KR20240020336A - Protospacer Adjacent Motif-independent mutant Cas9 protein - Google Patents

Abstract

It relates to a mutant Cas9 protein that is independent of the PAM sequence. It was confirmed that Cas9-PAMless1 exerts cleavage activity on the target gene without dependence on the PAM sequence, and PAM sequence-independent DNA cleavage activity was also confirmed in reversion mutants. In addition, not only was it confirmed that there was no preference for a specific base at the PAM position of the DNA substrate cleaved by Cas9-PAMless1, but also the PAM-less activity of Cas9-PAMless1 was confirmed on Lambda DNA. Therefore, it is expected that Cas9-PAMless1 of the present invention, which can diversify and expand the scope of target sequence selection, can be used for gene therapy and the development of a diagnostic system based on gene scissors.

Description

Mutant Cas9 protein independent of PAM sequence {Protospacer Adjacent Motif-independent mutant Cas9 protein}

It relates to a mutant Cas9 protein that is independent of the PAM sequence.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 system is an immune system of prokaryotes and archaea that provides resistance to external invaders, mostly viruses, and is usually classified into type I, type II, and type III. The CRISPR/Cas9 system creates a double-strand break (DSB) on the target DNA by Cas9 and guide RNA (gRNA) that make up the system, and the cell recognizes this as a damaged site and causes non-homologous end joining (non-homologous end joining). -Induces the conventional DNA repair process by homologous end joining (NHEJ) or homology-directed repair (HDR). Since normalization through mutation or gene replacement is possible during this process, this can be used to induce genome editing.

The Cas9 gene scissors of the CRISPR/Cas9 system uses gRNA to form a complementary bond with the target DNA sequence. Therefore, the construction of gene scissors for targets with various base sequences can be easily performed at the gRNA design level.

However, when actually using Cas9 gene scissors, the Cas9 protein must first bind to a short specific DNA sequence called PAM (protospacer adjacent motif) before binding to the target DNA sequence with gRNA, so the location where DNA cutting is to occur is necessary. When selecting a target sequence, there is a problem that limits the selection range of a base sequence suitable as a target.

Since the recognition of the PAM sequence is by the Cas9 protein itself, regardless of the gRNA, the PAM sequence recognized by a specific Cas9 is fixed. As a way to overcome this, either improve Cas9 so that it can recognize other PAM sequences, or modify the existing Cas9 Research is being conducted to discover new types of Cas9 homologs that are different from the PAM recognition sequence they have.

In order to solve the above problem, the present inventors developed a mutant Cas9 protein that allows Cas9 itself to have PAM-less recognition activity. In other words, a new variant of Cas9 was developed that can bind to the target sequence no matter what base sequence is in the PAM sequence position.

Republic of Korea Patent No. 10-1897213

The object of the present invention is to provide PAM (protospacer adjacent motif) sequence-independent activity;

To provide a mutant Cas9 protein, characterized in that the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2 is deleted.

Another object of the present invention is to provide a recombinant vector containing a nucleic acid encoding the mutant Cas9 protein of the present invention.

Another object of the present invention is to provide a protein complex comprising the mutant Cas9 protein of the present invention and gRNA.

Another object of the present invention is to produce dCas9-PAMless1 by applying the previously known manufacturing principle of deactivated Cas9 (dCas9), which is inactivated by DNA cutting, and to use it as a base-editor gene scissors or to regulate gene expression. The goal is to provide a protein complex that can be used as the DNA-binding domain of a protein that can act as a transcription factor.

Another object of the present invention is to provide a composition for gene correction comprising the mutant Cas9 protein of the present invention as an active ingredient.

Another object of the present invention is to provide a method for producing a mutant Cas9 protein, comprising the step of deleting the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above object of the present invention, the present invention has PAM (protospacer adjacent motif) sequence-independent activity;

Provided is a mutant Cas9 protein, characterized in that the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2 is deleted.

Additionally, the present invention provides a recombinant vector comprising a nucleic acid encoding the mutant Cas9 protein of the present invention.

The present invention also provides a protein complex comprising a mutant Cas9 protein of the present invention and a gRNA.

Additionally, the present invention provides a composition for gene correction, comprising the mutant Cas9 protein of the present invention as an active ingredient.

Additionally, the present invention provides a method for producing a mutant Cas9 protein, comprising the step of deleting the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2.

Additionally, the present invention provides the use of a gene editing composition containing the mutant Cas9 protein of the present invention as an active ingredient for gene editing.

In addition, the present invention provides a use for manufacturing a composition for gene editing comprising the mutant Cas9 protein of the present invention as an active ingredient.

Additionally, the present invention provides a gene editing method using a composition for gene editing containing the mutant Cas9 protein of the present invention as an active ingredient.

In one embodiment of the present invention, the mutant Cas9 protein may be characterized in that one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence shown in SEQ ID NO: 2 are replaced with another amino acid. However, it is not limited to this.

In another embodiment of the present invention, the mutant Cas9 protein may be characterized in that one or more amino acids selected from the group consisting of S1109, R1333, and R1335 in the amino acid sequence shown in SEQ ID NO: 2 are further substituted with another amino acid. Not limited.

In another embodiment of the present invention, the other amino acid may be characterized as being selected from the group consisting of lysine (K), arginine (R), alanine (A), and asparagine (N), but is not limited thereto. .

In another embodiment of the present invention, the mutation may include insertion of a DNA binding domain derived from the NHP6A protein, but is not limited thereto.

In another embodiment of the present invention, the DNA binding domain may be characterized as comprising amino acids D17 to A93 in the amino acid sequence represented by SEQ ID NO: 9, but is not limited thereto.

In another embodiment of the present invention, the mutant Cas9 protein may further include MBP (maltose binding protein) or a His-tag, but is not limited thereto.

In another embodiment of the present invention, the mutant Cas9 protein may be characterized by being represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 8, but is not limited thereto.

In another embodiment of the present invention, the PAM sequence is 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′-AAG-3 ′, 5′-TAG-3′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC-3′, It may be characterized as being any one sequence selected from the group consisting of 5′-GTA-3′, 5′-TAT-3′, and 5′-TAA-3′, but is not limited thereto.

In another embodiment of the present invention, the proteins expressed by the recombinant vector are 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′ -AAG-3′, 5′-TAG-3′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC It may be characterized by recognizing any one PAM sequence selected from the group consisting of -3′, 5′-GTA-3′, 5′-TAT-3′, and 5′-TAA-3′, but is limited to this. It doesn't work.

In another embodiment of the present invention, the recombinant vector may further include a nucleic acid encoding MBP (Maltose binding protein), or a nucleic acid encoding a His-tag, but is not limited thereto.

In another embodiment of the present invention, the recombinant vector includes a nucleic acid encoding a His-tag; Nucleic acid encoding MBP; and the nucleic acid encoding the mutant Cas9 protein of the present invention may be sequentially arranged, but is not limited thereto.

In another embodiment of the present invention, the recombinant vector may be characterized as comprising a nucleic acid encoding a protein represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4 to 8, but is not limited thereto.

In another embodiment of the present invention, the gRNA may be characterized as comprising a domain that binds complementary to a target sequence represented by one or more base sequences selected from the group consisting of SEQ ID NOs: 10 to 13, but is not limited thereto. No.

In another embodiment of the present invention, the production method further comprises the step of substituting one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence represented by SEQ ID NO: 2 with another amino acid. It may be characterized by, but is not limited to, this.

In another embodiment of the present invention, the production method may further include the step of inserting a DNA binding domain derived from the NHP6A protein, but is not limited thereto.

