Prime editing-based gene editing composition with improved editing efficiency and its uses

Abstract

The present invention relates to a prime-editing-based gene editing composition with improved gene editing efficiency, a gene editing method using the composition, a gene editing kit, and a method for producing a genetically modified mammal. The prime editor developed in the present invention exhibits significantly improved genome editing efficiency and target specificity, and it has been confirmed that mutant animal models created using the prime editor transmit mutations to subsequent generations and exhibit phenotypic changes. The improved prime editor or a gene editing composition containing the same can be useful in a variety of applications, such as the creation and research of humanized animal models, the field of genetic engineering, and as a means for treating genetic diseases.

Description

Technical Field

[0001] The present invention relates to a prime editing-based gene editing composition with improved gene editing efficiency, a gene editing method using the composition, a gene editing kit, and a method for producing a genetically modified mammal.

Background Art

[0002] The CRISPR-Cas system has evolved as various advanced genome editing tools such as nucleases, base editors, and gene transposases that can efficiently induce desired target mutations. In particular, cytosine base editors (CBEs) and adenine base editors (ABEs) developed based on the CRISPR system can efficiently replace C·G with T·A and A·T with G·C in various organisms including mice. Also, from recent research, CGBE1 has been reported as a C-to-G base editor capable of base editing from C to G in human cells. However, the generation of accurate target mutations such as the insertion, substitution, or cleavage of one or more bases remains difficult due to the limitation of gene editing caused by the low efficiency of homology-directed repair (HDR) in cells.

[0003] The Prime Editor (PE), a new concept of genome editing tool developed in response to such requirements, is a fusion protein composed of Cas9 nickase (Cas9 nickase-H840A) modified to cleave only one strand of the double-stranded DNA and reverse transcriptase (RT). The Prime Editor requires a pegRNA (prime editing guide RNA), a new form of guide RNA that encodes the desired editing sequence. The pegRNA has a base sequence (primer binding site, PBS) complementary to the non-target strand of the target gene and a reverse transcriptase template strand site (RT template) containing the base sequence to be edited. The Cas9 nickase of the Prime Editor cleaves the non-target strand of the target gene, and the reverse transcriptase synthesizes a new DNA strand containing the base sequence edited based on the RT template strand of the pegRNA. Subsequently, the conventional DNA base sequence is removed by the intracellular repair process, and the newly synthesized edited form of the DNA base sequence replaces it. Through such a sophisticated genome editing system, it is possible to generate target mutations including double-stranded DNA cleavage, base-to-base substitution without donor DNA, and insertion and deletion of small sizes. However, the Prime Editor also has an off-target problem, although less frequently than base editors. In particular, there is a problem that accurate editing is difficult due to additional base sequence insertion and deletion by reverse transcriptase. Furthermore, together with the above problems, currently developed base editors and prime editors have very low efficiency limits. For example, the most efficient PE3 among prime editors has a gene editing efficiency of only 20-50%.

[0004] The genetic material within the cells of living organisms, including humans, is constantly exposed to mutations. The accumulation of mutations induces various mutant phenotypes, some of which are associated with diseases. Therefore, sophisticated gene editing techniques using prime editing can be utilized as tools for the treatment of genetic diseases, for identifying pathogenic mutation factors, and are widely applicable to research on disease mechanisms. Accordingly, there is a need to develop a prime editor that is more sophisticated than known prime editors and has improved editing efficiency, as well as a gene editing technique using the same.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] To overcome the above problems of prime editing-based gene editing, the present inventors developed an improved prime editor using proximal dead sgRNA (dsgRNA) and / or chromatin-modulating peptides (CMPs), and completed the present invention by confirming its significantly enhanced genome editing efficiency and target specificity through specific experiments.

[0007] Therefore, an object of the present invention is to provide a composition for prime editing-based gene editing with improved gene editing efficiency.

[0008] Another object of the present invention is to provide a gene editing method using the composition for gene editing.

[0009] Another object of the present invention is to provide a kit for gene editing containing the composition for gene editing.

[0010] Furthermore, another object of the present invention is to provide a method for producing a genetically modified mammal other than humans using the above-described gene editing composition.

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

Means for Solving the Problems

[0012] In order to achieve the object of the present invention as described above, the present invention provides (a) a fusion protein or a nucleic acid encoding the same, which comprises i) a CRISPR / Cas9 protein or a variant thereof, and ii) a reverse transcriptase or a variant thereof, (b) a guide RNA or a nucleic acid encoding the same, wherein the guide RNA includes a pegRNA (prime editing guide RNA) and a dsgRNA (dead single guide RNA), and the dsgRNA has a length of 10 to 20 bp, and provides a gene editing composition.

[0013] As one embodiment of the present invention, the dsgRNA can bind to a position 5 to 70 nucleotides away from the pegRNA binding site to increase the chromatin accessibility of the fusion protein.

[0014] As another embodiment of the present invention, the gene editing composition may further include an sgRNA (single guide RNA) that binds complementarily to a non-target DNA strand to induce cleavage of the target DNA strand.

[0015] In addition, the present invention provides a fusion protein or a nucleic acid encoding the same, which comprises (a) i) a CRISPR / Cas9 protein or a variant thereof, ii) a reverse transcriptase or a variant thereof, and iii) chromatin-modulating peptides, and (b) a guide RNA or a nucleic acid encoding the same, wherein the guide RNA is a pegRNA (prime editing guide RNA) and a dsgRNA (dead single guide RNA), and provides a gene editing composition.

[0016] In one embodiment of the present invention, the chromatin-modulating peptide may be a high-mobility group nucleosome binding domain 1 (HN1), a histone H1 central globular domain (H1G), or a combination thereof.

[0017] In another embodiment of the present invention, the chromatin-modulating peptide may be linked to the CRISPR / Cas9 protein or reverse transcriptase directly by a chemical bond, indirectly by a linker, or a combination thereof.

[0018] In still another embodiment of the present invention, the fusion protein may have a structure of N-terminus - [HN1] - [Cas9] - [H1G] - [reverse transcriptase] - C-terminus, or N-terminus - [HN1] - [Cas9] - [reverse transcriptase] - [H1G] - C-terminus.

