WO2024053964A1 - Système d'amélioration de l'édition génique par le modification de la structure de l'arn guide de cas9 provenant de campylobacter jejuni - Google Patents

Système d'amélioration de l'édition génique par le modification de la structure de l'arn guide de cas9 provenant de campylobacter jejuni Download PDF

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WO2024053964A1
WO2024053964A1 PCT/KR2023/013166 KR2023013166W WO2024053964A1 WO 2024053964 A1 WO2024053964 A1 WO 2024053964A1 KR 2023013166 W KR2023013166 W KR 2023013166W WO 2024053964 A1 WO2024053964 A1 WO 2024053964A1
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sequence
engineered
guide rna
cas9
nucleic acid
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송동우
김석중
이혜림
오혜경
김운기
이재영
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주식회사 툴젠
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island

Definitions

  • the present invention is an invention in the field of CRISPR/Cas9 system technology.
  • the CRISPR/Cas system is a type of immune system found in prokaryotic organisms and includes Cas proteins, and guide RNA.
  • the detailed composition of the Cas protein or guide RNA is described in detail in the published document WO2018/231018 (International Publication No.).
  • Cas9 protein from Campylobacter jejuni also referred to as CjCas9, is one of the orthologs of Cas9 protein.
  • the CjCas9 has the smallest size among Cas9s and is known to exhibit double-strand DNA cleavage activity in eukaryotic cells.
  • the present invention relates to a Cas9 protein derived from Campylobacter jejuni and a guide RNA that can form a complex with the Cas9 protein.
  • the present specification is intended to provide an engineered guide RNA for the Cas9 protein from Campylobacter jejuni that has been modified to not contain more than four consecutive uridines.
  • the present specification seeks to provide various implementation forms of the engineered CRISPR/Cas9 system including the engineered guide RNA.
  • the present specification seeks to provide a gene editing method using the engineered CRISPR/Cas9 system. This specification seeks to provide uses of the engineered CRISPR/Cas9 system.
  • an engineered guide RNA capable of forming a complex with the Cas9 protein from Campylobacter jejuni represented by the following sequence:
  • the guide sequence can target a predetermined target sequence
  • the second sequence is AGUCCCUGAAGGGACU (SEQ ID NO: 6), or a sequence that is more than 80% identical to SEQ ID NO: 6,
  • the fourth sequence is UAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 7), or a sequence that is more than 80% identical to SEQ ID NO: 7,
  • the first sequence and the third sequence are selected from the following combinations:
  • the first sequence is 5'-GUUUC-3'
  • the third sequence is 5'-GAAA-3'
  • the first sequence is 5'-GUUCU-3'
  • the third sequence is 5'-AGAA-3'
  • the first sequence is 5'-GUCUU-3'
  • the third sequence is 5'-AAGA-3'
  • the first sequence is 5'-GCUUU-3'
  • the third sequence is 5'-AAAG-3'.
  • the sequence of the engineered scaffold is GUUUCAGUCCCUGAAGGGACUGGAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 2), GUUCUAGUCCCUGAAGGGACUGAGAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 3), GUCUUAGUCCCUGAAGGGACUGAAGAUAAAGAGUU It may be selected from UGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 4), and GCUUUAGUCCCUGAAGGGACUAAAGUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 5).
  • sequence of the engineered scaffold may be GUCUUAGUCCCUGAAGGGACUGAAGAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 4).
  • an engineered CRISPR/Cas9 complex comprising:
  • the CRISPR/Cas9 complex can target a predetermined target sequence of the engineered guide RNA.
  • DNA encoding the engineered guide RNA is provided herein.
  • the present specification provides a vector capable of expressing each component of the CRISPR/Cas9 system, including the following:
  • a nucleic acid encoding the Cas9 protein from Campylobacter jejuni A nucleic acid encoding the Cas9 protein from Campylobacter jejuni.
  • the vector may be a viral vector or a non-viral vector.
  • the vector may be one or more viral vectors selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus.
  • the vector may be included in a single vector.
  • the vector may be included in two or more vectors.
  • engineered CRISPR/Cas9 compositions comprising:
  • Cas9 protein derived from Campylobacter jejuni, or a nucleic acid encoding the Cas9 protein
  • the engineered guide RNA or a nucleic acid encoding the guide RNA.
  • the composition includes the Cas9 protein and the engineered guide RNA, and the Cas9 protein may bind to the engineered guide RNA to form a Cas9-gRNA complex.
  • the composition may be a composition containing a nucleic acid encoding the Cas9 protein and a nucleic acid encoding the engineered guide RNA.
  • the composition comprises the vector.
  • a target nucleic acid having a target sequence of a gene in a cell comprising:
  • the guide domain of the engineered guide RNA of the composition can target the target nucleic acid.
  • the gene editing efficiency of the CRISPR/Cas9 system derived from Campylobacter jejuni can be dramatically increased.
  • gene editing efficiency can be expected to be greatly increased.
  • Figure 1 schematically shows the engineered guide RNA disclosed herein and shows examples of four representative engineered guide RNAs.
  • Figure 2 shows the indel occurrence rate in the HIF1A-E4 gene of the CRISPR/CjCas9 system containing each of wild-type single guide RNA and engineered guide RNA targeting the HIF1A gene in a human cell line (HEK293T) according to Experimental Example 2.
  • NT is a negative control
  • Ori is a wild-type guide RNA
  • Modi-1 is an engineered guide RNA with an engineered scaffold sequence of SEQ ID NO: 2
  • Modi-2 is an engineered guide RNA with an engineered scaffold sequence of SEQ ID NO: 3.
  • the engineered guide RNA, Modi-3 refers to an engineered guide RNA having an engineered scaffold sequence of SEQ ID NO: 4
  • Modi-4 refers to an engineered guide RNA having an engineered scaffold sequence of SEQ ID NO: 5.
  • Figure 3 is a graph showing the expression efficiency of expression vectors for each of wild-type single guide RNA and engineered guide RNA targeting the HIF1A gene in a human cell line (HEK293T) according to Experimental Example 2.
  • NT is a negative control
  • Ori is a wild-type guide RNA
  • Modi-1 is an engineered guide RNA with an engineered scaffold sequence of SEQ ID NO: 2
  • Modi-2 is an engineered guide RNA with an engineered scaffold sequence of SEQ ID NO: 3.
  • the engineered guide RNA, Modi-3 refers to an engineered guide RNA having an engineered scaffold sequence of SEQ ID NO: 4
  • Modi-4 refers to an engineered guide RNA having an engineered scaffold sequence of SEQ ID NO: 5.
  • Figure 4 shows the indel generation efficiency of the CRISPR/CjCas9 system containing the engineered guide RNA of Modi-3 transfected with Low 240ng plasmid transfection and High 800ng plasmid transfection in a rat cell line (RT4-D6P2T) according to Experimental Example 3.
