KR102386498B1 - composition for gene editing based on CRISPR-CAS - Google Patents

Abstract

The present invention relates to a composition for improving cell permeability and gene editing efficiency of Cas protein and guide RNA. In the case of gene editing technology using CRISPR-Cas currently used, it is difficult to inject into cells in the form of a complex, and stability is proven even after injection It is not done, and the efficiency is low, and there is an off-target problem. However, when the composition for gene editing of the present invention is used, the intracellular delivery efficiency is remarkably high, it is possible to suppress off-target, and it is possible to secure stability, and thus it can be usefully used as a gene therapy.

Description

CRISPR-CAS-based gene editing composition {composition for gene editing based on CRISPR-CAS}

The present invention relates to a composition for improving cell permeability and gene editing efficiency of Cas protein and guide RNA, and more particularly, the composition of the present invention provides a higher gene by delivering the gene scissors in the form of a protein-based RNP complex into cells. Since the correction efficiency and off-target effect can be lowered, it can be used for clinical treatment or cell therapy.

Gene editing technologies include first-generation ZFNs; These include zinc-finger nucleases, second-generation transcription activator-like effector nucleases (TALENs), and third-generation gene scissors derived from the CRISPR/Cas system, Cas9 and Cpf1.

The CRISPR/Cas system originated from the adaptive immunity of microorganisms. It started from the immune system that remembers the fragments of the bacteriophage as DNA during bacteriophage infection and cuts them out with Cas9 (CRISPR associated protein 9: RNA-guided DNA endonuclease enzyme), a nuclease that plays a role in gene scissors when re-infected. . In this case, if a specific nucleotide sequence can be recognized by a guide RNA (gRNA) in the genome, a desired site can be easily cut and repaired. However, since the third-generation gene scissors are continuously expressed in cells, an off-targeting problem of cutting genes other than the target gene occurred.

In particular, it was necessary to verify the safety of the gene editing system using the Cas9 plasmid, which is an initial method, for antibiotic resistance and various immune responses when delivered in the body. Recently, a system for making and delivering protein-made gene scissors (Cas9) and guide RNA in vitro has been applied as an alternative. S et al., 2014).

Accordingly, the need to discover bio-applicable protein-based gene editing technology with low off-target risk is emerging. In order to develop novel gene scissors that can be stably applied to clinical and cell therapy, efficient intracellular delivery and reduced cytotoxicity are required. Because it is very limited inside and out, there is a limit to the existing gene editing technology discovery strategy.

It is an object of the present invention to provide a cell penetrating peptide for Cas protein-RNA complex (Ribonucleoprotein: RNP).

Another object of the present invention is to provide a composition for editing a gene comprising a Cas protein-RNA complex (Ribonucleoprotein: RNP).

Another object of the present invention is to provide a method for preparing a transformant other than a human through the composition.

In order to achieve the above object, the present invention provides a cell penetrating peptide for Cas protein-RNA complex (Ribonucleoprotein: RNP) represented by the following general formula (1).

[General formula 1]

Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly] _m -[His-Glu] _n

In the above formula, m is an integer of 3 to 7, and n is an integer of 5 to 15.

In Formula 1, n may be an integer of 9 to 15, and more preferably, n may be an integer of 10 to 12.

The cell-penetrating peptide for the Cas protein-RNA complex (Ribonucleoprotein: RNP) may be one represented by SEQ ID NO: 6.

In order to achieve the above object, the present invention provides a) Cas protein to which the cell-penetrating peptide represented by Formula 1 is bound; And b) guide RNA; provides a composition for gene editing comprising a complex (RNP) consisting of.

[General formula 1]

Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly] _m -[His-Glu] _n

In the above formula, m is an integer of 3 to 7, and n is an integer of 5 to 15.

In Formula 1, n may be an integer of 9 to 15, and more preferably, n may be an integer of 10 to 12.

The Cas protein may be represented by SEQ ID NO: 9.

The guide RNA may be in the form of a double RNA (dual RNA) or single-stranded guide RNA (sgRNA) including crRNA and tracrRNA.

The composition may induce a targeted mutation of a single-location or multi-location gene in a prokaryotic cell, a eukaryotic cell, or a non-human eukaryotic organism.

In order to achieve the above another object, the present invention provides 1) local injection, microinjection, electroporation and It provides a method for producing a transformant other than a human comprising; introducing any one selected from among lipofection methods.

In order to achieve the above another object, the present invention provides a transformant other than a human prepared by the above method.

The present invention relates to a composition for gene editing comprising a complex (RNP) of a Cas protein and guide RNA bound to a cell-permeable peptide. It is difficult, and even if injected, stability is not proven, efficiency is low, and there is an off-target problem. However, when the composition for gene editing of the present invention is used, the intracellular delivery efficiency is remarkably high, it is possible to suppress off-target, and it is possible to secure stability, and thus it can be usefully used as a gene therapy.

1A shows the pET28a vector into which AP-HE10-SpCas9 prepared in Example 1-6) was inserted and the pET28a vector into which CPP-SpCas9 prepared in Comparative Examples 2, 4, and 6 was inserted.
Figure 1b is a SDS-PAGE of AP-HE10-SpCas9 protein (SEQ ID NO: 15) purified through Example 6 and CPP-SpCas9 protein (SEQ ID NO: 23, 26, 31) purified through Comparative Examples 2, 4, 6 is the result of the analysis.
2A is a pET28a vector into which CPP-SpCas9 prepared in Comparative Examples 1, 3, 5, and 7 is inserted.
Figure 2b shows the results of SDS-PAGE analysis of CPP-SpCas9 proteins (SEQ ID NOs: 19, 21, 25, 29) purified through Comparative Examples 1, 3, 5, and 7;
Figure 2c shows the results of purifying the AP-SpCas9 protein prepared in Comparative Example 1 using various columns (purification technology), and analyzing each of them by SDS-PAGE.
3A is a pET28a vector into which CPP-AsCas12a prepared in Comparative Examples 8 to 11 is inserted.
Figure 3b is a result of SDS-PAGE analysis of the CPP-AsCas12a protein (SEQ ID NOs: 69, 70, 71, 72) purified through Comparative Examples 8 to 11.
Figure 3c shows the results of purifying the AP-AsCas12a protein prepared in Comparative Example 8 using various columns (purification technology), and analyzing each of them by SDS-PAGE.
4A is a pET28a vector into which CPP-LbCas12a prepared in Comparative Examples 12 to 16 is inserted.
4B is a result of SDS-PAGE analysis of CPP-LbCas12a proteins (SEQ ID NOs: 73, 74, 75, 76) prepared in Comparative Examples 11 to 15;
5 shows AP-HE-Cas9 (2 μM) (Example 6) alone or mixed with CQ (1, 10, 50, 100, 250, 500 μM) and control (2 μM), respectively. It is a graph measured by flow cytometry after treatment in wells.
Figure 6 is a photograph of intracellular fluorescence after treatment with AP-HE-Cas9 (2 μM) (Example 6) alone or mixed with CQ (500 μM) in each well.
7 is a result of measuring the gene editing efficiency by AP-HE-Cas9 RNP in HEK293T cells.
8 is a result of measuring the gene editing efficiency by AP-SpCas9 in HEK293T cells.
9 is a result of measuring the gene editing efficiency by AP RNP in HEK293T cells.
Figure 10 shows Cas9 (SEQ ID NO: 9), AP-Cas9 (SEQ ID NO: 19) of Comparative Example 1, or AP-HE-Cas9 (SEQ ID NO: 15) of Example 6 mixed with sgDNA to prepare RNP, which is a target DNA These are the results of agarose electrophoresis analyzed after treatment for 15, 30, and 60 minutes.
Figure 11 is Cas9 (SEQ ID NO: 9), TAT-Cas9 (SEQ ID NO: 29) of Comparative Example 7, TAT-HE-Cas9 (SEQ ID NO: 31) of Comparative Example 6, R9-Cas9 (SEQ ID NO: 25) of Comparative Example 5 , R9-HE-Cas9 (SEQ ID NO: 27) of Comparative Example 4, dNP2-Cas9 (SEQ ID NO: 21) of Comparative Example 3, or dNP2-HE-Cas9 (SEQ ID NO: 23) of Comparative Example 2 was mixed with sgDNA to obtain RNP These are the results of agarose electrophoresis after preparation and treatment with target DNA for 15 and 60 minutes.
12 shows each well of AP-HE-Cas9 (SEQ ID NO: 10 and SEQ ID NO: 15) prepared from Examples 1 and 6 at various concentrations (1, 2, 5 μM) and under various pH conditions (pH 7.4, 6.5, 6.0) is a graph measured by flow cytometry after treatment.
13a to 13g show AP-Cas9 prepared in Comparative Example 1 and AP-HE-Cas9 prepared in Examples 1, 2, 4, 6, and 8 in each well at various concentrations (1, 2, 5 μM) and various pH conditions (pH 7.4, 6.5, 6.0), and then measured by flow cytometry.

The present inventors overcame the limitations of gene editing technology through CRISPR-Cas and tried to develop an efficient gene editing technology that can replace it. As a result, in order to efficiently deliver the Cas protein-RNA complex (Ribonucleoprotein: RNP) into cells, a specific gene editing technology strategy using cell-penetrating peptides was devised, and using this, a Cas protein-RNA complex (Ribonucleoprotein) was introduced into the cell line. : The present invention was completed by making sure that it was delivered when RNP) was injected.

One aspect of the present invention relates to a cell-penetrating peptide for Cas protein-RNA complex (Ribonucleoprotein: RNP) represented by the following general formula (1).

[General formula 1]

Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly] _m -[His-Glu] _n

In the above formula, m may be an integer of 3 to 7, and n may be an integer of 5 to 15.

In the above formula, [Gly] _m serves to connect each peptide to each other with a linker, and m is not particularly limited, but preferably m may be 3 to 7. If the m is higher than 7, the length of the sequence may be excessively long and the cell permeation efficiency may be reduced, and if it is shorter than 3, sufficient flexibility may not be secured. More preferably, m in the above formula may be 4 to 6.

In the above formula, if n is an integer of 5 to 15, it can be used as a cell-penetrating peptide, but preferably an integer of 9 to 15, more preferably 10 to 12, 1.3 to 1.5 times in terms of masking and delivery efficiency more excellent More preferably, n may be an integer of 10.

The present invention provides a new Cas protein-RNA complex (Ribonucleoprotein: RNP), which is not easily introduced into cells, to have excellent permeability into cells, thereby increasing gene editing efficiency and reducing off-target to ensure stability. A protein capable of providing a protein-RNA complex (Ribonucleoprotein: RNP) was developed.

The cell penetrating peptide for Cas protein-RNA complex (Ribonucleoprotein: RNP) having the amino acid sequence represented by Formula 1 used in the present invention is the smallest peptide with the best delivery efficiency and masking effect, so any biological interference that may occur can be minimized.

According to one embodiment, the general formula 1 may be represented by any one of SEQ ID NOs: 10 to 17, preferably any one of SEQ ID NOs: 15 to 17, most preferably the amino acid sequence represented by SEQ ID NO: 15 may be displayed as Since this cell penetrating peptide for Cas protein-RNA complex (Ribonucleoprotein: RNP) contains an appropriate amount of HE, which is a transcription attenuation site (Attenuator), the effect of not interfering with the formation of Cas protein-RNA complex (Ribonucleoprotein: RNP) at all Have. Unlike the conventional cell-permeable peptide, the Cas protein-RNA complex (Ribonucleoprotein: RNP) to which the cell-permeable peptide of the present invention is coupled has the best intracellular delivery efficiency and gene editing efficiency.

Another aspect of the present invention is a) a cell-penetrating peptide represented by the general formula 1 is bound to the Cas protein; And b) guide RNA; relates to a composition for gene editing comprising a complex (RNP) consisting of.

[General formula 1]

Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly] _m -[His-Glu] _n

In the above formula, m is an integer of 3 to 7, and n is an integer of 5 to 15.

In the above formula, [Gly] _m serves to connect each peptide to each other with a linker, and m is not particularly limited, but preferably m may be 3 to 7. If the m is higher than 7, the length of the sequence may be excessively long and the cell permeation efficiency may be reduced, and if it is shorter than 3, sufficient flexibility may not be secured. More preferably, m in the above formula may be 4 to 6.

In the above formula, if n is an integer of 5 to 15, it can be used as a cell-penetrating peptide, but preferably an integer of 9 to 15, more preferably 10 to 12, 1.3 to 1.5 times in terms of masking and delivery efficiency more excellent More preferably, n may be an integer of 10.

The Cas protein or gene information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI). Specifically, the Cas protein may be a Cas9 protein. In addition, the Cas protein may be a Cas protein derived from the genus Campylobacter, more specifically, a Campylobacter jejuni, and more specifically, a Cas9 protein. As a more specific example, it may be an amino acid sequence set forth in SEQ ID NO: 9, or a protein having homology while having the activity of the protein of the sequence. In addition, the protein comprises at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In addition, the Cas protein is used in the present invention as a concept including all variants capable of acting as an endonuclease or nickase activated in cooperation with guide RNA in addition to the native protein. In the case of an activated endonuclease or nickase, it can bring about target DNA cleavage, and can be used to bring about genome editing. In addition, in the case of an inactivated mutant, it can be used to regulate transcription or to isolate a desired DNA.

The variant of the Cas protein may be a mutant form of Cas9 in which a catalytic aspartate residue or a histidine residue is changed to any other amino acid. Specifically, the other amino acid may be, but is not limited to, alanine.

More specifically, in the Cas protein, specifically, the Cas9 protein derived from C. jejuni ( campylobacter jejuni ), catalytic aspartic acid (D) at position 8 or histidine residue at position 559 (histidine, H) is substituted with another amino acid it may have been Specifically, the other amino acid may be, but is not limited to, alanine. That is, the Cas9 nuclease protein made by introducing a mutation into only one active site of the Cas9 nuclease protein can act as a nickase when it binds to the guide RNA. When two nickases are used, they are included in the category of RGEN because they can cause double strand breakage (DSB) by cleaving both DNA strands.

As used herein, the term "inactivated Cas protein" refers to a Cas nuclease protein in which all or part of the function of a nuclease is inactivated. The inactivated Cas is also called dCas.

As used herein, the term “cleavage” includes breakage of the covalent backbone of a nucleotide molecule.

The a) Cas protein to which the cell-penetrating peptide represented by Formula 1 is bound is a form that has been developed to act in a cell, and preferably, the cell-penetrating peptide represented by Formula 1 is represented by SEQ ID NO: 9 It may be bound to or linked to a Cas protein. The cell-penetrating peptide is preferably represented by SEQ ID NOs: 10 to 17, more preferably SEQ ID NOs: 15 to 17, and most preferably, may be a cell-penetrating peptide represented by SEQ ID NO: 15. The Cas protein to which the cell-penetrating peptide represented by SEQ ID NO: 15 is bound may be represented by SEQ ID NO: 15.

If other peptides other than the cell-penetrating peptide represented by the general formula 1 are linked, as in the experimental examples to be described later, there is a problem that the gene editing efficiency and intracellular delivery efficiency are significantly reduced, as well as the intracellular delivery efficiency. Even if it is high, the problem of increasing the off-target ratio may occur.

