KR102151064B1 - Gene editing composition comprising sgRNAs with matched 5' nucleotide and gene editing method using the same - Google Patents

Abstract

It is related to a technology for enhancing the efficiency of gene editing of a highly specific Cas9 variant using a guide RNA containing a matched 5'nucleotide, a complex of a guide RNA including a highly specific Cas9 variant and a matched 5'nucleotide, a high specificity Cas9 A composition for gene editing comprising a variant and a matched 5'nucleotide; And there is provided a gene editing method using the composition for gene editing.

Description

Gene editing composition comprising sgRNAs with matched 5'nucleotide and gene editing method using the same}

It is related to a technology for enhancing the efficiency of gene editing of a highly specific Cas9 variant using a guide RNA containing a matched 5'nucleotide, a complex of a guide RNA including a highly specific Cas9 variant and a matched 5'nucleotide, a high specificity Cas9 A composition for gene editing comprising a variant and a matched 5'nucleotide; And there is provided a gene editing method using the composition for gene editing.

CRISPR-Cas9 RNA-guided endonuclease, derived from the adaptive immune system in bacteria and archaea, has been repurposed for targeted genome editing in a variety of cells and organisms. These CRISPR-Cas9 RNA-guided endonucleases cleave chromosomal DNA in a targeted manner to generate site-specific DNA double strand break (DSB), and non-homologous end binding (NHEJ). Repair through -joining induces insertion or deletion (indels) at the target site. Unfortunately, cleavage of target DNA at sites with high sequence homology with the target site can induce mutations and chromosomal rearrangements at unwanted genomic sites (off-target effect). S. Both pyogenes Cas9 nuclease and guide RNA (sgRNA) have been modified to minimize or eliminate these off-target effects. In particular, a highly specific Cas9 variant having an off-target effect that is minimal or undetectable in human cells has been developed, for example enhanced Cas9-1.1 (eCas9-1.1) (Slaymaker, IM et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84-88 (2016 )) and Cas9 high-fidelity variant 1 (Cas9 -HF1) (Kleinstiver, BP et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529 , 490-495 (2016)). These highly specific Cas9 variants include alanine substitutions to attenuate non-specific ionic interactions between the Cas9 protein and the non-target or target DNA strand.

There is a need to develop a technology to further enhance the gene editing activity and specificity of such highly specific Cas9 variants.

The present specification provides a technique capable of further enhancing on-target specific gene editing activity while maintaining low gene editing activity for the off-target site of the high specific Cas9 variants. More specifically, it provides a technique for increasing the gene editing efficiency (eg, indel frequency) of Cas9 variants using a guide RNA (eg, sgRNA) comprising a matched 5′ nucleotide.

One example provides a complex of a guide RNA (eg, sgRNA) comprising a highly specific Cas9 variant and a matched 5′ nucleotide.

Another example provides a composition for gene editing comprising a guide RNA (eg, sgRNA) comprising a highly specific Cas9 variant and a matched 5′ nucleotide.

Another example provides a method for gene editing using a guide RNA (eg, sgRNA) comprising a highly specific Cas9 variant and a matched 5′ nucleotide. For example, the method includes a guide RNA (e.g., sgRNA) comprising the high specificity Cas9 variant and a matched 5'nucleotide into a target gene or a target site (including a PAM sequence, 10 to 30 nucleotides). , 10 or 25 nucleotides, 15 to 30 nucleotides, 15 or 25 nucleotides, 17 to 30 nucleotides, or 17 or 25 nucleotides, such as 20 nucleotides). The step may be performed by administering, injecting, or introducing the complex to a subject.

The high specificity Cas9 variant refers to a Cas9 variant in which one or more amino acids are substituted with alanine to enhance specificity for a target site.For example, based on the amino acid sequence (SEQ ID NO: 4) of Streptococcus pyogenes Cas9 protein, K848, K1003 , R1060, N497, R661, Q695, and Q926 may mean a Cas9 variant in which at least one amino acid selected from the group consisting of alanine is substituted. In one embodiment, the high specificity Cas9 variant is eCas9-1.1 in which K848A, K1003A, and R1060A mutations are introduced into Streptococcus pyogenes Cas9 protein (SEQ ID NO: 4), or Cas9-Introduced with N497A, R661A, Q695A, and Q926A mutations. HF1, or a combination thereof.

In the present specification, the 5'nucleotide matched with the guide RNA refers to a nucleotide comprising a base (matching) that matches a nucleotide (referred to as a 5'nucleotide) located at the 5'end of the target sequence targeted by each guide RNA. it means.

Another example provides a genetically modified cell comprising a gene corrected by the gene editing method.

Another example provides a genetically modified animal obtained from the genetically modified cell.

The methods, complexes and compositions are eukaryotic cells (e.g., isolated human cells, or cells of non-human eukaryotic animals or eukaryotic plants) or eukaryotic organisms (e.g., humans, or non-human eukaryotic animals or eukaryotic plants) It may be applied to.

Other examples include (1) a guide RNA (e.g., sgRNA) and (2) an RNA cleavage enzyme having a self-cleaving activity fused to the 5'end of the guide RNA or 1 to 6 tRNAs. to provide.

Another example is matching with a target sequence, comprising fusing 1 to 6 tRNAs or RNA cleavage enzymes having self-cleaving activity to the 5'end, 3'end, or both ends of the guide RNA (e.g., sgRNA). It provides a method for producing a guide RNA comprising the 5'terminal nucleotide. The step of fusing in the preparation method may include expressing a DNA encoding a guide RNA (eg, sgRNA) and an RNA cleavage enzyme having a self-cleaving activity or a DNA encoding 1 to 6 tRNAs in one vector. have.

The RNA cleavage enzyme (ribozyme) having the self-cleaving activity is a hammerhead ribozyme (eg, Type I hammerhead ribozyme, Type II hammerhead ribozyme, Type III hammerhead ribozyme, etc.), VS (Varkud satellite) ribozyme, lead Zyme (Leadzyme), hairpin ribozyme (hairpin ribozyme) may be one or more selected from the group consisting of, but is not limited thereto.

