it achieves highly efficient precise editing (up to 45.9%) in the mouse liver, significantly reducing low-density lipoprotein cholesterol and total cholesterol in plasma, without any obvious hepatocellular damage or toxicity.

[0138] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0139] Example 1: Construction of the CC-PE gene editing system

[0140] 1. Design and construction of plasmid vectors

[0141] 1.1 Design principles and guidelines for sPE-P3P4 and sPE-N5N6:

[0142] To improve the existing unsplit PE2 system (nCas9-RT fusion) Figure 1 A) The flexibility of pegRNA allows nCas9 and RT to fuse with coiled-coil peptide pairs P3-P4 or N5-N6, respectively, to form nCas9-P3 / P4-RT or nCas9-N5 / N6-RT. This allows nCas9 and RT to polymerize at the target site, forming a coiled-coil peptide-mediated PE system (hereinafter referred to as CC-PE). Figure 1 B).

[0143] Referencing the unsplit PE2 system, where nCas9 and RT are connected via a linker;

[0144] First, this invention fuses the coiled helical peptide P3, as shown in SEQ ID NO: 3, or the coiled helical peptide N5, as shown in SEQ ID NO: 4, with the C-terminus of nCas9 (120 bp downstream of the C-terminus of nCas9), and names them nCas9-P3 or nCas9-N5, respectively; and fuses the coiled helical peptide P4, as shown in SEQ ID NO: 5, or the coiled helical peptide N6, as shown in SEQ ID NO: 6, with the N-terminus of RT (120 bp upstream of the N-terminus of RT), and names them P4-RT or N6-RT, respectively; the relevant amino acid sequences or nucleotide sequences are shown in Table 1 below:

[0145] Table 1

[0146]

[0147] Subsequently, P4-RT will be recruited by nCas9-P3 into nCas9-P3 / P4-RT, and N6-RT will be recruited by nCas9-N5 into nCas9-N5 / N6-RT; nCas9-P3 / P4-RT and nCas9-N5 / N6-RT will be named sPE-P3P4 and sPE-N5N6, respectively; the directly split PE (sPE) system without affinity modules (P3-P4 pair or N5-N6 pair) will be used as a counterpart to sPE-P3P4 and sPE-N5N6, and named sPE-ctrl. Figure 2 ).

[0148] 1.2 Target Sequence pegRNA Design Principles: Based on the human B2M gene, VEGFA gene, HEK3 gene, and DMD gene, different pegRNA sequences were designed, as shown in Table 2:

[0149] Table 2

[0150]

[0151] 1.3 Construction of plasmids expressing nCas9, RT, and pegRNA

[0152] Based on the different genes and corresponding pegRNAs described in Table 2, plasmids expressing nCas9, RT, and pegRNA were constructed. The P3 or N5 sequence was introduced 120 bp downstream of the C-terminus of nCas9 using primers to construct nCas9-P3 or nCas9-N5. The P4 or N6 sequence was introduced 120 bp upstream of the N-terminus of RT using primers to construct P4-RT or N6-RT. The Spacer sequence, PBS, and RT sequence were introduced into the U6-gRNA backbone linearized by Bbs1 and HindIII restriction endonucleases using primers to construct the corresponding pegRNAs.

[0153] 1.4 Plasmid transfection

[0154] Plasmids expressing nCas9, RT, and pegRNA were co-transfected into HEK293T cells. Cells were collected for analysis three days after treatment.

[0155] For HEK293T cells, PEI was used for transfection in 24-well plates when the cell density reached 40-60%. Prior to transfection, the medium was replaced with 2% FBS. PEI (Sigma, 408727; final concentration: 1 μg / μL) was gently mixed with the 2% FBS medium and incubated at room temperature for 5 minutes. For each well, 3 μg of PEI was mixed with 25 μl of 2% FBS medium.

[0156] Add the plasmid to 2% FBS medium. In this experiment, for each well, mix approximately 1 μg of plasmid with 25 μL of medium containing 2% FBS (fetal bovine serum). The plasmid composition is: 250 ng of pegRNA and 750 ng of PE2 (or 375 ng each of sPE).