  • This is a graph compared to the CRISPR/CjCas9 system containing wild-type guide RNA.
  • ORI-Low is a wild-type guide RNA infected with Low 240ng plasmid transfection
  • ORI-High is a wild-type guide RNA infected with High 800ng plasmid transfection
  • Modi-3-Low is an engineered guide RNA of SEQ ID NO. 4.
  • Modi-3-High is a transfection of an engineered guide RNA with a scaffold sequence using Low 240ng plasmid transfection.
  • Modi-3-Low is a transfection of an engineered guide RNA with an engineered scaffold sequence of SEQ ID NO: 4 using High 800ng plasmid. It was infected by transfection.
  • Figure 5 is a graph comparing the expression level of engineered guide RNA of Modi-3 transfected with Low 240ng plasmid transfection and High 800ng plasmid transfection in rat cell line (RT4-D6P2T) according to Experimental Example 3 with that of wild type guide RNA. am.
  • ORI-Low is a wild-type guide RNA infected with Low 240ng plasmid transfection
  • ORI-High is a wild-type guide RNA infected with High 800ng plasmid transfection
  • Modi-3-Low is an engineered guide RNA of SEQ ID NO. 4.
  • Modi-3-High is a transfection of an engineered guide RNA with a scaffold sequence using Low 240ng plasmid transfection.
  • Modi-3-Low is a transfection of an engineered guide RNA with an engineered scaffold sequence of SEQ ID NO: 4 using High 800ng plasmid. It was infected by transfection.
  • the term "about” refers to 30, 25, 20, 15, 10, 9, 8, 7 for a reference amount, level, value, number, frequency, percent, dimension, size, amount, weight or length. means a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight or length that varies by , 6, 5, 4, 3, 2, 1 or 0%.
  • NLS refers to a peptide of a certain length that acts as a kind of “tag” attached to the protein that is the transport target when transporting substances from outside the cell nucleus into the inside of the cell through nuclear transport. It means sequence.
  • the NLS is the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 23); NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS with sequence KRPAATKKAGQAKKKK (SEQ ID NO: 24)); c-myc NLS with amino acid sequence PAAKRVKLD (SEQ ID NO: 25) or RQRRNELKRSP (SEQ ID NO: 26); hRNPA1 M9 NLS with sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 27); The sequence of the IBB domain from importin-alpha RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 28); the sequences VSRKRPRP (SEQ ID NO: 29) and PPKKARED (SEQ ID NO: 30) of the myoma T protein; Sequence
  • amino acid sequence in this specification it is written in the direction from the N-terminal to the C-terminal using amino acid single-letter notation or triple-letter notation.
  • RNVP refers to a peptide in which arginine, asparagine, valine, and proline are linked in order from the N-terminal to the C-terminal.
  • Thr-Leu-Lys it means a peptide in which Threonine, Leucine, and Lysine are sequentially connected from the N-terminal to the C-terminal.
  • amino acids that cannot be expressed with the above one-letter notation they are written using other letters and are further supplemented and explained.
  • the notation method for each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine (Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); and Valine (Val, V).
  • A, T, C, G and U used in this specification are interpreted as understood by those skilled in the art. Depending on the context and technology, it may be appropriately interpreted as a base, nucleoside, or nucleotide on DNA or RNA.
  • a base when referring to a base, it can be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), respectively, and when referring to a nucleoside, it can be interpreted as Each can be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G), or uridine (U), and when referring to nucleotides in the sequence, each nucleoside above is used. It should be interpreted to mean the containing nucleotide.
  • operably linked means that, in gene expression technology, a specific component is linked to another component so that the specific component can function in the intended manner.
  • a promoter sequence when operably linked to a coding sequence, it means that the promoter is linked to affect transcription and/or expression of the coding sequence in the cell.
  • the above term includes all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.
  • target gene or target nucleic acid
  • target gene or “target nucleic acid” basically refers to a gene or nucleic acid in a cell that is the target of gene editing.
  • the target gene or target nucleic acid may be used interchangeably and may refer to the same target.
  • the target gene or target nucleic acid may refer to either a gene or nucleic acid unique to the target cell or a gene or nucleic acid derived from an external source, and is not particularly limited as long as it can be the subject of gene editing.
  • the target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA.
  • the above term includes all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.
  • target sequence refers to a specific sequence recognized by the CRISPR/Cas complex to cleave a target gene or target nucleic acid.
  • the target sequence may be appropriately selected depending on the purpose.
  • target sequence refers to a sequence contained in a target gene or target nucleic acid sequence and has complementarity with a spacer sequence contained in the guide RNA or engineered guide RNA provided herein.
  • the spacer sequence is determined considering the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the effector protein of the CRISPR/Cas system.
  • the target sequence may refer only to a specific strand that binds complementary to the guide RNA of the CRISPR/Cas complex, or may refer to the entire target double strand including the specific strand portion, which is interpreted appropriately depending on the context.
  • the above term includes all meanings that can be recognized by a person skilled in the art, and can be appropriately interpreted depending on the context.
  • a vector refers collectively to all substances capable of transporting genetic material into a cell, unless otherwise specified.
  • a vector may be, but is not limited to, a DNA molecule containing the genetic material of interest, such as a nucleic acid encoding an effector protein of a CRISPR/Cas system, and/or a nucleic acid encoding a guide RNA.
  • the above terms include all meanings recognized by those skilled in the art, and can be appropriately interpreted depending on the context.
  • the CRISPR/Cas system is a type of immune system found in prokaryotic organisms and includes Cas proteins, and guide RNA.
  • the detailed composition of the Cas protein or guide RNA is described in detail in the published document WO2018/231018 (International Publication No.).
  • the term “Cas protein” is a general term for nucleases that can be interpreted as being used in the CRISPR/Cas system. Below, we briefly describe the DNA cutting process of the most commonly used CRISPR/Cas9 system.
  • the Cas9 protein corresponds to Class 2, Type II in the CRISPR/Cas system classification, and is suitable for Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., and Streptoma. Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptospor There is Cas9 protein derived from Streptosporangium roseum.
  • RNA that has the function of inducing the CRISPR/Cas9 complex to recognize a specific sequence contained in the target nucleic acid is called a guide RNA.
  • the composition of the guide RNA is divided functionally, it can be roughly divided into 1) a scaffold sequence portion, and 2) a guide sequence portion.
  • the scaffold sequence part is a part that interacts with the Cas9 protein and is a part that allows it to bind with the Cas9 protein to form a complex.
  • the scaffold sequence portion includes tracrRNA and crRNA repeat sequence portions, and the scaffold sequence is determined depending on which Cas9 protein is used.