Since the U6 promoter commonly used to express sgRNA in eukaryotic cells requires guanosine (G) nucleotides to initiate transcription, sgRNAs typically contain a "G" nucleotide at the 5'end. Most (average 75%) DNA target sites contain mismatched nucleotides at this position (i.e., nucleotides containing bases (A, T, or C) other than G). When the present inventors use a highly specific Cas9 variant with a guide RNA containing G at the 5'end and a target sequence that does not contain G at the 5'end, a mismatch between the guide RNA and the target sequence at the 5'end occurs. Thus, it was confirmed for the first time that the gene editing efficiency by the highly specific Cas9 variant at the target sequence site is lowered, and the 5'end of the guide RNA and the 5'end of the target sequence are matched in the gene editing efficiency of the highly specific Cas9 variant. It is suggested that whether or not it has an important role.

In addition, by using sgRNA containing a matched 5'nucleotide produced by linking to a self-cleaving ribozyme, in eukaryotic cells including humans, the highly specific Cas9 variant is low in the off-target site. It was confirmed that the gene editing activity in the on-target site can be remarkably improved without sacrificing high-specificity showing activity.

Accordingly, an example of the present invention provides a complex of a guide RNA (eg, sgRNA) comprising a highly specific Cas9 variant and a matched 5′ nucleotide.

Another example provides a composition for gene editing comprising a guide RNA (eg, sgRNA) comprising a highly specific Cas9 variant and a matched 5′ nucleotide.

Another example provides a method for gene editing using a guide RNA (eg, sgRNA) comprising a highly specific Cas9 variant and a matched 5′ nucleotide. For example, the method includes a guide RNA (e.g., sgRNA) comprising a highly specific Cas9 variant and a matched 5'nucleotide into a target gene or a target site located within the target gene (13 to 30 nucleotides, including the PAM sequence, 13 or 25 nucleotides, 15 to 30 nucleotides, 15 or 25 nucleotides, 20 to 30 nucleotides, or 20 or 25 nucleotides), and the contacting step may include the step of contacting the complex. (Eukaryotic cells or eukaryotic organisms) may be performed by administration, injection, or introduction.

The high specificity Cas9 variant refers to a Cas9 variant in which at least one amino acid residue other than alanine is substituted with alanine to enhance specificity for a target site, and is also referred to herein as a high-fidelity Cas9 variant. For example, the highly specific Cas9 variant is based on the amino acid sequence (SEQ ID NO: 4) of the Streptococcus pyogenes Cas9 protein, and at least one amino acid selected from the group consisting of K848, K1003, R1060, N497, R661, Q695, and Q926 is substituted with alanine. May mean a Cas9 variant. In one embodiment, the high specificity Cas9 variant is eCas9-1.1 in which K848A, K1003A, and R1060A mutations are introduced into Streptococcus pyogenes Cas9 protein (SEQ ID NO: 4), or Cas9-Introduced with N497A, R661A, Q695A, and Q926A mutations. HF1, or a combination thereof.

In the present specification, the matched 5'nucleotide of the guide RNA corresponds to the nucleotide (referred to as 5'nucleotide) located at the 5'end of the target sequence targeted by the guide RNA (the target sequence of the strand where the PAM sequence is located). It means a nucleotide comprising a base (matching the 5'nucleotide). In addition, the mismatch 5'nucleotide refers to a nucleotide containing a base that does not match the 5'nucleotide of the target sequence targeted by the guide RNA (which does not match the 5'nucleotide of the target sequence of the strand where the PAM sequence is located).

Another example provides a genetically modified cell comprising a gene corrected by the gene editing method.

Another example provides a genetically modified animal obtained from the genetically modified cell.

The methods, complexes and compositions are eukaryotic cells (e.g., isolated human cells, or cells of non-human eukaryotic animals or eukaryotic plants) or eukaryotic organisms (e.g., humans, or non-human eukaryotic animals or eukaryotic plants) It may be applied to.

Other examples include (1) a guide RNA (e.g., sgRNA) and (2) an RNA cleavage enzyme having a self-cleaving activity fused to the 5'end of the guide RNA or 1 to 6 tRNAs. to provide.

Another example is matching with a target sequence, comprising fusing 1 to 6 tRNAs or RNA cleavage enzymes having self-cleaving activity to the 5'end, 3'end, or both ends of the guide RNA (e.g., sgRNA). It provides a method for producing a guide RNA comprising the 5'terminal nucleotide. The step of fusing in the preparation method may include expressing a DNA encoding a guide RNA (eg, sgRNA) and an RNA cleavage enzyme having a self-cleaving activity or a DNA encoding 1 to 6 tRNAs in one vector. have.

The RNA cleavage enzyme (ribozyme) having the self-cleaving activity is a hammerhead ribozyme (eg, Type I hammerhead ribozyme, Type II hammerhead ribozyme, Type III hammerhead ribozyme, etc.), VS (Varkud satellite) ribozyme, lead Zyme (Leadzyme), hairpin ribozyme (hairpin ribozyme) may be one or more selected from the group consisting of, but is not limited thereto.

Another example provides a method for enhancing gene editing efficiency of the highly specific Cas9 variant, comprising introducing a guide RNA comprising a matched 5'terminal nucleotide into a eukaryotic cell or a eukaryotic organism together with the highly specific Cas9 variant. The enhancement of the gene editing efficiency may mean an increase in the gene editing efficiency (eg, indel frequency) at the on-target site and/or a decrease in the gene calibration efficiency (eg, indel frequency) at the off-target site.

The eukaryotic cell may be an isolated human cell, or a eukaryotic animal cell other than a human or a cell of a eukaryotic plant, and the eukaryotic organism may be a human, or a eukaryotic animal or a eukaryotic plant other than a human.

As used herein, the term "gene editing" results in a double-stranded DNA cleavage at a target site within a target gene, resulting in a mutation (deletion, substitution, and/or It means an action that causes insertion, etc.). In one example, the gene correction as described above generates a stop codon at the target site, or by generating a codon encoding an amino acid different from the wild type, thereby knocking out the target gene or not producing a protein. It may be in various forms, such as introducing a mutation into a non-coding DNA sequence, but is not limited thereto.

In the present specification, the gene correction may be performed in vitro or in vivo.

As used herein, the term "base sequence" refers to a sequence of a nucleotide containing a corresponding base, and may be used with the same meaning as a nucleotide sequence or a nucleic acid sequence.