[0157] Add the PEI mixture dropwise to the plasmid mixture, mix gently with a pipette, and let stand at room temperature for 20 minutes;

[0158] A mixture of PEI and plasmid was added dropwise to HEK293T cells. Twelve hours post-transfection, cells were cultured in 10% FBS medium. Three days after transfection, cells were digested into single cells, and the transfected positive cells were sorted by flow cytometry.

[0159] 2. Sanger sequencing and amplicon deep sequencing

[0160] Sorted cells were lysed using NP40 under the following conditions: 56°C for 60 minutes followed by 96°C for 10 minutes. Subsequently, PCR amplification of the target site was performed using PCR polymerase. The amplified products were purified and sent to a sequencing company for Sanger sequencing. For amplicon library preparation, primers were designed near the target site, specifically amplifying fragments within 190 bp. Two rounds of PCR amplification were required. The first round of PCR used high-throughput primers, with the cell lysis products as templates, for 30 cycles in a 25 μL reaction volume. The second round of PCR used barcoded primers, with the first round PCR products as templates, for 12 cycles. The second round PCR products were purified using a gel extraction kit, and the product concentration was quantified. The constructed library was then sent to a sequencing company for deep sequencing.

[0161] The primer sequences designed for different genes in this invention for high-throughput sequencing and Sanger sequencing are shown in Table 3:

[0162] Table 3

[0163]

[0164]

[0165] Note: HT: high-throughput primers; det: first-generation PCR amplification primers; Seq: Sanger sequencing primers.

[0166] 3. Data Analysis

[0167] For Sanger sequencing results, the location and height of the bimodal peaks were analyzed. For amplicon deep sequencing results, the efficiency of precise editing near the gRNA and the efficiency of random insertions and deletions were analyzed. High-throughput data were analyzed using PE-Analyzer.

[0168] Example 2: Detection of the base deletion ability of sPE-P3P4 and sPE-N5N6

[0169] (2.1) The design, construction and transfection of plasmids related to sPE-P3P4 and sPE-N5N6 are the same as in Example 1.

[0170] Based on the pegRNA sequences of B2M-Deletion and VEGFA-Deletion described in Table 2, the corresponding plasmids expressing nCas9, RT, and pegRNA were co-transfected into HEK293T cells. The purpose was to delete the 4GAAT base fragment at the human B2M locus and delete one T base at the VEGFA locus. Cells were collected for analysis three days after treatment.

[0171] (2.2) Sanger sequencing and amplicon deep sequencing are the same as in Example 1.

[0172] Sanger sequencing showed that sPE-N5N6, designed based on the B2M-Deletion pegRNA sequences described in Table 2, produced higher efficiency than sPE-ctrl and was comparable to the efficiency of the unresolved PE2. Figure 3 ).

[0173] Deep sequencing results showed that, based on the B2M-Deletion pegRNA sequences described in Table 2, the editing efficiency of sPE-P3P4 and sPE-N5N6 at the human B2M locus (4-bp (GAAT base) deletion) was significantly higher than that of sPE-ctrl. Figure 4 This is similar to the unsplit PE2 system, which is consistent with the results of the Sanger sequencing mentioned above.

[0174] Furthermore, based on the VEGFA-Deletion pegRNA sequences described in Table 2, the designed sPE-P3P4 and sPE-N5N6, while maintaining the same activity as the unsplit PE2 at the VEGFA (+1 T deletion) site, exhibited significantly higher precise deletion efficiency than the directly split PE (sPE-ctrl). Figure 5 ).

[0175] Example 3: Detection of the base substitution ability of sPE-P3P4 and sPE-N5N6

[0176] (3.1) The design, construction and transfection of plasmids related to sPE-P3P4 and sPE-N5N6 are the same as in Example 1.

[0177] Based on the pegRNA sequence of HEK3-Substitution described in Table 2, plasmids expressing nCas9, RT, and pegRNA were co-transfected into HEK293T cells to replace one T base with an A base at the HEK3 locus. Cells were collected for analysis three days after treatment.

[0178] (3.2) Amplicon deep sequencing is the same as in Example 1.

[0179] Deep sequencing results showed that, based on the HEK3-Substitution pegRNA sequences described in Table 2, the designed sPE-P3P4 and sPE-N5N6 had significantly higher base substitution efficiency at the HEK3 (+1 T to A) site than the directly split sPE-ctrl and the PE2 (+1 T to A) site which remained unsplit. Figure 6 ).