It was confirmed that Cas9-PAMless1 exerts cleavage activity on the target gene without dependence on the PAM sequence, and PAM sequence-independent DNA cleavage activity was also confirmed in reversion mutants. In addition, it was confirmed that there was no preference for a specific base at the PAM site of the DNA substrate cleaved by Cas9-PAMless1, and the PAM-less activity of Cas9-PAMless1 was confirmed at the target site of long DNA such as Lambda DNA. Therefore, the PAM-less activity of Cas9-PAMless1 can diversify and expand the scope of target sequence selection by reducing the limitations of the target sequence, so Cas9-PAMless1 can be used for gene therapy and the development of a diagnostic system based on gene scissors. It is expected that it will be possible.

Figure 1a is a schematic diagram of a PAM-independent variant Cas9 gene scissors (hereinafter, “Cas9-PAMless1”) - expression vector plasmid according to an embodiment of the present invention. Cas9-PAMless1 protein is expressed as a fusion protein with a His-tag and MBP domain located at the N-terminus.
Figure 1b is a diagram showing the base sequence of the Cas9-PAMless1 gene. Start codon and stop codon are indicated in red, and restriction sites required for the synthesis of recombinant genes are underlined.
Figure 1c is a diagram showing the amino acid sequence of Cas9-PAMless1 protein. The positions of the eight mutations (red), the linker ( bold ), and the amino acid sequence of the inserted NHP6A domain (yellow) are indicated.
Figure 2 is a schematic diagram of the target plasmid (pTarget plasmid) for confirming the target sequence-specific DNA cleavage activity of Cas9-PAMless1.
Figure 3 is a diagram showing the purification process of MBP-Cas9-PAMless protein and the results of confirming the purity of the purified MBP-Cas9-PAMless protein. ① is the elution profile of the NTA Ni-chelating column of the MBP-Cas9-PAMless fusion protein. Fractions marked with black boxes were used in the next chromatography step. ② is the elution profile of the size-exclusion column of the MBP-Cas9-PAMless fusion protein. Fractions marked with black boxes were used for the concentration step. ③ is the result of confirming the purity of the final purified MBP-Cas9-PAMless fusion protein.
Figure 4 shows the results of confirming the DNA cutting activity of Cas9-PAMless1 at various concentrations against the T1 target sequence with a 5'-GGG-3' PAM sequence, in order to confirm the DNA cutting activity of Cas9-PAMless1.
Figures 5a to 5c show 14 different PAM-less (or PAM-independent) activities of Cas9-PAMless1, Cas9-PAMless1-r1333/r1335, and Cas9-PAMless1-s1109/r1333/r1335. This is the result of an experiment confirming DNA cutting activity for the T1 target sequence with the PAM sequence.
Figure 6 is a schematic diagram of pTarget-T1-N5, a random PAM library.
Figure 7 shows the results of verifying the PAM-less activity of Cas9-PAMless1 by Dideoxy sequencing. After performing a cleavage experiment of Cas9-PAMless1 on random PAM library substrates, the PAM sequences of the cleaved substrates were analyzed to confirm the preference of Cas9-PAMless1 for each base at the five randomized nucleotide positions in the library, and T1 -Compared with the N5 library.
Figure 8 shows the results of analyzing the composition ratio of each NNN combination of the pTarget-T1-N5 plasmids that underwent cleavage using next-generation sequencing to verify the PAM-less activity of Cas9-PAMless1. The composition ratios of 64 possible PAM sequence combinations were normalized to have a total of 100, and compared with the results of wild-type (WT) SpCas9, a control gene scissors. If there is no preference for a specific PAM sequence, the composition ratio value of each PAM sequence combination appears close to 1.5625.
Figure 9a shows the results of analyzing the composition ratio of each NNN combination of pTarget-T1-N5 plasmids without cleavage over time using next-generation sequencing to verify the PAM-less activity of Cas9-PAMless1. If there is no preference for a specific PAM sequence, the composition ratio value of each PAM sequence combination appears close to 1.5625.
Figure 9b is a graph showing the change in composition ratio over time of each NNN combination of pTarget-T1-N5 plasmids without cleavage to verify the PAM-less activity of Cas9-PAMless1. In the case of WT SpCas9, the well-known preferred PAM sequence (red box), 5'-NGG-3', can be seen decreasing rapidly, whereas in Cas9-PAMless1, the composition ratio of all PAM sequence combinations does not change within a narrow range. maintain.
Figure 10a shows the results of analyzing the DNA cleavage activity specificity of Cas9-PAMless1 for target sequences located in Lambda DNA. While WT SpCas9 showed cleavage activity only against targets with the 5'-TGG-3' PAM sequence, Cas9-PAMless1 showed even cleavage activity against all three targets with different PAM sequences. For the DNA samples on the right, the restriction enzyme site located close to the target was treated with restriction enzyme for size comparison.
Figure 10b shows the results of analyzing the cleavage activity of the Cas9-PAMless1/SgRNA complex when Cas9-PAMless1 and two or more types of SgRNA (single guide RNA) are used. As complexes with different SgRNAs with different target sequences were formed, it was shown that target sequences at two or more positions could be cut in one reaction, and the complex was confirmed to have the same effect as double digestion using restriction enzymes.
Figure 11 is a schematic diagram showing the PAM-less activity of Cas9-PAMless1 compared to WT SpCas9.

The present inventors designed and produced a new variant Cas9 gene scissors with PAM-less recognition activity, confirmed the PAM-independent (PAM-less) activity of the present invention, and completed the present invention.

In one example of the present invention, it was confirmed that Cas9-PAMless1 specifically recognizes the target sequence and effectively causes a DNA cleavage reaction (see Example 3).

In another example of the present invention, it was confirmed that Cas9-PAMless1 exerts cleavage activity on the target gene without dependence on the PAM sequence, and reversion mutations (Cas9-PAMless-r1333/r1335; and Cas9-PAMless-s1109/ r1333/r1335) was confirmed to have a similar level of PAM sequence-independent DNA cleavage activity as Cas9-PAMless1 (see Example 4).

In another example of the present invention, it was confirmed that there was no preference for a specific base at the PAM position of the DNA substrate cleaved by Cas9-PAMless1, and the change in the normalized ratio for the PAM sequence combination of Cas9-PAMless1 was confirmed. It was confirmed that this occurred within a narrow range over time (see Example 5).

In another example of the present invention, the PAM-less activity of Cas9-PAMless1 on Lambda DNA was confirmed (see Example 6).

Hereinafter, the present invention will be described in detail.

The present invention has PAM (protospacer adjacent motif) sequence-independent activity;

Provided is a mutant Cas9 protein, characterized in that the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2 is deleted.

In the present invention, PAM (protospacer adjacent motif) is a sequence recognized by the Cas9 protein before binding to the target DNA sequence. Although the PAM sequence affects the binding of the Cas9 protein to the target DNA sequence, the mutant Cas9 protein of the present invention is not limited by recognition of the PAM sequence and can bind to and cleave the target DNA sequence.

The PAM sequence may be 5'-NNN-3', and N may be a base of adenine (A), cytosine (C), thymine (T), or guanine (G), but is not limited thereto. More preferably, the PAM sequence is 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′-AAG-3′, 5′ -TAG-3′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC-3′, 5′-GTA It may be any one sequence selected from the group consisting of -3′, 5′-TAT-3′, and 5′-TAA-3′, but is not limited thereto.

In the present invention, PAM-less (PAM less) activity may mean that the recognition ability for a specific PAM is not superior to the recognition ability for other PAMs, or that it shows an equivalent level of recognition ability or cognitive activity for two or more sequences. .