The Cas protein or a nucleic acid encoding the same may further include a nuclear localization signal (NLS) for localizing the Cas protein in the nucleus.

In addition, the nucleic acid encoding the Cas protein may additionally include a nuclear localization signal (NLS) sequence. Accordingly, the expression cassette including the nucleic acid encoding the Cas protein may include an NLS sequence in addition to regulatory sequences such as a promoter sequence for expressing the Cas protein. However, it is not limited thereto.

The Cas protein may be linked with a tag advantageous for isolation and/or purification. For example, a small peptide tag such as a His tag, a Flag tag, an S tag, or a Glutathione S-transferase (GST) tag, a Maltose binding protein (MBP) tag, etc. may be linked depending on the purpose, but is not limited thereto.

In the present invention, RNP is a) a cell-penetrating peptide represented by Formula 1 is bound to Cas protein; and b) a target DNA-specific guide RNA; refers to a ribonucleic acid protein in the form of a binding complex.

In the present invention, the RNP is a) a cell-penetrating peptide represented by Formula 1 is bound to a Cas protein or a nucleic acid encoding the same; and b) a target DNA-specific guide RNA or DNA encoding the guide RNA; but may be applied to cells in the form of, but not limited to. In this case, the guide RNA or DNA encoding the same and a) a Cas protein or a nucleic acid encoding the cell-penetrating peptide represented by Formula 1 bound thereto; may be simultaneously or sequentially applied to cells.

Accordingly, the present invention is a) Cas protein to which the cell-penetrating peptide represented by Formula 1 is bound; And b) guide RNA; to be delivered to cells in the form of an RNP complex composed of, is most preferable in terms of stability, gene editing efficiency, and delivery efficiency. In particular, it has the advantage that there is no risk of genetically modified organisms because it is not mediated by a DNA carrier.

According to one embodiment, the general formula 1 may be represented by any one of SEQ ID NOs: 10 to 17, preferably any one of SEQ ID NOs: 15 to 17, most preferably the amino acid sequence represented by SEQ ID NO: 15 may be displayed as The cell penetrating peptide for the Cas protein-RNA complex represented by SEQ ID NO: 15 is the smallest peptide with the best delivery and masking effects, so it is possible to minimize any biological interference that may occur.

The “guide RNA” refers to a target DNA-specific RNA, and may bind to a Cas protein to guide the Cas protein to the target DNA. The guide RNA can be prepared to be specific for any target to be cleaved.

In the present invention, the guide RNA includes two RNAs, that is, a dual RNA comprising crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components; or a form comprising a first site comprising a sequence capable of base pairing with a complementary strand of a target DNA and a second site comprising a sequence that interacts with a Cas protein, more specifically crRNA and tracrRNA It may be a single-chain guide RNA (sgRNA) in which a major part of the fusion is formed.

The length of the sequence capable of forming a base pair with the complementary chain of the target DNA sequence of the guide RNA is 17 to 23 bp, 18 to 23 bp, 19 to 23 bp, more specifically 20 to 23 bp, even more specifically, 21 to 23 bp may be, but is not limited thereto. This applies to both double RNA and sgRNA, and more specifically to sgRNA.

In addition, the guide RNA may have 1 to 3 additional nucleotides, more specifically 2 or 3 nucleotides in front of the 5' region of the sequence capable of base pairing with the complementary strand of the target DNA sequence. Examples of the nucleotides include A, T, G, and C. The guide RNA may have 1 to 3 guanine (G), more specifically, 2 or 3 G. This applies to both double RNA and sgRNA, and more specifically to sgRNA. However, it is not limited to the examples described above.

The sgRNA may include a portion having a sequence complementary to a sequence in the target DNA (also called a spacer region, a target DNA recognition sequence, a base pairing region, etc.) and a hairpin structure for binding Cas protein. More specifically, it may include a portion having a sequence complementary to a sequence in the target DNA, a hairpin structure for Cas protein binding, and a terminator sequence. The structure described above may be sequentially present in the order of 5' to 3'. However, the present invention is not limited thereto.

Any type of guide RNA may be used in the present invention as long as the guide RNA includes main portions of crRNA and tracrRNA and a complementary portion of target DNA.

The guide RNA may include a first site capable of forming a base pair with a complementary strand of a target DNA sequence; and a second portion having a stem or loop structure having a length of 13 to 18 bp (preferably 5 to 10 bp).

That is, the guide RNA may be appropriately selected depending on the target sequence or the type of endonuclease to form a complex and/or a microorganism derived therefrom. For example, the guide RNA may be at least one selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single-stranded guide RNA (sgRNA), and depending on the type of endonucleotide, CRISPR RNA (crRNA) and a double-stranded complex of trans-activating crRNA (tracrRNA), or single-stranded guide RNA (sgRNA). The sgRNA may include a portion of crRNA and tracrRNA.

The composition is for correcting a target DNA (or gene) present at a single site or multiple sites in a prokaryotic cell, a eukaryotic cell or a non-human eukaryotic organism.

In the present invention, "gene editing" refers to generating a double-stranded DNA cleavage at a target site in a target gene to cause mutation (deletion, substitution, and/or insertion, etc.) of one or more nucleotides. means action. In one example, the gene correction as described above generates a stop codon at the target site, or generates a codon encoding an amino acid different from the wild type, thereby inactivating (knock-out) the target gene, or does not produce 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 invention, a 'target gene' refers to a gene that is a target of gene editing, and a 'target site or target region' refers to a region where gene correction by Cas (or Cas9) in the target gene occurs. means

In the present invention, the 'target sequence' may be a nucleotide sequence of a site including a nucleotide (nt) to which a guide RNA hybridizes with a target gene or a target site of the target gene.

The prokaryotic cells and eukaryotic cells are isolated cells, and the eukaryotic cells are cells of yeast, mold, protozoa, plants, higher plants and insects, or amphibians, or CHO, HeLa, HEK293, and COS-1 and The cells may be isolated from the same mammal. The eukaryotic cells are cultured cells (in vitro), transplanted cells (graft cells) and primary cell cultures (in vitro and ex vivo), and in vivo, which are commonly used in the art. It may be a cell, or a cell isolated from a mammal including a human (mammalian cell).

The eukaryotic organism is a eukaryotic cell (eg, fungus such as yeast, eukaryotic and/or eukaryotic plant-derived cells (eg, embryonic cells, stem cells, somatic cells, germ cells, etc.), etc.), eukaryotic animals (eg, monkeys other than humans) It may be any one selected from the group consisting of primates such as primates, dogs, pigs, cattle, sheep, goats, mice, rats, etc.), and eukaryotic plants (eg, algae such as green algae, corn, soybeans, wheat, rice, etc.) .

Another aspect of the present invention is 1) local injection of the composition for gene editing into isolated prokaryotic cells, eukaryotic cells or eukaryotic organisms other than humans, microinjection, electroporation, and lipofection method. It relates to a method for producing a transformant other than a human comprising; introducing any one selected from among.

When a non-human transformant was prepared by the method for producing a non-human transformant of the present invention, a high success rate was shown, and it was confirmed that a desired transformant could be obtained. The success rate through the composition for gene editing of the present invention can be said to be a remarkably improved effect considering that a transformant can be obtained successfully compared to using other compositions for gene editing.

The present invention may provide a pharmaceutical composition containing the composition for gene editing. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and may be administered, for example, by intravenous injection, local injection, and intraperitoneal injection.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

<Examples 1 to 8> Preparation of a composition for gene editing comprising AP-HE-SpCas9 protein

1) Preparation of pET28a vector into which AP-HE-SpCas9 is inserted

In order to clone the plasmid DNA (SEQ ID NOs: 41 to 48; FIG. 1A) to express the AP-HE-SpCas9 protein (SEQ ID NOs: 10 to 17), pET28a, a protein expression vector, was cut with NheI and EcoRI restriction enzymes and then NheI-SpCas9- DNA (SEQ ID NO: 49) encoding EcoRI was inserted with a ligase. In addition, in order to insert AP-[HE] _m (SEQ ID NOs: 1 to 8) at the front, the vector was cut with NdeI and NheI restriction enzymes and then NdeI-AP-HE-NheI-encoding DNA (SEQ ID NOs: 32 to 39) was ligated Enzyme was inserted. The colonies obtained after transformation of each of the prepared plasmid DNAs into DH5α Escherichia coli were inoculated in LB medium, and then cultured in a shaking incubator at 37°C and 200 RPM for 12 hours. After the culture was completed, E. coli was recovered, plasmid DNA was isolated therefrom, and DNA sequencing was requested to Cosmogene Tech to confirm that the vector was prepared correctly.

2) Expression and purification of AP-HE-SpCas9 protein in E. coli

1) Each plasmid DNA prepared through step was transformed into Escherichia coli BL21(DE3)star pLysS strain. Each colony was inoculated into 50 ml of LB broth containing chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml) antibiotics, and incubated at 37° C. for 10 hours, and transferred to 500 ml of fresh LB broth. Cultivation was performed using a spectrophotometer until the value at OD _600nm was between 0.4 and 0.6. After adding IPTG to a concentration of 0.2 mM, the temperature was lowered to 20 °C, and incubated for 14 hours at 150 rpm. After the culture was completed, the culture solution was recovered, the supernatant was discarded by centrifugation, and the lysate (0.5M NaCl, 5mM imidazole, 20mM Tris-HCl, pH 8.0) was added to the pellet to re-turbid. The resuspended solution was treated with an ultrasonic cell disrupter (VCX-130 (Sonics & Materials)) and then centrifuged, and the supernatant was separated. The separated supernatant was filtered using a 0.45 μm filter, purified using 1M imidazole solution using an AKTA prime protein purification machine, and AP-HE-SpCas9 protein (SEQ ID NOs: 10 to 10) using a PD-10 desalting column. 17) was finally isolated. Each of these was confirmed through 12% SDS-PAGE (Fig. 1b).

1a is a pET28a vector into which AP-HE10-SpCas9 prepared in Example 1-6) is inserted, and FIG. 1b is an AP-HE10-SpCas9 protein (SEQ ID NO: 15) purified in Example 6 by SDS-PAGE. It is the result of analysis. According to this, it can be seen that the final separation and purification was properly performed.

<Comparative Examples 1 to 7> Preparation of pET28a vector into which CPP-SpCas9 is inserted

1) Preparation of pET28a vector into which CPP-SpCas9 is inserted

In order to clone the plasmid DNA (Fig. 1a, 2) to express the CPP-SpCas9 protein, pET28a, a protein expression vector, was cut with NheI and EcoRI restriction enzymes, and then the DNA encoding NheI-SpCas9-EcoRI (SEQ ID NO: 40) was ligated. inserted into In addition, in order to insert CPP (AP, dNP2-HE, dNP2, R9-HE, R9, TAT-HE, TAT) at the front, the vector was cut with NdeI and NheI restriction enzymes and then NdeI-CPP-NheI-encoding DNA was ligated. Enzyme was inserted. After transforming the prepared plasmid DNA into DH5α E. coli, the obtained colonies were inoculated in LB medium, and then cultured in a shaking incubator at 37°C and 200 RPM for 12 hours. After the culture was completed, E. coli was recovered, plasmid DNA was isolated therefrom, and DNA sequencing was requested to Cosmogene Tech to confirm that the vector was prepared correctly.

In Table 1, Ala Ser is a restriction enzyme site (AS: Nhel).

division name SEQ ID NO: order Comparative Example 1 AP 18 (protein) ArgArgArgTrpCysLysArgArgArgAlaSer 49(gene) ATGGGCAGCAGCCATCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCGCCGGCGCTGGTGCAAACGCCGCCGG Comparative Example 2 dNP2-HE10 22 (protein) LysIleLysLysValLysLysLysGlyArgLysGlySerLysIleLysLysValLysLysLysGlyArgLysAlaSerGlyGlyGlyGlyGlyHisHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGlu 53(gene) ATGGGCGGTTCTCATCATCATCATCATCATCATCATATGAAGATCAAGAAGGTTAAAAAAAAGGGTCGCAAGGGCTCTAAAATTAAAAAAGTCAAGAAGAAAGGAAGAAAAGCTAGCGGTGGTGGTGGAGGTCACGAACATGAACATGAACATGAACACGAGCACGAGCATGAGCACGAACACGAACACGAA Comparative Example 3 dNP2 20 (protein) LysIleLysLysValLysLysLysGlyArgLysGlySerLysIleLysLysValLysLysLysGlyArgLysAlaSer 51(gene) ATGGGCAGCAGCCATCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAGATCAAGAAGGTTAAAAAAAAGGGTCGCAAGGGCTCTAAAATTAAAAAAGTCAAGAAGAAAGGAAGAAAA Comparative Example 4 R9-HE10 26 (protein) ArgArgArgArgArgArgArgArgArgAlaSerGlyGlyGlyGlyGlyHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGlu 57(gene) ATGGGCGGTTCTCATCATCATCATCATCATCATATGAGACGAAGACGAAGACGTAGACGTAGAGCTAGCGGTGGTGGTGGAGGTCACGAACATGAACATGAACATGAACACGAGCACGAGCATGAGCACGAACACGAACACGAA Comparative Example 5 R9 24 (protein) ArgArgArgArgArgArgArgArgArgAlaSer 55(gene) ATGGGCAGCAGCCATCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAGACGAAGACGAAGACGTAGACGTAGA Comparative Example 6 TAT-HE10 30 (protein) TyrGlyArgLysLysArgArgGlnArgArgArgArgAlaSerGlyGlyGlyGlyGlyHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGluHisGlu 61(gene) ATGGGCGGTTCTCATCATCATCATCATCATCATCATATGTATGGACGCAAGAAGCGCCGCCAGCGCCGCCGCGCTAGCGGTGGTGGTGGAGGTCACGAACATGAACATGAACATGAACACGAGCACGAGCATGAGCACGAACACGAACACGAA Comparative Example 7 TAT 28 (protein) TyrGlyArgLysLysArgArgGlnArgArgArgArgAlaSer 59(gene) ATGGGCAGCAGCCATCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGTATGGACGCAAGAAGCGCCGCCAGCGCCGCCGC

2) Expression and purification of CPP-SpCas9 protein in E. coli

1) Each plasmid DNA prepared in step 1) was transformed into the E. coli BL21(DE3)star pLysS strain, and the obtained colonies were LB liquid containing chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml) antibiotics. It was inoculated into 50 ml of medium and cultured at 37 ° C. for 10 hours, and then transferred to 500 ml of new LB liquid medium. Cultivation was performed using a spectrophotometer until the value at OD _600nm was between 0.4 and 0.6. After adding IPTG to a concentration of 0.2 mM, the temperature was lowered to 20 °C, and incubated for 14 hours at 150 rpm. After the culture was completed, the culture solution was recovered, the supernatant was discarded by centrifugation, and the lysate (0.5M NaCl, 5mM imidazole, 20mM Tris-HCl, pH 8.0) was added to the pellet to re-turbid. The resuspended solution was treated with an ultrasonic cell disrupter (VCX-130 (Sonics & Materials)) and then centrifuged, and the supernatant was separated. The separated supernatant was filtered using a 0.45 μm filter, purified using 1M imidazole solution using an AKTA prime protein purification machine, and CPP-SpCas9 protein (SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31) were finally isolated. This was confirmed through 12% SDS-PAGE (Fig. 1b, Fig. 2).