  • the guide sequence portion is a portion that can bind complementary to a nucleotide sequence portion of a certain length in the target nucleic acid.
  • the guide sequence portion is a base portion that can be artificially modified and is determined by the target nucleotide sequence of interest.
  • the Cas9 protein When the CRISPR/Cas9 complex contacts the target nucleic acid, the Cas9 protein recognizes a nucleotide sequence of a certain length, and a part of the guide RNA (part of the guide sequence) binds complementary to the part adjacent to the PAM sequence, CRISPR/Cas9
  • the target nucleic acid is cleaved by the complex.
  • the nucleotide sequence of a certain length recognized by the Cas9 protein is called a protospacer-adjacent motif (PAM) sequence, which is a sequence determined depending on the type or origin of the Cas9 protein.
  • PAM protospacer-adjacent motif
  • the Cas9 protein from Streptococcus pyogenes can recognize the 5'-NGG-3' sequence in the target nucleic acid.
  • N is one of adenosine (A), thymidine (T), cytidine (C), and guanosine (G).
  • the guide sequence portion of the guide RNA must bind complementary to the sequence portion adjacent to the PAM sequence, so the guide sequence portion must be a sequence of the target nucleic acid, specifically a sequence adjacent to the PAM sequence. It is decided according to the part.
  • the CRISPR/Cas9 complex cleaves the target nucleic acid, any position within the PAM sequence portion of the target nucleic acid and/or the sequence portion that binds complementary to the guide sequence is cleaved.
  • target strand non-target strand
  • the CRISPR/Cas9 complex has cleavage activity on double-stranded DNA.
  • the strand containing the protospacer that binds to the guide sequence portion is called the target strand (TS).
  • a strand that is complementary to the target strand and has a protospacer that does not bind to the guide sequence portion is called a non-target strand (NTS).
  • the guide sequence portion may bind complementary to the protospacer sequence portion included in the target strand (TS) of double-stranded DNA.
  • the guide sequence and the protospacer sequence included in the non-target strand (NTS) of double-stranded DNA are equivalent sequences. Specifically, the only difference is that the guide sequence is an RNA sequence, and the protospacer sequence included in the non-target strand (NTS) is a corresponding DNA sequence.
  • Cas9 protein from Campylobacter jejuni also referred to as CjCas9, is one of the orthologs of Cas9 protein.
  • the CjCas9 has the smallest size among Cas9s and is known to exhibit double-strand DNA cleavage activity in eukaryotic cells.
  • Campylobacter jejuni-derived Cas9 protein The gene editing efficiency of Campylobacter jejuni-derived Cas9 protein is low.
  • the Cas9 protein derived from Campylobacter jejuni shows double-strand DNA cleavage activity, and its relatively small size is advantageous for commercialization.
  • the Cas9 protein derived from Campylobacter jejuni is known to have lower gene editing efficiency in cells than Cas9 derived from Streptococcus pyogenes, and the efficiency is particularly low when the Cas9 protein and guide RNA are vectorized and introduced. There is.
  • Guide RNA contains four consecutive uridines
  • the guide RNA for the Cas9 protein derived from Campylobacter jejuni includes a sequence of four consecutive uridines in the repeat portion of the crRNA among the scaffold sequences. Accordingly, when vectorizing the guide RNA, four thymi A DNA sequence with contiguous Deans is included in the vector. RNA polymerase, which transcribes the vector and produces RNA, recognizes the sequence of five consecutive thymidines (5'-TTTTT-3') as a termination signal and stops polymerization. Although the DNA sequence (5'-TTTT-3') containing four consecutive thymidines contained in the vectorized guide RNA is not a termination signal, it is recognized similarly to a termination signal, and transcriptional pausing or immature termination can occur. It has been reported.
  • the guide RNA may not be expressed properly when introduced into cells in vector form. This causes the Cas9 protein-guide RNA complex derived from Campylobacter jejuni to not be sufficiently formed within the cell, thereby reducing the gene editing efficiency of the CRISPR/CjCas9 system expression vector.
  • the engineered CRISPR/Cas9 system comprises a Cas9 protein (CjCas9) from Campylobacter jejuni and an (engineered) guide RNA, and is characterized in that the guide RNA is engineered.
  • the guide RNA comprises a guide domain and an engineered scaffold, and the engineered scaffold has a 5'-UUUU-3' sequence portion adjacent to the guide domain, compared to the guide RNA of the corresponding wild-type CjCas9 protein.
  • the 5'-AAAA-3' sequence part that binds complementary to this to form a Tetraloop is modified.
  • the engineered CRISPR/Cas9 system has the following characteristics: 1) when introduced into cells in the form of an expression vector, it exhibits a higher expression level compared to wild-type guide RNA, and 2) the activity of the CRISPR/Cas9 system is increased. .
  • the engineered CRISPR/Cas9 system disclosed herein includes an engineered guide RNA.
  • the engineered guide RNA consists of a guide domain and an engineered scaffold sequentially linked from the 5' end to the 3' end.
  • the guide domain is a part that can target a nucleic acid having a target sequence, and allows the engineered CRISPR/Cas9 system to exhibit target-specific nucleic acid cleavage activity.
  • the guide domain is appropriately designed according to the target sequence.
  • the engineered scaffold is a part that interacts with the Cas9 protein to form a complex.
  • the engineered scaffold is designed not to have more than four consecutive uridine (U) elements, and thus exhibits high expression levels when using the vector.
  • the engineered CRISPR/Cas9 system disclosed herein includes the Cas9 protein.
  • the Cas9 protein is a Cas9 protein derived from Campylobacter jejuni.
  • the Cas9 protein refers collectively to wild-type Cas9 protein, modified Cas9 protein, and fusion protein in which an additional domain is fused to the Cas9 protein.
  • the engineered CRISPR/Cas9 system disclosed herein is characterized in that the scaffold portion is engineered compared to the guide RNA of the CRISPR/Cas9 system found in nature. Specifically, the 5'-UUUU-3' sequence contained in the repeat portion of the wild-type crRNA and the 5'-AAAA-3' sequence contained in the antirepeat portion of the wild-type tracrRNA that binds complementary to it are appropriately changed. there is. Therefore, the engineered guide RNA does not have a continuous region of four or more uridines in the sequence.
  • RNA polymerase recognizes four consecutive thymidine (T) regions, preventing transcriptional pausing or immature termination.
  • T thymidine
  • the engineered CRISPR/Cas9 system disclosed herein has the effect of improving gene cleavage activity and/or efficiency itself due to the engineered scaffold.
  • the guide RNA engineered with the above-mentioned scaffold portion not only increases the expression level of the above-mentioned guide RNA, but also exhibits the characteristic of increasing the DNA cutting efficiency itself by interaction with CjCas9. Accordingly, the engineered CRISPR/Cas9 system shows higher gene editing activity than the wild-type CRISPR/Cas9 system regardless of the form in which it is used (ribonucleoprotein, vector, and/or composition).