In the present invention, PAM less activity may mean binding to any two or more of the PAM sequences disclosed in an embodiment of the present invention at an equal level. For example, as a result of next-generation sequencing, the activity for a specific PAM Assuming that the recognition (combining) level is 1, this may mean that the recognition (combining) level for other PAMs is 0.9 to 2.2.

In the present invention, the deletion is 50 to 150, 50 to 120, 50 to 110, 60 to 150, 60 to 60 amino acids including amino acids L1226 to A1323 of the amino acid sequence shown in SEQ ID NO: 2. 120, 60 to 110, 70 to 150, 70 to 120, 70 to 110, 80 to 150, 80 to 120, 80 to 110, 90 to 150, 90 to 120, 90 to 110, 90 to 100, 95 to 100, or 98 amino acids may be deleted, but are not limited thereto.

In the present invention, the combination of the alphabet 1 (base or amino acid) + number (N) may mean the Nth base or amino acid from the 5' or N-terminus of the base sequence or amino acid sequence to which the above expression corresponds. For example, L1226 may mean L, the 1226th amino acid from the N terminus of the amino acid sequence.

In the present invention, the mutant Cas9 protein may include a point mutation, but is not limited thereto. Specifically, the mutant Cas9 protein may have one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence shown in SEQ ID NO: 2 substituted with another amino acid, but is not limited thereto. In addition, in the mutant Cas9 protein, one or more amino acids selected from the group consisting of S1109, R1333, and R1335 in the amino acid sequence shown in SEQ ID NO: 2 may be further substituted with another amino acid, but is not limited thereto. The type of the other amino acid is not limited, but is preferably an amino acid selected from the group consisting of lysine (K), arginine (R), alanine (A), and asparagine (N), but is not limited thereto.

In the present invention, the point mutation may be made using site-directed mutagenesis and gene synthesis, and the mutation position and substituted amino acid may be any one or more selected from the group consisting of the following. Not limited to: S1109K, D1135R, E1219R, D1332K, R1333A, R1335A, T1337K, and S1338N.

In the present invention, alphabet 1 (amino acid) + number (N) + alphabet 2 (amino acid) refers to a mutation in which alphabet 1, the Nth amino acid from the N terminus of the amino acid sequence to which the above expression corresponds, is replaced by alphabet 2, a different amino acid. It can mean. For example, S1109K refers to a mutation in which S, the 1109th amino acid from the N terminus of the amino acid sequence, is replaced with K.

In the present invention, the mutation may include insertion of a DNA binding domain derived from the NHP6A protein, but is not limited thereto. The DNA binding domain may include amino acids D17 to A93 in the amino acid sequence represented by SEQ ID NO: 9, but is not limited thereto. Additionally, the DNA binding domain may be nonsequence-specific, but is not limited thereto.

A Ser-Thr linker may be located at the N-terminus of the DNA binding domain, and a Gly-Ala-Ala-Ala-Gly-Ser-Thr linker may be located at the C-terminus, but are not limited thereto. The DNA binding domain can be inserted into the SalI and NotI restriction enzyme sites of the SpCas9 gene, but is not limited thereto.

In the present invention, the mutant Cas9 protein may further include MBP (Maltose binding protein) or His-tag, but is not limited thereto. Additionally, the mutant Cas9 protein may be represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 8, but is not limited thereto. In the present invention, MBP (Maltose binding protein) can increase the solubility and stability of recombinant proteins, but is not limited thereto.

In the present invention, the “CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) system or Cas9 protein” is a type of immune system and gene correction tool that exists in prokaryotes, and is a means of quickly eliminating invading foreign DNA. Recognize and cut. CRISPR refers to a structure in which repetitive nucleotide sequences of 20-50 bp are arranged sequentially. Each pair of repetitive sequences in the CRISPR-Cas9 system is separated by an insertion sequence (spacer) with a unique sequence, and the insertion sequences are similar to parts of the bacteriophage genome, and the RNA made therefrom has a guide gRNA (guide RNA). After binding to the invading phage DNA, the Cas9 protein (Cas nucleic acid internal hydrolase) cuts the phage DNA. Using this principle, genes can be inserted or removed from embryos, cells, etc. Since Cas9 is a component of the immune system of prokaryotes, gene knockout in eukaryotic cells is performed by creating a cell line expressing the Cas9 enzyme and then transfecting gRNA, or simultaneously inserting Cas9 and gRNA into the cell. This may be accomplished by transfection, but is not limited thereto.

In the present invention, the gRNA refers to RNA that guides the Cas9 protein to the target sequence. In the present invention, the target sequence refers to a region where gRNA binds complementary, and the complementary binding may be complete complementarity or may be sufficient to cause hybridization so that the CRISPR-Cas9 system can operate, but is not limited thereto. The gRNA may include a domain that binds complementary to a target sequence represented by one or more base sequences selected from the group consisting of SEQ ID NOs: 10 to 13, but is not limited thereto.

In the present invention, the gRNA includes three regions. Specifically, the first region of the 5' end complementary to the target site within the chromosomal sequence; a second region forming a stem-loop structure; and a third region at the 3' end that remains essentially single stranded. The first region is for the gRNA to guide the Cas9 protein to the target sequence and is different for each gRNA, and the second and third regions may be the same for all gRNAs, but are not limited thereto.

In the present invention, the first region of the gRNA is complementary to the target sequence within the chromosomal sequence so that the first region of the gRNA can base pair with the target sequence. The first region may include, but is not limited to, about 10 to 25, or more than 25 nucleotides.

In the present invention, the second region of gRNA includes secondary structures such as hairpins and loops. The loop may vary from about 3 to about 10 nucleotides in length, and the stem may vary from about 6 to about 20 nucleotides in length, but is not limited thereto. Additionally, the overall length of the second region may vary from about 16 to about 60 nucleotides, but is not limited thereto.

In the present invention, the third region of the gRNA has no complementarity to any chromosomal sequence in the cell and has no complementarity to the remaining portion of the gRNA. The length of the third region may vary, but is not limited to, ranging from about 5 to about 60 nucleotides in length.

The present invention provides a recombinant vector comprising a nucleic acid encoding a mutant Cas9 protein of the present invention. The proteins expressed by the recombinant vector are 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′-AAG-3′, 5′ -TAG-3′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC-3′, 5′-GTA Any PAM sequence selected from the group consisting of -3′, 5′-TAT-3′, and 5′-TAA-3′ can be recognized, but is not limited thereto. In addition, the recombinant vector may further include a nucleic acid encoding MBP (maltose binding protein) or a nucleic acid encoding a His-tag, but is not limited thereto. Additionally, the recombinant vector includes a nucleic acid encoding a His-tag; Nucleic acid encoding MBP; And the nucleic acid encoding the mutant Cas9 protein of claim 1 may be arranged sequentially, but is not limited thereto. Additionally, the recombinant vector may include a nucleic acid encoding a protein represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4 to 8, but is not limited thereto.

In the present invention, a “recombinant vector” is a genetic construct containing essential regulatory elements operably linked to express the gene insert, and may be a plasmid vector, cosmid vector, bacteriophage vector, or viral vector. , but is not limited to this.

In the present invention, the recombinant vector contains a nucleic acid encoding a mutant Cas9 protein. The nucleic acid encoding the mutant Cas9 protein is preferably present in a suitable expression construct. In the expression construct, the nucleic acid encoding the mutant Cas9 protein is preferably operatively linked to the promoter.

In the present invention, “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, thereby Regulatory sequences regulate the transcription and/or translation of these other nucleic acid sequences.