Figure 1a shows the vectors prepared from Comparative Examples 2, 4, and 6, Figure 2a shows the pET28a vector into which CPP-SpCas9 prepared from Comparative Examples 1, 3, 5, and 7 is inserted, Figure 1b is CPP-SpCas9 protein (SEQ ID NOs: 23, 26, 31) purified through Comparative Examples 2, 4, 6 is the result of SDS-PAGE analysis, Figure 2b is purified through Comparative Examples 1, 3, 5, 7 CPP-SpCas9 protein (SEQ ID NO: 19, 21, 25, 29) is the result of SDS-PAGE analysis. According to this, it can be seen that the final separation and purification was properly performed.

Figure 2c shows the analysis by SDS-PAGE when the AP-SpCas9 protein prepared in Comparative Example 1 was purified by a column (purification technique), and it can be seen that purification according to the present method is most preferable.

<Comparative Examples 8 to 15> Preparation of pET28a vector into which CPP-AsCas12a and CPP-LbCas12a are inserted

1) Preparation of pET28a vector into which CPP-AsCas12a and CPP-LbCas12a are inserted

To clone the plasmid DNA ( FIGS. 3 and 4 ) to express CPP-AsCas12a and CPP-LbCas12a proteins, pET28a, a protein expression vector, was cut with NheI and EcoRI restriction enzymes and then NheI-AsCas12a (or LbCas12a)-EcoRI-encoding DNA (SEQ ID NOs: 63, 64) was inserted as a ligase. In addition, to insert CPP (AP, dNP2, R9, TAT) at the front, the vector was cut with NdeI and NheI restriction enzymes, and then DNA encoding NdeI-CPP-NheI was inserted using a ligase. The resulting plasmid DNA was transformed into DH5α Escherichia coli, and the obtained colonies were inoculated in LB medium, and then cultured in a shaking incubator at 37° C. and 200 RPM for 12 hours. After the culture was completed, E. coli was recovered, plasmid DNA was isolated therefrom, and DNA sequencing was requested to Cosmogene Tech to confirm that the vector was accurately prepared.

2) Expression and purification of CPP-AsCas12a and CPP-LbCas12a proteins in E. coli

1) Each plasmid DNA prepared in step 1) was transformed into the E. coli BL21(DE3)star pLysS strain, and the obtained colonies were LB liquid containing chloramphenicol (34 μg/ml) and ampicillin (50 μg/ml) antibiotics. It was inoculated into 50 ml of medium and cultured at 37 ° C. for 10 hours, and then transferred to 500 ml of new LB liquid medium. Cultivation was performed using a spectrophotometer until the value at OD _600nm was between 0.4 and 0.6. After adding IPTG to a concentration of 0.2 mM, the temperature was lowered to 20 °C, and incubated for 14 hours at 150 rpm. After the culture was completed, the culture solution was recovered, the supernatant was discarded by centrifugation, and the lysate (0.5M NaCl, 5mM imidazole, 20mM Tris-HCl, pH 8.0) was added to the pellet to re-turbid. The resuspended solution was treated with an ultrasonic cell disrupter (VCX-130 (Sonics & Materials)) and then centrifuged, and the supernatant was separated. The separated supernatant was filtered through a 0.45㎛ filter, purified using 1M imidazole solution using an AKTA prime protein purification machine, and CPP (AP, dNP2, R9, TAT)- using a PD-10 desalting column AsCas12a (SEQ ID NOs: 69, 70, 71, 72) (comparative examples 8-11 in that order) and CPP (AP, dNP2, R9, TAT)-LbCas12a protein (SEQ ID NOs: 73, 74, 75, 76) (comparison in order) Examples 12-15) were finally isolated. This was confirmed through 12% SDS-PAGE (Fig. 1b, Fig. 2).

3A is a pET28a vector into which CPP-AsCas12a prepared in Comparative Examples 8 to 11 is inserted, and FIG. 4A is a pET28a vector into which CPP-LbCas12a prepared in Comparative Examples 12 to 15 is inserted. Figure 3b is a result of SDS-PAGE analysis of CPP-AsCas12a protein (SEQ ID NO: 69, 70, 71, 72) purified through Comparative Examples 8 to 11, Figure 4b is CPP- LbCas12a protein (SEQ ID NOs: 73, 74, 75, 76) was analyzed by SDS-PAGE. According to this, it can be seen that the final separation and purification was properly performed.

FIG. 3c shows that the AP-AsCas12a protein prepared in Comparative Example 8 was purified by various columns (purification technology), and each was analyzed by SDS-PAGE. As a result, it was confirmed that the protein was well purified without impurities.

<Test Example 1> Delivery of protein into HEK293 T cell line

Cell permeability was confirmed through an experiment in which the AP-HE-SpCas9 protein purified in Example 6 was delivered into the human HEK293 T cell line. The HEK293 T cells are cultured using DMEM, and the cells are dispensed by adding 25 μl of DMEM medium containing 2.5×10 ⁵ cells to each well of a 96-well plate in which 155 μl of DMEM medium has been dispensed. did. Then, the cells and proteins were incubated together under various conditions so that the total volume was 200 μl by mixing D-PBS and the protein to be treated to an appropriate concentration in 20 μl.

AP-HE-Cas9 (2 μM) mixed with CQ (1, 10, 50, 100, 250, 500 μM), AP-HE-Cas9 (2 μM) alone and control (2 μM) After treatment in each well, 5% CO ₂ Incubated at 37 ° C in a cell incubator for 2 hours. After centrifugation, the cultured cells were washed twice with PBS buffer and treated with a trypsin solution for 5 minutes to remove proteins attached to the cell surface. Here, it was neutralized with DMEM solution, washed again with PBS buffer, and then intracellular fluorescence was measured using a flow cytometry (flow cytometry, FACS machine, BD science FACS canto II) to confirm the delivery efficiency. At this time, CQ is chloroquine, which is a lysosomal degradation inhibitor.

Figure 5 shows AP-HE-Cas9 (2 μM) (Example 6) alone or mixed with CQ (1, 10, 50, 100, 250, 500 μM) and control (2 μM) After treatment in each well, it is a graph measured with a flow cytometer. According to this, the cell permeation efficiency of the AP-HE-Cas9 protein according to the present invention increases as the concentration increases, and when mixed with CQ, the concentration of CQ increases. Accordingly, it can be seen that the cell permeability increases.

<Test Example 2> Delivery of AP-HE-SpCas9 into the cancer cell host HeLa cell line

Through Test Example 1, it was confirmed that the AP-HE-SpCas9 protein (Example 6) of the present invention was well delivered into cells. However, since it is not possible to analyze where it is located after delivery into the cell, we tried to analyze the location inside the cell through a microscope.

In each well of a 6-well plate, lay a 24 mm ² square cover glass for a microscope in advance, dispense 1×10 ⁵ HeLa cells, and incubate in DMEM medium for 24 hours to allow the cells to adhere to the cover glass. made to be After removing the DMEM medium, 900 μl of new DMEM medium was added. Here, 50 μl of the AP-HE-SpCas9 protein prepared in Example 6 was mixed with D-PBS so as to have concentrations of 0.5 μM, 1 μM, and 2 μM, was added. Then, under 37 °C conditions for 2 hours, 5% CO ₂ Incubated in a cell incubator. After culture was completed, proteins except for adherent cells and DMEM medium were removed, washed twice with PBS buffer, and cells were fixed with 1 ml of 4% paraformaldehyde phosphate buffer solution (Wako), followed by PBS again. After washing with a buffer, F-actin was stained with a green fluorescent material (Alexa fluor 488 conjugated phalloidin) (invitrogen), and the nucleus was stained with Hoechst 33342 (invitrogen). After washing twice with PBS buffer, it was mounted on a slide glass, and then using a fluorescence microscope (Eclipse 50i, Nikon) or a confocal microscope (TCS SP5, Leica), the AP-HE-Cas9 of the present invention (implementation) Example 6) The protein position was confirmed.

Figure 6 is a photograph of intracellular fluorescence after treatment with AP-HE-Cas9 (2 μM) (Example 6) alone or mixed with CQ (500 μM) in each well. It was confirmed that the AP-HE-Cas9 (Example 6) protein of the present invention was well delivered into the cell, and was located in the cell.

<Test Example 3> Cas9 protein-RNA complex (Ribonucleoprotein: RNP) gene editing efficacy

1) Preparation of AP-HE Cas9 protein-RNA complex (Ribonucleoprotein: RNP)

Cas9 protein-RNA complex (Ribonucleoprotein: RNP) was obtained by mixing the AP-HE-SpCas9 protein (5 μM) and sgRNA (5 μM) of Example 6 in a 1:1 ratio, followed by reaction at room temperature for 10 minutes to obtain AP A -HE-Cas9 protein-RNA complex was prepared, and was subsequently designated as AP-HE-Cas9 RNP. crRNP is a collective term for Cas9 protein-RNA complex (Ribonucleoprotein: RNP) regardless of the type of Cas9 protein.

2) Gene editing

When the AP-HE-SpCas9 protein of Example 6 was prepared in the form of a Cas9 protein-RNA complex (Ribonucleoprotein: RNP), it was tried to determine whether it exhibits gene editing efficacy in cells. To this end, the RFP/GFP reporter system and T7 endonuclease 1 assay, which express both RFP and GFP when a specific gene is cut, were used. The murine HEK293 T cell line was cultured in DMEM medium. 400 μl DMEM medium was dispensed into each well of a 24-well plate, and 50 μl DMEM medium containing 2.5×10 ⁵ cells in each was mixed. CQ (50 μM) and AP-HE-Cas9 RNP were added to the appropriate concentration with D-PBS present in the 50 μl medium (day 0). At this time, the final volume was set to 500 μl.

On the first day of culture (day 1), 5 μM of AP-HE-Cas9 RNP was treated in each well of the HEK293 T cell line, and incubated at 37° C. in a 5% CO ₂ cell incubator for 6 hours, and then replaced with fresh DMEM medium. .

The next day (day 2), 5 μM of AP-HE-Cas9 RNP was treated in each well and reacted at 37° C. in a 5% CO ₂ cell incubator for 6 hours, and then replaced with fresh DMEM medium. On the third and second days of culture, AP-HE RNp was treated in the same manner as on the second day, and after reaction for 6 hours, the culture medium was recovered and centrifuged to remove the supernatant. The remaining pellet was washed twice with PBS buffer and treated with trypsin for 5 minutes to remove proteins attached to the cell surface. After neutralization by the addition of RPMI solution, it was washed once again with PBS buffer, and gene editing efficiency was checked using flow cytometry (flow cytometry, FACS machine, BD science FACS canto II) and T7 endonuclease 1 assay. At this time, as a control, 250 ng of CCR5 sgRNA vector and 250 ng of SpCas9 vector were mixed into the cells once daily.

7 is a result of measuring the gene editing efficiency by AP-HE-Cas9 RNP in HEK293T cells. In spite of this, it can be seen that the gene was efficiently corrected with 6.5 indels (%) in the cell.

<Test Example 4> AP-SpCas9 gene correction in HEK293 T cell line

It was attempted to determine whether AP-SpCas9 of Comparative Example 1 exhibits gene editing efficacy in cells. To this end, the RFP/GFP reporter system and T7 endonuclease 1 assay, which express both RFP and GFP when a specific gene is cut, were used. The murine HEK293 T cell line was cultured in DMEM medium. 400 μl DMEM medium was dispensed into each well of a 24-well plate, and 50 μl DMEM medium containing 2.5×10 ⁵ cells in each was mixed. CQ (50 μM) and AP-Cas9 of Comparative Example 1 were added to the appropriate concentration with D-PBS present in the 50 μl medium (day 0). At this time, the final volume was set to 500 μl.

At this time, the HEK293 T cells were transformed with lipofectamine with an sgRNA plasmid targeting the CCR5 gene before AP-Cas9 of Comparative Example 1 was added.

On the first day of culture, AP-Cas9 plasmid (Comparative Example 1-1) and sgRNA plasmid targeting CCR5 gene were delivered to HEK293 T cells through lipofectamine, and then cultured at 37 ° C. in a 5% CO ₂ cell incubator for 24 hours. . On day 2, the DMEM medium was newly replaced, and 5 μM of AP-SpCas9 protein (Comparative Example 1) was treated in each well, and reacted at 37° C. in a 5% CO ₂ cell incubator for 6 hours. After 6 hours, it was replaced with fresh DMEM medium. On the 3rd day, 5 μM of AP-SpCas9 protein (Comparative Example 1) was treated in each well, and reacted at 37° C. in a 5% CO ₂ cell incubator for 6 hours, and then replaced with a new DMEM medium. On the 4th day, the AP-SpCas9 was treated in the same way as on the 3rd day, and after reacting for 6 hours, the culture medium was recovered and centrifuged to remove the supernatant. The remaining pellet was washed twice with PBS buffer and treated with trypsin for 5 minutes to remove proteins attached to the cell surface. After neutralization by the addition of RPMI solution, it was washed once again with PBS buffer, and gene editing efficiency was checked using flow cytometry (flow cytometry, FACS machine, BD science FACS canto II) and T7 endonuclease 1 assay.

8 is a result of measuring the gene editing efficiency by AP-SpCas9 in HEK293T cells. According to this, when cells were treated with AP-SpCas9 and sgRNA plasmid according to the present invention, it was confirmed that indel (%) was 1.8 to 4.0. .

<Test Example 5> AP-RNP (Ribonucleoprotein) gene correction in HEK293 T cell line

1) Preparation of AP-HE Cas9 protein-RNA complex (Ribonucleoprotein: RNP)

Cas9 protein-RNA complex (Ribonucleoprotein: RNP) was obtained by mixing AP-SpCas9 protein (5 μM) and sgRNA (5 μM) of Comparative Example 1 in a ratio of 1:1, followed by reaction at room temperature for 10 minutes to form AP Cas9 protein -RNA complexes were prepared, and then denoted as AP-Cas9 RNP. crRNP is a collective term for Cas9 protein-RNA complex (Ribonucleoprotein: RNP) regardless of the type of Cas9 protein.

2) Gene editing

When the AP-SpCas9 protein of Comparative Example 1 was prepared in the form of a Cas9 protein-RNA complex (Ribonucleoprotein: RNP), it was attempted to determine whether it exhibits gene editing efficacy in cells. To this end, the RFP/GFP reporter system and T7 endonuclease 1 assay, which express both RFP and GFP when a specific gene is cut, were used. The murine HEK293 T cell line was cultured in DMEM medium. 400 μl DMEM medium was dispensed into each well of a 24-well plate, and 50 μl DMEM medium containing 2.5×10 ⁵ cells in each was mixed. D-PBS and CQ (50 μM) and AP RNP in the 50 μl medium were added to the appropriate concentration (day 0). At this time, the final volume was set to 500 μl.

On the first day of culture (day 1), 5 μM of AP-Cas9 RNP was treated in each well of the HEK293 T cell line, and incubated at 37 ° C. in a 5% CO ₂ cell incubator for 6 hours, and then replaced with fresh DMEM medium.