  • the engineered scaffold disclosed herein can be divided into a first region, a second region, a third region, and a fourth region from the 5' end to the 3' end.
  • Each of the above regions may correspond to each part of the scaffold of the wild-type guide RNA for the Cas9 protein derived from Campylobacter jejuni (hereinafter referred to as wild-type scaffold), which will be described below.
  • wild-type scaffold for the Cas9 protein derived from Campylobacter jejuni
  • the wild-type guide RNA refers to guide RNA including naturally occurring crRNA and tracrRNA.
  • a single guide RNA in which the crRNA and tracrRNA are connected by a linker e.g., a linker with a 5'-GAAA-3' or 5'-GA-3' sequence
  • the wild-type scaffold refers to the naturally occurring scaffold of guide RNA for the Cas9 protein from Campylobacter jejuni.
  • the wild-type scaffold may be represented by the sequence GUUUUAGUCCCUGAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 1).
  • the wild-type scaffold can be divided as follows:
  • the first region of the wild type is 5'-GUUUU-3'
  • the second region of the wild type is 5'-AGUCCCUGAAGGGACU-3' (SEQ ID NO: 6)
  • the third region of the wild type is 5'-AAAA-3'
  • the fourth region of the wild type is 5'-UAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC-3' (SEQ ID NO: 7).
  • the first region of the engineered scaffold disclosed herein is characterized by being modified to not have four consecutive uridines (U). Specifically, the first region is represented by a sequence selected from 5'-GUUUC-3', 5'-GUUCU-3', 5'-GUCUU-3', and 5'-GCUUU-3'.
  • the second region of the engineered scaffold disclosed herein is expressed in a sequence identical to or similar to the sequence of the second region of the wild type.
  • the second region is 5'-AGUCCCUGAAGGGACU-3' (SEQ ID NO: 6) and 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87 % or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more , or expressed as a 100% identical, equivalent, or homologous sequence.
  • the third region of the engineered scaffold disclosed herein is a sequence that can bind complementary to the first region to form a stem-loop structure.
  • the third region is determined depending on the sequence in which the first region is expressed. Specifically, when the first region is expressed as 5'-GUUUC-3', the third region is expressed as 5'-GAAA-3', and the first region is expressed as 5'-GUUCU-3' In this case, the third region is expressed as 5'-AGAA-3', and if the first region is expressed as 5'-GUCUU-3', the third region is expressed as 5'-AAGA-3' And, when the first region is expressed as 5'-GCUUU-3', the third region is expressed as 5'-AAAG-3'.
  • the fourth region of the engineered scaffold disclosed herein is expressed in a sequence identical to or similar to the sequence of the fourth region of the wild type. Specifically, the fourth region is 5'-UAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC-3' (SEQ ID NO: 7) and 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87 % or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more , or expressed as a 100% identical, equivalent, or homologous sequence.
  • the engineered guide RNA provided herein can interact with the Cas9 protein to form a complex and can target nucleic acids with a target sequence.
  • the engineered guide RNA includes a guide domain and an engineered scaffold.
  • the engineered scaffold is a composition in which the guide RNA interacts with the Cas9 protein to form a complex.
  • the guide domain is configured to target a nucleic acid having a target sequence so that the Cas9-guide RNA complex can exhibit target-specific nucleic acid cleavage activity.
  • the guide domain is designed to target a nucleic acid having a target sequence.
  • the engineered guide RNA consists of a guide domain and an engineered scaffold sequentially linked from the 5' end to the 3' end.
  • the engineered scaffold of the guide RNA allows the guide RNA to interact with the Cas9 protein to form a complex.
  • the engineered scaffold is as described in the “Engineered Scaffold” section.
  • the programmable guide RNA includes a guide domain.
  • the guide domain is capable of targeting nucleic acids of a target sequence and is involved in activating the target-specific nucleic acid cleavage effect of the CRISPR/Cas9 system.
  • the guide domain may bind complementary to the target nucleic acid.
  • the guide domain is artificially designed to target the nucleic acid of the gene or target sequence to be edited.
  • the guide domain In order for the engineered CRISPR/Cas9 system to cleave a specific nucleic acid, the guide domain must first be able to bind to the nucleic acid of the target sequence. Accordingly, the guide domain may have a sequence complementary to the target sequence, or in some cases, may have a sequence equivalent to the target sequence.
  • the relationship between the above-described guide domain and the target sequence varies depending on the type of nucleic acid having the target sequence and/or the location of the target sequence within the nucleic acid.
  • the guide domain when the nucleic acid of the target sequence is a single-stranded nucleic acid, the guide domain may have a sequence complementary to the target sequence. In another embodiment, when the nucleic acid of the target sequence is a double-stranded nucleic acid and the target sequence is located on the same strand as the strand where the PAM sequence of the CRISPR / Cas9 system is located, the guide domain is equivalent to the target sequence ( equivalent) may be a sequence.
  • the guide domain is complementary to the target sequence It may be a ranking.
  • the guide domain is 1nt, 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt , 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt long.
  • the guide domain may be the length between two numerical ranges selected in the immediately preceding sentence.
  • the guide domain may be 18nt to 22nt long.
  • the guide domain is located toward the 5' end of the engineered scaffold, and the engineered scaffold is located toward the 3' end of the guide domain.
  • the guide RNA is characterized in that the guide domain and the engineered scaffold are sequentially connected from the 5' end to the 3' end.
  • the guide RNA is expressed as [Structural Formula 2]:
  • the engineered CRISPR/Cas9 system includes a Cas9 protein, specifically a Cas9 protein from Campylobacter jejuni.
  • the Cas9 protein encompasses wild-type Cas9 protein, Cas9 protein with one or more sequence modifications, Cas9 protein with altered function, Cas9 protein containing other additional modifications, and fusion protein containing the Cas9 protein.
  • the Cas9 protein should be interpreted as including all of the various forms of Cas9 protein described in the “Cas9 protein” section.
  • the Cas9 protein of the engineered CRISPR/Cas9 system provided herein may be a wild-type Cas9 protein.
  • the wild-type Cas9 protein may be a Cas9 protein derived from Campylobacter jejuni.
  • the engineered CRISPR/Cas9 system provided herein may include a modified Cas9 protein.
  • the modified Cas9 means that at least part of the sequence is modified from the wild-type or codon-optimized Cas9 protein sequence.
  • the Cas9 protein modification may be made in individual amino acid units or in functional domain units of the protein.
  • the modification of the protein may be one or more amino acids, peptides, polypeptides, proteins, and/or domains individually substituted, removed, and/or added to the wild-type or codon-optimized Cas9 protein sequence.