In the present invention, the promoter bound to the nucleic acid encoding the mutant Cas9 protein can be any promoter capable of initiating the expression of the nucleic acid encoding the mutant Cas9 protein, specifically in animal cells, and more specifically in mammalian cells. It is advisable to select one of the RNA polymerase III (pol III) promoters that transcribe the transcript, which can operate to regulate the transcription of the nucleic acid encoding the mutant Cas9 protein. The promoter includes a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell, for example, U6 promoter, T7 promoter, H1 promoter, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia nia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, human IL-4 Promoters of genes, promoters of human lymphotoxin genes, promoters of human GM-CSF genes, inducible promoters, cancer cell specific promoters (e.g. TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and AFP promoter), or tissue-specific promoters (e.g., albumin promoter). The vector can be manufactured using genetic recombination technology well known in the art, and enzymes generally known in the art can be used for site-specific DNA cutting and ligation.

The present invention also provides a protein complex comprising a mutant Cas9 protein of the present invention and a gRNA. The gRNA may include a domain that binds complementary to a target sequence represented by one or more base sequences selected from the group consisting of SEQ ID NOs: 10 to 13, but is not limited thereto.

Additionally, the present invention provides a composition for gene correction, comprising the mutant Cas9 protein of the present invention as an active ingredient. Additionally, a kit containing the composition for gene editing may be provided. The composition for gene correction can be used in vitro, but is not limited thereto. The kit may include instructions for deleting amino acids at positions 1226 to 1323 from the N terminus in the amino acid sequence shown in SEQ ID NO: 2, but is not limited thereto.

The composition for gene editing according to the present invention can be applied for a diagnostic system. For example, it can be useful in a diagnostic system by detecting subtle nucleotide sequence mutations caused by viruses, bacterial infections, cancer, or other diseases. Additionally, it can be used to develop diagnostic reagents using gene sequence-specific restriction enzyme activity.

In the present invention, “gene editing” refers to nucleic acid molecules (one or more, e.g., 1-100,000bp, 1-10,000bp, 1-1000, 1-100bp, It may mean loss, alteration, and/or restoration (correction) of gene function by deletion, insertion, or substitution of (1-70bp, 1-50bp, 1-30bp, 1-20bp, or 1-10bp), It is not limited to this. Gene The Cas9 protein or the gene editing composition may specifically cleave DNA independent of the PAM sequence, but is not limited thereto. The term “cleavage” refers to a breakage of the covalent backbone of a nucleotide molecule.

Additionally, the present invention provides a method for producing a mutant Cas9 protein, comprising the step of deleting the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2. The production method may further include substituting one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence shown in SEQ ID NO: 2 of the mutant Cas9 protein with another amino acid, It is not limited to this. In addition, the manufacturing method may further include the step of inserting a DNA binding domain derived from the NHP6A protein, but is not limited thereto.

In the present invention, the method for producing a mutant Cas9 protein may use the pMJ806 vector as a basic backbone, but is not limited thereto. The method for producing the Cas9 protein includes (a) substituting one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence shown in SEQ ID NO: 2 with another amino acid;

(b) deleting the amino acid at positions 1226 to 1323 from the N terminus in the amino acid sequence represented by SEQ ID NO: 2; and

(c) may include the step of inserting a DNA binding domain derived from the NHP6A protein, but is not limited thereto. A recombinant vector can be produced using the above manufacturing method, but is not limited thereto.

In the present invention, the deletion may be performed using gene synthesis, and the substitution may be performed using gene synthesis or site-directed mutagenesis, but is not limited thereto.

In the present invention, the method for producing the mutant Cas9 protein is

(d) mixing the recombinant vector and competent cells; and

(e) may further include the step of heat shocking the mixture, but is not limited thereto. The competent cell may be used for transformation of an E. coli host, but is not limited thereto. Additionally, the competent cells can be produced using methods well known in the art, preferably using the CaCl ₂ method, but are not limited thereto. A transformant into which pM-Cas9-PAMless1 has been introduced can be produced using the above production method, but is not limited thereto.

In the present invention, the method for producing the mutant Cas9 protein is

(f) culturing the transformant and expressing the protein; and

(g) may further include the step of purifying the expressed protein, but is not limited thereto.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Construction of recombinant expression vector

1-1. Construction of pM-Cas9-PAMless1

To construct the Cas9-PAMless1 expression vector, the pMJ806 vector (Addgene, cat#39312) containing the SpCas9 (Cas9 derived from Streptococcus pyogenes) gene was used as a basic backbone. The base sequence and amino acid sequence of the SpCas9 gene are shown in Table 1 below. As can be seen in the schematic diagram of the Cas9-PAMless1 expression vector shown in Figure 1a, the SpCas9 gene contained in the vector is a maltose binding protein (MBP) domain and a fusion protein for the purpose of increasing the solubility and stability of the expressed protein. expressed in the form In addition, the expressed fusion protein has a His-tag at the N-terminus so that it can be purified through affinity chromatography using the Ni-chelating principle. Figures 1b and 1c describe the gene base sequence and amino acid sequence of the Cas9-PAMless1 protein, respectively.