The next day (day 2), 5 μM of AP-Cas9 RNP was treated in each well and reacted at 37° C. in a 5% CO ₂ cell incubator for 6 hours, and then replaced with fresh DMEM medium. On the third and second days of culture, AP-Cas9 RNP was treated in the same manner as on the second day, and after reacting for 6 hours, the culture medium was recovered and centrifuged to remove the supernatant. The remaining pellet was washed twice with PBS buffer and treated with trypsin for 5 minutes to remove proteins attached to the cell surface. After neutralization by addition of RPMI solution, it was washed once again with PBS buffer, and gene editing efficiency was checked using flow cytometry (flow cytometry, FACS machine, BD science FACS canto II) and T7 endonuclease 1 assay. At this time, as a control, 250 ng of CCR5 sgRNA vector and 250 ng of SpCas9 vector were mixed into the cells once daily.

9 is a result of measuring the gene editing efficiency by AP-Cas9 RNP in HEK293T cells. According to this, when the AP-Cas9 protein-RNA complex (Ribonucleoprotein: RNP) according to the present invention is directly treated in the cell, indel (%) is 0.1 to 1.8, which indicates that using the AP-HE-Cas9 RNP represented by the general formula 1 of the present invention, particularly the AP-HE-Cas9 RNP of Example 6, is about 3 to 6 times more efficient. there is.

<Test Example 6> Gene editing efficacy of AP-HE-SpCas9 prepared in Example 6 in vitro

The AP-HE-SpCas9 protein prepared in Example 6 of the present invention contains 10 HE, a transcription attenuation site (attenuator), and functions to suppress the + charge of the cell-permeable peptide. The efficiency of cleavage of the target DNA in vitro using the AP-HE-SpCas9 protein of Example 1 of the present invention was confirmed. First, 50 nM of Cas9 (SEQ ID NO: 9), AP-Cas9 (SEQ ID NO: 19) of Comparative Example 1 or AP-HE-Cas9 (SEQ ID NO: 15) of Example 6 and 50 nM of sgRNA were mixed, and then, at room temperature for 15 minutes During the reaction, a Cas9 protein-RNA complex (Ribonucleoprotein: RNP) was prepared. Thereafter, 300 ng of target DNA was reacted at 37° C. for 15, 30 or 60 minutes to determine whether the target DNA was cut through agarose electrophoresis (0.8% agarose gel).

Figure 10 shows Cas9 (SEQ ID NO: 9), AP-Cas9 (SEQ ID NO: 19) of Comparative Example 1, or AP-HE-Cas9 (SEQ ID NO: 15) of Example 6 mixed with sgDNA to prepare RNP, which is a target DNA These are the results of agarose electrophoresis analyzed after treatment for 15, 30, and 60 minutes. According to this, it can be confirmed that the complex (RNP) of AP-HE-Cas9 (SEQ ID NO: 15) and sgDNA of Example 6 of the present invention exhibits the best cleavage efficiency. That is, it can be seen that the protein with the HE sequence added (AP-HE-Cas9) has superior gene editing efficiency than the protein without the HE sequence (AP-Cas9).

<Test Example 7> Gene editing efficacy of Comparative Examples in vitro

The AP-HE-SpCas9 protein prepared in Example 6 of the present invention contains 10 HE, a transcription attenuation site (attenuator), and functions to suppress the + charge of the cell-permeable peptide. Whether the target DNA was cleaved in vitro using the AP-HE-SpCas9 protein of Example 6 of the present invention was confirmed. First, Cas9 (SEQ ID NO: 9), TAT-Cas9 (SEQ ID NO: 29) of Comparative Example 7, TAT-HE-Cas9 (SEQ ID NO: 31) of Comparative Example 6, R9-Cas9 (SEQ ID NO: 25) of Comparative Example 5 (SEQ ID NO: 25), comparison R9-HE-Cas9 (SEQ ID NO: 27) of Example 4, dNP2-Cas9 (SEQ ID NO: 21) of Comparative Example 3 or dNP2-HE-Cas9 (SEQ ID NO: 23) of Comparative Example 2 50 nM and sgRNA 50 nM were mixed and , and reacted at room temperature for 15 minutes to prepare a Cas9 protein-RNA complex (Ribonucleoprotein: RNP). Thereafter, 300 ng of target DNA was reacted at 37° C. for 15 or 60 minutes to determine whether the target DNA was cut through Agarose electrophoresis (0.8% agarose gel).

Figure 11 is Cas9 (SEQ ID NO: 9), TAT-Cas9 (SEQ ID NO: 29) of Comparative Example 7, TAT-HE-Cas9 (SEQ ID NO: 31) of Comparative Example 6, R9-Cas9 (SEQ ID NO: 25) of Comparative Example 5 , R9-HE-Cas9 (SEQ ID NO: 27) of Comparative Example 4, dNP2-Cas9 (SEQ ID NO: 21) of Comparative Example 3, or dNP2-HE-Cas9 (SEQ ID NO: 23) of Comparative Example 2 was mixed with sgDNA to obtain RNP These are the results of agarose electrophoresis after preparation and treatment with target DNA for 15 and 60 minutes. According to this, in the complexes of CPP-SpCas9 protein and sgDNA of Comparative Examples 2 to 7, it was confirmed that some uncut target DNA remained. Taken together, it can be seen that the complex (RNP) of AP-HE-Cas9 (SEQ ID NO: 15) and sgDNA of Example 6 of the present invention exhibits the best cleavage efficiency.

That is, it can be seen that the gene editing effect is rather reduced in CPPs (cell-permeable peptides) such as TAT, R9, dNP2 to which the HE sequence is added.

<Test Example 8> Efficacy of gene editing according to the length of HE, a transcriptional weakening site

It was attempted to confirm the gene editing efficiency of target DNA in vitro using the AP-HE-Cas9 protein prepared in Examples 1 to 8 of the present invention. 50 nM of AP-HE-SpCas9 protein prepared in Examples 1 to 8 of the present invention and 50 nM of sgRNA were mixed, and reacted at room temperature for 15 minutes to prepare a Cas9 protein-RNA complex (Ribonucleoprotein: RNP).

In each well of a 6-well plate, lay a 24 mm ² square cover glass for a microscope in advance, dispense 1×10 ⁵ HeLa cells, and incubate in DMEM medium for 24 hours to allow the cells to adhere to the cover glass. made to be After removing the DMEM medium, 900 μl of new DMEM medium was added. The AP-HE-Cas9 protein prepared in Examples 1 to 8 was added to each of the Hela cells at a concentration of 1, 2, and 5 μM, and under various pH conditions (pH 7.4, 6.5, 6.0) and 37 ° C for 1 hour, Cultivated in a 5% CO ₂ cell incubator. After centrifugation, the cultured cells were washed twice with PBS buffer and treated with a trypsin solution for 5 minutes to remove proteins attached to the cell surface. Here, it was neutralized with DMEM solution, washed again with PBS buffer, and then intracellular fluorescence was measured using a flow cytometry (flow cytometry, FACS machine, BD science FACS canto II) to confirm the delivery efficiency.

12 shows each well of AP-HE-Cas9 (SEQ ID NO: 10 and SEQ ID NO: 15) prepared from Examples 1 and 6 at various concentrations (1, 2, 5 μM) and under various pH conditions (pH 7.4, 6.5, 6.0) is a graph measured by flow cytometry after treatment. According to this, it can be seen that AP-HE10-Cas9 (SEQ ID NO: 15) of Example 6 shows a significantly higher masking effect than AP-HE5-Cas9 (SEQ ID NO: 10) of Example 1 under the same conditions.

In addition, it can be confirmed that AP-HE10-Cas9 (SEQ ID NO: 15) of Example 6 has a higher transfer efficiency than AP-HE5-Cas9 (SEQ ID NO: 10) of Example 1 at pH 7.4 and 6.5 conditions.

13a to 13g show AP-Cas9 prepared in Comparative Example 1 and AP-HE-Cas9 prepared in Examples 1, 2, 4, 6, and 8 in each well at various concentrations (1, 2, 5 μM) and various pH conditions (pH 7.4, 6.5, 6.0), and then measured by flow cytometry. At this time, the AP-HE10-Cas9 (SEQ ID NO: 15) of Example 6 is '10HE', the AP-HE5-Cas9 (SEQ ID NO: 10) of Example 1 is '5HE', and the AP-HE6-Cas9 of Example 2 ( SEQ ID NO: 11) is '6HE', AP-HE8-Cas9 (SEQ ID NO: 13) of Example 4 is '8HE', and AP-HE5-Cas9 (SEQ ID NO: 17) of Example 8 is '12HE'.

According to this, under the same conditions, AP-HE10-Cas9 (SEQ ID NO: 15) of Example 6 was obtained from AP-HE5-Cas9 (SEQ ID NO: 10) of Example 1, AP-HE6-Cas9 (SEQ ID NO: 11) of Example 2, It was confirmed that the masking effect was significantly higher than that of AP-HE8-Cas9 (SEQ ID NO: 13) of Example 4.

In addition, at pH 7.4 to 6.0 conditions, AP-HE10-Cas9 (SEQ ID NO: 15) of Example 6 is AP-HE5-Cas9 (SEQ ID NO: 10) of Example 1, AP-HE6-Cas9 (SEQ ID NO: 10) of Example 2 11), it can be seen that the transfer efficiency is higher than that of AP-HE8-Cas9 (SEQ ID NO: 13) of Example 4.

On the other hand, AP-HE10-Cas9 (SEQ ID NO: 15) of Example 6 and AP-HE5-Cas9 (SEQ ID NO: 17) of Example 8 showed no significant difference in masking and delivery efficiency even though the HE sequence length was changed.