[0019] In yet another embodiment of the present invention, the fusion protein may further comprise nuclear localization signal (NLS) sequences at the N-terminus and C-terminus, respectively.

[0020] As yet another embodiment of the present invention, the CRISPR / Cas9 protein variant may be a nickase.

[0021] As yet another embodiment of the present invention, either the RuvC domain or the HNH domain of the CRISPR / Cas9 protein variant may be inactivated.

[0022] As yet another embodiment of the present invention, the reverse transcriptase or its variant may be derived from Moloney murine leukemia virus (M-MLV).

[0023] As yet another embodiment of the present invention, the fusion protein may consist of the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

[0024] As yet another embodiment of the present invention, the dsgRNA may be composed of 10 to 20 nucleotides in length.

[0025] As yet another embodiment of the present invention, the dsgRNA may bind to a position 5 to 70 nucleotides away from the pegRNA binding site to improve the chromatin accessibility of the fusion protein.

[0026] As yet another embodiment of the present invention, the gene editing composition is characterized in that the gene editing efficiency and target specificity are enhanced.

[0027] Furthermore, the present invention provides (a) a protein or a nucleic acid encoding the same, comprising (i) a CRISPR / Cas9 protein or its variant, (ii) a reverse transcriptase or its variant, and (iii) chromatin-modulating peptides, (b) a guide RNA or a nucleic acid encoding the same. Provided is a gene editing composition, wherein the guide RNA is pegRNA (prime editing guide RNA) and dsgRNA (dead single guide RNA).

[0028] In one embodiment of the present invention, the i) CRISPR / Cas9 protein or a variant thereof and the ii) reverse transcriptase or a variant thereof are fused and transmitted into cells, or i) and ii) are separately expressed and transmitted into cells in the form of plasmid DNA, RNA, or protein.

[0029] The present invention also provides a gene editing method, which includes contacting the gene editing composition with a target region containing a target nucleic acid sequence in vitro or ex vivo.

[0030] The present invention also provides a gene editing kit containing the gene editing composition.

[0031] The present invention also includes introducing the gene editing composition into mammalian cells excluding human cells to obtain genetically recombinant mammalian cells, and transplanting the obtained genetically recombinant mammalian cells into the oviduct of a mammalian surrogate mother excluding human. A method for producing a genetically recombinant mammalian excluding human is provided.

[0032] In one embodiment of the present invention, the mammalian cells can be mammalian embryonic cells.

Advantages of the Invention

[0033] In the present invention, a prime editor with improved performance was developed using dead sgRNA (dsgRNA) and / or chromatin-modulating peptides (CMPs). Its significantly enhanced genome editing efficiency and target specificity were confirmed, and a mutant animal model of the target gene was created to confirm the transmission of mutations to the next generation and phenotypic changes. Therefore, the gene editing composition containing the improved prime editor according to the present invention can be usefully utilized for various applications such as the creation and research of humanized animal models, the field of genetic engineering technology, and treatment means for genetic diseases.

Brief Description of the Drawings

[0034]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Mode for Carrying Out the Invention

[0035] The inventors of the present invention developed a prime editor with improved conventional problems using proximal dsgRNA (dead sgRNA) and / or chromatin-modulating peptides (CMPs), and completed the present invention by confirming its excellent editing efficiency and target specificity.

[0036] Therefore, the present invention provides a gene editing composition comprising: (a) a fusion protein or a nucleic acid encoding the same, comprising i) a CRISPR / Cas9 protein or a variant thereof, and ii) a reverse transcriptase or a variant thereof; and (b) a guide RNA or a nucleic acid encoding the same, wherein the guide RNA includes a pegRNA (prime editing guide RNA) and a dsgRNA (dead single guide RNA), and the dsgRNA is 10 to 20 nt in length.

[0037] The gene editing composition may further include an sgRNA (single guide RNA) that binds complementarily to a non-target DNA strand to induce cleavage of the target DNA strand. Preferably, the guide RNA may mean a nicking sgRNA in the present invention.

[0038] The term "gene editing" used in the present invention can be used with the same meaning as gene correction, genome editing, etc. Gene editing means a mutation (substitution, insertion, or deletion) that induces a mutation in one or more bases at a target site within a target gene. Preferably, the gene editing may not involve double-stranded DNA cleavage of the target gene, and more preferably, it may be performed via prime editing.

[0039] In one embodiment of the present invention, the mutation or gene editing that induces a mutation in one or more bases inactivates the target gene by generating a stop codon at the target site or generating a codon that encodes an amino acid different from the wild type. Alternatively, inactivate the gene by substituting the start codon with another amino acid or edit the gene mutation, inactivate the gene or edit the gene mutation by frameshift due to insertion or deletion, introduce a mutation into a non-coding DNA sequence that does not produce a protein, or change the DNA of a sequence different from the wild type that induces a disease to the same sequence as the wild type. It can be in various forms such as this, but is not limited thereto.

[0040] In the present invention, the term "base sequence" means the sequence of nucleotides containing the base and can be used with the same meaning as nucleotide sequence, nucleic acid sequence, or DNA sequence.

[0041] In the present invention, the "target gene" means a gene to be subjected to gene editing, and the "target site (or target region)" means a site where gene editing or correction by a target-specific nuclease occurs within the target gene. In one example, when the target-specific nuclease includes an RNA-guided engineered nuclease (RGEN), it may be located adjacent to the 5'-end and / or 3'-end of a sequence (PAM sequence) recognized by the RNA-guided nuclease within the target gene.

[0042] Furthermore, the present invention provides a fusion protein or a nucleic acid encoding the same, which comprises (a) i) a CRISPR / Cas9 protein or a variant thereof, ii) a reverse transcriptase or a variant thereof, and iii) a chromatin-modulating peptide, (b) a guide RNA or a nucleic acid encoding the same. The guide RNA is a pegRNA (prime editing guide RNA) and a dsgRNA (dead single guide RNA), and the present invention provides a gene editing composition.

[0043] The gene editing composition may further comprise an sgRNA (single guide RNA) that binds complementarily to a non-target DNA strand to induce cleavage of the target DNA strand. Preferably, the guide RNA may mean a nicking sgRNA in the present invention.