division order SpCas9 DNA
(SEQ ID NO: 1) ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAA ATGAGATGGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCT GATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGG ATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCC TTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTC TTTCAGGTAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGA TGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATAATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTCATGA ACATATTGCAAATTTAGCTGGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATC TCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCT GAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACT GCTTTGATTAAGAAATATCCAAAACTTGCAAATGGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAAAA GTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAAAAAAA ATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGGCTCTGCCAAGCAAATATGGGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTA GATGAGATTATTGAGCAAATCAGGGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACG CATTGATTTGAGTCAGCTAGGAGGTGACTAA SpCas9 protein
(SEQ ID NO: 2) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Cas9-PAMless1 DNA
(Deletion+8 point mutations+
insertion)
(SEQ ID NO: 3) atggataagaaatactcaataggcttagatatcggcacaaatagcgtcggatgggcggtgatcactgatgaatataaggttccgtctaaaaagttcaaggttctgggaaatacagaccgccacagtatcaaaaaaaatcttataggggctctttttttgacagtggagagacagcggaagcgactcgtctcaaacggacagctcgtagaaggtatac acgtcggaagaatcgtatttgttatctacaggagattttttcaaatgagatggcgaaagtagatgatagtttctttcatcgacttgaagagtcttttttggtggaagaagacaagaagcatgaacgtcatcctatttttggaaatatagtagatgaagttgcttatcatgagaaatatccaactatctatcatctgcgaaaaaaattggtagattctctgata aagcggatttgcgcttaatctatttggccttagcgcatatgattaagtttcgtggtcattttttgattgagggagatttaaatcctgataatagtgatgtggacaaactatttatccagttggtacaaacctacaatcaattatttgaagaaaaccctattaacgcaagtggagtagatgctaaagcgattctttctgcacgattgagtaaatcaagacg attagaaaatctcattgctcagctccccggtgagaagaaaaatggcttatttgggaatctcattgctttgtcattgggtttgacccctaattttaaatcaaattttgatttggcagaagatgctaaattacagctttcaaaagatacttacgatgatgatttagataatttattggcgcaaattggagatcaatatgctgatttgtttttggcagctaagaattta tcagatgctattttactttcagatatcctaagagtaaatactgaaataactaaggctcccctatcagcttcaatgattaaacgctacgatgaacatcatcaagacttgactcttttaaaagctttagttcgacaacaacttccagaaaagtataaagaaatctttttttgatcaatcaaaaaacggatatgcaggttatattgatgggggagctagccaagaagaattttataa atttatcaaaccaattttagaaaaaatggatggtactgaggaattattggtgaaactaaatcgtgaagatttgctgcgcaagcaacggacctttgacaacggctctattccccatcaaattcacttgggtgagctgcatgctattttgagaagacaagaagacttttatccatttttaaaagacaatcgtgagaagattgaaaaaatcttgacttttcgaattccttattatgtt ggtccattggcgcgtggcaatagtcgttttgcatggatgactcggaagtctgaagaaacaattaccccatggaattttgaagaagttgtcgataaaggtgcttcagctcaatcatttattgaacgcatgacaaactttgataaaaatcttccaaatgaaaaagtactaccaaaacatagttttgctttatgagtattttacggtttataacgaattgacaaa ggtcaaatatgttactgaaggaatgcgaaaaccagcatttcttttcaggtgaacagaagaaagccattgttgatttactcttcaaaacaaatcgaaaagtaaccgttaagcaattaaaagaagattatttcaaaaaaatagaatgttttgatagtgttgaaatttcaggagttgaagatagatttaatgcttcattaggtacctaccatgatttgctaaaaattattaaagataa agattttttggataatgaagaaaatgaagatatcttagaggatattgttttaacattgaccttatttgaagatagggagatgattgaggaaagacttaaaacatatgctcacctctttgatgataaggtgatgaaacagcttaaacgtcgccgttatactggttggggacgtttgtctcgaaaattgattaatggtattagggataagcaatctggcaaaacaatattag attttttgaaatcagatggttttgccaatcgcaattttatgcagctgatccatgatgatagtttgacatttaaagaagacattcaaaaagcacaagtgtctggacaaggcgatagtttacatgaacatattgcaaatttagctggtagccctgctattaaaaaaggtattttacagactgtaaaagttgttgatgaattggtcaaagtaatggggcggcataagccaga aaatatcgttattgaaatggcacgtgaaaatcagacaactcaaaagggccagaaaaattcgcgagagcgtatgaaacgaatcgaagaaggtatcaaagaattaggaagtcagattcttaaagagcatcctgttgaaaatactcaattgcaaaatgaaaagctctatctctattatctccaaaatggaagagacatgtatgtggaccaagaattagatattaatcgtttaagt gattatgatgtcgatcacattgttccacaaagtttccttaaagacgattcaatagacaataaggtcttaacgcgttctgataaaaatcgtggtaaatcggataacgttccaagtgaagaagtagtcaaaaagatgaaaaactattggagacaacttctaaacgccaagttaatcactcaacgtaagtttgataatttaacgaaagctgaacgtggagg tttgagtgaacttgataaagctggttttatcaaacgccaattggttgaaactcgccaaatcactaagcatgtggcacaaattttggatagtcgcatgaatactaaatacgatgaaaatgataaacttattcgagaggttaaagtgattaccttaaaatctaaattagtttctgacttccgaaaagatttccaattctataaagtacgtgagattaacaattaccat catgcccatgatgcgtatctaaatgccgtcgttggaactgctttgattaagaaatatccaaaacttgaatcggagtttgtctatggtgattataaagtttatgatgttcgtaaaatgattgctaagtctgagcaagaaataggcaaagcaaccgcaaaatatttcttttactctaatatcatgaacttcttcaaaacagaaattacacttgcaa atggagagattcgcaaacgccctctaatcgaaactaatggggaaactggagaaattgtctgggataaagggcgagattttgccacagtgcgcaaagtattgtccatgccccaagtcaatattgtcaagaaaacagaagtacagacaggcggattctccaaggagaagattttaccaaaaagaaattcggacaagcttattgctcgtaaaaaagactgggatccaaaa aaatatggtggttttcgtagtccaacggtagcttattcagtcctagtggttgctaaggtggaaaaagggaaatcgaagaagttaaaatccgttaaagagttactagggatcacaattatggaaagaagttcctttgaaaaaaatccgattgactttttagaagctaaaggatataaggaagttaaaaaagacttaatcatttaaactacctaaatatagtctttttgagttaga aaacggtcgtaaacggatgctggctagtgccggacgcttacaaaaaggaaatgagtcgacagacccaaatgcccctaagagggctttgtccgcctacatgtttttcgctaacgaaaacagagatattgttcgttctgaaaatccagatatcacatttggacaagtcggcaagaagttgggtgagaagtggaaggctctaacgccagagg aaaagcagccttacgaggccaaggcccaggccgataagaagagatatgaatccgaaaaggagttatataacgccactttggctggtgcggccgcaggttcgacatttaaatattttgatacaacaattaaagctaaagcatataagaacacaaaagaagtactagatgccactcttatccatcaatccatcactggtctttatgaaacacgcattgatttgagtcagctaggagg tgactaa

First, new mutations were introduced into eight amino acid positions of the SpCas9 gene using site-directed mutagenesis and gene synthesis. The positions of the mutations and substituted amino acids are as follows: S1109K, D1135R, E1219R, D1332K, R1333A, R1335A, T1337K, and S1338N (Figure 1c).

Next, 98 amino acids from L1226 to A1323 were deleted using gene synthesis and insertion of a new DNA binding domain using specific site mutagenesis was performed on E1225. SalI and NotI restriction enzyme sites were prepared behind and in front of F1324, respectively (Figures 1b and 1c).

Next, the nonsequence-specific DNA binding domain of the yeast NHP6A protein was used to create a mutant Cas9. NHP6A is known to bind to DNA and participate in various biological processes such as DNA recombination, DNA repair, and regulation of gene expression at the transcription level (ref). The role of the inserted NHP6A DNA binding domain is to mediate non-sequence-specific interactions with DNA during the PAM sequence recognition process of Cas9-PAMless1, thereby preventing it from having a preference for a specific PAM sequence. A gene fragment containing the DNA binding domain of the NHP6A protein from D17 to A93 was prepared by gene synthesis and inserted into the SalI and NotI restriction enzyme sites of the previously created SpCas9 mutant gene. As a result, a Ser-Thr linker is located at the N-terminus of the inserted NHP6A protein, and a linker consisting of 7 amino acids (Gly-Ala-Ala-Ala-Gly-Ser-Thr) is located at the C-terminus (Figure 1c) .

Additionally, the amino acid sequences of the mutant Cas9 protein and NHP6A protein used in the experiment are shown in Tables 2 and 3 below.

Mutant Cas9 protein
(SEQ ID NO) amino acid sequence Deletion of 98 amino acids
(SEQ ID NO: 4) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD Deletion + 5 point mutations
(SEQ ID NO: 5) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFRSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGRLQKGNEFKYFDTTIKRKRYKNTKEVLDATLIHQSITGLYE TRIDLSQLGGD Deletion + 8 point mutations
(SEQ ID NO: 6) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKEKILPKRNSDKLIARKKDWDPKKYGGFRSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGRLQKGNEFKYFDTTIKAKAYKNTKEVLDATLIHQSITGLY ETRIDLSQLGGD Deletion+5 point mutations+
insertion
(SEQ ID NO: 7) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFRSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGRLQKGNESTDPNAPKRALSAYMFFANENRDIVRSENPDITFGQVG KKLGEKWKALTPEEKQPYEAKAQADKKRYESEKELYNATLAGAAAGSTFKYFDTTIKRKRYKNTKEVLDATLIHQSITGLYETRIDLSQLGGD Deletion + 8 point mutations +
insertion
(SEQ ID NO: 8) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFD DKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKEKILPKRNSDKLIARKKDWDPKKYGGFRSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGRLQKGNESTDPNAPKRALSAYMFFANENRDIVRSENPDITFGQV GKKLGEKWKALTPEEKQPYEAKAQADKKRYESEKELYNATLAGAAAGSTFKYFDTTIKAKAYKNTKEVLDATLIHQSITGLYETRIDLSQLGGD

division
(SEQ ID NO) amino acid sequence NHP6A protein
(SEQ ID NO: 9) DPNAPKRALSAYMFFANENRDIVRSENPDITFGQVGKKLGEKWKALTPEEKQPYEAKAQADKKRYESEKELYNATLA

1-2. Construction of target plasmid

As shown in Figure 2, the DNA substrate used to confirm the DNA cleavage activity of Cas9-PAMless1 for a specific base sequence is a chemically synthesized DNA duplex fragment in the EcoRI/SalI restriction enzyme site of the pUC19X plasmid. It was produced by inserting it. Target plasmids (pTarget plasmids) created in this way were designed to have an insertion fragment consisting of a specific target sequence and a specific PAM sequence. For example, the target sequence of pTarget-T1-GGG is 5′-TAA AAG GGC GAT CTA ATG AA-3′, and the PAM sequence is 5′-GGG-3′.