That is, Example 6 having 10 HE sequences showed superior masking efficiency than Examples 2 and 4 having 6 or 8 HE sequences under various pH conditions, but AP-HE of Examples 6 and 9 -Cas9 showed almost no difference. Through this, it can be confirmed that increasing the HE after 10 repetitions has no significant effect, so it can be seen that AP-HE10-Cas9 of Example 6 is the most preferable.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> composition for gene editing based on CRISPR-CAS <130> HPC8944 <150> KR 10-2018-0170463 <151> 2018-12-27 <160> 84 <170> KoPatentIn 3.0 <210> 1 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; AP-HE5 <400> 1 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu 20 25 <210> 2 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE6 <400> 2 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu 20 25 <210> 3 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE7 <400> 3 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 <210> 4 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE8 <400> 4 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 <210> 5 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE9 <400> 5 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu <210> 6 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE10 <400> 6 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu 35 <210> 7 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE11 <400> 7 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu His Glu 35 <210> 8 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> ppenetrating peptide; AP-HE12 <400> 8 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu His Glu His Glu 35 40 <210> 9 <211> 1409 <212> PRT <213> Artificial Sequence <220> <223> Cas9 <400> 9 Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly 1 5 10 15 Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr 20 25 30 Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser 35 40 45 Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys 50 55 60 Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala 65 70 75 80 Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn 85 90 95 Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val 100 105 110 Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu 115 120 125 Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu 130 135 140 Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys 145 150 155 160 Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala 165 170 175 Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp 180 185 190 Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val 195 200 205 Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly 210 215 220 Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg 225 230 235 240 Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu 245 250 255 Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys 260 265 270 Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp 275 280 285 Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln 290 295 300 Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu 305 310 315 320 Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu 325 330 335 Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr 340 345 350 Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu 355 360 365 Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly 370 375 380 Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu 385 390 395 400 Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp 405 410 415 Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln 420 425 430 Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe 435 440 445 Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr 450 455 460 Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg 465 470 475 480 Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn 485 490 495 Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu 500 505 510 Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro 515 520 525 Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr 530 535 540 Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser 545 550 555 560 Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg 565 570 575 Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu 580 585 590 Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala 595 600 605 Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp 610 615 620 Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu 625 630 635 640 Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys 645 650 655 Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg 660 665 670 Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly 675 680 685 Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser 690 695 700 Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser 705 710 715 720 Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly 725 730 735 Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile 740 745 750 Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys 755 760 765 Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg 770 775 780 Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met 785 790 795 800 Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys 805 810 815 Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu 820 825 830 Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp 835 840 845 Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser 850 855 860 Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp 865 870 875 880 Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys 885 890 895 Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr 900 905 910 Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser 915 920 925 Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg 930 935 940 Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr 945 950 955 960 Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 965 970 975 Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr 980 985 990 Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 995 1000 1005 Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1010 1015 1020 Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met 1025 1030 1035 1040 Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe 1045 1050 1055 Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1060 1065 1070 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr 1075 1080 1085 Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys 1090 1095 1100 Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln 1105 1110 1115 1120 Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp 1125 1130 1135 Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly 1140 1145 1150 Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1155 1160 1165 Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly 1170 1175 1180 Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe 1185 1190 1195 1200 Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys 1205 1210 1215 Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met 1220 1225 1230 Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1235 1240 1245 Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1250 1255 1260 Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln 1265 1270 1275 1280 His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser 1285 1290 1295 Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1300 1305 1310 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile 1315 1320 1325 Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys 1330 1335 1340 Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu 1345 1350 1355 1360 Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu 1365 1370 1375 Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro 1380 1385 1390 Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr 1395 1400 1405 Ala <210> 10 <211> 1437 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE5-Cas9 <400> 10 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu Ala Ser Leu Asp Ser Thr 20 25 30 Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala 35 40 45 Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly 50 55 60 Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys 65 70 75 80 Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly 85 90 95 Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys 100 105 110 Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr 115 120 125 Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe 130 135 140 Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His 145 150 155 160 Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His 165 170 175 Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser 180 185 190 Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met 195 200 205 Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp 210 215 220 Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn 225 230 235 240 Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys 245 250 255 Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu 260 265 270 Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu 275 280 285 Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp 290 295 300 Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp 305 310 315 320 Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu 325 330 335 Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile 340 345 350 Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met 355 360 365 Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala 370 375 380 Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp 385 390 395 400 Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln 405 410 415 Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly 420 425 430 Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys 435 440 445 Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly 450 455 460 Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu 465 470 475 480 Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro 485 490 495 Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met 500 505 510 Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val 515 520 525 Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn 530 535 540 Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu 545 550 555 560 Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr 565 570 575 Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys 580 585 590 Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val 595 600 605 Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser 610 615 620 Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr 625 630 635 640 Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn 645 650 655 Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu 660 665 670 Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His 675 680 685 Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr 690 695 700 Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys 705 710 715 720 Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala 725 730 735 Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys 740 745 750 Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His 755 760 765 Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile 770 775 780 Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg 785 790 795 800 His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr 805 810 815 Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu 820 825 830 Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val 835 840 845 Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln 850 855 860 Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu 865 870 875 880 Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp 885 890 895 Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly 900 905 910 Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn 915 920 925 Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe 930 935 940 Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys 945 950 955 960 Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys 965 970 975 His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu 980 985 990 Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys 995 1000 1005 Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu 1010 1015 1020 Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val 1025 1030 1035 1040 Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val 1045 1050 1055 Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser 1060 1065 1070 Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn 1075 1080 1085 Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile 1090 1095 1100 Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val 1105 1110 1115 1120 Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met 1125 1130 1135 Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe 1140 1145 1150 Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala 1155 1160 1165 Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro 1170 1175 1180 Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys 1185 1190 1195 1200 Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met 1205 1210 1215 Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys 1220 1225 1230 Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr 1235 1240 1245 Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala 1250 1255 1260 Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1265 1270 1275 1280 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro 1285 1290 1295 Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr 1300 1305 1310 Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile 1315 1320 1325 Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His 1330 1335 1340 Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe 1345 1350 1355 1360 Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1365 1370 1375 Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala 1380 1385 1390 Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp 1395 1400 1405 Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg 1410 1415 1420 Lys Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1425 1430 1435 <210> 11 <211> 1439 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE6-Cas9 <400> 11 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu Ala Ser Leu Asp 20 25 30 Ser Thr Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro 35 40 45 Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser 50 55 60 Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys 65 70 75 80 Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu 85 90 95 Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg 100 105 110 Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile 115 120 125 Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp 130 135 140 Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys 145 150 155 160 Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala 165 170 175 Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val 180 185 190 Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala 195 200 205 His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn 210 215 220 Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr 225 230 235 240 Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp 245 250 255 Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu 260 265 270 Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly 275 280 285 Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn 290 295 300 Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr 305 310 315 320 Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala 325 330 335 Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser 340 345 350 Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala 355 360 365 Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu 370 375 380 Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe 385 390 395 400 Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala 405 410 415 Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met 420 425 430 Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu 435 440 445 Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His 450 455 460 Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro 465 470 475 480 Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg 485 490 495 Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala 500 505 510 Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu 515 520 525 Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met 530 535 540 Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His 545 550 555 560 Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val 565 570 575 Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu 580 585 590 Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val 595 600 605 Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe 610 615 620 Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu 625 630 635 640 Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu 645 650 655 Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu 660 665 670 Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr 675 680 685 Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg 690 695 700 Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg 705 710 715 720 Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly 725 730 735 Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr 740 745 750 Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser 755 760 765 Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys 770 775 780 Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met 785 790 795 800 Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn 805 810 815 Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg 820 825 830 Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His 835 840 845 Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr 850 855 860 Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn 865 870 875 880 Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu 885 890 895 Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn 900 905 910 Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met 915 920 925 Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg 930 935 940 Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu 945 950 955 960 Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile 965 970 975 Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr 980 985 990 Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys 995 1000 1005 Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val 1010 1015 1020 Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala 1025 1030 1035 1040 Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu 1045 1050 1055 Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1060 1065 1070 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr 1075 1080 1085 Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly 1090 1095 1100 Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu 1105 1110 1115 1120 Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu 1125 1130 1135 Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly 1140 1145 1150 Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu 1155 1160 1165 Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp 1170 1175 1180 Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys 1185 1190 1195 1200 Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr 1205 1210 1215 Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu 1220 1225 1230 Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro 1235 1240 1245 Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala 1250 1255 1260 Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys 1265 1270 1275 1280 Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly 1285 1290 1295 Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1300 1305 1310 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg 1315 1320 1325 Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn 1330 1335 1340 Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His 1345 1350 1355 1360 Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe 1365 1370 1375 Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu 1380 1385 1390 Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg 1395 1400 1405 Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys 1410 1415 1420 Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1425 1430 1435 <210> 12 <211> 1441 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE7-Cas9 <400> 12 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu Ala Ser 20 25 30 Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly 35 40 45 Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr 50 55 60 Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser 65 70 75 80 Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys 85 90 95 Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala 100 105 110 Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn 115 120 125 Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val 130 135 140 Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu 145 150 155 160 Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu 165 170 175 Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys 180 185 190 Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala 195 200 205 Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp 210 215 220 Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val 225 230 235 240 Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly 245 250 255 Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg 260 265 270 Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu 275 280 285 Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys 290 295 300 Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp 305 310 315 320 Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln 325 330 335 Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu 340 345 350 Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu 355 360 365 Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr 370 375 380 Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu 385 390 395 400 Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly 405 410 415 Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu 420 425 430 Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp 435 440 445 Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln 450 455 460 Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe 465 470 475 480 Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr 485 490 495 Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg 500 505 510 Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn 515 520 525 Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu 530 535 540 Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro 545 550 555 560 Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr 565 570 575 Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser 580 585 590 Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg 595 600 605 Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu 610 615 620 Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala 625 630 635 640 Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp 645 650 655 Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu 660 665 670 Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys 675 680 685 Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg 690 695 700 Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly 705 710 715 720 Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser 725 730 735 Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser 740 745 750 Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly 755 760 765 Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile 770 775 780 Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys 785 790 795 800 Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg 805 810 815 Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met 820 825 830 Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys 835 840 845 Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu 850 855 860 Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp 865 870 875 880 Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser 885 890 895 Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp 900 905 910 Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys 915 920 925 Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr 930 935 940 Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser 945 950 955 960 Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg 965 970 975 Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr 980 985 990 Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 995 1000 1005 Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr 1010 1015 1020 Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 1025 1030 1035 1040 Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1045 1050 1055 Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met 1060 1065 1070 Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe 1075 1080 1085 Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1090 1095 1100 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr 1105 1110 1115 1120 Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys 1125 1130 1135 Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln 1140 1145 1150 Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp 1155 1160 1165 Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly 1170 1175 1180 Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val 1185 1190 1195 1200 Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly 1205 1210 1215 Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe 1220 1225 1230 Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys 1235 1240 1245 Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met 1250 1255 1260 Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1265 1270 1275 1280 Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1285 1290 1295 Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln 1300 1305 1310 His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser 1315 1320 1325 Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1330 1335 1340 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile 1345 1350 1355 1360 Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys 1365 1370 1375 Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu 1380 1385 1390 Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu 1395 1400 1405 Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro 1410 1415 1420 Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr 1425 1430 1435 1440 Ala <210> 13 <211> 1443 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE8-Cas9 <400> 13 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 Ala Ser Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg Lys Val Gly Ile 35 40 45 His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile 50 55 60 Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val 65 70 75 80 Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile 85 90 95 Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala 100 105 110 Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg 115 120 125 Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala 130 135 140 Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val 145 150 155 160 Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val 165 170 175 Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg 180 185 190 Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr 195 200 205 Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu 210 215 220 Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln 225 230 235 240 Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala 245 250 255 Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser 260 265 270 Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn 275 280 285 Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn 290 295 300 Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser 305 310 315 320 Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly 325 330 335 Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala 340 345 350 Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala 355 360 365 Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp 370 375 380 Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr 385 390 395 400 Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile 405 410 415 Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile 420 425 430 Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg 435 440 445 Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro 450 455 460 His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu 465 470 475 480 Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile 485 490 495 Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn 500 505 510 Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro 515 520 525 Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe 530 535 540 Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val 