As used herein,

The term'target gene' refers to a gene that is subject to gene correction,

The term'target site or target region' refers to a site where gene correction by Cas9 in a target gene occurs, and in one example, the 5'end of the sequence (PAM sequence) recognized by Cas9 in the target gene and/ Or a gene site located adjacent to the 3'end and having a maximum length of about 50 bp or about 40 bp (either double stranded or double stranded, single stranded),

The'target sequence' is about 15 to about 30, about 15 to about 35, about 17 to about 23, or about 18 to about 30, about 15 to about 35, about 18 to about, hybridization of the guide RNA in the target gene or the target site of the target gene. It may be a nucleotide sequence of a site containing 22, for example, about 20 nucleotides (nt).

In addition, the term'targeting sequence' included in the guide RNA is about 15 to about 30, about 15 to about 35, about 17 to about 23, or about 18 to about 22 consecutive in the target site. It may be a site of a guide RNA including a base sequence complementary to a base sequence of a site including a dog, for example, about 20 nucleotides (nt) (hybridizable). The nucleotide sequence of the target site including the nucleotide sequence complementary to the targeting sequence may be referred to as a'target sequence', and the target sequence is the 5'end of the PAM sequence recognized by the RNA-guide nuclease and/ Or it may mean a nucleotide sequence of about 15 nt to about 30 nt, about 15 nt to about 25 nt, about 17 nt to about 23 nt, or about 18 nt to about 22 nt, such as about 20 nt in length, located adjacent to the 3'end. .

The Cas9 protein may be one or more selected from all Cas9s capable of recognizing a specific sequence (PAM) of a target gene and having nucleotide cleavage activity to cause indel (insertion and/or deletion, Indel) in the target gene.

The Cas9 protein can cause double strand break (DSB) by recognizing a specific nucleotide sequence in the genome of prokaryotic cells and/or animal and plant cells (eg, eukaryotic cells) including human cells. The double helix cleavage may cut a double helix of DNA to create a blunt end or a cohesive end. DSB can be efficiently repaired in cells by homologous recombination or non-homologous end-joining (NHEJ) mechanisms, and in this process, desired mutations can be introduced at target sites.

The Cas9 protein is used together with a target DNA specific guide RNA for guiding to a target site of genomic DNA. The guide RNA is transcribed in vitro or extracellularly (e.g., transcribed from an oligonucleotide double strand or plasmid template), or recombinantly produced in vivo or in cells by a recombinant vector (expression vector). However, it is not limited thereto. The Cas9 protein may act in the form of ribonucleic acid protein (RNP) by forming a complex with a guide RNA after delivery outside the body (or cells) or in the body (cells).

The Cas9 protein is a major protein component of the CRISPR/Cas system, and is a protein capable of forming an activated endonuclease or nickase.

Cas9 protein or gene information can be obtained from a known database such as GenBank of NCBI (National Center for Biotechnology Information). For example, the Cas9 protein,

Streptococcus sp. ( Streptococcus sp.), for example, a Cas9 protein derived from Streptococcus pyogenes (eg, SwissProt Accession number Q99ZW2 (NP_269215.1));

Campylobacter genus, such as Campylobacter jejuni ( Campylobacter jejuni ) derived Cas9 protein;

Streptococcus genus, for example, Streptococcus Thermo filler's (Streptococcus thermophiles) or Streptococcus aureus (Streptocuccus aureus ) derived Cas9 protein;

Cas9 protein from Neisseria meningitidis ;

Cas9 protein from the genus Pasteurella , for example Pasteurella multocida ;

Cas Cas9 protein from the genus Francisella , for example, Francisella novicida

It may be one or more selected from the group consisting of, but is not limited thereto.

The Cas9 protein may be isolated from a microorganism or artificially or non-naturally produced such as a recombinant method or a synthetic method. The Cas9 protein may be used in the form of an mRNA transcribed in vitro or a protein produced in advance, or contained in a recombinant vector for expression in target cells or in vivo. In one example, the Cas9 protein may be a recombinant protein made by recombinant DNA (recombinant DNA; rDNA). Recombinant DAN refers to a DNA molecule that has been artificially made by a gene recombination method such as molecular cloning to contain heterologous or homogeneous genetic material obtained from various organisms. For example, when recombinant DNA is expressed in an appropriate organism to produce Cas9 protein ( in vivo or in vitro ), recombinant DNA is reconstituted by selecting a codon optimized for expression in the organism from among the codons encoding the protein to be produced. It may have a nucleotide sequence.

The Cas9 protein used herein may be a mutated Cas9 in a mutated form. The mutant Cas9 protein may mean that it has been mutated to lose the endonuclease activity that cuts the DNA double strand, for example, a mutant target-specific nuclea that has lost endonuclease activity and mutated to have a nickase activity. It may be one or more selected from among mutant target-specific nucleases that have been mutated so as to lose both endonuclease activity and nickase activity. When the mutant Cas9 protein has a nickase activity, the strand in which the base conversion has occurred simultaneously or sequentially regardless of the sequence or simultaneously with the base conversion by the deaminase (eg, cytidine to uradine conversion) or A nick may be introduced on the opposite strand (eg, the opposite strand of the strand in which the base transformation has occurred) (eg, a nick is introduced between the 3 nucleotide and the 4 nucleotide in the 5′ end direction of the PAM sequence). Such mutations (eg, amino acid substitutions, etc.) of the Cas9 protein may occur at least in the catalytically active domain (eg, RuvC catalytic domain) of the nuclease. In one example, when the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes (SwissProt Accession number Q99ZW2 (NP_269215.1); SEQ ID NO: 4), the mutation is a catalytic aspartate residue; for example, In the case of SEQ ID NO: 4, aspartic acid at position 10 (D10), etc.), glutamic acid at position 762 of SEQ ID NO: 4 (E762), histidine at position 840 (H840), asparagine at position 854 (N854), position 863 Asparagine at position (N863), aspartic acid at position 986 (D986), and the like may include a mutation substituted with one or more arbitrary other amino acids selected from the group consisting of. In this case, any other amino acid to be substituted may be alanine, but is not limited thereto.