  • the Cas9 protein is one or more amino acids, peptides, and/or polypeptides in the RuvC domain, REC1 domain, REC2 domain, HNH domain, and/or PI domain included in the wild-type Cas9 protein. /or may be added.
  • the Cas9 protein included in the engineered CRISPR/Cas9 system provided herein may have the same function as the wild-type Cas9 protein.
  • the Cas9 protein included in the engineered CRISPR/Cas9 system provided herein may have an altered function compared to the wild-type Cas9 protein.
  • the change may be a modification of all or part of the function, loss of all or part of the function, and/or addition of an additional function.
  • the Cas9 protein is not particularly limited as long as it is a change that can be applied to the Cas protein of the CRISPR/Cas system by a person skilled in the art. At this time, the change may be made using known technology.
  • the Cas9 protein may be modified to cleave only one strand of the double strands of the target nucleic acid. Furthermore, the Cas9 protein can cleave only one strand of the double strands of the target nucleic acid, and may be modified to perform base editing or prime editing on the strand that is not cut. In one embodiment, the Cas9 protein may be modified so that it cannot cleave the entire double strand of the target nucleic acid. Furthermore, the Cas9 protein cannot cleave all double strands of the target nucleic acid, and may be modified to perform base editing, prime editing, or gene expression control functions for the target nucleic acid. .
  • the engineered CRISPR/Cas9 system provided herein may include a Cas9 fusion protein.
  • the Cas9 fusion protein refers to a protein in which additional amino acids, peptides, polypeptides, proteins, and/or domains are fused to the wild-type or modified Cas9 protein.
  • the Cas9 protein may be a base editor and/or reverse transcriptase fused to the wild-type Cas9 protein.
  • the base editor may be adenosine deaminase and/or cytidine deaminase.
  • the reverse transcriptase may be Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, and/or a variant thereof.
  • M-MLV Moloney Murine Leukemia Virus
  • the Cas9 protein fused with the reverse transcriptase can function as a prime editor.
  • the Cas9 protein may be a fusion of various enzymes that may be involved in the gene expression process within cells to the wild-type Cas9 protein. At this time, the Cas9 protein to which the enzyme is fused can cause various quantitative and qualitative changes in gene expression in cells.
  • the Cas9 protein may include a Nuclear Localization Sequence (NLS) or a Nuclear Export Sequence (NES).
  • NLS nuclear Localization Sequence
  • NES Nuclear Export Sequence
  • the NLS may be any one of those exemplified in the NLS section of the “Definition of Terms”, but is not limited thereto.
  • the Cas9 protein may include a tag.
  • the tag may be one of those exemplified in the tag section of “Definition of Terms”, but is not limited thereto.
  • the base sequence (nucleotide sequence) of a certain length within the target gene or target nucleic acid that can be recognized by the Cas9 protein is called the Protospacer Adjacent Motif (PAM) sequence.
  • the PAM sequence is a unique sequence determined by the Cas9 protein.
  • Cas9 protein recognizes the PAM sequence of a certain length, and 2) the spacer sequence portion binds complementary to the sequence portion surrounding the PAM sequence, Cas9 protein/guide RNA complex ( CRISPR/Cas9 complex) cleaves the target gene or target nucleic acid. Therefore, when determining the target sequence of the CRISPR/Cas9 complex, there is a constraint that the target sequence must be determined within a sequence adjacent to the PAM sequence.
  • the Cas9 system of the CRISPR/Cas9 system is based on the Cas9 protein derived from Campylobacter jejuni, it can recognize the PAM sequence recognized by the Cas9 protein derived from Campylobacter jejuni.
  • the PAM sequence of the Cas9 protein may be 5'-NNNNRYAC-3'.
  • the N is each independently one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G).
  • the R is either deoxyadenosine (A) or deoxyguanosine (G).
  • the Y is either deoxycytidine (T) or deoxycytidine (C).
  • the PAM sequence of the Cas9 protein may be different from the PAM sequence of Cas9 derived from wild-type Campylobacter jejuni.
  • the engineered CRISPR/Cas9 complex is a complex of Cas9 protein and engineered guide RNA, and directly exhibits target-specific nucleic acid cleavage activity.
  • the Cas9 protein is the same as described in the “Cas9 protein” section.
  • the engineered guide RNA is the same as described in the “Engineered Guide RNA” section.
  • vectors capable of expressing each component of the engineered CRISPR/Cas9 system are disclosed.
  • the vector can achieve a predetermined purpose by expressing the engineered CRISPR/Cas9 system in target cells.
  • the expression vector is not particularly limited as long as it can express each component of the engineered CRISPR/Cas9 system.
  • the expression vector includes a nucleic acid encoding the Cas9 protein, a nucleic acid encoding an engineered guide RNA, and may include other additional components such as a promoter.
  • the expression vector may be DNA and/or mRNA, but is not limited thereto.
  • the expression vector for each component of the engineered CRISPR/Cas9 system contains nucleic acid encoding the Cas9 protein.
  • the Cas9 protein is the same as described in the “Cas9 protein” section.
  • the expression vector for each component of the engineered CRISPR/Cas9 system includes a nucleic acid encoding the engineered guide RNA.
  • the engineered guide RNA is as described in the “Engineered Guide RNA” section.
  • the engineered CRISPR/Cas9 system component expression vector may include other components necessary to express each component of the engineered CRISPR/Cas9 system in other cells.
  • the other components include a promoter, enhancer, intron, polyadenylation signal, Kozak consensus sequence, Internal Ribosome Entry Site (IRES), splice acceptor, 2A sequence and/ Alternatively, it may include a replication origin.
  • the promoter sequence can be designed differently depending on the corresponding RNA transcription factor or expression environment, and is not limited as long as it can properly express the components of the CRISPR/Cas system within the cell.
  • the promoter may be SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, CMV immediate early promoter region (CMVIE), etc.
  • CMV cytomegalovirus
  • RSV rous sarcoma virus
  • enhanced U6 promoter e.g., Xia et al. , Nucleic Acids Res. 2003 Sep 1;31(17)
  • human H1 promoter H1
  • 7SK human H1 promoter
  • the origin of replication may be an f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, Adeno origin of replication, AAV origin of replication, and/or BBV origin of replication, but is not limited thereto.
  • the expression vector may be a viral vector.
  • the viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus. In one embodiment, the viral vector may be an adeno-associated virus.
  • the expression vector may be a non-viral vector.
  • the non-viral vector may be one or more selected from the group consisting of plasmid, phage, naked DNA, DNA complex, and mRNA.
  • the plasmid is pcDNA series, pS456, p326, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19.
  • the phage may be selected from the group consisting of ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, and M13.
  • the encoding nucleic acid may be a PCR amplicon.