[0044] In the present invention, the chromatin regulatory peptide means a chromosomal protein or a fragment thereof that interacts with nucleosomes and / or chromosomal proteins to facilitate nucleosome rearrangement and / or chromatin remodeling. More specifically, the chromatin regulatory peptide can be, but is not limited to, a high-mobility group nucleosome binding domain 1 (HN1) or a fragment thereof, a histone H1 central globular domain (H1G) or a fragment thereof, or a combination thereof.

[0045] The high-mobility group nucleosome binding domain (HMGN) is a chromosomal protein that regulates the structure and function of chromatin, and the histone H1 central globular domain (H1G) is a domain that constitutes histone H1, also known as "linker histone". Histone H1 regulates the compaction state of the nucleosome array, affects the morphology, and the central globular domain is known to bind near the entry / exit site of the linker DNA on the nucleosome.

[0046] The chromatin regulatory peptide can be linked to the CRISPR / Cas9 protein or reverse transcriptase directly by a chemical bond, indirectly by a linker, or by a combination thereof. Specifically, at least one chromatin regulatory peptide can be linked to the N-terminus, C-terminus, and / or internal position of the CRISPR / Cas9 protein. Preferably, the fusion protein of the present invention comprises two chromatin regulatory peptides linked to the CRISPR / Cas9 protein or reverse transcriptase. More preferably, in the fusion protein, HMGN1 (HN1) can be linked to the N-terminus of the Cas9 protein or a variant thereof, and the histone H1 central globular domain (H1G) can be linked to the C-terminus of the Cas9 protein or a variant thereof or the C-terminus of the reverse transcriptase. Most preferably, the fusion protein can consist of the configuration of i) N-terminus - [HN1] - [Cas9] - [H1G] - [reverse transcriptase] - C-terminus, or ii) N-terminus - [HN1] - [Cas9] - [reverse transcriptase] - [H1G] - C-terminus.

[0047] In the present invention, the fusion protein may further comprise at least one nuclear localization signal, at least one cell-penetrating domain, at least one marker domain, or a combination thereof, and preferably may further comprise nuclear localization signal (NLS) sequences at the N-terminus and C-terminus, respectively, but is not limited thereto.

[0048] The fusion protein according to the present invention may consist of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. At this time, the fusion protein may include an amino acid sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95%, 96%, 97%, 98%, 99% or more with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0049] In the present invention, the "Cas9 (CRISPR associated protein 9) protein" is a protein that plays an important role in the immunological defense of certain bacteria against DNA viruses and is widely used in the application of genetic engineering. Since the main function of the protein is to cleave DNA, it can be applied when modifying the genome of cells. Specifically, CRISPR / Cas9 is a third-generation gene scissors that recognizes, cleaves, and edits a specific base sequence to be used, and is useful for easily, quickly, and efficiently performing operations such as inserting a specific gene into a target site of the genome or stopping the activity of a specific gene. The Cas9 protein or gene information can be obtained from a known database such as GenBank of NCBI (National Center for Biotechnology Information), but is not limited thereto. In addition, the Cas9 protein can be appropriately ligated with additional domains according to its purpose. In the present invention, the Cas9 protein may include all mutants of Cas9 as long as it has the function of a nuclease for gene editing, not just wild-type Cas9.

[0050] In the present invention, the Cas9 mutant may mean a mutant that has been mutated to lose the endonuclease activity of cleaving double-stranded DNA. For example, the Cas9 mutant may be one or more selected from a Cas9 protein mutated to lose endonuclease activity and have nickase activity, and a Cas9 protein mutated to lose both endonuclease activity and nickase activity, and preferably may be Cas9 nickase.

[0051] The Cas9 nickase may be one in which a mutation has occurred and inactivated it in the catalytic active domain of the nuclease (e.g., the RuvC or HNH domain of Cas9). Specifically, it may contain a mutation in which one or more selected from the group consisting of aspartic acid at position 10 (D10), glutamic acid at position 762 (E762), histidine at position 840 (H840), asparagine at position 854 (N854), asparagine at position 863 (N863), and aspartic acid at position 986 (D986) are substituted with any other amino acid. Preferably, the Cas9 nickase of the present invention may contain, but is not limited to, a mutation in which histidine at position 840 is substituted with alanine (H840A).

[0052] The origin of the Cas9 protein or its variant is not limited. As non-limiting examples, it may be derived from Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, Legionella pneumophila, Listeria innocua, or Streptococcus mutans.

[0053] The Cas9 protein or its variant can be isolated from microorganisms or can be artificial or non-naturally occurring, such as by recombinant or synthetic methods. The Cas9 can be used in the form of pre-transcribed mRNA or pre-produced protein in vitro, or in the form contained in a recombinant vector for expression in target cells or in vivo. In one embodiment, the Cas9 can be a recombinant protein made by recombinant DNA (rDNA). Recombinant DNA means a DNA molecule artificially made by genetic recombination methods such as molecular cloning because it contains heterologous or homologous genetic materials obtained from various organisms.

[0054] In the present invention, the term "reverse transcriptase" refers to an enzyme having the ability to synthesize DNA using RNA as a template. In the present invention, the reverse transcriptase can include all variants of the reverse transcriptase as long as it has the function of synthesizing DNA using RNA as a template as described above. The reverse transcriptase or its variant can preferably be derived from Moloney murine leukemia virus (M-MLV), but is not limited thereto.

[0055] The term "guide RNA" used in the present invention means an RNA containing a targeting sequence capable of hybridizing to a specific base sequence (target sequence) within a target site in a target gene, and binds to a nuclease protein such as Cas in vitro or in vivo (or in cells), and serves to guide it to the target gene (or target site).

[0056] The guide RNA can be appropriately selected according to the type of nuclease forming the complex and / or the microorganism from which it is derived. For example, the guide RNA of the present invention can be pegRNA, dead sgRNA, or nicking sgRNA.