For reference, the pUC19X plasmid used in this example contains nucleotides 1882 to 1887 of pUC19 so that its DNA cleavage activity can be easily confirmed on an agarose gel. This is a plasmid in which a new XhoI restriction enzyme site was introduced using site-specific mutagenesis.

Example 2. Preparation of transformant into which pM-Cas9-PAMless1 was introduced

The host used for transformation of pM-Cas9-PAMless1 is E. coli Rosetta2 (DE3) strain, which has a T7 RNA polymerase gene in its genome that can express proteins using the T7 promoter.

Competent cells required for transformation of E. coli hosts can be obtained using the CaCl ₂ method well known in the art (Sambrook J, Fritsch E, Maniatis T. Molecular cloning: a laboratory manual 1989. 2. New York: Cold Spring Harbor Laboratory; 1989.) It was produced using .

Mix 50 μL of the solution in which the produced competent cells are dispersed and 1 μL of the pM-Cas9-PAMless1 expression vector and leave at 0°C for about 40 minutes. Then, heat shock was applied at 42°C for 90 seconds and then again at 0°C for 2 minutes. It was left unattended. About 1 mL of sterilized liquid LB (Luria Bertani) medium was added here, and then cultured with shaking at 37°C and 220 rpm for about 45 minutes. Thereafter, the culture was spread on LB solid medium to which kanamycin (Kan) was added at a final concentration of 30 μg/mL, and then cultured overnight at 37°C to obtain a single colony.

For reference, the LB medium used in this example consists of Tryptone 12 g/L, Yeast extract 5 g/L, and sodium chloride 10 g/L.

Example 3. Protein expression and activity confirmation experiment using pM-Cas9-PAMless1/Rosetta2 (DE3)

3-1. pM-Cas9-PAMless1 protein expression by transformants

pM-Cas9-PAMless1/Rosetta2 (DE3) (transformant into which pM-Cas9-PAMless1 was introduced) prepared in Example 2 was cultured in TB (Terrific-Broth) medium. TB medium consists of 20 g/L of Tryptone, 24 g/L of yeast extract, 4 mL/L of 100% glycerol, and 100 mL/L of phosphate buffer (0.17 M KH ₂ PO ₄ , 0.72 MK ₂ HPO ₄ ).

First, to prepare an overnight culture for mass culture, E. coli obtained from a single colony was inoculated into 5 mL of TB/Kan medium (final concentration of Kan = 30 μg/mL) and then incubated at 37°C at a speed of 200 rpm on a shaker. Grow it for a day.

For mass culture, add 3 mL of the overnight culture inoculated the previous day to 400 mL of new TB/Kan medium in a 2 L flask, grow at 37°C on a shaker at a speed of 200 rpm, and measure the recombinant transformant at 600 nm. When the absorbance reached 0.8, the temperature was lowered to 16°C. And to induce transcription from the promoter of the T7 polymerase gene, IPTG was added to the medium to a final concentration of 0.2 mM.

3-2. Purification of pM-Cas9-PAMless1 protein

The purification process for Cas9-PAMless1 protein expressed from the transformant is as follows:

(1) E. coli cells expressing protein are collected by centrifugation at 4°C for 10 minutes.

(2) Cells were redispersed using 80 mL of lysis buffer (50mM Tris-Cl, pH7.5, 300mM NaCl, 10% (v/v) glycerol, 0.1% tween 20, and 2mM β-Mercaptoethanol). Then, 50 units of benzonase and PMSF (phenylmethylsulfonyl fluoride) at a final concentration of 100 μM are added.

(3) The redispersed E. coli cells are sonicated for 2 hours at a low temperature of 4°C.

(4) Insoluble portions of cells and cell lysates are precipitated by centrifugation at high speed for 30 minutes at 4°C.

(5) Filter the centrifuged supernatant and mix with 5 mL of Ni-NTA resin previously equilibrated with lysis buffer at 4°C for 1 hour.

(6) Put the solution mixed with the resin into a purification column, wait until the Ni-NTA resin settles, and then pass it through at a speed of 2 mL/min.

(7) Wash the column with 50 mL of Washing buffer 1 (50mM Tri-Cl, pH7.5, 300mM NaCl, 10mM Imidazole, 2mM β-Mercaptoethanol).

(8) Wash the column with 50 mL of Washing buffer 2 (50mM Tri-Cl, pH7.5, 300mM NaCl, 20mM Imidazole, 2mM β-Mercaptoethanol).

(9) Elute the protein with 15 mL of Elution buffer (50mM Tri-Cl, pH7.5, 300mM NaCl, 300mM Imidazole, 2mM β-Mercaptoethanol).

(10) Concentrate the eluted protein at 4°C using a centrifugal filter (Amicon 50,000 MWCO tube). Make the final volume less than 1 mL.

(11) Equilibrate the size exclusion (HiLoad 16/600 Superdex 200 prep grade) column in advance with size exclusion buffer (40mM HEPES, pH7.5, 600mM NaCl, 1mM TCEP), then inject and separate the concentrated protein. .

(12) Proteins purified from size exclusion chromatography are concentrated at 4°C using a centrifugal filter. Purified proteins are stored at -20℃ (short-term storage) or -80℃ (long-term storage) with 50% (v/v) glycerol at a final concentration of 100 μM or more.

Figure 3 shows the purification process of Cas9-PAMless1 protein and the purity of the protein purified through this process.

3-3. Confirmation of DNA cutting activity of Cas9-PAMless1

To confirm the DNA cleavage activity of the purified protein, a complex of Cas9-PAMless1 and a chemically synthesized single guide RNA (SgRNA) (purchased from IDT DNA) was prepared, and then a target plasmid containing the base sequence targeted by the SgRNA was prepared. A DNA cleavage reaction was performed using DNA as a substrate. The reaction conditions are as follows.

(1) Conditions for formation of Cas9-PAMless1/SgRNA complex: Cas9-PAMless1 protein and SgRNA were mixed in CutSmart™ buffer (purchased from New England Biolab) and left at 25°C for 10 minutes to prepare Cas9-PAMless1/SgRNA complex. do.

(2) Mix the Cas9-PAMless1/SgRNA complex corresponding to the required concentration with 300 ng of pTarget plasmid in a reaction volume of 15 μL and react at 37°C for 16 hours in CutSmart™ buffer conditions.

(3) To easily check the activity of the DNA cleavage reaction, add 10 units of XhoI restriction enzyme to the reaction mixture and react at 37°C for 2 hours.

(4) To remove protein components, add 0.4 unit of Proteinase K to the reaction material and react at 25°C for 2 hours.

(5) Check the DNA cleavage reaction that occurred in the reactant on an agarose gel.

As a result, bands of DNA cut by Cas9-PAMless1 were detected in all ranges regardless of the concentration range, confirming that Cas9-PAMless1 has excellent DNA cutting activity (Figure 4).