  • engineered CRISPR/Cas9 compositions comprising each component of the engineered CRISPR/Cas9 system and/or nucleic acids encoding each component.
  • each component of the engineered CRISPR/Cas9 system is described in the “Cas9 Protein” and “Engineered Guide RNA” sections.
  • the form of the engineered CRISPR/Cas9 composition is not particularly limited as long as the CRISPR/Cas9 system can function for its intended purpose.
  • the gene editing method may be delivering, injecting, and/or administering the engineered CRISPR/Cas9 system to the gene editing target in an appropriate delivery form and using an appropriate delivery method.
  • the gene editing method may involve contacting or inducing the engineered CRISPR/Cas9 complex to contact a nucleic acid having a target sequence.
  • the gene editing target may be an individual or tissue, and may be referred to as a target individual or target tissue.
  • the target entity may be a plant, animal, non-human animal, and/or human.
  • the target object may be a mammal.
  • the target tissue may be non-human animal tissue and/or human tissue.
  • the gene editing target may refer to a cell and may be referred to as a target cell.
  • the target cell may be a prokaryotic cell.
  • the target cell may be a eukaryotic cell.
  • the eukaryotic cells may be plant cells, animal cells, non-human animal cells, and/or human cells.
  • the delivery form may be a ribonucleoprotein particle in which Cas9 protein and engineered guide RNA are combined. This may be in the form of a protein-nucleic acid complex as described in the section “Engineered CRISPR/Cas9 Complexes”.
  • the delivery form may be a vector capable of expressing each engineered component of CRISPR/Cas9. This may be explained in the section “Vectors capable of expressing each component of engineered CRISPR/Cas9”.
  • the delivery form may be a composition comprising each component of the engineered CRISPR/Cas9 system and/or a nucleic acid encoding each component. This may be the composition described in the section “Engineered CRISPR/Cas9 Compositions”.
  • the delivery method is not particularly limited as long as the guide RNA engineered into the cell or the nucleic acid encoding it, and the Cas9 protein or the nucleic acid encoding it can be delivered into the cell using any of the above delivery methods.
  • the delivery method may be electroporation, gene gun, sonoporation, magnetofection, and/or transient cell compression or squeezing.
  • the delivery method may be delivering at least one component included in the CRISPR/Cas9 system using nanoparticles.
  • the delivery method may be a known method that can be appropriately selected by a person skilled in the art.
  • the nanoparticle delivery method may be the method disclosed in (WO 2019/089820 A1), but is not limited thereto.
  • the delivery method may be delivering the Cas9 protein or the nucleic acid encoding it and/or the engineered guide RNA or the nucleic acid encoding it using nanoparticles.
  • the delivery method is cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation, lipofection, PEI (Polyethyleneimine)-mediated transfection, and DEAE-dextran-mediated transfection. , and/or nanoparticle-mediated nucleic acid delivery (see Panyam et. , al Adv Drug Deliv Rev. 2012 Sep 13.pii: S0169-409X(12)00283-9.
  • the components of the CRISPR/Cas9 system may be in any one of the above delivery forms.
  • the components of the CRISPR/Cas9 system may be in the form of mRNA encoding each component, but are not limited thereto.
  • the gene editing method includes delivering an engineered guide RNA or a nucleic acid encoding it, and a Cas9 protein or a nucleic acid encoding it into a cell, wherein the constructs can be delivered simultaneously into the cell or sequentially with a time difference. there is. At this time, there is no limitation as to which configuration is delivered first, as long as the purpose of gene editing can be achieved.
  • indels may occur in the target gene or target nucleic acid.
  • the indel may occur inside and/or outside the target sequence portion and/or the protospacer sequence portion.
  • the indel refers to a mutation in which some nucleotides are deleted, certain nucleotides are inserted, and/or the insertion and deletion are mixed in the nucleotide sequence of the nucleic acid before gene editing.
  • the gene or nucleic acid is inactivated.
  • one or more nucleotides in the target gene or target nucleic acid may be deleted and/or added.
  • base editing may occur within the target gene or target nucleic acid.
  • one or more nucleotides in the target gene or target nucleic acid may be replaced with another nucleotide.
  • knock-in may occur in the target gene or target nucleic acid.
  • the knock-in refers to the insertion of an additional nucleic acid sequence into the target gene or target nucleic acid sequence.
  • a donor containing the additional nucleic acid sequence is required in addition to the CRISPR/Cas9 complex.
  • the CRISPR/Cas9 complex cleaves a target gene or target nucleic acid within a cell, repair of the cleaved target gene or target nucleic acid occurs.
  • the donor participates in the repair process so that the additional nucleic acid sequence can be inserted into the target gene or target nucleic acid.
  • the gene editing method may additionally include introducing a donor into the target cell.
  • the donor includes an exogeneous DNA sequence for insertion into the intracellular genome, and the donor induces insertion of the exogeneous DNA sequence into the target gene or target nucleic acid.
  • the above-described delivery form and/or delivery method may be used.
  • the removal means removing a certain base sequence (nucleotide sequence) within the target gene or target nucleic acid over a certain length. Compared to the indel effect described above, the removal refers to an effect that can entirely remove a specific region of a gene, for example, the first exon region.
  • the gene editing method includes a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a second engineered Cas12f1 guide RNA or a nucleic acid encoding the target gene or target nucleic acid. It includes introducing into cells containing. As a result, the gene editing results in the removal of a specific sequence portion within the target gene or target nucleic acid.
  • the gene editing method disclosed herein uses the engineered CRISPR/Cas9 system.
  • the guide RNA included in the engineered CRISPR/Cas9 system does not have a continuous region of four or more uridines in the sequence. Therefore, when the CRISPR/Cas9 system engineered according to the above gene editing method is vectorized and delivered to the editing target, RNA polymerase within the cell recognizes four consecutive thymidine (T) regions, leading to transcriptional pausing or immature termination. The probability is very low or non-existent.
  • the gene editing method is characterized by an increase in the expression level of the guide RNA included in the engineered CRIPSR/Cas9 system within the cell being edited.
  • the engineered CRISPR/Cas9 system disclosed herein has the feature that the gene cutting activity itself is increased due to the engineered scaffold. Therefore, when the engineered CRISPR/Cas9 system is used in a gene editing method, regardless of the form of use (ribonucleoprotein, vector, and/or composition), gene editing is higher than when using the wild-type CRISPR/Cas9 system. indicates activity.
  • An engineered guide RNA scaffold modified from RNA having a nucleic acid sequence of GUUUUAGUCCCUGAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 1) to not have four consecutive uridines.