[0057] The guide RNA has a spacer region (also referred to as a Spacer region, Target DNA recognition sequence, base pairing region, etc.) which is a portion having a sequence (targeting sequence) complementary to a target sequence within a target gene (target site), and may include a hairpin structure for Cas9 protein binding. More specifically, it may include a portion containing a sequence complementary to the target sequence within the target gene, a hairpin structure for Cas protein binding, and a terminator sequence. Additionally, the pegRNA has a base sequence (primer binding site, PBS) complementary to the non-target strand of the target gene and a reverse transcriptase template strand site (RT template) containing the base sequence to be edited.

[0058] The targeting sequence of the guide RNA, which is capable of hybridizing with the target sequence of the guide RNA, means a nucleotide sequence having a sequence complementarity of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% with the nucleotide sequence of the DNA strand where the target sequence is located (i.e., the DNA strand where the PAM sequence (5’-NGG-3’ (N is A, T, G, or C)) is located) or its complementary strand, and is capable of complementary binding with the nucleotide sequence of the complementary strand.

[0059] The guide RNA can be used in the form of RNA (or included in the composition), or can be used (or included in the composition) in the form of a plasmid containing DNA encoding it.

[0060] In the present invention, the dsgRNA can be composed of 10 - 20 nt (nucleotides) in length, preferably 11 - 19 nt, 12 - 18 nt, 13 - 17 nt, 13 - 16 nt, and most preferably 14 - 15 nt in length, but is not limited thereto.

[0061] In addition, the dsgRNA can bind to a pegRNA binding site, preferably at a position 5 to 70 nt (nucleotides), preferably 6 to 65 nt, most preferably 7 to 62 nt away from the spacer region of the pegRNA, to increase the chromatin accessibility of the fusion protein.

[0062] As used in the present invention, the term "Chromatin accessibility" refers to the physical compaction level of chromatin, which is a complex mainly composed of DNA and related proteins composed of histones, transcription factors (TFs), chromatin-modifying enzymes, and chromatin-remodelling complexes. The eukaryotic genome is generally compressed into nucleosomes containing ~147 bp of DNA surrounding a histone octamer, but the occupancy of nucleosomes is not uniform in the genome and varies depending on the tissue and cell type. Nucleosomes generally deplete at genomic positions where cis-regulatory elements (enhancers and promoters) that interact with transcription regulators (e.g., transcription factors) are present, generating accessible chromatin. Regarding gene editing (gene correction), there is a significant difference in the activity of gRNAs targeting open genomic regions compared to closed genomic regions, and since the efficiency of Cas9 is affected by local chromatin accessibility, a positive correlation between chromatin accessibility and CRISPR-Cas9-mediated gene editing efficiency is known.

[0063] In another aspect of the present invention, the present invention provides a gene editing method comprising the step of contacting the gene editing composition with a target region containing a target nucleic acid sequence in vitro or ex vivo.

[0064] The composition for gene editing can preferably be applied to eukaryotic cells, and the eukaryotic cells can preferably be derived from mammals including primates such as humans and rodents such as mice, but are not limited thereto.

[0065] In another aspect of the present invention, the present invention provides a gene editing kit containing the composition for gene editing.

[0066] In the present invention, the kit may include, together with the composition for gene editing, all substances (reagents) necessary for performing gene editing such as a buffer and deoxyribonucleotide-5-triphosphate (dNTP). Also, the optimal amounts of the reagents used in specific reactions of the kit can be easily determined by those skilled in the art who have acquired the disclosures in this specification.

[0067] In yet another aspect of the present invention, there is provided a method for producing a genetically modified mammal excluding humans, including the steps of injecting the composition for gene editing into mammalian cells excluding humans to obtain genetically modified mammalian cells, and transplanting the obtained genetically modified mammalian cells into the fallopian tubes of a surrogate mother of a mammal excluding humans.

[0068] By introducing the gene editing composition of the present invention, gene editing efficiencies (e.g., base substitution, insertion, or deletion) of 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100% can be achieved in mammalian cells.

[0069] In the present invention, the step of introducing the composition for gene editing into the mammalian cells is: i) transfecting the cells with a plasmid vector or viral vector encoding the prime editing fusion protein, pegRNA, nsgRNA, and dsgRNA according to the present invention, ii) directly injecting a mixture of mRNA, pegRNA, nsgRNA, and dsgRNA encoding the fusion protein into the cells, or injecting each of them directly, or iii) directly injecting a mixture or complex form of ribonucleoprotein of the fusion protein, pegRNA, nsgRNA, and dsgRNA into the cells can be performed.

[0070] The direct injection may mean that each of the mRNA and guide RNA or ribonucleoprotein in ii) or iii) passes through the cell membrane and / or nuclear membrane and is transmitted to the genome without using a recombinant vector, and can be performed, for example, by nanoparticles, electroporation, lipofection, microinjection, etc.

[0071] The mammalian cells into which the gene editing composition is introduced can be embryos of mammals including primates such as humans and rodents such as mice, and preferably embryos of mammals other than humans. For example, the embryo can be obtained by collecting a fertilized embryo obtained by mating a superovulated female mammal and a male mammal from the oviduct of the female mammal. The embryo to which the base editing composition is applied (injected) can be a fertilized 1-cell stage embryo (zygote).

[0072] The obtained genetically recombinant mammalian cells can be cells in which base substitution, insertion, or deletion mutations have occurred in the target gene by the introduction of the gene editing composition.

[0073] The genetically recombinant mammalian cells, preferably the mammal that receives the transplantation of genetically recombinant embryonic cells into the oviduct, can be a mammal of the same species (surrogate mother) as the mammal from which the embryonic cells are derived.

[0074] Further, the present invention provides a genetically recombinant mammal produced by the above method.

[0075] Hereinafter, preferred embodiments are presented to assist in the understanding of the present invention. However, the following embodiments are merely provided to more easily understand the present invention, and the content of the present invention is not limited by the following embodiments.

[0076] [Example] Example 1. Experimental Method 1-1. Construction of Plasmid DNA Using NEBuilder® HiFi DNA Assembly Master Mix (E2621L, NEB), the HN1 (High-mobility group nucleosome binding domain 1) and H1G (Histone H1 central globular domain) oligos were fused to both sides of nCas9 in pCMV-PE2 (#132775, Addgene), and a chromatin-modulating peptide prime editor was constructed according to the manufacturer's protocol.