[0180] Example 4: Detection of base insertion ability of sPE-P3P4 and sPE-N5N6

[0181] (4.1) The design, construction and transfection of plasmids related to sPE-P3P4 and sPE-N5N6 are the same as in Example 1.

[0182] Based on the pegRNA sequences of VEGFA-Insertion and HEK3-Insertion described in Table 2, the corresponding plasmids expressing nCas9, RT, and pegRNA were co-transfected into HEK293T cells. The aim was to insert one C base into the VEGFA locus and one A base into the HEK3 locus. Cells were collected for analysis three days after treatment.

[0183] (4.2) Amplicon deep sequencing is the same as in Example 1.

[0184] Deep sequencing results showed that, based on the VEGFA-Insertion pegRNA sequences described in Table 2, the designed sPE-P3P4 exhibited significantly higher base insertion efficiency at the VEGFA (+1C insertion) site than PE2, which remained unsplit at the VEGFA (+1C insertion) site; while the base insertion efficiency of sPE-N5N6 at the VEGFA (+1C insertion) site was comparable to that of PE2; furthermore, the precise editing efficiency of the directly split sPE-ctrl system at the VEGFA (+1C insertion) site was similar to that of the unsplit PE2. Figure 7 This is consistent with previously reported results.

[0185] Based on the HEK3-Insertion pegRNA sequences described in Table 2, the designed sPE-P3P4 and sPE-N5N6 showed significantly higher base insertion efficiency at the HEK3 (+1 A insertion) site than the directly split sPE-ctrl, with an average efficiency increase of more than 4 times; and showed no significant difference compared to PE2, which remained unsplit at the HEK3 (+1 A insertion) site. Figure 8 ).

[0186] Example 5: Detection of the precise insertion efficiency of sPE-P3P4 and sPE-N5N6 at gene therapy-related DMD sites.

[0187] (5.1) The design, construction and transfection of plasmids related to sPE-P3P4 and sPE-N5N6 are the same as in Example 1.

[0188] Based on the DMD-Insertion pegRNA sequence and the DMD-nick sgRNA sequence with an additional nick described in Table 2, the corresponding plasmids expressing nCas9, RT, and pegRNA were co-transfected into HEK293T cells to insert one AC base into the DMD locus. Cells were collected for analysis three days after treatment.

[0189] (5.2) Sanger sequencing and amplicon deep sequencing are the same as in Example 1.

[0190] Sanger sequencing ( Figure 9 A) and amplicon deep sequencing ( Figure 9 B) The results show that the base insertion efficiency of sPE-P3P4 and sPE-N5N6 designed according to the DMD-Insertion pegRNA sequence and the DMD-nick sgRNA sequence with extra gaps in Table 2 is significantly better than that of sPE-ctrl which is directly split, and does not affect the efficiency of maintaining the unsplit PE at the DMD (+1 AC insertion) site.

[0191] Example 6 examines whether sPE-P3P4 and sPE-N5N6 lead to excessive undesired insertion / deletion edit products.

[0192] Based on the experimental methods described in Examples 1-4 above, the inventors compared the total insertion and deletion frequencies of CC-PE with PE2 and sPE-ctrl at three gene loci: HEK3 (+1 T to A), B2M (+1 GAAT deletion), and VEGFA (+1 C insertion). The results are as follows: Figure 10-12 As shown, where,

[0193] (1) At the HEK3 (+1 T to A) locus, the total unexpected insertion / deletion frequency of sPE-P3P4 (1.04%) and sPE-N5N6 (1.20%) was more than 3-fold lower than that of PE2 (3.73%) and sPE-ctrl (4.50%). Figure 10 A); further analysis revealed the specific frequencies of unintended insertions and deletions generated by CC-PEs (sPE-P3P4 or sPE-N5N6), PE2, and sPE-ctrl. Consistently, at the HEK3 (+1 T to A) locus, the frequency of unintended insertions (A) by CC-PEs was high. Figure 10 B) and deletion ( Figure 10 CF) frequency is lower than PE2;