  • Example 1 an engineered guide RNA scaffold represented by the following sequence:
  • the first region is selected from 5'-GUUUC-3', 5'-GUUCU-3', 5'-GUCUU-3', and 5'-GCUUU-3',
  • the second region is AGUCCCUGAAGGGACU (SEQ ID NO: 6), or the sequence of SEQ ID NO: 6 and at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, Matches at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or , is a homologous or corresponding sequence,
  • the third region is selected from 5'-GAAA-3', 5'-AGAA-3', 5'-AAGA-3', and 5'-AAAG-3',
  • the fourth region is UAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 7), or the sequence of SEQ ID NO: 7 and at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, Matches at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or , is a homologous or corresponding sequence.
  • the engineered guide RNA scaffold is GUUUCAGUCCCUGAAGGGACUGGAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 2), GUUCUAGUCCCUGAAGGGACUGAGAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 3), GUCUUAGUCCCUGAAGG GACUGAAGAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 4), and GCUUUAGUCCCUGAAGGGACUAAAGUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGC (SEQ ID NO: 5)
  • An engineered guide RNA scaffold having a sequence from a group of nucleic acid sequences.
  • Engineered guide RNA represented by the following sequence:
  • the guide domain is artificially designed to target a target nucleic acid having a predetermined target sequence
  • the engineered guide RNA scaffold is any one of the engineered guide RNA scaffolds selected from Examples 1 to 4.
  • the guide domain is 1nt, 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, Engineered guide RNA having a length of 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, 27nt, 28nt, 29nt, or 30nt.
  • the guide domain has a sequence equivalent to the predetermined target sequence, or has a complementary sequence to the predetermined target sequence.
  • Programmable guide RNA is a sequence equivalent to the predetermined target sequence, or has a complementary sequence to the predetermined target sequence.
  • the target nucleic acid having the predetermined target sequence is a double-stranded nucleic acid
  • the target nucleic acid includes a target strand and a nontarget strand
  • the target sequence of the target nucleic acid can be specified only by the sequence of the target strand or the sequence of the non-target strand,
  • the statement that the guide domain targets a target nucleic acid of the predetermined target sequence has the meaning selected from the following:
  • the guide domain may complementarily bind, and/or hybridize, with the target strand of the target nucleic acid
  • the sequence of the guide domain includes a sequence complementary to all or part of the sequence of the target strand of the target nucleic acid
  • the sequence of the guide domain is identical, matches, homologs, and/or comprises an equivalent sequence to all or part of the sequence of the non-target strand of the target nucleic acid;
  • sequence of the guide domain is any one selected from the following:
  • All or part of the sequence of the target sequence contained in the non-target strand of the target nucleic acid and the remaining bases except for 1, 2, 3, 4, or 5 nucleotide bases are identical (identical) or match ( match, homolog, and/or equivalent sequence;
  • a sequence in which all or part of the target sequence contained in the target strand of the target nucleic acid is complementary to the remaining bases except for 1, 2, 3, 4, or 5 nucleotide bases.
  • Example 10 the amino acid sequence of (SEQ ID NO: 39), or the amino acid sequence of SEQ ID NO: 39 and at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86% , at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or Cas9 protein expressed as a sequence that is at least 99% identical, corresponding, or equivalent.
  • the Cas9 protein according to any one of Examples 10 to 11, wherein the Cas9 is modified to have a nickase function.
  • the Cas9 protein according to any one of Examples 10 to 12, wherein the Cas9 is modified so as not to have nucleic acid cleavage activity.
  • the Cas9 protein according to any one of Examples 10 to 13, wherein the Cas9 is fused to a domain selected from the following:
  • the Cas9 protein according to any one of Examples 10 to 14, wherein the Cas9 protein includes one or more Nuclear Localization Signals at the N-terminus and/or C-terminus.
  • the one or more Nuclear Localization Signals are each independently PKKKRKV (SEQ ID NO: 23), KRPAATKKAGQAKKKK (SEQ ID NO: 24), PAAKRVKLD (SEQ ID NO: 25), RQRRNELKRSP (SEQ ID NO: 26), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 27) , RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 28), VSRKRPRP (SEQ ID NO: 29), PPKKARED (SEQ ID NO: 30), PQPKKKPL (SEQ ID NO: 31), SALIKKKKKMAP (SEQ ID NO: 32), DRLRR (SEQ ID NO: 33), PKQKKRK (SEQ ID NO: 34), Has an amino acid sequence selected from RKLKKKIKKL (SEQ ID NO: 35), REKKKFLKRR (SEQ ID NO: 36),
  • the guide domain of the engineered guide RNA is artificially designed to target a nucleic acid of a predetermined target sequence
  • the engineered scaffold of the engineered guide RNA can interact with the Cas9 protein to form a complex.
  • a nucleic acid encoding the Cas9 protein selected from Examples 10 to 16;
  • the guide domain of the engineered guide RNA is artificially designed to target a nucleic acid of a predetermined target sequence
  • the engineered scaffold of the engineered guide RNA can interact with the Cas9 protein to form a complex.
  • nucleic acid encoding the Cas9 protein and the nucleic acid encoding the engineered guide RNA are each independently operably linked to a promoter capable of expressing them.
  • the promoter is SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, CMV immediate early promoter region (CMVIE) Such as cytomegalovirus (CMV) promoter, rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), enhanced U6 promoter (e.g., Xia et al. al., Nucleic Acids Res. 2003 Sep 1;31(17)), human H1 promoter (H1), and 7SK, each independently selected from an expression vector.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMVIE CMV immediate early promoter region
  • CMV CMV immediate early promoter region
  • the viral vector is one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus and herpes simplex virus. vector.
  • non-viral vector is one or more selected from the group consisting of plasmid, phage, naked DNA, DNA complex, and mRNA.
  • Example 25 specific nucleic acid encoding guide RNA
  • nucleic acid encoding the engineered guide RNA is GTTTCAGTCCCTGAAGGGACTGGAAATAAAGAGTTTGCGGGACTCTGCGGGGTTTACAATCCCCTAAAACCGC (SEQ ID NO: 9), GTTCTAGTCCCTGAAGGGACTGAGAATAAAGAGTTTGCGGGACTCTGCGGGGGTTACAATCCCCTAAAACCGC (SEQ ID NO: 10), GTCTTAGTCCCTGAAG GGACTGAAGATAAAGAGTTTGCGGGACTCTGCGGGGTTACAATCCCCTAAAACCGC (SEQ ID NO: 11), and GCTTTAGTCCCTGAAGGGACTAAAGTAAAGAGTTTGCGGGACTCTGCGGGGGTTACAATCCCCTAAAACCGC (SEQ ID NO: 12)
  • An engineered CRISPR/Cas9 composition comprising:
  • the guide domain of the engineered guide RNA is artificially designed to target a nucleic acid of a predetermined target sequence
  • the engineered scaffold of the engineered guide RNA can interact with the Cas9 protein to form a complex.