Example 4. Confirmation of PAM-independent (PAM-less) activity of Cas9-PAMless1

4-1. Confirmation of PAM-less activity of Cas9-PAMless1

It is known that the PAM sequence preferred by WT SpCas9 is 5′-NGG-3′. In comparison, to confirm the PAM sequence-independence (PAM-less activity) of Cas9-PAMless1, the DNA cleavage activity of Cas9-PAMless1 was confirmed for 14 different PAM sequences. The PAM sequences used for this purpose were as follows: 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′-AAG-3′; 5′-TAG-3′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC-3′, 5′ -GTA-3′, 5′-TAT-3′, and 5′-TAA-3′. The target sequences of each DNA substrate were all identical: T1, 5′-TAA AAG GGC GAT CTA ATG AA-3′ (Table 4).

As a result of confirming the cleavage activity using 200 nM Cas9-PAMless1/SgRNA complex under the same experimental conditions as in Example 3-3, WT cleaved the target by preferring a DNA substrate with 5′-NGG-3′ as the PAM sequence. Unlike -SpCas9, Cas9-PAMless1 was confirmed to exhibit the same level of DNA cleavage activity for all DNA substrates with different PAM sequences (Figure 5a). From the above results, it was confirmed that Cas9-PAMless1 of the present invention can exert cleavage activity on the target gene independently of the PAM sequence.

target name Target sequence (5’→3’) sequence number Remarks (position) T1 TAAAAGGGCGATCTAATGAA 10 - X-TAC GCTTATCGGAAGTGAACGAA TAC ^# 11 24458 nt to 24480 nt of lambda DNA X-TGG CGCCTCGAGTGAAGCGTTAT TGG ^# 12 33495 nt to 33517 nt of lambda DNA N-ATC GCAATTAATGTGCATCGATT ATC ^# 13 34685 nt to 34707 nt of lambda DNA ^# PAM sequence

4-2. Confirm the effect of each mutation on the PAM-less activity of Cas9-PAMless1

Amino acid S1109 of WT-SpCas9 is reported to participate in the formation of a phosphate-lock loop, which is known to be important in the unwinding process of the DNA substrate for recognition of the DNA target sequence after recognition of the PAM sequence (Anders C, Niewoehner O, Duerst A, Jinek M. 2014. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513(7519):569-73). In addition, R1333 and R1335 are known to be amino acids that directly interact with the guanine bases of the PAM sequence (Anders C, Niewoehner O, Duerst A, Jinek M. 2014. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513(7519):569-73).

Therefore, in order to confirm the relationship between the mutation occurring at the above position and the PAM-independent DNA cleavage activity of Cas9-PAMless1, revertant mutants for the mutation at each position were created. Specifically, these are Cas9-PAMless-r1333/r1335 (R1333, revertant of R1335); and Cas9-PAMless-s1109/r1333/r1335 (revertants of S1109, R1333, and R1335) were expressed and purified in the same manner as Cas9-PAMless1, and DNA cleavage activity was confirmed under the same conditions as in Example 4-1.

As a result, as shown in Figures 5b and 5c, the revert mutants were found to have a similar level of PAM sequence-independent DNA cleavage activity as Cas9-PAMless1. In other words, the above results suggest that amino acid substitution at positions S1109, R1333, or R1335 does not affect the PAM-independent DNA cleavage activity of Cas9-PAMless1.

Example 5. Confirmation of PAM-less activity of Cas9-PAMless1 using random PAM library

5-1. Construction of DNA substrate with random PAM library

A DNA substrate was prepared by randomly combining the base sequences at the 3′ end of the target sequence where the PAM sequence is located. Specifically, a DNA oligo in which nucleotides can be randomly inserted into the 5 nucleotide positions at the 3′ end of the target sequence was chemically synthesized. A DNA duplex was made using this as a substrate, and the plasmid library, pTarget-T1-N5, was created by inserting it into the EcoRI/SalI restriction enzyme site of the pUC19 plasmid (Figure 6).

5-2. Preparation of samples for analysis of DNA cleavage activity of Cas9-PAMless1 on random PAM library substrates

Under the same conditions as in Example 3-3, 500 ng of pTarget-T1-N5 was used as a DNA substrate, and the DNA cleavage activity of 200 nM Cas9-PAMless1/SgRNA complex was confirmed. Since the type of plasmid DNA used as the DNA substrate was different, ScaI restriction enzyme was used instead of XhoI restriction enzyme. To confirm the PAM sequence of the cut DNA, an experiment was performed in which linker DNA was linked to the end of the cut DNA using the Quick blunting™ kit (New England Biolab) as follows:

(1) DNA cleaved by Cas9-PAMless1 is separated and extracted from an agarose gel using a gel-isolation kit.

(2) The extracted DNA is mixed with linker DNA, dNTP, and Blunt enzyme mix under 1' Blunting buffer conditions in a reaction volume of 50 μL, and then reacted at 25°C for 1 hour.

(3) After the reaction is completed, the reaction sample is left at 70°C for 1 hour to deactivate the enzyme.

(4) Add 9 μL of 40 μM duplex linker DNA, 10 μL of 10 ´ T4 ligase buffer, 4 μL of T4 DNA ligase, and 27 μL of dH ₂ O to the reaction sample and react at 25°C for 16 hours.

(5) After electrophoresis of the reaction sample on an agarose gel, the DNA is separated and extracted using a gel-isolation kit.

5-3. Confirmation of PAM-less activity of Cas9-PAMless1 using dideoxy sequencing

A PCR reaction was performed using the DNA sample prepared in Example 5-2 as a template. One of the PCR primers used at this time binds complementary to the substrate DNA, and the other binds to the newly connected linker DNA portion.

To confirm the sequence of the DNA product amplified through PCR reaction, a sequencing primer that can read the base sequence from the 3′ end of the target sequence was used, and through this, the pTarget-T1-N5 DNA substrate cleaved by Cas9-PAMless1 The base sequence of this PAM position was confirmed.

When confirming the base sequence of the PAM position through dideoxy sequencing, it is possible to qualitatively confirm the composition of each base position by analyzing the mixture of cut DNA.

Dideoxy sequencing results are shown in Figure 7. It was found that there was no preference for a specific base at the PAM position of the DNA substrate cleaved by Cas9-PAMless1. In other words, this demonstrates the PAM-less activity of the PAM-independent Cas9 gene scissors according to the present invention.

5-4. Confirmation of PAM-less activity of Cas9-PAMless1 using next generation sequencing

A PCR reaction was performed using the DNA sample prepared in Example 5-3 as a template. One of the PCR primers used at this time binds complementary to the substrate DNA, and the other binds to the newly connected linker DNA portion.

To confirm the sequence of the DNA product amplified through PCR reaction, a sequencing primer that can read the base sequence from the 3′ end of the target sequence was used, and through this, the pTarget-T1-N5 DNA substrate cleaved by Cas9-PAMless1 The base sequence of this PAM position was confirmed.

When confirming the base sequence of the PAM position using next-generation sequencing, it is possible to accurately confirm the base sequence of each DNA molecule in the mixture of cut DNA.

As a result of analyzing the preference for 64 PAM sequence candidates (64 combinations of 5′-NNN-3′) that DNA substrates cleaved by Cas9-PAMless1 can have, WT SpCas9 showed 5′-NGG-3 as expected. While it showed a high preference for the PAM sequence, Cas9-PAMless1, a mutant gene scissors according to the present invention, was confirmed to have no preference for a specific sequence among 64 combinations of sequences (FIG. 8).