In another example, the mutant Cas9 protein may be mutated to recognize a PAM sequence different from that of the wild-type Cas9 protein. For example, the Cas9 protein is one or more of aspartic acid at position 1135 (D1135) at position 1135, arginine at position 1335 (R1335), and threonine at position 1337 (T1337) of the Cas9 protein derived from Streptococcus pyyogens, such as all three. Is substituted with another amino acid, and may be mutated to recognize an NGA different from the PAM sequence (NGG) of wild-type Cas9 (N is any base selected from A, T, G, and C).

In one example, the mutant Cas9 protein is of the amino acid sequence (SEQ ID NO: 4) of the Cas9 protein derived from Streptococcus pyogenes,

(1) D10, H840, or D10 + H840;

(2) D1135, R1335, T1337, or D1135 + R1335 + T1337; or

(3) both (1) and (2) residues

The amino acid substitution may have occurred in

As used herein, the'other amino acids' are alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, Arginine, histidine, lysine, among all known variants of the above amino acids, refers to an amino acid selected from amino acids except for amino acids that the wild-type protein has at its original mutation position. In one example, the'other amino acid' may be alanine, valine, glutamine, or arginine.

In one example, the mutant Cas9 protein is a modified Cas9 protein that has lost endonuclease activity (e.g., has nickase activity, loses both endonuclease activity and nickase activity), or a PAM different from wild-type Cas9 It may be to recognize the sequence. For example, the mutant Cas9 protein, in the Cas9 protein (SEQ ID NO: 4) derived from Streptococcus pyogenes ,

(1) A mutation (e.g., substitution with another amino acid) is introduced at the D10 or H840 position, resulting in loss of endonuclease activity and a modified Cas9 having nickase activity, or a Cas9 protein derived from Streptococcus pyogenes Modified Cas9 protein in which mutations (eg, substitutions with other amino acids) are introduced at both D10 and H840 positions to lose both endonuclease activity and nickase activity;

(2) a modified Cas9 protein that recognizes a PAM sequence different from the wild type by introducing a mutation (eg, substitution with another amino acid) in one or more of D1135, R1335 and T1337 or both; or

(3) The mutations of (1) and (2) are all introduced to have nickase activity and recognize a PAM sequence different from the wild type, or lose both endonuclease activity and nickase activity and recognize a PAM sequence different from the wild type. Modified Cas9 protein

Can be

For example, the mutation at the D10 position of the CAs9 protein is a D10A mutation (means a mutation in which D, which is the 10th amino acid of the Cas9 protein, is substituted with A; hereinafter, the mutation introduced into Cas9 is indicated in the same manner). The mutation at the H840 position may be an H840A mutation, and the mutation at the D1135, R1335, and T1337 positions may be D1135V, R1335Q, and T1337R, respectively.

The Cas9 protein is in the form of a protein, a nucleic acid molecule encoding the same (eg, DNA or mRNA), a ribonucleic acid protein bound to a guide RNA, a nucleic acid molecule encoding the ribonucleic acid protein, or a recombinant vector containing the nucleic acid molecule Can be used as

The nucleic acid molecule encoding the Cas9 protein may be in a form capable of being transferred into the nucleus, acting, and/or expressed in the nucleus.

The Cas9 protein may be in a form that is easy to be introduced into cells. For example, the Cas9 protein or a gene encoding it may be linked to a cell penetrating peptide and/or a protein transduction domain or a gene encoding it. The protein transduction domain may be poly-arginine or a TAT protein derived from HIV, but is not limited thereto. Since various types of cell penetrating peptides or protein transduction domains are known in the art in addition to the examples described above, those skilled in the art are not limited to the above examples and can apply various examples.

In addition, the Cas9 protein, and/or a nucleic acid molecule encoding them may further include a nuclear localization signal (NLS; for example, cccaagaaga agaggaaagtc (SEQ ID NO: 6)). Accordingly, the expression cassette including the Cas9 protein-encoding nucleic acid molecule may further include a regulatory sequence such as a promoter sequence for expressing the Cas9 protein, and, optionally, an NLS sequence. Such NLS sequences are well known in the art.

The Cas9 protein, and/or a nucleic acid molecule encoding the same may be linked to a tag for isolation and/or purification or a nucleic acid sequence encoding the tag. As an example, the tag may be appropriately selected from the group consisting of small peptide tags such as His tag, Flag tag, S tag, GST (Glutathione S-transferase) tag, MBP (Maltose binding protein) tag, etc. It doesn't work.

In the present specification, the term "guide RNA" refers to RNA comprising a targeting sequence capable of hybridizing to a specific nucleotide sequence (target sequence) within a target site within a target gene, and in vitro or in vivo ( Or cells) by binding to the Cas9 protein and leading it to the target gene (or target site).

The guide RNA may be appropriately selected according to the type of Cas9 protein to form the complex and/or the microorganism derived therefrom.

For example, the guide RNA,

CRISPR RNA (crRNA) comprising a site capable of hybridizing with a target sequence (targeting sequence);

Trans- activating crRNA (tracrRNA) containing a site that interacts with the Cas9 protein; And

A single guide RNA (sgRNA) in the form of fusion of the main regions of the crRNA and tracrRNA (eg, a crRNA site including a targeting sequence and a site of tracrRNA interacting with a nuclease)

It may be one or more selected from the group consisting of,

Specifically, it may be a dual RNA (dual RNA) including CRISPR RNA (crRNA) and a trans- activating crRNA (tracrRNA), or a single guide RNA (sgRNA) including a major site of crRNA and tracrRNA.

The sgRNA is a portion having a sequence (targeting sequence) complementary to the target sequence in the target gene (target site) (also referred to as a spacer region, target DNA recognition sequence, base pairing region, etc.) and a hairpin structure for binding Cas protein It may include. More specifically, a portion including a sequence (targeting sequence) complementary to a target sequence in a target gene, a hairpin structure for binding to a Cas protein, and a Terminator sequence may be included. The structure described above may be sequentially present in the order of 5'to 3', but is not limited thereto. Any type of guide RNA can be used in the present invention as long as the guide RNA includes a major portion of crRNA and tracrRNA and a complementary portion of the target DNA.