  • Example 27 wherein the engineered CRISPR/Cas9 composition includes the Cas9 protein and the engineered guide RNA, and the Cas9 protein combines with the engineered guide RNA to form a ribonucleoprotein.
  • Example 27 wherein the engineered CRISPR/Cas9 composition comprises a nucleic acid encoding the Cas9 protein and a nucleic acid encoding the engineered guide RNA.
  • Example 27 wherein the composition comprises the CRISPR/Cas9 complex of Example 17, or the vector of any one of Examples 18 to 26.
  • a method of gene editing a cell containing a target nucleic acid including:
  • the guide domain of the engineered guide RNA of the composition is capable of targeting the target nucleic acid.
  • a gene editing method for cells containing a target nucleic acid including:
  • the guide domain of the engineered guide RNA of the composition is capable of targeting the target nucleic acid.
  • Example 33 limited to eukaryotic/prokaryotic cells
  • Example 34 limited to cell types
  • Example 36 limited method performance environment
  • a plasmid containing both the nucleic acid sequence encoding the CjCas9 protein between the inverted tandem repeat (ITR) of AAV2 and the nucleic acid encoding the CjCas9 guide RNA containing the wild-type scaffold was prepared using Gibson assembly (NEB/M5520AA). It was prepared by cloning (pAAV-EFS-CjCas9-U6-sgRNA vector). Afterwards, in the sequence encoding the scaffold of the wild-type CjCas9 guide RNA (SEQ ID NO: 8), the four consecutive T's were replaced with C one by one, and the paired part was modified to A>G to produce an engineered guide.
  • Four types of vectors for RNA were additionally prepared.
  • each engineered guide RNA is schematically shown in Figure 1, and each sequence is summarized in Table 1 below.
  • a guide domain sequence targeting the target sequence CATGAGGAAATGAGAGAAATGCTTACACAC (SEQ ID NO: 16) to be used in Experimental Examples 2 and 3 above was synthesized, and this was cloned into the 5' end of the scaffold sequence in the above vector using the BSPQ1 Site, resulting in the final A vector to be transfected into cells was prepared.
  • the target sequence includes the PAM sequence recognized by the CjCas9 protein.
  • Human HEK293T cells (CRL-3216, ATCC) or rat RT4-D6P2T cells (CRL-2768TM, ATCC) were cultured and used. The cells were subcultured at 2-3 day intervals using Dulbecco's Modified Eagle Medium (DMEM) (WelGene) supplemented with 10% fetal bovine serum (WelGene) and 1 ⁇ penicillin/streptomycin (WelGene), and maintained. .
  • DMEM Dulbecco's Modified Eagle Medium
  • WelGene fetal bovine serum
  • WelGene 1 ⁇ penicillin/streptomycin
  • RNA extracted in Experimental Example 1.3 1000 ng of RNA extracted in Experimental Example 1.3 was used to prepare cDNA using a cDNA Reverse-transcription kit (ThermoFisher). qRT-PCR was performed in QuantStudio 3 using SYBR Green Master Mix on 10 ng - 15 ng of cDNA obtained, according to the manufacturer's protocol (Thermo Fisher). Using the CT value information obtained as a result of qRT-PCR, the expression of each engineered guide RNA was compared relative to the expression level of the guide RNA containing the wild-type scaffold.
  • Primer information used for (engineered) guide RNA qRT-PCR analysis Primer name Sequence (5'-3') SEQ ID NO HIF1_sgRNA_qRT_F CATGAGGAAATGAGAGAAATG 17 sgRNA-primer-R GCGGTTTTAGGGGATTGTAAC 18
  • the genomic DNA extracted in Experimental Example 1.3 was analyzed and the indel occurrence rate was measured to evaluate the gene editing efficiency of each vector.
  • the specific process is as follows:
  • Primer information used in the indel analysis process Cell line Primer name Sequence (5'-3') SEQ ID NO HEK293 hHIF1a_F acactctttccctacacgacgctcttccgatctACATGGGATTAACTCAGG 19 hHIF1a_R gtgactggagttcagacgtgtgctcttccgatctTTTGCCTTGGGTAAGTAC 20 RT4-D6P2T rHIF1a_F acactctttccctacacgacgctcttccgatctCCACATATGAAGAGCACTTATGGG 21 rHIF1a_R gtgactggagttcagacgtgtgctctccgatctGTAGTAACAATATCTGACTGAAA 22
  • the plasmids of each example prepared according to Experimental Example 1.1 were transfected into HEK293T cells cultured according to Experimental Example 1.2 according to Experimental Example 1.3, and then according to Experimental Examples 1.4 to 1.6, the effect of improving guide RNA expression and target nucleic acid Editing efficiency was measured.
  • the plasmids of each example prepared according to Experimental Example 1.1 were transfected into RT4-D6P2T cells cultured according to Experimental Example 1.2 according to Experimental Example 1.3, and then according to Experimental Examples 1.4 to 1.6, the effect of improving guide RNA expression and Target nucleic acid editing efficiency was measured.
  • the engineered guide RNA for the Cas9 protein derived from Campylobacter jejuni modified to not contain four or more uridines and the CRISPR/CjCas9 system containing the same provided herein can be used for gene editing purposes.

Abstract

La présente invention concerne un ARN guide modifié pour une protéine Cas9 provenant de Campylobacter jejuni, l'ARN guide modifié étant modifié afin de ne pas comporter quatre uridines consécutives ou plus. Un système CRISPR/Cas9 comprenant l'ARN guide modifié est caractérisé en ce que : 1) il présente un niveau d'expression élevé dans les cellules en raison de l'absence de pause transcriptionnelle ou de terminaison immature lorsqu'il est exprimé dans un vecteur d'expression, et 2) il présente une efficacité accrue de clivage de l'ADN grâce aux interactions entre l'ARN guide modifié et la protéine Cas9. Par conséquent, le système CRISPR/Cas9 comprenant l'ARN guide modifié, de la présente invention, présente une activité d'édition génique plus élevée que le système CRISPR/Cas9 de type sauvage.
PCT/KR2023/013166 2022-09-06 2023-09-04 Système d'amélioration de l'édition génique par le modification de la structure de l'arn guide de cas9 provenant de campylobacter jejuni WO2024053964A1 (fr)

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US20210388348A1 (en) * 2020-05-12 2021-12-16 University Of Massachusetts Modified guide rnas for crispr genome editing

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US20140273232A1 (en) * 2012-12-12 2014-09-18 The Broad Institute, Inc. Engineering of systems, methods and optimized guide compositions for sequence manipulation
KR20190037145A (ko) * 2017-09-28 2019-04-05 주식회사 툴젠 유전자 발현 조절을 위한 인위적인 게놈 조작
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