545 550 555 560 Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu 565 570 575 Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe 580 585 590 Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr 595 600 605 Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys 610 615 620 Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe 625 630 635 640 Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp 645 650 655 Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile 660 665 670 Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg 675 680 685 Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu 690 695 700 Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile 705 710 715 720 Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu 725 730 735 Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp 740 745 750 Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly 755 760 765 Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro 770 775 780 Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu 785 790 795 800 Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met 805 810 815 Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu 820 825 830 Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile 835 840 845 Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu 850 855 860 Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu 865 870 875 880 Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro 885 890 895 Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg 900 905 910 Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val 915 920 925 Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu 930 935 940 Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly 945 950 955 960 Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu 965 970 975 Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met 980 985 990 Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val 995 1000 1005 Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln 1010 1015 1020 Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala 1025 1030 1035 1040 Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys 1045 1050 1055 Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg 1060 1065 1070 Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys 1075 1080 1085 Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr 1090 1095 1100 Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly 1105 1110 1115 1120 Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1125 1130 1135 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu 1140 1145 1150 Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn 1155 1160 1165 Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr 1170 1175 1180 Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala 1185 1190 1195 1200 Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu 1205 1210 1215 Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile 1220 1225 1230 Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile 1235 1240 1245 Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys 1250 1255 1260 Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala 1265 1270 1275 1280 Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu 1285 1290 1295 Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val 1300 1305 1310 Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu 1315 1320 1325 Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu 1330 1335 1340 Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu 1345 1350 1355 1360 Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1365 1370 1375 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr 1380 1385 1390 Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu 1395 1400 1405 Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala 1410 1415 1420 Asp Pro Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp Val Pro 1425 1430 1435 1440 Asp Tyr Ala <210> 14 <211> 1445 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE9-Cas9 <400> 14 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu Ala Ser Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg Lys Val 35 40 45 Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu 50 55 60 Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 65 70 75 80 Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His 85 90 95 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 100 105 110 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 115 120 125 Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 130 135 140 Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe 145 150 155 160 Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn 165 170 175 Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 180 185 190 Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 195 200 205 Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu 210 215 220 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 225 230 235 240 Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 245 250 255 Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 260 265 270 Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys 275 280 285 Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 290 295 300 Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 305 310 315 320 Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 325 330 335 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 340 345 350 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 355 360 365 Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 370 375 380 Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu 385 390 395 400 Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly 405 410 415 Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 420 425 430 Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 435 440 445 Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 450 455 460 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 465 470 475 480 Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 485 490 495 Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 500 505 510 Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile 515 520 525 Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln 530 535 540 Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 545 550 555 560 Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 565 570 575 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 580 585 590 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 595 600 605 Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 610 615 620 Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp 625 630 635 640 Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile 645 650 655 Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 660 665 670 Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 675 680 685 Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 690 695 700 Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 705 710 715 720 Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 725 730 735 Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile 740 745 750 His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val 755 760 765 Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly 770 775 780 Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 785 790 795 800 Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 805 810 815 Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 820 825 830 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 835 840 845 Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 850 855 860 Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp 865 870 875 880 Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile 885 890 895 Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu 900 905 910 Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 915 920 925 Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 930 935 940 Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 945 950 955 960 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 965 970 975 Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser 980 985 990 Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 995 1000 1005 Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp 1010 1015 1020 Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His 1025 1030 1035 1040 Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr 1045 1050 1055 Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp 1060 1065 1070 Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr 1075 1080 1085 Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu 1090 1095 1100 Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr 1105 1110 1115 1120 Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala 1125 1130 1135 Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys 1140 1145 1150 Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1155 1160 1165 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys 1170 1175 1180 Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val 1185 1190 1195 1200 Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys 1205 1210 1215 Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn 1220 1225 1230 Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp 1235 1240 1245 Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly 1250 1255 1260 Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu 1265 1270 1275 1280 Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His 1285 1290 1295 Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu 1300 1305 1310 Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile 1315 1320 1325 Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys 1330 1335 1340 Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln 1345 1350 1355 1360 Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro 1365 1370 1375 Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr 1380 1385 1390 Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1395 1400 1405 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser 1410 1415 1420 Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp 1425 1430 1435 1440 Val Pro Asp Tyr Ala 1445 <210> 15 <211> 1447 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE10-Cas9 <400> 15 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu Ala Ser Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg 35 40 45 Lys Val Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile 50 55 60 Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp 65 70 75 80 Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp 85 90 95 Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser 100 105 110 Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg 115 120 125 Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser 130 135 140 Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu 145 150 155 160 Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe 165 170 175 Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile 180 185 190 Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu 195 200 205 Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His 210 215 220 Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys 225 230 235 240 Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn 245 250 255 Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg 260 265 270 Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly 275 280 285 Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly 290 295 300 Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys 305 310 315 320 Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu 325 330 335 Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn 340 345 350 Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu 355 360 365 Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu 370 375 380 His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu 385 390 395 400 Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr 405 410 415 Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe 420 425 430 Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val 435 440 445 Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn 450 455 460 Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu 465 470 475 480 Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys 485 490 495 Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu 500 505 510 Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu 515 520 525 Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser 530 535 540 Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro 545 550 555 560 Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr 565 570 575 Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg 580 585 590 Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu 595 600 605 Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp 610 615 620 Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val 625 630 635 640 Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys 645 650 655 Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile 660 665 670 Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met 675 680 685 Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val 690 695 700 Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser 705 710 715 720 Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile 725 730 735 Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln 740 745 750 Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala 755 760 765 Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu 770 775 780 Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val 785 790 795 800 Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile 805 810 815 Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys 820 825 830 Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu 835 840 845 Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln 850 855 860 Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr 865 870 875 880 Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp 885 890 895 His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys 900 905 910 Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro 915 920 925 Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu 930 935 940 Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala 945 950 955 960 Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg 965 970 975 Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu 980 985 990 Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg 995 1000 1005 Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg 1010 1015 1020 Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His 1025 1030 1035 1040 Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys 1045 1050 1055 Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val 1060 1065 1070 Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys 1075 1080 1085 Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys 1090 1095 1100 Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile 1105 1110 1115 1120 Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp 1125 1130 1135 Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val 1140 1145 1150 Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu 1155 1160 1165 Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp 1170 1175 1180 Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1185 1190 1195 1200 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser 1205 1210 1215 Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu 1220 1225 1230 Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys 1235 1240 1245 Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu 1250 1255 1260 Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly 1265 1270 1275 1280 Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 1285 1290 1295 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys 1300 1305 1310 Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 1315 1320 1325 Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 1330 1335 1340 Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg 1345 1350 1355 1360 Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 1365 1370 1375 Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 1380 1385 1390 Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser 1395 1400 1405 Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly 1410 1415 1420 Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro 1425 1430 1435 1440 Tyr Asp Val Pro Asp Tyr Ala 1445 <210> 16 <211> 1449 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE11-Cas9 <400> 16 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu His Glu Ala Ser Leu Asp Ser Thr Ala Pro Lys Lys 35 40 45 Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr 50 55 60 Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile 65 70 75 80 Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn 85 90 95 Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe 100 105 110 Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg 115 120 125 Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile 130 135 140 Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu 145 150 155 160 Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro 165 170 175 Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro 180 185 190 Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala 195 200 205 Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg 210 215 220 Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val 225 230 235 240 Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu 245 250 255 Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser 260 265 270 Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu 275 280 285 Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser 290 295 300 Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp 305 310 315 320 Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn 325 330 335 Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala 340 345 350 Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn 355 360 365 Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr 370 375 380 Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln 385 390 395 400 Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn 405 410 415 Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr 420 425 430 Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu 435 440 445 Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe 450 455 460 Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala 465 470 475 480 Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg 485 490 495 Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly 500 505 510 Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser 515 520 525 Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly 530 535 540 Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn 545 550 555 560 Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr 565 570 575 Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly 580 585 590 Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val 595 600 605 Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys 610 615 620 Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser 625 630 635 640 Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu 645 650 655 Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu 660 665 670 Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg 675 680 685 Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp 690 695 700 Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg 705 710 715 720 Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys 725 730 735 Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe 740 745 750 Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln 755 760 765 Lys Ala Gln Val Ser Gly Gly Gly Asp Ser Leu His Glu His Ile Ala 770 775 780 Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val 785 790 795 800 Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu 805 810 815 Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly 820 825 830 Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys 835 840 845 Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln 850 855 860 Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp 865 870 875 880 Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp 885 890 895 Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp 900 905 910 Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn 915 920 925 Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln 930 935 940 Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr 945 950 955 960 Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile 965 970 975 Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln 980 985 990 Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu 995 1000 1005 Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp 1010 1015 1020 Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr 1025 1030 1035 1040 His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu 1045 1050 1055 Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr 1060 1065 1070 Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile 1075 1080 1085 Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe 1090 1095 1100 Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro 1105 1110 1115 1120 Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly 1125 1130 1135 Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn 1140 1145 1150 Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser 1155 1160 1165 Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp 1170 1175 1180 Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr 1185 1190 1195 1200 Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu 1205 1210 1215 Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1220 1225 1230 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu 1235 1240 1245 Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu 1250 1255 1260 Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln 1265 1270 1275 1280 Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr 1285 1290 1295 Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu 1300 1305 1310 Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile 1315 1320 1325 Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala 1330 1335 1340 Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro 1345 1350 1355 1360 Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn 1365 1370 1375 Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg 1380 1385 1390 Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His 1395 1400 1405 Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu 1410 1415 1420 Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Glu Phe 1425 1430 1435 1440 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1445 <210> 17 <211> 1451 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-HE12-Cas9 <400> 17 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu His Glu His Glu Ala Ser Leu Asp Ser Thr Ala Pro 35 40 45 Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp Lys 50 55 60 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 65 70 75 80 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 85 90 95 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 100 105 110 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 115 120 125 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 130 135 140 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 145 150 155 160 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 165 170 175 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 180 185 190 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 195 200 205 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 210 215 220 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 225 230 235 240 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 245 250 255 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 260 265 270 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 275 280 285 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 290 295 300 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 305 310 315 320 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 325 330 335 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 340 345 350 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 355 360 365 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 370 375 380 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 385 390 395 400 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 405 410 415 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 420 425 430 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 435 440 445 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 450 455 460 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 465 470 475 480 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 485 490 495 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 500 505 510 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 515 520 525 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 530 535 540 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 545 550 555 560 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 565 570 575 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 580 585 590 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 595 600 605 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 610 615 620 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 625 630 635 640 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 645 650 655 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 660 665 670 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 675 680 685 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 690 695 700 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 705 710 715 720 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 725 730 735 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 740 745 750 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 755 760 765 Ile Gln Lys Ala Gln Val Ser Gly Gly Gly Asp Ser Leu His Glu His 770 775 780 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 785 790 795 800 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 805 810 815 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 820 825 830 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 835 840 845 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 850 855 860 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 865 870 875 880 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 885 890 895 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 900 905 910 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 915 920 925 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 930 935 940 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 945 950 955 960 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 965 970 975 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 980 985 990 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 995 1000 1005 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 1010 1015 1020 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1025 1030 1035 1040 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1045 1050 1055 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1060 1065 1070 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1075 1080 1085 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1090 1095 1100 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1105 1110 1115 1120 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1125 1130 1135 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1140 1145 1150 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1155 1160 1165 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1170 1175 1180 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1185 1190 1195 1200 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1205 1210 1215 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1220 1225 1230 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1235 1240 1245 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1250 1255 1260 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1265 1270 1275 1280 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1285 1290 1295 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1300 1305 1310 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1315 1320 1325 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1330 1335 1340 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1345 1350 1355 1360 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1365 1370 1375 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1380 1385 1390 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1395 1400 1405 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1410 1415 1420 Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1425 1430 1435 1440 Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1445 1450 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; AP <400> 18 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser 1 5 10 <210> 19 <211> 1420 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; AP-Cas9 <400> 19 Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser Leu Asp Ser Thr Ala 1 5 10 15 Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp 20 25 30 Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp 35 40 45 Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val 50 55 60 Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala 65 70 75 80 Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg 85 90 95 Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu 100 105 110 Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe 115 120 125 His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu 130 135 140 Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu 145 150 155 160 Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr 165 170 175 Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile 180 185 190 Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn 195 200 205 Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln 210 215 220 Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala 225 230 235 240 Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile 245 250 255 Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile 260 265 270 Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu 275 280 285 Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp 290 295 300 Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe 305 310 315 320 Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu 325 330 335 Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile 340 345 350 Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu 355 360 365 Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln 370 375 380 Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu 385 390 395 400 Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr 405 410 415 Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln 420 425 430 Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu 435 440 445 Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys 450 455 460 Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr 465 470 475 480 Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr 485 490 495 Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val 500 505 510 Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe 515 520 525 Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu 530 535 540 Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val 545 550 555 560 Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys 565 570 575 Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys 580 585 590 Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val 595 600 605 Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr 610 615 620 His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu 625 630 635 640 Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe 645 650 655 Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu 660 665 670 Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly 675 680 685 Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln 690 695 700 Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn 705 710 715 720 Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu 725 730 735 Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu 740 745 750 His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu 755 760 765 Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His 770 775 780 Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr 785 790 795 800 Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu 805 810 815 Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu 820 825 830 Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn 835 840 845 Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser 850 855 860 Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp 865 870 875 880 Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys 885 890 895 Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr 900 905 910 Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp 915 920 925 Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala 930 935 940 Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His 945 950 955 960 Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn 965 970 975 Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu 980 985 990 Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile 995 1000 1005 Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly 1010 1015 1020 Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr 1025 1030 1035 1040 Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu 1045 1050 1055 Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile 1060 1065 1070 Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg 1075 1080 1085 Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1090 1095 1100 Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro 1105 1110 1115 1120 Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser 1125 1130 1135 Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg 1140 1145 1150 Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr 1155 1160 1165 Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser 1170 1175 1180 Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu 1185 1190 1195 1200 Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly 1205 1210 1215 Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser 1220 1225 1230 Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1235 1240 1245 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn 1250 1255 1260 Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu 1265 1270 1275 1280 Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu 1285 1290 1295 Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu 1300 1305 1310 Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg 1315 1320 1325 Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr 1330 1335 1340 Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr 1345 1350 1355 1360 Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr 1365 1370 1375 Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu 1380 1385 1390 Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys 1395 1400 1405 Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1410 1415 1420 <210> 20 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; dNP2 <400> 20 Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Gly Ser Lys Ile Lys 1 5 10 15 Lys Val Lys Lys Lys Gly Arg Lys 20 <210> 21 <211> 1435 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; dNP2-Cas9 <400> 21 Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Gly Ser Lys Ile Lys 1 5 10 15 Lys Val Lys Lys Lys Gly Arg Lys Ala Ser Leu Asp Ser Thr Ala Pro 20 25 30 Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gly Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1410 1415 1420 Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1425 1430 1435 <210> 22 <211> 51 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; dNP2-HE <400> 22 Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Gly Ser Lys Ile Lys 1 5 10 15 Lys Val Lys Lys Lys Gly Arg Lys Ala Ser Gly Gly Gly Gly Gly His 20 25 30 Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu His 35 40 45 Glu His Glu 50 <210> 23 <211> 1460 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; dNP2-HE10-Cas9 <400> 23 Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Gly Ser Lys Ile Lys 1 5 10 15 Lys Val Lys Lys Lys Gly Arg Lys Ala Ser Gly Gly Gly Gly Gly His 20 25 30 Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu His 35 40 45 Glu His Glu Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg Lys Val Gly 50 55 60 Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp 65 70 75 80 Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys 85 90 95 Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser 100 105 110 Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr 115 120 125 Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg 130 135 140 Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met 145 150 155 160 Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu 165 170 175 Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile 180 185 190 Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu 195 200 205 Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile 210 215 220 Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile 225 230 235 240 Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile 245 250 255 Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn 260 265 270 Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys 275 280 285 Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys 290 295 300 Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro 305 310 315 320 Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu 325 330 335 Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile 340 345 350 Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp 355 360 365 Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys 370 375 380 Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln 385 390 395 400 Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys 405 410 415 Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr 420 425 430 Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro 435 440 445 Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn 450 455 460 Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile 465 470 475 480 Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln 485 490 495 Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys 500 505 510 Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly 515 520 525 Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr 530 535 540 Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser 545 550 555 560 Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys 565 570 575 Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn 580 585 590 Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala 595 600 605 Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys 610 615 620 Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys 625 630 635 640 Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg 645 650 655 Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys 660 665 670 Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp 675 680 685 Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu 690 695 700 Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln 705 710 715 720 Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu 725 730 735 Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe 740 745 750 Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His 755 760 765 Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser 770 775 780 Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser 785 790 795 800 Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu 805 810 815 Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu 820 825 830 Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg 835 840 845 Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln 850 855 860 Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys 865 870 875 880 Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln 885 890 895 Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val 900 905 910 Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr 915 920 925 Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu 930 935 940 Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys 945 950 955 960 Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly 965 970 975 Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val 980 985 990 Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg 995 1000 1005 Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys 1010 1015 1020 Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe 1025 1030 1035 1040 Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp 1045 1050 1055 Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro 1060 1065 1070 Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val 1075 1080 1085 Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala 1090 1095 1100 Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile 1105 1110 1115 1120 Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn 1125 1130 1135 Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr 1140 1145 1150 Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1155 1160 1165 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg 1170 1175 1180 Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys 1185 1190 1195 1200 Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val 1205 1210 1215 Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu 1220 1225 1230 Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro 1235 1240 1245 Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu 1250 1255 1260 Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg 1265 1270 1275 1280 Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu 1285 1290 1295 Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr 1300 1305 1310 Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe 1315 1320 1325 Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser 1330 1335 1340 Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val 1345 1350 1355 1360 Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala 1365 1370 1375 Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala 1380 1385 1390 Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1395 1400 1405 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly 1410 1415 1420 Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg 1425 1430 1435 1440 Ala Asp Pro Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp Val 1445 1450 1455 Pro Asp Tyr Ala 1460 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; R9 <400> 24 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 25 <211> 1420 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; R9-Cas9 <400> 25 Arg Arg Arg Arg Arg Arg Arg Arg Arg Ala Ser Leu Asp Ser Thr Ala 1 5 10 15 Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp 20 25 30 Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp 35 40 45 Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val 50 55 60 Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala 65 70 75 80 Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg 85 90 95 Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu 100 105 110 Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe 115 120 125 His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu 130 135 140 Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu 145 150 155 160 Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr 165 170 175 Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile 180 185 190 Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn 195 200 205 Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln 210 215 220 Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala 225 230 235 240 Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile 245 250 255 Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile 260 265 270 Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu 275 280 285 Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp 290 295 300 Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe 305 310 315 320 Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu 325 330 335 Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile 340 345 350 Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu 355 360 365 Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln 370 375 380 Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu 385 390 395 400 Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr 405 410 415 Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln 420 425 430 Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu 435 440 445 Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys 450 455 460 Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr 465 470 475 480 Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr 485 490 495 Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val 500 505 510 Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe 515 520 525 Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu 530 535 540 Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val 545 550 555 560 Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys 565 570 575 Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys 580 585 590 Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val 595 600 605 Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr 610 615 620 His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu 625 630 635 640 Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe 645 650 655 Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu 660 665 670 Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly 675 680 685 Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln 690 695 700 Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn 705 710 715 720 Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu 725 730 735 Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu 740 745 750 His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu 755 760 765 Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His 770 775 780 Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr 785 790 795 800 Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu 805 810 815 Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu 820 825 830 Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn 835 840 845 Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser 850 855 860 Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp 865 870 875 880 Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys 885 890 895 Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr 900 905 910 Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp 915 920 925 Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala 930 935 940 Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His 945 950 955 960 Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn 965 970 975 Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu 980 985 990 Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile 995 1000 1005 Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly 1010 1015 1020 Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr 1025 1030 1035 1040 Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu 1045 1050 1055 Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile 1060 1065 1070 Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg 1075 1080 1085 Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp 1090 1095 1100 Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro 1105 1110 1115 1120 Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser 1125 1130 1135 Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg 1140 1145 1150 Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr 1155 1160 1165 Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser 1170 1175 1180 Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu 1185 1190 1195 1200 Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly 1205 1210 1215 Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser 1220 1225 1230 Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1235 1240 1245 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn 1250 1255 1260 Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu 1265 1270 1275 1280 Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu 1285 1290 1295 Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu 1300 1305 1310 Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg 1315 1320 1325 Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr 1330 1335 1340 Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr 1345 1350 1355 1360 Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr 1365 1370 1375 Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu 1380 1385 1390 Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys 1395 1400 1405 Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1410 1415 1420 <210> 26 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; R9-HE10 <400> 26 Arg Arg Arg Arg Arg Arg Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu 35 <210> 27 <211> 1445 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; R9-HE10-Cas9 <400> 27 Arg Arg Arg Arg Arg Arg Arg Arg Arg Ala Ser Gly Gly Gly Gly Gly 1 5 10 15 His Glu His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg Lys Val 35 40 45 Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile Gly Leu 50 55 60 Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr 65 70 75 80 Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His 85 90 95 Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 100 105 110 Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 115 120 125 Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 130 135 140 Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe 145 150 155 160 Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn 165 170 175 Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 180 185 190 Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 195 200 205 Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu 210 215 220 Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe 225 230 235 240 Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 245 250 255 Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 260 265 270 Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys 275 280 285 Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 290 295 300 Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln 305 310 315 320 Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 325 330 335 Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 340 345 350 Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 355 360 365 Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 370 375 380 Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu 385 390 395 400 Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly 405 410 415 Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 420 425 430 Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 435 440 445 Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 450 455 460 Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg 465 470 475 480 Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 485 490 495 Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 500 505 510 Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile 515 520 525 Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln 530 535 540 Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu 545 550 555 560 Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 565 570 575 Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 580 585 590 Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 595 600 605 Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 610 615 620 Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp 625 630 635 640 Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile 645 650 655 Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 660 665 670 Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 675 680 685 Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 690 695 700 Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys 705 710 715 720 Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 725 730 735 Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile 740 745 750 His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val 755 760 765 Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly 770 775 780 Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp 785 790 795 800 Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 805 810 815 Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 820 825 830 Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 835 840 845 Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 850 855 860 Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp 865 870 875 880 Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile 885 890 895 Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu 900 905 910 Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 915 920 925 Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 930 935 940 Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg 945 950 955 960 Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 965 970 975 Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser 980 985 990 Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 995 1000 1005 Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp 1010 1015 1020 Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His 1025 1030 1035 1040 Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr 1045 1050 1055 Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp 1060 1065 1070 Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr 1075 1080 1085 Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu 1090 1095 1100 Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr 1105 1110 1115 1120 Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala 1125 1130 1135 Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys 1140 1145 1150 Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1155 1160 1165 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys 1170 1175 1180 Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val 1185 1190 1195 1200 Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys 1205 1210 1215 Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn 1220 1225 1230 Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp 1235 1240 1245 Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly 1250 1255 1260 Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu 1265 1270 1275 1280 Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His 1285 1290 1295 Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu 1300 1305 1310 Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile 1315 1320 1325 Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys 1330 1335 1340 Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln 1345 1350 1355 1360 Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro 1365 1370 1375 Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr 1380 1385 1390 Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1395 1400 1405 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser 1410 1415 1420 Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro Tyr Asp 1425 1430 1435 1440 Val Pro Asp Tyr Ala 1445 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; TAT <400> 28 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 29 <211> 1422 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; TAT-Cas9 <400> 29 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ser Leu Asp Ser 1 5 10 15 Thr Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala 20 25 30 Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 35 40 45 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 50 55 60 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 65 70 75 80 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 85 90 95 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 100 105 110 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 115 120 125 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 130 135 140 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 145 150 155 160 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 165 170 175 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 180 185 190 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 195 200 205 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 210 215 220 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 225 230 235 240 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 245 250 255 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 260 265 270 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 275 280 285 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 290 295 300 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 305 310 315 320 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 325 330 335 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 340 345 350 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 355 360 365 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 370 375 380 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 385 390 395 400 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 405 410 415 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 420 425 430 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 435 440 445 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 450 455 460 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 465 470 475 480 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 485 490 495 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 500 505 510 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 515 520 525 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 530 535 540 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 545 550 555 560 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 