For example, the Cas9 protein is a trans- activating crRNA (tracrRNA; Cas9 protein that interacts with two guide RNAs, namely, a CRISPR RNA (crRNA) having a nucleotide sequence hybridizable with a target site of a target gene and a Cas9 protein for target gene correction. Interact), and these crRNA and tracrRNA can be used in the form of a double-stranded crRNA:tracrRNA complex bonded to each other, or a single guide RNA (sgRNA) form by being linked through a linker. In one example, when using the Cas9 protein derived from Streptococcus pyogenes , the sgRNA is at least a part or all of the crRNA comprising a hybridizable nucleotide sequence of the crRNA and a portion of the tracrRNA comprising at least a site that interacts with the Cas9 protein of the tracrRNA of Cas9 Alternatively, all may be to form a hairpin structure (stem-loop structure) through a nucleotide linker (in this case, the nucleotide linker may correspond to a loop structure).

The guide RNA, specifically crRNA or sgRNA, includes a sequence complementary to the target sequence in the target gene (targeting sequence), and at least one at the 5'end of the crRNA or sgRNA upstream region, specifically the crRNA of sgRNA or dualRNA, such as , 1-10, 1-5, or 1-3 additional nucleotides. The additional nucleotide may be guanine (G), but is not limited thereto.

The specific sequence of the guide RNA can be appropriately selected according to the type of Cas9 (ie, the derived microorganism), which can be easily known to those of ordinary skill in the art to which this invention belongs.

In one example, when using the Cas9 protein derived from Streptococcus pyogenes as a target-specific nuclease, crRNA can be expressed by the following general formula 1:

5'-(N _cas9 ) _l -(GUUUUAGAGCUA)-(X _cas9 ) _m -3' (general formula 1)

In the general formula 1,

N _cas9 is a targeting sequence, that is, a site determined according to the sequence of the target site of the target gene (can be hybridized with the target sequence of the target site), and l is the number of nucleotides contained in the targeting sequence. As represented, it may be an integer of 15 to 30, 17 to 23, or 18 to 22, such as 20

The site containing 12 consecutive nucleotides (GUUUUAGAGCUA) (SEQ ID NO: 1) adjacent to each other in the 3'direction of the targeting sequence is an essential part of crRNA,

X _cas9 is a site containing m nucleotides located at the 3'end of the crRNA (that is, adjacent to the 3'direction of the essential part of the crRNA), where m is an integer of 8 to 12, such as 11 days. And, the m nucleotides may be the same or different from each other, and each may be independently selected from the group consisting of A, U, C and G.

In one example, the X _cas9 may include UGCUGUUUUG (SEQ ID NO: 2), but is not limited thereto.

In addition, the tracrRNA can be represented by the following general formula 2:

5'-(Y _cas9 ) _p -(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC)-3' (general formula 2)

In the above general formula 2,

The site marked with 60 nucleotides (UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC) (SEQ ID NO: 3) is an essential part of tracrRNA,

Y _cas9 is a site containing p nucleotides located adjacent to the 5'end of the essential part of the tracrRNA, p may be an integer of 6 to 20, such as an integer of 8 to 19, and the p nucleotides are the same. Or may be different, and each may be independently selected from the group consisting of A, U, C and G.

In addition, the sgRNA is a crRNA portion including the targeting sequence and essential site of the crRNA and the tracrRNA portion including the essential portion (60 nucleotides) of the tracrRNA to form a hairpin structure (stem-loop structure) through an oligonucleotide linker. (In this case, the oligonucleotide linker corresponds to the loop structure). More specifically, the sgRNA is a double-stranded RNA molecule in which a crRNA portion including a targeting sequence of crRNA and an essential portion and a tracrRNA portion containing an essential portion of tracrRNA are bonded to each other, and the 3'end of the crRNA site and 5 of the tracrRNA site 'It may have a hairpin structure whose ends are linked through an oligonucleotide linker.

In one example, the sgRNA can be represented by the following general formula 3:

5'-(N _cas9 ) _l -(GUUUUAGAGCUA)-(oligonucleotide linker)-(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC)-3' (general formula 3)

In the general formula 3, (N _cas9 ) _l is the targeting sequence as described in the above general formula 1.

The oligonucleotide linker included in the sgRNA may include 3 to 5, such as 4 nucleotides, and the nucleotides may be the same or different from each other, and each independently selected from the group consisting of A, U, C and G Can be. In one embodiment, the oligonucleotide linker may include the nucleic acid sequence of GAAA, but is not limited thereto.

The crRNA or sgRNA may further include 1 to 3 guanine (G) at the 5'end (ie, the 5'end of the targeting sequence site of the crRNA).

The tracrRNA or sgRNA may further include a terminating site comprising 3 to 7, 3 to 5, or 5 to 7 uracils (U) at the 3'end of the essential portion (60 nt) of the tracrRNA. have.

The target sequence of the guide RNA is adjacent to the 5'of the PAM (Protospacer Adjacent Motif sequence (5'-NGG-3' in the case of S. pyogenes Cas9 (N is A, T, G, or C)) on the target DNA. It may be from about 17 to about 23 or from about 18 to about 22, such as 20 contiguous nucleic acid sequences located therein.

The targeting sequence of the guide RNA hybridizable with the target sequence of the guide RNA is the DNA strand where the target sequence is located (i.e., the PAM sequence (5'-NGG-3' (N is A, T, G, or C)) Located DNA strand) or a nucleotide sequence of a complementary strand thereof and 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% of sequence complementarity. It means a nucleotide sequence, and complementary binding to the nucleotide sequence of the complementary strand is possible.

In the present specification, the nucleic acid sequence of the target site (target sequence) is represented by the nucleic acid sequence of the strand where the PAM sequence is located among two DNA strands of the corresponding gene site of the target gene. At this time, since the DNA strand to which the guide RNA is actually bound may be a complementary strand of the strand where the PAM sequence is located, the targeting sequence included in the guide RNA is a target sequence except for changing T to U due to RNA characteristics. It may have the same nucleic acid sequence as. Accordingly, in the present specification, the targeting sequence and the target sequence of the guide RNA are represented by the same nucleic acid sequence except that T and U are mutually altered.

The guide RNA may be used in the form of RNA (or included in the composition), or may be used in the form of a plasmid including DNA encoding it (or included in the composition).

The technology that can further enhance the on-target specific gene editing activity while maintaining low gene editing activity for the off-target site of the highly specific Cas9 variants provided herein can be widely applied in the field of genetic engineering and medicine. have.