[0077] Also, to create pegRNA expression vectors for inducing specific mutations in the Igf2, Adamts20, Casp1, and Hoxd13 genes, spacer, prime binding site, and reverse transcriptase template oligos sequences were inserted into the BsaI enzyme cleavage site of the pU6-pegRNA-GG-receptor vector (#132777, Addgene). The nsgRNA and dsgRNA expression vectors were inserted into the pRG2-GG vector (#104174, Addgene). Additionally, the sequences of pegRNAs, nsgRNAs, and dsgRNAs specific to each target gene used in this example are presented in Tables 1-3 below.

[0078] JPEG0007700222000001.jpg239170JPEG0007700222000002.jpg200170JPEG0007700222000003.jpg193170

[0079] 1-2. Lipofection and electroporation HEK293T cells, NIH / 3T3 cells (ATCC, CRL-1658), and C2C12 cells (ATCC, CRL-1772) into which a reporter system expressing tdTomato at the AAVS1 locus was introduced were cultured under conditions of 5% CO2 and 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM; LM001-05, Welgene) supplemented with 10% FBS (S 001-01, Welgene) or BCS (26170-043, Gibco). In this example, according to the protocol of the manufacturing company, 50 μL of Opti-MEM (31985070, Gibco) containing 0.5 μg of pegRNA, 2.15 μg of PE2, 0.22 μg of nsgRNA, and 1 μL of Lipofectamine 2000 reagent (11668019, Thermo Fisher Scientific) was added to 2×10 4 cells for treatment and transfection, and then the cells were cultured for 11 days.

[0080] Electroporation was performed on 1×10 5 each of NIH / 3T3 cells and C2C12 cells. Each cell line was mixed with plasmids of 3 μg of PE2 or CMP-PE, 0.7 μg of pegRNA, 0.3 μg of nsgRNA, and 0.25 μg of dsgRNA, and transfected using a Neon TM Transfection System (MPK1096, Thermo Fisher Scientific) according to the protocol of the manufacturing company. Then, the cells were collected after 72 hours of culture, and targeted deep sequencing was performed.

[0081] 1 - 3. Production of DNA Amplicons After transfection, genomic DNA was extracted from tdTomato-expressing reporter HEK293T, NIH / 3T3, C2C12 cells, and mouse embryos using DNeasy Blood & Tissue Kits (69506, Qiagen). Subsequently, the target sequences edited using Phusion TM High-Fidelity DNA polymerase (F-530XL, Thermo Fisher Scientific) and Sungen (SG-PT02, Sun genetics) were amplified.

[0082] 1 - 4. In vitro Transcription Transcripts of PE2 and CMP-PE were produced using mMESSAGE mMACHINE T7 Ultra Kit (AM1345, Invitrogen) and purified using MEGAclear TM Transcription Clean-Up Kit (AM1908, Invitrogen). According to the manufacturer's protocol, transcription of pegRNA, nsgRNA, and dsgRNA was induced using T7 RNA polymerase (M0251, NEB), and the transcribed RNAs were purified using Expin TM CleanUp SV (113 - 150, GeneAll). Subsequently, the purified RNAs were quantified using NanoDrop One UV-Vis (Thermo Fisher Scientific).

[0083] 1 - 5. Experimental Animals The in vivo experiments in this example were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. C57BL / 6N and ICR mice were used for embryo donation, and surrogate mothers were housed in a specific pathogen-free laboratory.

[0084] 1 - 6. Microinjection Female C57BL / 6N mice were injected with HyperOva (KYD-010-EX-x5, CARD) and hCG (CG10-1vl, Sigma) at 48-hour intervals to induce ovarian hyperstimulation. After injection of the hCG hormone, the female mice were mated with male C57BL / 6N wild-type mice. Fertilized one-cell stage embryos were obtained from the ampulla and cultured in M2 medium (M7167, Sigma) until two pronuclei appeared. The prime editing mixture for microinjection was prepared at a concentration of 100 ng of PE2 or CMP-PE mRNA, 65 ng of pegRNA, 32.5 ng of nsgRNA, and 22 ng of dsgRNA under 100 μL of Tris-EDTA (pH 7.4). After microinjection, the embryos were cultured in KSOM medium (MR-121-D, Millipore) in an incubator at 37 °C for 4 days for development into blastocysts. Subsequently, a part of the two-cell stage embryos was transplanted into the oviducts of pseudopregnant surrogate mothers.

[0085] 1-7. Genotyping and targeted deep sequencing Each target was amplified by nested PCR using specific primers. For the library composed of PCR amplicons, base sequence analysis was performed using the iSeq TM 100 sequencing system (Illumina, Inc.). Subsequently, the sequence analysis data were analyzed using the CRISPR REGN Tools program (http: / / www.rgenome.net / ) and the EUN program (https: / / daeunyoon.com / ).

[0086] 1-8. Whole genome sequencing and variant calling Genomic DNA was isolated from the ears of mice (C57BL / 6N) using the DNeasy Blood & Tissue kit (69506, Qiagen). After that, according to the manufacturer's instructions, the genomic DNA was sheared using a Covaris S2 ultrasonic device system, and a paired-end DNA library was prepared using the Truseq Nano DNA sample prep kit. Deep coverage (30x) whole-genome sequencing was performed by 101 base paired-end sequencing on the Illumina Novaseq 6000 platform (Illumina), and the sequence reads were aligned to the mouse reference genome GRCm38 / mm10 using BWA-MEM. Single nucleotide variants and small insertions / deletions (indels) were detected using GATK4 HaplotypeCaller, and known variants present in dbSNP were annotated against mouse v142 (dbSNP142) using ANNOVAR. New variants not present in dbSNP142 were further analyzed to confirm their positions at off-target sites. The putative off-target sites were compared with the candidates of Cas-OFFinder considering a maximum of 7-bp or 2-bp bulge + 5-bp mismatch. All variants were manually confirmed by visualizing the read plots.