Specifically, when the preference of Cas9-PAMless1 for the PAM sequence was checked, it was found to be 0.9316 to 2.1420, with the lowest and highest preferences being only 2.3 times higher. On the other hand, in the case of the wild type, the preference for the PAM sequence was found to be 0.1123 to 6.3579, and the difference between the two boundary values was about 57 times, indicating that the recognition (binding) activity for a specific PAM sequence was excessive. In other words, the difference between the respective threshold values of Cas9-PAMless1 and the wild type is about 25 times, and Cas9-PAMless1 of the present invention has a significantly low PAM recognition activity, making it a gene scissors with excellent cutting activity for target genes regardless of the PAM sequence. It was reaffirmed that it can be used.

5-5. Analysis of the catalytic rate of PAM-less activity of Cas9-PAMless1 using next-generation sequencing.

A DNA cleavage experiment was performed under the same reaction conditions as in Example 3-3 using 100 nM of Cas9-PAMless1/SgRNA complex and 200 ng of pTarget-T1-N5 as DNA substrates. The reaction times used at this time were 20 minutes, 50 minutes, 100 minutes, 200 minutes, 500 minutes, and 1540 minutes, respectively, to compare catalyst cleavage rates. Each reaction was terminated by adding a final concentration of 15mM EDTA.

The PAM sequences of pTarget-T1-N5 DNA that did not undergo cleavage were confirmed using primers that bind upstream and downstream of the target sequence. To confirm the base sequence of the PAM site of the pTarget-T1-N5 DNA substrate that was not cleaved by Cas9-PAMless1, next-generation sequencing was performed on the DNA product amplified through PCR reaction.

The normalized ratios for each of the 64 possible PAM sequence combinations at the 5'-NNN-3' position located at the 3' end of the target sequence are shown in Figures 9a and 9b. At this time, WT SpCas9 was used as a control. As a result, it was confirmed that the change in the normalization ratio of Cas9-PAMless1 occurred in a narrow range over time compared to WT SpCas9. This is in contrast to the rapid decrease in the ratio of the preferred PAM sequence (5'-NGG-3') in WT SpCas9, reaffirming that Cas9-PAMless1 does not prefer a specific sequence and does not recognize it as a PAM.

Example 6. Confirmation of PAM-less activity of Cas9-PAMless1 on Lambda DNA

In order to confirm whether Cas9-PAMless1 can accurately recognize and cut the target sequence regardless of the PAM sequence in a DNA substrate much larger than a plasmid DNA substrate less than 3 kbp in length, lambda DNA characterized by a long length was used. The cleavage activity was analyzed. Specifically, Lambda DNA, a 48.5 kbp linear DNA, was prepared as a DNA substrate with a much longer length than the 2.7 kbp plasmid DNA substrate used in other examples, and 200 nM of 200 nM was prepared under the same reaction conditions as in Example 3-3. DNA cleavage experiments were performed with Cas9-PAMless1/SgRNA complex and 400 ng of Lambda DNA substrate in a reaction volume of 30 μL.

Cleavage activity was confirmed for target sequences with various PAM sequences in Lambda DNA. The different target sequences at the three selected positions have different PAM sequences: TAC, TGG, and ATC. To make it easy to confirm the location of the cut DNA fragment, each target sequence overlaps with or is close to the cutting site of the XbaI, XhoI, and NheI restriction enzymes. For comparison, the cleavage reaction of WT SpCas9, which has 5'-NGG-3' as the PAM recognition sequence, was performed under the same conditions.

As a result, as shown in Figure 10a, Cas9-PAMless1 showed similar DNA cleavage activity for all three target sequences having TAC, TGG, and ATC as PAM sequences, as expected. On the other hand, it was confirmed that WT SpCas9 showed DNA cleavage activity only at positions with TGG as the PAM sequence. Additionally, according to Figure 10b, when using Cas9-PAMless1 and two or more types of SgRNA, it was shown that the Cas9-PAMless1/SgRNA complex was able to cleave target sequences at two or more positions. According to these results, it was confirmed that the same effect as performing double digestion using restriction enzymes can be obtained when using the Cas9-PAMless1/SgRNA complex.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims (20)

It has PAM (protospacer adjacent motif) sequence-independent activity;
A mutant Cas9 protein, characterized in that the amino acid at positions 1226 to 1323 amino acids from the N terminus in the amino acid sequence represented by SEQ ID NO: 2 is deleted.
According to paragraph 1,
The mutant Cas9 protein is characterized in that one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence shown in SEQ ID NO: 2 are substituted with another amino acid.
According to paragraph 2,
The mutant Cas9 protein is characterized in that one or more amino acids selected from the group consisting of S1109, R1333, and R1335 in the amino acid sequence shown in SEQ ID NO: 2 are further substituted with another amino acid.
According to paragraph 2 or 3,
A mutant Cas9 protein, wherein the other amino acid is an amino acid selected from the group consisting of lysine (K), arginine (R), alanine (A), and asparagine (N).
According to paragraph 1,
The mutation is a mutant Cas9 protein, characterized in that it includes the insertion of a DNA binding domain derived from the NHP6A protein.
According to clause 5,
The DNA binding domain is a mutant Cas9 protein, characterized in that it contains amino acids D17 to A93 in the amino acid sequence represented by SEQ ID NO: 9.
According to paragraph 1,
The mutant Cas9 protein is characterized in that the mutant Cas9 protein further includes MBP (maltose binding protein) or a His-tag.
According to paragraph 1,
The mutant Cas9 protein is characterized in that it is represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 8.
According to paragraph 1,
The PAM sequences are 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′-AAG-3′, 5′-TAG-3 ′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC-3′, 5′-GTA-3′, A mutant Cas9 protein, characterized in that it is any one sequence selected from the group consisting of 5′-TAT-3′ and 5′-TAA-3′.
A recombinant vector comprising a nucleic acid encoding the mutant Cas9 protein of claim 1.
According to clause 10,
The proteins expressed by the recombinant vector are 5′-AGG-3′, 5′-TGG-3′, 5′-CGG-3′, 5′-GGG-3′, 5′-AAG-3′, 5′ -TAG-3′, 5′-CAG-3′, 5′-GAG-3′, 5′-TGA-3′, 5′-AAC-3′, 5′-CAC-3′, 5′-GTA A recombinant vector characterized in that it recognizes any one PAM sequence selected from the group consisting of -3′, 5′-TAT-3′, and 5′-TAA-3′.
According to clause 10,
The recombinant vector further comprises a nucleic acid encoding MBP (maltose binding protein), or a nucleic acid encoding a His-tag.
According to clause 12,
The recombinant vector includes a nucleic acid encoding a His-tag; Nucleic acid encoding MBP; And a recombinant vector, characterized in that the nucleic acid encoding the mutant Cas9 protein of claim 1 is sequentially arranged.
According to clause 10,
The recombinant vector is characterized in that it contains a nucleic acid encoding a protein represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4 to 8.
A protein complex comprising the mutant Cas9 protein of claim 1 and gRNA.
According to clause 15,
The gRNA is a protein complex characterized in that it comprises a domain that binds complementary to a target sequence represented by one or more base sequences selected from the group consisting of SEQ ID NOs: 10 to 13.
A composition for gene editing comprising the mutant Cas9 protein of claim 1 as an active ingredient.
A method for producing a mutant Cas9 protein, comprising the step of deleting amino acids at positions 1226 to 1323 from the N terminus in the amino acid sequence represented by SEQ ID NO: 2.
According to clause 18,
The production method further comprises the step of substituting one or more amino acids selected from the group consisting of D1135, E1219, D1332, T1337, and S1338 in the amino acid sequence shown in SEQ ID NO: 2 with another amino acid. Mutant Cas9 protein. Manufacturing method.
According to clause 18,
The method of producing a mutant Cas9 protein further comprises the step of inserting a DNA binding domain derived from the NHP6A protein.