1 is a high-fidelity (high-fidelity) Cas9 mutants showing the gene editing activity in the target site having a non-G 5'nucleotide,
1a schematically shows wild-type Cas9 (Cas9-WT), engineered Cas9 variants, and sgRNA, and compared to Cas9-WT, the alanine substitutions introduced in eCas9-1.1 or Cas9-HF1 are indicated by blue or red asterisks, respectively. The red triangle indicates the cut position, the GX ₁₉ sgRNA indicates that it starts with a G matched with the protospacer (blue line), the gX ₁₉ sgRNA has a mismatched G at the 5′-end, and gX ₂₀ The sgRNA contains an additional guanine at the 5'-end, and the PAM (protospacer-adjacent motif) is NGG labeled with a red stripe (not H, G (A or C or T); D, C is not ( A or G or T)),
1b is a graph showing the frequency of Indel at the on-target site where the 5'nucleotide obtained using gX ₁₉ sgRNA or gX ₂₀ sgRNA in HeLa cells is not G (measured by targeted deep sequencing, PAM sequence is shown in blue) , Error bars, sem; n = 3).
Figure 2 shows a Hammerhead ribozyme-binding sgRNA,
2a schematically shows a sgRNA fused with a self-processing ribozyme, pre-sgRNA contains Hammerhead (HH) ribozyme at the 5'end, and pre-sgRNA is self-cutting (self -cleavage) is released as mature sgRNA, the red arrow indicates the site of self-cleavage,
2b is HH ribozyme-fused sgRNA with matched 5'nucleotides (HH-X ₂₀ ) or HH ribozyme-fused sgRNA with mismatched guanosine (HH-gX ₁₉ ) Cas9-WT or high-fidelity Cas9 Each combination with the mutant was tested for gene editing efficiency for 5 target sites in HeLa, and the Indel frequency was measured using targeted deep sequencing (PAM is indicated in blue, Error bars, sem; n = 3, Statistical significances were calculated by t-test.* P <0.05, ** P <0.01),
2c is a graph showing the mean indel frequencies ± sem at 5 target sites (* P <0.05, ** P <0.01),
2d is a graph showing the frequency of Indel at the on-target and off-target sites measured by co-transfecting HH-X ₂₀ sgRNA with Cas9-WT or Cas9 variant into HeLa cells and targeted deep sequencing (PAM sequence is Indicated in blue and mismatched bases in red; the specificity ratio measured in multiples of the ratio of the indel frequency in the on-target site to the indel frequency in the off-target site obtained using the Cas9 variant and Cas9-WT. One value ([Ratio of indel frequency at the on-target site to indel frequency at the off-target site obtained using the Cas9 variant]/[for the indel frequency at the off-target site obtained using Cas9-WT] Ratio of indel frequency in the on-target site]); Error bars, sem; n = 3; Significant level of indel frequency compared to mock transfected sample is marked with an asterisk (* P <0.05, ** P <0.01) ).

Hereinafter, the present invention will be described in more detail by way of examples, but these are only illustrative and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the embodiments described below may be modified within the scope of the essential gist of the invention.

Example 1: high-fidelity Cas9 Variant Coding plasmid and HH - Ribozyme -Construction of fused sgRNA encoding plasmid

As a plasmid encoding a Cas9 variant, a plasmid encoding eSpCas9-1.1 (p3seCas9-1.1; Addgene #104172) and N497A, R661A, Q695A into which K848A, K1003A, and R1060A mutations were introduced into Streptococcus pyogenes Cas9 protein (SEQ ID NO: 4) , And a plasmid encoding p3s-Cas9-HF1 into which the Q926A mutation was introduced (p3s-Cas9-HF1, Addgene #104173) was used, respectively.

HH (Hammerhead)-ribozyme sgRNA construct (see FIG. 2A) is ligated with an annealed oligonucleotide comprising an HH-ribozyme nucleic acid sequence and a protospacer sequence into a plasmid (pRG2, Addgene #104® sgRNA is expressed under U6 promoter control). It was cloned by doing.

The sgRNA has the following sequence:

5'-(target sequence)-(GUUUUAGAGCUA; SEQ ID NO: 1)-(nucleotide linker)-(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; SEQ ID NO: 3)-UUUU-3'

(The target sequence is a sequence obtained by converting "T" to "U" in the target site sequence (20nt) shown in Table 1 below,

gX ₁₉ sgRNA is an sgRNA constructed to include'G' at the 5'terminal base (indicated by the underline) in all target site sequences in Table 1,

X ₂₀ sgRNA is an sgRNA constructed to contain a base matching the 5'end base (indicated by the underline) of each target site sequence in Table 1,

The nucleotide linker has the nucleotide sequence of GAAA).

Target site sequence Locus Target site + PAM sequence (in bold and underlined; 5'→3') AAVS1 C TCCCTCCCAGGATCCTCTC TGG (SEQ ID NO: 7) CCR5 T CATCCTGATAAACTGCAAA AGG (SEQ ID NO: 8) HBB-02 C TTGCCCCACAGGGCAGTAA CGG (SEQ ID NO: 9) HBB-03 C ACGTTCACCTTGCCCCACA GGG (SEQ ID NO: 10) HBB-04 C CACGTTCACCTTGCCCCAC AGG (SEQ ID NO: 11) EMX1-05 T GTACTTTGTCCTCCGGTTG TGG (SEQ ID NO: 12)

Example 2: Cell culture and transfection ( transfection )

HeLa cells (ATCC, CCL-2) in 100 units/mL penicillin, 100 ug (microgram)/mL streptomycin, 0.1 mM non-essential amino acids, and 10% (w/v) fetal fetal blood?? (fetal bovine serum; FBS) was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented. 0.8 x 10 ⁵ HeLa cells were transfected with Cas9-encoding plasmid (0.5 ug) and sgRNA expression plasmid (0.5 ug) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Example 3: Targeted deep sequencing

For construction of the NGS library, on-target and off-target regions (see Table 1, FIGS. 1b, 2b, and 2d) were PCR amplified using Phusion polymerase (Thermo Fisher Scientific). Pooled PCR amplicons were sequenced according to the manufacturer's instructions using the MiniSeq with TruSeq HT Dual Index system (Illumina). Indel frequency Cas-Analyzer (Park, J., Lim, K., Kim, JS & Bae, S. Cas-analyzer:. An online tool for assessing genome editing results using NGS data Bioinformatics 33, 286-288 (2017) ) Was measured.