[0087] 1-9. DNase I digestion assay and qPCR The inventors of the present invention performed a DNase I digestion assay according to a conventionally known method. Specifically, after detaching the cells and repeatedly washing them with cold 1X PBS, the cells were spin-downed twice at 900 rpm for 5 minutes. Next, the cells were lysed using cold RSB buffer (10 mM Tris-HCl, 10 mM NaCl, and 3 mM MgCl2) + 0.1% IGEPAL CA-630 (I8896, Sigma), and the nuclei were pelleted by spin-down at 500 g for 10 minutes at 4°C. Next, the supernatant was removed, and the nuclei were cultured at 37°C for 20 - 30 minutes under conditions with or without treatment with DNase I (2 - 16 U). Thereafter, 50 mM EDTA was added to stop the reaction, and the DNA degraded by DNase I was purified using the DNeasy Blood & Tissue kit (69506, Qiagen). Real-time qPCR was performed using the KAPA SYBR FAST qPCR Master Mix Kit (KR0389, Kapa Biosystems), and the fraction of the complete genomic DNA was measured using the comparative C T method. The primer sequences used for the real-time qPCR are listed in Table 4 below. JPEG0007700222000004.jpg174170

[0088] 1 - 10. Statistical analysis The data are shown as the mean and standard deviation, and three or five independent repeated experiments were performed. The P value was calculated using an unpaired and two-sided Student's t-test.

[0089] Example 2. Verification of Prime Editing System in Mammalian Genome and Confirmation of Editing Efficiency The inventors first attempted to verify the applicability of the prime editing system in the mammalian genome. For this purpose, a reporter system that expresses tdTomato at the AAVS1 locus of HEK293T cells was used. Specifically, as shown in Figure 1a, a prime editor 3 (PE3) with a primer binding site (PBS) length of 8 nt and a reverse transcriptase (RT) template length of 17 nt (PBS8-RT17) was used to generate a stop codon by inserting a thymine (T) base into the tdTomato sequence. Also, the pegRNA (prime-editing guide RNA) was designed to remove the PAM sequence on the non-target strand to suppress editing in the edited strand. Subsequently, the inventors gated tdTomato-negative cells by flow cytometry to evaluate the editing efficiency of the tdTomato gene by the PE3.

[0090] As a result, as shown in Figure 1b, when inducing tdTomato gene editing using PE3 (tdTomato expressing HEK293T+PE3), it was confirmed that 32% of the cells were tdTomato-negative when compared with the positive control group expressing tdTomato (tdTomato expressing HEK293T). Such results suggest that the prime editor enables the generation of the desired target mutations in the system of the present invention.

[0091] Next, in order to create a mouse model using a prime editor, we attempted to design a target containing 8 metastatic mutations or one or more nucleotide insertions or deletions. First, as shown in Fig. 2a, we induced the generation of stop codons in two mouse genes, namely, Igf2 (insulin-like growth factor 2) and Adamts20 (a disintegrin and metalloproteinase domain with thrombospondin type-1 motifs 20). The Igf2 gene can induce a dwarf phenotype by mutation of the Igf2 allele inherited from the paternal line. Specifically, we generated a stop codon by inserting TA bases into exon4 of the Igf2 gene, inducing loss of gene function. Also, to suppress continuous editing of the strand to be edited, we replaced the nucleotides of the PAM sequence from NGG to NCG. On the other hand, Adamts20 is a gene involved in the development of melanocytes, and the generation of an early stop codon at the E584 site of the Adamts20 locus is known to be associated with a typical white belt phenotype. We induced the substitution of CG with TT in exon12 of the Adamts20 locus, inducing an early stop codon (E584*) and modification of the PAM sequence (from NGG to NAG). Also, to induce the above mutations in the mouse gene target, we used a PE3 system composed of PE (nCas9 fused with engineered M-MLV RT), pegRNA, and nicking sgRNA (nsgRNA). At this time, the nsgRNA was used for the purpose of improving the editing efficiency by promoting DNA repair activity through cleavage of the target DNA strand, that is, the non-edited strand.

[0092] Next, the inventors evaluated the editing efficiency with pegRNAs composed of various PBS lengths (8-14 nt) and RT template lengths (10-18 nt) to optimize prime editing at the Igf2 and Adamts20 target sites. At this time, PBS lengths with thymine at the 3'-end, which can be part of the transcription termination signal, and RT lengths with cytosine at the 5'-end, which can interfere with the pegRNA structure, were excluded. Subsequently, three types of plasmids encoding PE, pegRNA, and nsgRNA were transfected into NIH / 3T3 cells by electroporation, and the cells were collected after 72 hours of culture and subjected to targeted deep sequencing.

[0093] As a result of the experiment, as shown in Fig. 2b, it was confirmed that the PE3-mediated editing efficiency for the Igf2 and Adamts20 targets in NIH / 3T3 cells was less than 3%. From such results, it was determined that improvement of the editing efficiency is necessary in order to use prime editors for the generation of mouse models.

[0094] Example 3. Confirmation of Enhancement of Editing Efficiency of Prime Editor by dsgRNA As an attempt to improve the editing efficiency of prime editors, the inventors used dsgRNAs for resolving the chromatin structure of target sites based on the proxy-CRISPR idea, instead of catalytically dead endonucleases. A dsgRNA is a guide RNA 14-15 nt in length that exhibits inactivated catalytic activity and guides a Cas endonuclease to bind to a target site. Therefore, the inventors hypothesized that the prime editor plays the following two roles. One is to perform a prime editing function together with pegRNA at the target site, and the other is to regulate the chromatin adjacent to the target site with dsgRNA. The inventors designed proximal dsgRNAs adjacent to the Igf2 and Adamts20 target sites in the nucleotide position range of 7-62 nucleotides from the spacer of pegRNA. Subsequently, to confirm the editing efficiency, proximal dsgRNA was applied to various pegRNA lengths at the Igf2 and Adamts20 sites, and interestingly, it was revealed that the editing efficiency of PE3 using proximal dsgRNA was improved in most groups. Therefore, the inventors selected PBS9-RT14 pegRNA and PBS11-RT13 pegRNA, which showed the highest efficiency for the Igf2 and Adamts20 targets, respectively, and conducted subsequent experiments.