[0194] (2) At the human B2M (+1 GAAT deletion) locus, the unexpected insertion / deletion frequencies of sPE-P3P4 (0.93%) and sPE-N5N6 (1.40%) were comparable to those of PE2 (1.31%) and sPE-ctrl (1.00%). Figure 11 A); Further analysis revealed the specific frequencies of unintended insertions and deletions generated by CC-PEs (sPE-P3P4 or sPE-N5N6), PE2, and sPE-ctrl. Consistently, at the B2M (+1 GAAT deletion) locus, the frequency of unintended insertions (CC-PEs) was high. Figure 11 B) and deletion ( Figure 11 The CF frequency is comparable to that of PE2 and sPE-ctrl;

[0195] (3) At the VEGFA (+1 C insertion) locus, the unexpected insertion / deletion frequencies of sPE-P3P4 (3.19%) and sPE-N5N6 (4.03%) were comparable to those of PE2 (2.27%) and sPE-ctrl (4.77%). Figure 12 A); Further analysis revealed the specific frequencies of unexpected insertions and deletions generated by CC-PEs (sPE-P3P4 or sPE-N5N6), PE2, and sPE-ctrl. Consistently, at the VEGFA (+1 C insertion) locus, the frequency of unexpected insertions (...) by CC-PEs was... Figure 12 B) and deletion ( Figure 12 The CF frequency is comparable to PE2 and sPE-ctrl.

[0196] These data suggest that CC-PE does not produce excessive unwanted insertion and deletion byproducts, thus giving CC-PE potential for gene therapy applications.

[0197] Example 7 uses dual AAV to verify the flexible delivery of CC-PE in vivo and its precise editing effect.

[0198] To maximize the efficiency of in vivo lead editing, the inventors used the RNA structural group evopreQ1 (as shown in SEQ ID NO: 7) to modify the 3′ end of pegRNA (EpegRNA). This method has been reported to improve lead editing efficiency by enhancing pegRNA stability and preventing its degradation. Simultaneously, the PE3 system was used for editing. The PE3 system has an additional nick gRNA compared to PE2, which can create a nick in the non-edited strand, improving the efficiency of precise editing.

[0199] Because mouse prorotein convertase subtilisin / kexin type 9 (Pcsk9) has the potential to treat familial hypercholesterolemia, it was selected as a target gene for in vivo validation. The inventors designed pegRNAs and corresponding nicking sgRNAs targeting the first exon of the Pcsk9 gene, inserting a stop codon (TGA) or T to disrupt the function of Pcsk9.

[0200] The efficiency of precise editing was initially evaluated in vitro. The corresponding pegRNA was co-electroplated with unsplit / split PE3 cells into mouse embryonic fibroblasts (MEFs) using electroporation parameters of 1350V, 30ms, and 2 pulses. Deep sequencing results are shown below. Figure 13 As shown, the precise insertion efficiency mediated by sPE-P3P4 is significantly higher than that of directly split PE (TGA insertion: 35.6±1.1 vs 23.4±1.2; T insertion: 26.3±0.7 vs 21.5±0.6) and unsplit PE3 (TGA insertion: 35.6±1.1 vs 29.0±0.3; T insertion: 26.3±0.7 vs 19.0±0.1). Figure 13 A, 13B). TGA insertion is generally more efficient than T insertion. Figure 13 Avs Figure 13 B), in addition, it was found that the modified EpigRNA had higher plasmid editing activity ( Figure 13 A, 13B).

[0201] Therefore, the inventors chose to encapsulate sPE-P3P4 and TGA-inserted EpigRNA into AAV8, which has high affinity for hepatocytes and specifically targets the liver, to disrupt the Pcsk9 gene in vivo. The inventors constructed two AAV vectors: the first vector contained nCas9 (H840A) driven by the EF-1a short promoter (EFS), with a coiled-coil peptide P3 at the C-terminus; the second vector contained an EFS-driven RT (with a coiled-coil peptide P4 at the N-terminus), a Pcsk9-EpegRNA oriented by the U6 promoter, and the corresponding nickingsgRNA (ngRNA) expression cassette. Figure 14A), and also constructed an AAV vector that directly split the PE system as a control ( Figure 14 B); All of the above-mentioned carriers are within the packaging capacity of AAV4.8kb and are packaged as AAV8.