Example 4: Mismatch 5'end Nucleotides Gene editing efficiency test of guide RNA having

Since the U6 promoter commonly used to express sgRNA in eukaryotic cells requires a guanosine (G) nucleotide to initiate transcription, sgRNAs typically contain a G nucleotide at the 5'end. Most (average 75%) DNA target sites contain mismatches at this site, so gX ₁₉ sgRNA at these sites (Fig. 1A; in Fig. 1A, "g" refers to mismatched guanosine, "G" refers to matched Each represents guanosine, and each N or X is independently selected from nucleic acid bases including A, T, G, and C).The level of intracellular correction by the attenuated Cas9 variant can be lowered. .

In order to prove this hypothesis, using gX ₁₉ sgRNA, the gene editing activity of eCas9-1.1 and Cas9-HF1 at 5 gene sites where the 5'terminal nucleotide is not guanosine in HeLa cells was determined by wild-type Cas9 protein (Cas9- WT) and the results are shown in Fig. 1b. Gene editing activity was confirmed by measuring idel frequency with reference to the method described in Example 3.

As shown in Fig. 1B, Cas9-WT was not sensitive to nucleotide mismatch at the 5'end, and thus induces indels at a high frequency of 48% to 78% (average 70±5%). eCas9-1.1 showed very low indel frequencies of 1.4-45% (22±10%) in 4 of the 5 sites (33±14%). Cas9-HF1 was the least active among the three Cas9 nucleases, and showed an indel frequency of 0.2 to 20% (8.3±4.1%). At the CCR5 target site, both attenuated Cas9 proteins were inactive (showing an indel frequency of less than 1%). In addition, the gene editing activity (indel frequency) was tested using gX ₂₀ sgRNA with additional guanosine at the 5'end. These sgRNAs have nucleotides matched to the 5'end. gX ₂₀ sgRNA increased the gene editing activity of the Cas9 variant at the AAVS1 and HBB-02 sites, but decreased the gene editing activity of the Cas9 variant at the other three sites compared to the gX ₁₉ sgRNA.

Example 5: matched 5' Nucleotides Included sgRNA Fabrication and gene editing efficiency test of Cas9 variant using the same

In order to expand the number of gene sites that can be targeted with the high fidelity Cas9 variant, a self-cleaving ribozyme was used to produce sgRNA in which the 5'nucleotide matches the target DNA sequence. Each sgRNA is fused to Hammerhead (HH) ribozyme at the 5'end (Gao, Y. & Zhao, Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Journal of integrative plant biology 56 , 343-349 (2014)), mature 20-nucleotide (X ₂₀ ) sgRNAs were generated after self-cleavage. The above process is schematically shown in FIG. 2A.

HH ribozyme-fused sgRNA with matched 5'nucleotides (designated HH-X ₂₀ ) or HH ribozyme-fused sgRNA with mismatched 5'guanosine nucleotides (designated HH-gX ₁₉ ) with Cas9-WT or Gene editing activity in HeLa cells was tested in combination with a high fidelity Cas9 variant, and the results are shown in FIGS. 2B and 2C. Gene editing activity was confirmed by measuring idel frequency with reference to the method described in Example 3.

As shown in FIG. 2B, the two Cas9 variants tested (eCas9-1.1 and Cas9-HF1) were all able to exhibit corrective activity at all five target sites by using HH-X ₂₀ sgRNA. Indel incidence HH-HH-gX X ₂₀ or ₁₉ X obtained with the _HH-20 sgRNA use eCas9-1.1 (69 ± 5%) or Cas9-HF1 (59 ± 7% ) It was almost similar to the results obtained using Cas9-WT (71±6% or 70±3%, respectively) with sgRNA (Fig. 2c). Cas9 variants eCas9-1.1 and Cas9-HF1 showed low corrective activity when used with HH-gX ₁₉ sgRNA, and this result indicates that the high fidelity variant is a nucleotide matched to the 5'end rather than the fusion with the ribozyme itself. Shows that it can maintain high gene editing activity due to the presence of

Next, the frequency of mutations at known off-target positions in HeLa cells were measured, and the target specificities of the two Cas9 variants when used with HH-X ₂₀ sgRNAs were compared (for CCR5 specific sgRNAs, off- Target location is unknown, so it is excluded from analysis). The obtained results are shown in Fig. 2d. As shown in FIG. 2D, in most of the off-target sites where each on-target site and 1 to 3 nucleotides differ, both Cas9 variants showed lower indel frequencies than Cas9-WT. Cas9-HF1 was able to differentiate between three off-target sites (one HBB-03 off-target site and two HBB-04 off-target sites) with one nucleotide mismatch.

Summarizing the above results, it shows that unlike wild-type proteins, the newly developed high specificity Cas9 variant often has low calibration efficiency at the target site having a mismatch at the 5'end, and these results show that 5'nucleotides are prokaryotic cells. Or it is the first to show that it contributes to the high specificity of CRISPR-Cas9 in eukaryotic cells such as human cells. By matching the first nucleotide of the sgRNA with the target DNA sequence by the self-degrading activity of HH-ribozyme fusion, high specificity of genome editing can be achieved without adversely affecting on-target calibration efficiency. As an alternative to using HH-ribozyme fusion, tRNA fusion (Port, F. & Bullock, SL Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs.Nature methods 13 , 852-854 (2016)) Or by chemical synthesis (Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.Nature biotechnology 33 , 985-989 (2015)), a matched nucleotide other than G at the 5'end ( 5′ non-G nucleotide) can be prepared and used in combination with the above two high-fidelity Cas9 variants. Cas9 variant ribonucleic acid proteins (Kim, S., Kim, D., Cho, SW, pre-assembled (assembled (made) ex vivo or extracellularly) compared to the case using Cas9- and sgRNA-encoding plasmids. Kim, J. & Kim, JS Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.Genome research (2014)) can further improve genome-wide target specificity.