[0095] Next, in order to screen for dsgRNAs with high editing efficiency, additional proximal dsgRNAs for the Igf2, Adamts20, Casp1 (4 bp deletion), Hoxd13 (substitution from G to T), Angpt1 (substitution from CGG to TGA), and Ksr2 (insertion of TGAT) genes were designed and tested. For this purpose, plasmids encoding PE, pegRNA, nsgRNA, and their respective proximal dsgRNAs were transfected into NIH / 3T3 and C2C12 cells by electroporation, and then targeted deep sequencing was performed.

[0096] As a result, as shown in Fig. 3, it was confirmed that the proximal dsgRNA selectively improved the editing efficiency in most targets compared to PE3. From such results, it was found that the gene editing efficiency by the application of dsgRNA varies depending on the position of the proximal dsgRNA, and in order to induce effective target mutations, a screening process for the optimal dsgRNA for each target and cell type is necessary.

[0097] Example 4. Confirmation of Enhancement of Editing Efficiency of Prime Editor Bound with CMP As another attempt to improve the editing efficiency of prime editors, the inventors manipulated prime editors using high-mobility group nucleosome binding domain 1 (HN1) and histone H1 central globular domain (H1G), which are chromatin-modulating peptides (CMPs). Specifically, the HN1 and H1G were added to the protein in which the Cas9 nickase (nCas9) of the prime editor was fused with reverse transcriptase to produce two versions. As shown in Fig. 4a, the fusion protein with HN1 bound to the N-terminal side of nCas9 and H1G bound to the C-terminal side was designated as CMP-PE-V1, and the fusion protein in which HN1 was bound to the N-terminal side of nCas9, the engineered M-MLV RT was bound to the C-terminal side of nCas9, and H1G was bound to the C-terminal of the RT was designated as CMP-PE-V2. Also, the amino acid sequences of the CMP-PE-V1 and CMP-PE-V2 are shown in Figs. 4b and 4c, respectively.

[0098] The inventors transmitted the aforementioned CMP-PE3-V1 (pegRNA / nsgRNA and CMP-PE-V1) or CMP-PE3-V2 (pegRNA / nsgRNA and CMP-PE-V2) to two types of mouse cell lines respectively, and compared the editing efficiency with the case where PE3 not conjugated with CMP was introduced into the cells. As a result, as shown in Fig. 4b, it was confirmed that in most target sites, in the case of CMP-PE3-V1, the editing efficiency was much higher than that of PE3. In particular, the editing efficiency by CMP-PE3-V1 was 2.55 times higher in the case of Igf2 and 3.92 times higher in the case of Adamts20 in NIH / 3T3 cells. Also, in HEK293T cells, which are human cells, the editing efficiency was confirmed by applying a prime editor improved targeting the HEK3 sequence and a dead sgRNA. The results are shown in Fig. 4e. It was confirmed that the editing efficiency was improved in CMP-PE-V1 or CMP-PE-V2, which are prime editing methods improved compared to PE3. Also, when the dead sgRNA was applied together with the improved prime editor, it was confirmed that the efficiency was improved compared to the case where only PE3 or the improved editor was treated (CMP-PE3-V2 + dsgRNA(-11)). Such results suggest that the prime editor engineered using chromatin-modulating peptides HN1 and H1G significantly improves the editing efficiency.

[0099] Example 5. Confirmation of Uprising Effect of Editing Efficiency of Prime Editor Applied with dsgRNA and CMP Furthermore, based on the results of Examples 3 and 4 above, the inventors co-transmitted CMP-PE3-V1 and dsgRNA (CMP-PE3-V1 + dsgRNA) to mouse cells and analyzed whether a synergistic effect on the editing efficiency appeared.

[0100] As a result, as shown in Fig. 5, when CMP-PE3-V1 and dsgRNA were co-delivered into mouse cells (CMP-PE3-V1 + dsgRNA), the editing efficiencies at the Igf2, Adamts20, and Hoxd13 target sites were increased by up to 4.20-fold, 5.11-fold, and 3.56-fold, respectively, compared to the PE3 control group. Such synergistic effects showed different levels depending on the cell line and target. Also, from the above results, when only CMP-PE3 was introduced into cells (CMP-PE3-V1), the efficiency of the prime editor was significantly improved in all the targets and two cell types tested. In contrast, when only dsgRNA was introduced into cells (PE3 + dsgRNA), or when CMP-PE3-V1 and dsgRNA were co-introduced (CMP-PE3 + dsgRNA), it was found that the editing efficiency could vary depending on the target site and cell type.

[0101] Furthermore, the inventors attempted to induce the generation of targeted mutations by injecting the advanced prime editor system into mouse embryos by microinjection and analyze its efficiency. Specifically, among the designed mouse targets, the Igf2 target site with relatively low undesired mutations was selected. As a result, as shown in Fig. 6a, a relatively high level of editing efficiency was shown for the Igf2 target in the embryos injected with CMP-PE3-V1 and the embryos injected with CMP-PE3-V1 + dsgRNA. Especially in the case of CMP-PE3-V1 + dsgRNA, the desired mutations were observed in 21 out of 22 embryos (95%) at the target site of the Igf2 gene, confirming that CMP-PE3-V1 + dsgRNA showed a very high editing efficiency compared to PE3 and PE3 + dsgRNA.

[0102] In addition, to verify the editing efficiency in the Adamts20, Hoxd13, Angpt1, Ksr2, and Ar genes in mouse embryos, tests were conducted using CMP-PE-V1 and proximal dsgRNA. As a result of targeted deep sequencing, as shown in Figure 6b, it was confirmed that in embryos injected with CMP-PE-V1 and proximal dsgRNA, the editing efficiency was improved at the Adamts20, Hoxd13, and Angpt1 targets, except for the Ksr2 and Ar targets, compared to the case of injecting PE3. Collectively, such results suggest that prime editing with enhanced editing efficiency is possible in mouse embryos using proximal dsgRNA and chromatin regulatory peptides.