[0202] like Figure 14 The flowchart shown in C describes the intraperitoneal injection of either dual AAV8sPE-P3P4 or sPE-ctrl (5 × 10⁻⁶ each). 11 The viral genome was injected into 2-week-old mice (n=3 per group); four weeks after injection, total plasma and liver were collected from mice in the AAV8-injected group and the untreated control group; 12 samples were collected from the liver of each mouse injected with AAV8 (3 mice per group) or each mouse in the control group not injected with AAV8 (2 mice per group), and genomic DNA was extracted for the following analysis:

[0203] (1) Sanger sequencing showed that sPE-P3P4 mediated higher plasmid editing efficiency in vivo compared to sPE-ctrl. Figure 15 );

[0204] (2) Amplicon deep sequencing further confirmed that sPE-P3P4 and sPE-ctrl successfully inserted the TGA stop codon into the Pcsk9 gene in vivo. Figure 16 A); the average precise insertion efficiency of sPE-P3P4 (35.8%, n=3; range from 25.7% to 45.9%) was significantly higher than that of sPE-ctrl (17.6%, n=3; range from 10.9% to 24.3%). Figure 16 B); the average unexpected insertion / deletion efficiencies of sPE-P3P4 and sPE-ctrl were 1.1% (n=3) and 0.7% (n=3), respectively. Figure 16 C).

[0205] (3) The absence of Pcsk9 leads to a decrease in low-density lipoprotein cholesterol. Compared with the untreated control group, mice injected with dual AAV8 sPE-P3P4 showed significantly lower levels of total cholesterol and low-density lipoprotein cholesterol, which is completely consistent with the function of Pcsk9. Figure 17 A and B). Importantly, mice injected with dual AAV8 sPE-P3P4 did not show elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating no significant hepatocellular damage. Figure 17 (C and D).

[0206] In conclusion, these data strongly demonstrate that CC-PE can effectively perform precise in vivo editing.

[0207] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A heterodimeric gene editor complex, characterized in that, The complex includes: (a) A first fusion protein comprising a fused Cas9 nuclease and a first coiled-helical peptide element; and (b) A second fusion protein comprising a reverse transcriptase and a second coiled-coil peptide element fused together; The first fusion protein and the second fusion protein form a heterodimeric gene editor through the first coiled helical peptide element and the second coiled helical peptide element.

2. The gene editor complex according to claim 1, characterized in that, The first fusion protein has a structure of Formula I from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: Y1-X1-L1-Z1(I) in, Y1 is either absent or a signal peptide; X1 is a Cas9 nuclease element; L1 is either absent or linked to a peptide element; Z1 is the first coiled helical peptide element; "-" indicates a peptide bond or peptide linker; and / or The second fusion protein has the structure of Formula II from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: Y2-Z2-L2-X2(II) in, Y2 is either absent or a signal peptide; Z2 is the second coiled helical peptide element; L2 is either absent or linked to a peptide element; X2 is a reverse transcriptase peptide element; "-" indicates a peptide bond or peptide linker.

3. A polynucleotide encoding the heterodimeric gene editor complex of claim 1, characterized in that, The polynucleotides include: (i) a first polynucleotide encoding the first fusion protein; and (ii) A second polynucleotide, which encodes the second fusion protein.

4. A carrier, characterized in that, It includes a first carrier and a second carrier, the first carrier containing the first fusion protein and the second carrier containing the second fusion protein, wherein the first fusion protein and the second fusion protein are as defined in claim 1.

5. A genetically engineered cell, characterized in that, The cell contains the polynucleotide of claim 3 or the carrier of claim 4.

6. A composition, characterized in that, It comprises the gene editor complex of claim 1, or the vector of claim 4.

7. The use of the composition of claim 6 in the editing of a predetermined target gene.

8. A gene editing method, characterized in that, Including the following steps: (1) Provide the target nucleic acid sequence to be edited and the heterodimeric gene editor complex of claim 1; and (2) In the presence of the heterodimeric gene editor complex, gene editing is performed on the provided nucleic acid target sequence to be edited.

9. A reagent kit, characterized in that, The kit includes: (a) a first container, and a first carrier located within the first container; and (b) A second container, and a second carrier located in the second container; the first carrier contains the first fusion protein, and the second carrier contains the second fusion protein, the first fusion protein and the second fusion protein being as defined in claim 1.

10. The use of the gene editor complex of claim 1 or the vector of claim 4 in the preparation of reagents and / or drugs for gene editing.

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