<110> INSTITUTE FOR BASIC SCIENCE <120> Gene editing composition comprising sgRNAs with matched 5' nucleotide and gene editing method using the same <130> DPP20181455KR <150> KR 10-2017-0064332 <151> 2017-05-24 <160> 12 <170> KopatentIn 3.0 <210> 1 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Essential part of crRNA <400> 1 guuuuagagc ua 12 <210> 2 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> 3'-terminal part of crRNA <400> 2 ugcuguuuug 10 <210> 3 <211> 60 <212> RNA <213> Artificial Sequence <220> <223> Essential part of tracrRNA <400> 3 uagcaaguua aaauaaggcu aguccguuau caacuugaaa aaguggcacc gagucggugc 60 60 <210> 4 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> Cas9 from Streptococcus pyogenes <400> 4 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1010 1015 1020 Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 1025 1030 1035 1040 Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1045 1050 1055 Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1060 1065 1070 Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1075 1080 1085 Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1090 1095 1100 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 1105 1110 1115 1120 Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1125 1130 1135 Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1140 1145 1150 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1155 1160 1165 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1170 1175 1180 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1185 1190 1195 1200 Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1205 1210 1215 Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1220 1225 1230 Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1250 1255 1260 Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 1265 1270 1275 1280 Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1285 1290 1295 His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 1300 1305 1310 Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1315 1320 1325 Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1330 1335 1340 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 1345 1350 1355 1360 Asp Leu Ser Gln Leu Gly Gly Asp 1365 <210> 5 <211> 4107 <212> DNA <213> Artificial Sequence <220> <223> Cas9-coding sequence <400> 5 atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg ctgggccgtg 60 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgaccgc 120 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag 180 gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc 240 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccaccgc 300 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc 360 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag 420 aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc cctggcccac 480 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 540 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc 600 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag caagagccgc 660 cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac 720 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 780 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 840 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc 900 ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct gagcgccagc 960 atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc 1020 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc 1080 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg 1140 gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc 1200 aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac 1260 gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc 1320 gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaacagc 1380 cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa cttcgaggag 1440 gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa cttcgacaag 1500 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1560 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgccttcctg 1620 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc 1680 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc 1740 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg 1860 ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc 1920 cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc 1980 cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa gaccatcctg 2040 gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac 2100 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg 2160 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc 2220 gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg 2280 atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaacag ccgcgagcgc 2340 atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc 2400 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc 2460 gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga cgtggaccac 2520 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gacccgcagc 2580 gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag 2640 aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt cgacaacctg 2700 accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat caagcgccag 2760 ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag ccgcatgaac 2820 accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagagc 2880 aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac 2940 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag 3000 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcgcaag 3060 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc 3120 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc 3180 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc 3240 gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg 3300 cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga caagctgatc 3360 gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc 3420 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg 3480 aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa ccccatcgac 3540 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag 3600 tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc cggcgagctg 3660 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc 3720 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag 3780 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagcgcgtg 3840 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca ccgcgacaag 3900 cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc 3960 cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac cagcaccaag 4020 gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga gacccgcatc 4080 gacctgagcc agctgggcgg cgactaa 4107 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NLS <400> 6 cccaagaaga agaggaaagt c 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Target sequence on AAVS1 and PAM <400> 7 ctccctccca ggatcctctc tgg 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Target sequence on CCR5 and PAM <400> 8 tcatcctgat aaactgcaaa agg 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Target sequence on HBB-02 and PAM <400> 9 cttgccccac agggcagtaa cgg 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Target sequence on HBB-03 and PAM <400> 10 cacgttcacc ttgccccaca ggg 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Target sequence on HBB-04 and PAM <400> 11 ccacgttcac cttgccccac agg 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Target sequence on EMX1-05 and PAM <400> 12 tgtactttgt cctccggttg tgg 23

Claims (10)

Including sgRNA (single-guide RNA) comprising a highly specific Cas9 variant and a matched 5'nucleotide,
The highly specific Cas9 variant is one in which at least one amino acid residue other than alanine of the Cas9 protein is substituted with alanine,
The sgRNA comprising the matched 5'nucleotide is an sgRNA comprising a base matched with the 5'terminal nucleotide of the target sequence,
Composition for gene editing. The method of claim 1, wherein the highly specific Cas9 variant is a Cas9 variant in which at least one amino acid selected from the group consisting of K848, K1003, R1060, N497, R661, Q695, and Q926 of the Streptococcus pyogenes Cas9 protein is substituted with alanine. Dragon composition. The gene of claim 2, wherein the high specificity Cas9 variant is eCas9-1.1 into which K848A, K1003A, and R1060A mutations are introduced into Streptococcus pyogenes Cas9 protein, or Cas9-HF1 into which N497A, R661A, Q695A, and Q926A mutations are introduced. Orthodontic composition. The composition of claim 1, wherein the sgRNA comprises a base matching the 5'terminal nucleotide of the target sequence, and is an sgRNA represented by the following sequence general formula:
5'-(N _cas9 ) _l -(SEQ ID NO: 1)-(oligonucleotide linker)-(SEQ ID NO: 3)-3'
In the above sequence general formula, (N _cas9 ) _l is a targeting sequence capable of hybridizing with the target sequence, l is the number of nucleotides included in the targeting sequence, and is an integer of 15 to 30,
The oligonucleotide linker contains 3 to 5 nucleotides each independently selected from the group consisting of A, U, C and G. The composition for gene editing according to any one of claims 1 to 4, which is for use in eukaryotic cells or eukaryotic organisms. Any one of claims 1 to 4, comprising the step of administering the composition for genetic modification of any one of the isolated eukaryotic cells or eukaryotic organisms other than human, genome editing method. The sgRNA according to any one of claims 1 to 4, wherein the sgRNA comprising a base matching the 5'terminal nucleotide of the target sequence,
(1) sgRNA and
(2) RNA cleavage enzyme or 1 to 6 tRNAs having self-cleaving activity fused to the 5'end, 3'end, or both ends of the (1) sgRNA
It will be produced from a fusion RNA molecule comprising a, gene editing composition. The method of claim 7, wherein the RNA cleavage enzyme having self-cleavage activity is 1 selected from the group consisting of hammerhead ribozyme, VS (Varkud satellite) ribozyme, leadzyme, and hairpin ribozyme. More than a species, a composition for gene editing. delete delete

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