[0103] Example 6. Confirmation of Improvement of Target Site Chromatin Accessibility by dsgRNA and CMP The inventors performed DNaseI digestion analysis and qPCR to confirm the chromatin state of each target site in order to confirm whether CMP-PE-V1 and dsgRNA unwind the chromatin structure of the target site and improve chromatin accessibility. At this time, a gene located at Chr3:71,026,628-71,026,685 (mouse genome build mm9) was used as a negative control group (closed chromatin), and the Col6a1 gene was used as a positive control group (open chromatin). As a result of the experiment, as shown in Figures 7a and 7b, in the NIH / 3T3 cell line, Igf2, Adamts20, and Hoxd13 appeared as relatively closed chromatin, and in C2C12, all targets except Igf2 were analyzed to be in an open chromatin state. Such results suggest that the chromatin structure state is different even though the target sequences are the same in the two cell lines.

[0104] Furthermore, at the target site of Igf2, a representative gene confirmed to have a closed chromatin structure, it was analyzed whether CMP-PE-V1 or dsgRNA changes the chromatin state. As a result, as shown in Fig. 7c, when compared with PE3, it was confirmed that the chromatin structure in the closed state was gradually changed to the open state by CMP-PE-V1, dsgRNA, or CMP-PE-V1 + dsgRNA. Such results are direct evidence demonstrating that the use of CMP-PE-V1 or dsgRNA can unwind the closed chromatin structure, improve chromatin accessibility, and enhance the efficiency of prime editing.

[0105] Example 7. Generation of Mouse Model with Induced Target Mutation and Analysis of Off-Target Effect Next, the inventors used PBS9-RT14 and dsgRNA+7, which have a relatively low frequency of undesired mutations, to induce the generation of the target mutation of Igf2 as shown in Fig. 8a in mouse embryos by microinjection, and transplanted the mouse embryos into surrogate mothers. Subsequently, as a result of observing and analyzing the offspring born from the surrogate mothers, as shown in Fig. 8b, it was confirmed that substitution from G to C and insertion of TA occurred at the Igf2 locus with an editing frequency of up to 47% (2 out of 10). In addition, as a result of analyzing whether the Igf2 mutation as described above is transmitted to the next generation, as shown in Fig. 8c, since 7 out of 9 F1 offspring born from Igf2 mutant mice have the same mutation, it was found that the target mutation can be transmitted to the next generation through germ cells.

[0106] To evaluate the off-target effect of the prime editor, the inventors used Cas-OFFinder to identify potential off-target sites in the mouse genome for pegRNAs and nsgRNAs of the Igf2 target with up to three nucleotide mismatches each. As a result, as shown in Fig. 9a, no potential off-target mutations were detected when compared to the wild type. Also, to confirm the off-target effect in the generated Igf2 mutant mice, whole-genome sequencing (WGS) was performed. As a result, as shown in Figs. 9b and 9c, a single off-target site of the nsgRNA was discovered, but it was confirmed by Sanger sequencing using genomic DNA that the site was a false positive.

[0107] Finally, to confirm the phenotype of the Igf2 mutant mice, Igf2 p+ / m- male (F1) was mated with wild-type female mice. As a result, as shown in Figs. 10a and 10b, the Igf2 p- / m+ mice carrying the mutation of the Igf2 gene inherited from the paternal allele showed a dwarfism phenotype consistent with the desired mutant genotype. Such results suggest that the improved prime editing system according to the present invention can be effectively applied to the generation of mouse models.

[0108] The above description of the present invention is for illustrative purposes, and those of ordinary skill in the technical field to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical idea and essential features of the present invention. Therefore, it must be understood that the above-described embodiments are illustrative in all respects and not restrictive.

Claims

1. (a) (i) A CRISPR / Cas9 protein or a variant thereof, (ii) A reverse transcriptase, and (iii) A fusion protein comprising chromatin-modulating peptides or a nucleic acid encoding the same, (b) A guide RNA or a nucleic acid encoding the same, wherein the CRISPR / Cas9 protein variant is a nickase in which either the RuvC domain or the HNH domain is inactivated, the guide RNA comprises a pegRNA (prime editing guide RNA) and a dsgRNA (dead single guide RNA), the chromatin-modulating peptides are a high-mobility group nucleosome binding domain 1 (HNH1) and a histone H1 central globular domain (H1G), the fusion protein has the structure of N-terminal - [HNH1] - [Cas9] - [H1G] - [reverse transcriptase] - C-terminal, or N-terminal - [HNH1] - [Cas9] - [reverse transcriptase] - [H1G] - C-terminal, the dsgRNA binds to a position 5 to 70 nt away from the pegRNA binding site to improve the chromatin accessibility of the fusion protein, and is A composition for gene editing of mammalian cells.

2. The composition for gene editing according to claim 1, wherein the fusion protein further comprises a nuclear localization signal (NLS) sequence at the N-terminus and the C-terminus, respectively.

3. The composition for gene editing according to claim 1, wherein the reverse transcriptase or a variant thereof is derived from Moloney murine leukemia virus (M-MLV).

4. The composition for gene editing according to claim 1, wherein the fusion protein consists of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

5. The dsRNA for gene editing according to claim 1, characterized in that it is composed of 10 to 20 nucleotides (nt) in length.

6. The composition for gene editing according to claim 1, further comprising a sgRNA (single guide RNA) that binds complementarily to a non-target DNA strand and induces cleavage of the target DNA strand.

7. The composition for gene editing according to claim 1, characterized in that the gene editing efficiency and target specificity are enhanced.

8. A gene editing method, comprising the step of contacting the composition for gene editing according to claim 1 with a target region containing a target nucleic acid sequence in vitro or ex vivo.

9. A kit for gene editing, comprising the composition for gene editing according to claim 1.

10. Introducing the composition for gene editing according to claim 1 into mammalian cells excluding human cells to obtain genetically recombinant mammalian cells; Transplanting the obtained genetically recombinant mammalian cells into the fallopian tube of a mammalian surrogate mother excluding human; a method for producing a genetically recombinant mammal excluding human, comprising the steps.

11. The production method according to claim 10, characterized in that the mammalian cells are mammalian embryonic cells.

Images