565 570 575 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 580 585 590 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 595 600 605 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 610 615 620 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 625 630 635 640 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 645 650 655 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 660 665 670 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 675 680 685 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 690 695 700 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 705 710 715 720 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 725 730 735 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 740 745 750 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 755 760 765 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 770 775 780 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 785 790 795 800 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 805 810 815 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 820 825 830 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 835 840 845 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 850 855 860 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 865 870 875 880 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 885 890 895 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 900 905 910 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 915 920 925 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 930 935 940 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 945 950 955 960 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 965 970 975 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 980 985 990 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 995 1000 1005 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 1010 1015 1020 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 1025 1030 1035 1040 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys 1045 1050 1055 Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser 1060 1065 1070 Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1075 1080 1085 Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1090 1095 1100 Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1105 1110 1115 1120 Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly 1125 1130 1135 Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 1140 1145 1150 Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1155 1160 1165 Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly 1170 1175 1180 Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1185 1190 1195 1200 Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1205 1210 1215 Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1220 1225 1230 Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser 1235 1240 1245 Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1250 1255 1260 Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1265 1270 1275 1280 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His 1285 1290 1295 Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val 1300 1305 1310 Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1315 1320 1325 His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu 1330 1335 1340 Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1345 1350 1355 1360 Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp 1365 1370 1375 Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 1380 1385 1390 Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys 1395 1400 1405 Arg Lys Val Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1410 1415 1420 <210> 30 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide; TAT-HE10 <400> 30 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ser Gly Gly Gly 1 5 10 15 Gly Gly His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu His Glu 35 <210> 31 <211> 1447 <212> PRT <213> Artificial Sequence <220> <223> penetrating peptide-linkage-Cas protein; TAT-HE-Cas9 <400> 31 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Ser Gly Gly Gly 1 5 10 15 Gly Gly His Glu His Glu His Glu His Glu His Glu His Glu His Glu 20 25 30 His Glu His Glu His Glu Leu Asp Ser Thr Ala Pro Lys Lys Lys Arg 35 40 45 Lys Val Gly Ile His Gly Val Pro Ala Ala Asp Lys Lys Tyr Ser Ile 50 55 60 Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp 65 70 75 80 Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp 85 90 95 Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser 100 105 110 Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg 115 120 125 Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser 130 135 140 Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu 145 150 155 160 Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe 165 170 175 Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile 180 185 190 Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu 195 200 205 Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His 210 215 220 Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys 225 230 235 240 Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn 245 250 255 Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg 260 265 270 Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly 275 280 285 Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly 290 295 300 Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys 305 310 315 320 Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu 325 330 335 Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn 340 345 350 Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu 355 360 365 Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu 370 375 380 His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu 385 390 395 400 Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr 405 410 415 Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe 420 425 430 Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val 435 440 445 Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn 450 455 460 Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu 465 470 475 480 Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys 485 490 495 Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu 500 505 510 Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu 515 520 525 Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser 530 535 540 Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro 545 550 555 560 Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr 565 570 575 Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg 580 585 590 Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu 595 600 605 Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp 610 615 620 Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val 625 630 635 640 Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys 645 650 655 Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile 660 665 670 Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met 675 680 685 Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val 690 695 700 Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser 705 710 715 720 Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile 725 730 735 Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln 740 745 750 Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala 755 760 765 Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu 770 775 780 Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val 785 790 795 800 Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile 805 810 815 Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys 820 825 830 Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu 835 840 845 Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln 850 855 860 Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr 865 870 875 880 Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp 885 890 895 His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys 900 905 910 Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro 915 920 925 Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu 930 935 940 Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala 945 950 955 960 Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg 965 970 975 Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu 980 985 990 Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg 995 1000 1005 Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg 1010 1015 1020 Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His 1025 1030 1035 1040 Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys 1045 1050 1055 Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val 1060 1065 1070 Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys 1075 1080 1085 Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys 1090 1095 1100 Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile 1105 1110 1115 1120 Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp 1125 1130 1135 Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val 1140 1145 1150 Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu 1155 1160 1165 Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp 1170 1175 1180 Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1185 1190 1195 1200 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser 1205 1210 1215 Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu 1220 1225 1230 Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys 1235 1240 1245 Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu 1250 1255 1260 Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly 1265 1270 1275 1280 Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 1285 1290 1295 Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys 1300 1305 1310 Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 1315 1320 1325 Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 1330 1335 1340 Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg 1345 1350 1355 1360 Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 1365 1370 1375 Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 1380 1385 1390 Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser 1395 1400 1405 Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly 1410 1415 1420 Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val Glu Phe Tyr Pro 1425 1430 1435 1440 Tyr Asp Val Pro Asp Tyr Ala 1445 <210> 32 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE5 <400> 32 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgagcatg agcacgaaca cgaacacgaa 120 120 <210> 33 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE6 <400> 33 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgagcacg agcatgagca cgaacacgaa 120 cacgaa 126 <210> 34 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE7 <400> 34 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacacg agcacgagca tgagcacgaa 120 cacgaacacg aa 132 <210> 35 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE8 <400> 35 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacatg aacacgagca cgagcatgag 120 cacgaacacg aacacgaa 138 <210> 36 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE9 <400> 36 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacatg aacatgaaca cgagcacgag 120 catgagcacg aacacgaaca cgaa 144 <210> 37 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE10 <400> 37 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgaacatg aacatgaaca tgaacacgag 120 cacgagcatg agcacgaaca cgaacacgaa 150 <210> 38 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE11 <400> 38 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacacg aacatgaaca tgaacatgaa 120 cacgagcacg agcatgagca cgaacacgaa cacgaa 156 <210> 39 <211> 162 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP-HE12 <400> 39 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgaacatg aacacgaaca tgaacatgaa 120 catgaacacg agcacgagca tgagcacgaa cacgaacacg aa 162 <210> 40 <211> 4230 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; Cas9 <400> 40 agcctggaca gcaccgctcc caaaaagaaa aggaaggtgg gcattcacgg cgtgcctgcg 60 gccgacaaaa agtacagcat cggccttgat atcggcacca atagcgtggg ctgggccgtt 120 atcacagacg aatacaaggt acccagcaag aagttcaagg tgctggggaa tacagacagg 180 cactctatca agaaaaacct tatcggggct ctgctgtttg actcaggcga gaccgccgag 240 gccaccaggt tgaagaggac cgcaaggcga aggtacaccc ggaggaagaa caggatctgc 300 tatctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacagg 360 ctggaggaga gcttccttgt cgaggaggat aagaagcacg aacgacaccc catcttcggc 420 aacatagtcg acgaggtcgc ttatcacgag aagtacccca ccatctacca cctgcgaaag 480 aaattggtgg atagcaccga taaagccgac ttgcgactta tctacttggc tctggcgcac 540 atgattaagt tcaggggcca cttcctgatc gagggcgacc ttaaccccga caacagtgac 600 gtagacaaat tgttcatcca gcttgtacag acctataacc agctgttcga ggaaaaccct 660 attaacgcca gcggggtgga tgcgaaggcc atacttagcg ccaggctgag caaaagcagg 720 cgcttggaga acctgatagc ccagctgccc ggtgaaaaga agaacggcct cttcggtaat 780 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggcagaa 840 gatgccaagc tgcagttgag taaggacacc tatgacgacg acttggacaa tctgctcgcc 900 caaatcggcg accagtacgc tgacctgttc ctcgccgcca agaacctttc tgacgcaatc 960 ctgcttagcg atatccttag ggtgaacaca gagatcacca aggcccccct gagcgccagc 1020 atgatcaaga ggtacgacga gcaccatcag gacctgaccc ttctgaaggc cctggtgagg 1080 cagcaactgc ccgagaagta caaggagatc tttttcgacc agagcaagaa cggctacgcc 1140 ggctacatcg acggcggagc cagccaagag gagttctaca agttcatcaa gcccatcctg 1200 gagaagatgg atggcaccga ggagctgctg gtgaagctga acagggaaga tttgctccgg 1260 aagcagagga cctttgacaa cggtagcatc ccccaccaga tccacctggg cgagctgcac 1320 gcaatactga ggcgacagga ggatttctac cccttcctca aggacaatag ggagaaaatc 1380 gaaaagattc tgaccttcag gatcccctac tacgtgggcc ctcttgccag gggcaacagc 1440 cgattcgctt ggatgacaag aaagagcgag gagaccatca ccccctggaa cttcgaggaa 1500 gtggtggaca aaggagcaag cgcgcagtct ttcatcgaac ggatgaccaa tttcgacaaa 1560 aacctgccta acgagaaggt gctgcccaag cacagcctgc tttacgagta cttcaccgtg 1620 tacaacgagc tcaccaaggt gaaatatgtg accgagggca tgcgaaaacc cgctttcctg 1680 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaacag gaaggtgacc 1740 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gctttgatag cgtggaaata 1800 agcggcgtgg aggacaggtt caacgccagc ctgggcacct accacgactt gttgaagata 1860 atcaaagaca aggatttcct ggataatgag gagaacgagg atatactcga ggacatcgtg 1920 ctgactttga ccctgtttga ggaccgagag atgattgaag aaaggctcaa aacctacgcc 1980 cacctgttcg acgacaaagt gatgaaacaa ctgaagagac gaagatacac cggctggggc 2040 agactgtcca ggaagctcat caacggcatt agggacaagc agagcggcaa gaccatcctg 2100 gatttcctga agtccgacgg cttcgccaac cgaaacttca tgcagctgat tcacgatgac 2160 agcttgacct tcaaggagga catccagaag gcccaggtta gcggccaggg cgactccctg 2220 cacgaacata ttgcaaacct ggcaggctcc cctgcgatca agaagggcat actgcagacc 2280 gttaaggttg tggacgaatt ggtcaaggtc atgggcaggc acaagcccga aaacatagtt 2340 atagagatgg ccagagagaa ccagaccacc caaaagggcc agaagaacag ccgggagcgc 2400 atgaaaagga tcgaggaggg tatcaaggaa ctcggaagcc agatcctcaa agagcacccc 2460 gtggagaata cccagctcca gaacgagaag ctgtacctgt actacctgca gaacggcagg 2520 gacatgtacg ttgaccagga gttggacatc aacaggcttt cagactatga cgtggatcac 2580 atagtgcccc agagctttct taaagacgat agcatcgaca acaaggtcct gacccgctcc 2640 gacaaaaaca ggggcaaaag cgacaacgtg ccaagcgaag aggtggttaa aaagatgaag 2700 aactactgga ggcaactgct caacgcgaaa ttgatcaccc agagaaagtt cgataacctg 2760 accaaggccg agaggggcgg actctccgaa cttgacaaag cgggcttcat aaagaggcag 2820 ctggtcgaga cccgacagat cacgaagcac gtggcccaaa tcctcgacag cagaatgaat 2880 accaagtacg atgagaatga caaactcatc agggaagtga aagtgattac cctgaagagc 2940 aagttggtgt ccgactttcg caaagatttc cagttctaca aggtgaggga gatcaacaac 3000 taccaccatg cccacgacgc atacctgaac gccgtggtcg gcaccgccct gattaagaag 3060 tatccaaagc tggagtccga atttgtctac ggcgactaca aagtttacga tgtgaggaag 3120 atgatcgcta agagcgaaca ggagatcggc aaggccaccg ctaagtattt cttctacagc 3180 aacatcatga actttttcaa gaccgagatc acacttgcca acggcgaaat caggaagagg 3240 ccgcttatcg agaccaacgg tgagaccggc gagatcgtgt gggacaaggg cagggacttc 3300 gccaccgtga ggaaagtcct gagcatgccc caggtgaata ttgtgaaaaa aactgaggtg 3360 cagacaggcg gctttagcaa ggaatccatc ctgcccaaga ggaacagcga caagctgatc 3420 gcccggaaga aggactggga ccctaagaag tatggaggct tcgacagccc caccgtagcc 3480 tacagcgtgc tggtggtcgc gaaggtagag aaggggaaga gcaagaaact gaagagcgtg 3540 aaggagctgc tcggcataac catcatggag aggtccagct ttgagaagaa ccccattgac 3600 tttttggaag ccaagggcta caaagaggtc aaaaaggacc tgatcatcaa actccccaag 3660 tactccctgt ttgaattgga gaacggcaga aagaggatgc tggcgagcgc tggggaactg 3720 caaaagggca acgaactggc gctgcccagc aagtacgtga attttctgta cctggcgtcc 3780 cactacgaaa agctgaaagg cagccccgag gacaacgagc agaagcagct gttcgtggag 3840 cagcacaagc attacctgga cgagataatc gagcaaatca gcgagttcag caagagggtg 3900 attctggccg acgcgaacct ggataaggtc ctcagcgcct acaacaagca ccgagacaaa 3960 cccatcaggg agcaggccga gaatatcata cacctgttca ccctgacaaa tctgggcgca 4020 cctgcggcat tcaaatactt cgataccacc atcgacagga aaaggtacac tagcactaag 4080 gaggtgctgg atgccacctt gatccaccag tccattaccg gcctgtatga gaccaggatc 4140 gacctgagcc agcttggagg cgactctagg gcggacccaa aaaagaaaag gaaggtggaa 4200 ttctacccat acgatgttcc agattacgct 4230 <210> 41 <211> 4353 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE5-Cas9 <400> 41 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgagcatg agcacgaaca cgaacacgaa 120 gctagcctgg acagcaccgc tcccaaaaag aaaaggaagg tgggcattca cggcgtgcct 180 gcggccgaca aaaagtacag catcggcctt gatatcggca ccaatagcgt gggctgggcc 240 gttatcacag acgaatacaa ggtacccagc aagaagttca aggtgctggg gaatacagac 300 aggcactcta tcaagaaaaa ccttatcggg gctctgctgt ttgactcagg cgagaccgcc 360 gaggccacca ggttgaagag gaccgcaagg cgaaggtaca cccggaggaa gaacaggatc 420 tgctatctgc aggagatctt cagcaacgag atggccaagg tggacgacag cttcttccac 480 aggctggagg agagcttcct tgtcgaggag gataagaagc acgaacgaca ccccatcttc 540 ggcaacatag tcgacgaggt cgcttatcac gagaagtacc ccaccatcta ccacctgcga 600 aagaaattgg tggatagcac cgataaagcc gacttgcgac ttatctactt ggctctggcg 660 cacatgatta agttcagggg ccacttcctg atcgagggcg accttaaccc cgacaacagt 720 gacgtagaca aattgttcat ccagcttgta cagacctata accagctgtt cgaggaaaac 780 cctattaacg ccagcggggt ggatgcgaag gccatactta gcgccaggct gagcaaaagc 840 aggcgcttgg agaacctgat agcccagctg cccggtgaaa agaagaacgg cctcttcggt 900 aatctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggca 960 gaagatgcca agctgcagtt gagtaaggac acctatgacg acgacttgga caatctgctc 1020 gcccaaatcg gcgaccagta cgctgacctg ttcctcgccg ccaagaacct ttctgacgca 1080 atcctgctta gcgatatcct tagggtgaac acagagatca ccaaggcccc cctgagcgcc 1140 agcatgatca agaggtacga cgagcaccat caggacctga cccttctgaa ggccctggtg 1200 aggcagcaac tgcccgagaa gtacaaggag atctttttcg accagagcaa gaacggctac 1260 gccggctaca tcgacggcgg agccagccaa gaggagttct acaagttcat caagcccatc 1320 ctggagaaga tggatggcac cgaggagctg ctggtgaagc tgaacaggga agatttgctc 1380 cggaagcaga ggacctttga caacggtagc atcccccacc agatccacct gggcgagctg 1440 cacgcaatac tgaggcgaca ggaggatttc taccccttcc tcaaggacaa tagggagaaa 1500 atcgaaaaga ttctgacctt caggatcccc tactacgtgg gccctcttgc caggggcaac 1560 agccgattcg cttggatgac aagaaagagc gaggagacca tcaccccctg gaacttcgag 1620 gaagtggtgg acaaaggagc aagcgcgcag tctttcatcg aacggatgac caatttcgac 1680 aaaaacctgc ctaacgagaa ggtgctgccc aagcacagcc tgctttacga gtacttcacc 1740 gtgtacaacg agctcaccaa ggtgaaatat gtgaccgagg gcatgcgaaa acccgctttc 1800 ctgagcggcg agcagaagaa ggccatcgtg gacctgctgt tcaagaccaa caggaaggtg 1860 accgtgaagc agctgaagga ggactacttc aagaagatcg agtgctttga tagcgtggaa 1920 ataagcggcg tggaggacag gttcaacgcc agcctgggca cctaccacga cttgttgaag 1980 ataatcaaag acaaggattt cctggataat gaggagaacg aggatatact cgaggacatc 2040 gtgctgactt tgaccctgtt tgaggaccga gagatgattg aagaaaggct caaaacctac 2100 gcccacctgt tcgacgacaa agtgatgaaa caactgaaga gacgaagata caccggctgg 2160 ggcagactgt ccaggaagct catcaacggc attagggaca agcagagcgg caagaccatc 2220 ctggatttcc tgaagtccga cggcttcgcc aaccgaaact tcatgcagct gattcacgat 2280 gacagcttga ccttcaagga ggacatccag aaggcccagg ttagcggcca gggcgactcc 2340 ctgcacgaac atattgcaaa cctggcaggc tcccctgcga tcaagaaggg catactgcag 2400 accgttaagg ttgtggacga attggtcaag gtcatgggca ggcacaagcc cgaaaacata 2460 gttatagaga tggccagaga gaaccagacc acccaaaagg gccagaagaa cagccgggag 2520 cgcatgaaaa ggatcgagga gggtatcaag gaactcggaa gccagatcct caaagagcac 2580 cccgtggaga atacccagct ccagaacgag aagctgtacc tgtactacct gcagaacggc 2640 agggacatgt acgttgacca ggagttggac atcaacaggc tttcagacta tgacgtggat 2700 cacatagtgc cccagagctt tcttaaagac gatagcatcg acaacaaggt cctgacccgc 2760 tccgacaaaa acaggggcaa aagcgacaac gtgccaagcg aagaggtggt taaaaagatg 2820 aagaactact ggaggcaact gctcaacgcg aaattgatca cccagagaaa gttcgataac 2880 ctgaccaagg ccgagagggg cggactctcc gaacttgaca aagcgggctt cataaagagg 2940 cagctggtcg agacccgaca gatcacgaag cacgtggccc aaatcctcga cagcagaatg 3000 aataccaagt acgatgagaa tgacaaactc atcagggaag tgaaagtgat taccctgaag 3060 agcaagttgg tgtccgactt tcgcaaagat ttccagttct acaaggtgag ggagatcaac 3120 aactaccacc atgccccacga cgcatacctg aacgccgtgg tcggcaccgc cctgattaag 3180 aagtatccaa agctggagtc cgaatttgtc tacggcgact acaaagttta cgatgtgagg 3240 aagatgatcg ctaagagcga acaggagatc ggcaaggcca ccgctaagta tttcttctac 3300 agcaacatca tgaacttttt caagaccgag atcacacttg ccaacggcga aatcaggaag 3360 aggccgctta tcgagaccaa cggtgagacc ggcgagatcg tgtgggacaa gggcagggac 3420 ttcgccaccg tgaggaaagt cctgagcatg ccccaggtga atattgtgaa aaaaactgag 3480 gtgcagacag gcggctttag caaggaatcc atcctgccca agaggaacag cgacaagctg 3540 atcgcccgga agaaggactg ggaccctaag aagtatggag gcttcgacag ccccaccgta 3600 gcctacagcg tgctggtggt cgcgaaggta gagaagggga agagcaagaa actgaagagc 3660 gtgaaggagc tgctcggcat aaccatcatg gagaggtcca gctttgagaa gaaccccatt 3720 gactttttgg aagccaaggg ctacaaagag gtcaaaaagg acctgatcat caaactcccc 3780 aagtactccc tgtttgaatt ggagaacggc agaaagagga tgctggcgag cgctggggaa 3840 ctgcaaaagg gcaacgaact ggcgctgccc agcaagtacg tgaattttct gtacctggcg 3900 tcccactacg aaaagctgaa aggcagcccc gaggacaacg agcagaagca gctgttcgtg 3960 gagcagcaca agcattacct ggacgagata atcgagcaaa tcagcgagtt cagcaagagg 4020 gtgattctgg ccgacgcgaa cctggataag gtcctcagcg cctacaacaa gcaccgagac 4080 aaacccatca gggagcaggc cgagaatatc atacacctgt tcaccctgac aaatctgggc 4140 gcacctgcgg cattcaaata cttcgatacc accatcgaca ggaaaaggta cactagcact 4200 aaggaggtgc tggatgccac cttgatccac cagtccatta ccggcctgta tgagaccagg 4260 atcgacctga gccagcttgg aggcgactct agggcggacc caaaaaagaa aaggaaggtg 4320 gaattctacc catacgatgt tccagattac gct 4353 <210> 42 <211> 4359 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE6-Cas9 <400> 42 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgagcacg agcatgagca cgaacacgaa 120 cacgaagcta gcctggacag caccgctccc aaaaagaaaa ggaaggtggg cattcacggc 180 gtgcctgcgg ccgacaaaaa gtacagcatc ggccttgata tcggcaccaa tagcgtgggc 240 tgggccgtta tcacagacga atacaaggta cccagcaaga agttcaaggt gctggggaat 300 acagacaggc actctatcaa gaaaaacctt atcggggctc tgctgtttga ctcaggcgag 360 accgccgagg ccaccaggtt gaagaggacc gcaaggcgaa ggtacacccg gaggaagaac 420 aggatctgct atctgcagga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc 480 ttccacaggc tggaggagag cttccttgtc gaggaggata agaagcacga acgacacccc 540 atcttcggca acatagtcga cgaggtcgct tatcacgaga agtaccccac catctaccac 600 ctgcgaaaga aattggtgga tagcaccgat aaagccgact tgcgacttat ctacttggct 660 ctggcgcaca tgattaagtt caggggccac ttcctgatcg agggcgacct taaccccgac 720 aacagtgacg tagacaaatt gttcatccag cttgtacaga cctataacca gctgttcgag 780 gaaaacccta ttaacgccag cggggtggat gcgaaggcca tacttagcgc caggctgagc 840 aaaagcaggc gcttggagaa cctgatagcc cagctgcccg gtgaaaagaa gaacggcctc 900 ttcggtaatc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac 960 ctggcagaag atgccaagct gcagttgagt aaggacacct atgacgacga cttggacaat 1020 ctgctcgccc aaatcggcga ccagtacgct gacctgttcc tcgccgccaa gaacctttct 1080 gacgcaatcc tgcttagcga tatccttagg gtgaacacag agatcaccaa ggcccccctg 1140 agcgccagca tgatcaagag gtacgacgag caccatcagg acctgaccct tctgaaggcc 1200 ctggtgaggc agcaactgcc cgagaagtac aaggagatct ttttcgacca gagcaagaac 1260 ggctacgccg gctacatcga cggcggagcc agccaagagg agttctacaa gttcatcaag 1320 cccatcctgg agaagatgga tggcaccgag gagctgctgg tgaagctgaa cagggaagat 1380 ttgctccgga agcagaggac ctttgacaac ggtagcatcc cccaccagat ccacctgggc 1440 gagctgcacg caatactgag gcgacaggag gatttctacc ccttcctcaa ggacaatagg 1500 gagaaaatcg aaaagattct gaccttcagg atcccctact acgtgggccc tcttgccagg 1560 ggcaacagcc gattcgcttg gatgacaaga aagagcgagg agaccatcac cccctggaac 1620 ttcgaggaag tggtggacaa aggagcaagc gcgcagtctt tcatcgaacg gatgaccaat 1680 ttcgacaaaa acctgcctaa cgagaaggtg ctgcccaagc acagcctgct ttacgagtac 1740 ttcaccgtgt acaacgagct caccaaggtg aaatatgtga ccgagggcat gcgaaaaccc 1800 gctttcctga gcggcgagca gaagaaggcc atcgtggacc tgctgttcaa gaccaacagg 1860 aaggtgaccg tgaagcagct gaaggaggac tacttcaaga agatcgagtg ctttgatagc 1920 gtggaaataa gcggcgtgga ggacaggttc aacgccagcc tgggcaccta ccacgacttg 1980 ttgaagataa tcaaagacaa ggatttcctg gataatgagg agaacgagga tatactcgag 2040 gacatcgtgc tgactttgac cctgtttgag gaccgagaga tgattgaaga aaggctcaaa 2100 acctacgccc acctgttcga cgacaaagtg atgaaacaac tgaagagacg aagatacacc 2160 ggctggggca gactgtccag gaagctcatc aacggcatta gggacaagca gagcggcaag 2220 accatcctgg atttcctgaa gtccgacggc ttcgccaacc gaaacttcat gcagctgatt 2280 cacgatgaca gcttgacctt caaggaggac atccagaagg cccaggttag cggccagggc 2340 gactccctgc acgaacatat tgcaaacctg gcaggctccc ctgcgatcaa gaagggcata 2400 ctgcagaccg ttaaggttgt ggacgaattg gtcaaggtca tgggcaggca caagcccgaa 2460 aacatagtta tagagatggc cagagagaac cagaccaccc aaaagggcca gaagaacagc 2520 cgggagcgca tgaaaaggat cgaggagggt atcaaggaac tcggaagcca gatcctcaaa 2580 gagcaccccg tggagaatac ccagctccag aacgagaagc tgtacctgta ctacctgcag 2640 aacggcaggg acatgtacgt tgaccaggag ttggacatca acaggctttc agactatgac 2700 gtggatcaca tagtgcccca gagctttctt aaagacgata gcatcgacaa caaggtcctg 2760 acccgctccg acaaaaacag gggcaaaagc gacaacgtgc caagcgaaga ggtggttaaa 2820 aagatgaaga actactggag gcaactgctc aacgcgaaat tgatcaccca gagaaagttc 2880 gataacctga ccaaggccga gaggggcgga ctctccgaac ttgacaaagc gggcttcata 2940 aagaggcagc tggtcgagac ccgacagatc acgaagcacg tggcccaaat cctcgacagc 3000 agaatgaata ccaagtacga tgagaatgac aaactcatca gggaagtgaa agtgattacc 3060 ctgaagagca agttggtgtc cgactttcgc aaagatttcc agttctacaa ggtgagggag 3120 atcaacaact accaccatgc ccacgacgca tacctgaacg ccgtggtcgg caccgccctg 3180 attaagaagt atccaaagct ggagtccgaa tttgtctacg gcgactacaa agtttacgat 3240 gtgaggaaga tgatcgctaa gagcgaacag gagatcggca aggccaccgc taagtatttc 3300 ttctacagca acatcatgaa ctttttcaag accgagatca cacttgccaa cggcgaaatc 3360 aggaagaggc cgcttatcga gaccaacggt gagaccggcg agatcgtgtg ggacaagggc 3420 agggacttcg ccaccgtgag gaaagtcctg agcatgcccc aggtgaatat tgtgaaaaaa 3480 actgaggtgc agacaggcgg ctttagcaag gaatccatcc tgcccaagag gaacagcgac 3540 aagctgatcg cccggaagaa ggactgggac cctaagaagt atggaggctt cgacagcccc 3600 accgtagcct acagcgtgct ggtggtcgcg aaggtagaga aggggaagag caagaaactg 3660 aagagcgtga aggagctgct cggcataacc atcatggaga ggtccagctt tgagaagaac 3720 cccattgact ttttggaagc caagggctac aaagaggtca aaaaggacct gatcatcaaa 3780 ctccccaagt actccctgtt tgaattggag aacggcagaa agaggatgct ggcgagcgct 3840 ggggaactgc aaaagggcaa cgaactggcg ctgcccagca agtacgtgaa ttttctgtac 3900 ctggcgtccc actacgaaaa gctgaaaggc agccccgagg acaacgagca gaagcagctg 3960 ttcgtggagc agcacaagca ttacctggac gagataatcg agcaaatcag cgagttcagc 4020 aagagggtga ttctggccga cgcgaacctg gataaggtcc tcagcgccta caacaagcac 4080 cgagacaaac ccatcaggga gcaggccgag aatatcatac acctgttcac cctgacaaat 4140 ctgggcgcac ctgcggcatt caaatacttc gataccacca tcgacaggaa aaggtacact 4200 agcactaagg aggtgctgga tgccaccttg atccaccagt ccattaccgg cctgtatgag 4260 accaggatcg acctgagcca gcttggaggc gactctaggg cggacccaaa aaagaaaagg 4320 aaggtggaat tctacccata cgatgttcca gattacgct 4359 <210> 43 <211> 4365 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE7-Cas9 <400> 43 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacacg agcacgagca tgagcacgaa 120 cacgaacacg aagctagcct ggacagcacc gctcccaaaa agaaaaggaa ggtgggcatt 180 cacggcgtgc ctgcggccga caaaaagtac agcatcggcc ttgatatcgg caccaatagc 240 gtgggctggg ccgttatcac agacgaatac aaggtaccca gcaagaagtt caaggtgctg 300 gggaatacag acaggcactc tatcaagaaa aaccttatcg gggctctgct gtttgactca 360 ggcgagaccg ccgaggccac caggttgaag aggaccgcaa ggcgaaggta cacccggagg 420 aagaacagga tctgctatct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 480 agcttcttcc acaggctgga ggagagcttc cttgtcgagg aggataagaa gcacgaacga 540 caccccatct tcggcaacat agtcgacgag gtcgcttatc acgagaagta ccccaccatc 600 taccacctgc gaaagaaatt ggtggatagc accgataaag ccgacttgcg acttatctac 660 ttggctctgg cgcacatgat taagttcagg ggccacttcc tgatcgaggg cgaccttaac 720 cccgacaaca gtgacgtaga caaattgttc atccagcttg tacagaccta taaccagctg 780 ttcgaggaaa accctattaa cgccagcggg gtggatgcga aggccatact tagcgccagg 840 ctgagcaaaa gcaggcgctt ggagaacctg atagcccagc tgcccggtga aaagaagaac 900 ggcctcttcg gtaatctgat tgccctgagc ctgggcctga cccccaactt caagagcaac 960 ttcgacctgg cagaagatgc caagctgcag ttgagtaagg acacctatga cgacgacttg 1020 gacaatctgc tcgcccaaat cggcgaccag tacgctgacc tgttcctcgc cgccaagaac 1080 ctttctgacg caatcctgct tagcgatatc cttagggtga acacagagat caccaaggcc 1140 cccctgagcg ccagcatgat caagaggtac gacgagcacc atcaggacct gacccttctg 1200 aaggccctgg tgaggcagca actgcccgag aagtacaagg agatcttttt cgaccagagc 1260 aagaacggct acgccggcta catcgacggc ggagccagcc aagaggagtt ctacaagttc 1320 atcaagccca tcctggagaa gatggatggc accgaggagc tgctggtgaa gctgaacagg 1380 gaagattgc tccggaagca gaggaccttt gacaacggta gcatccccca ccagatccac 1440 ctgggcgagc tgcacgcaat actgaggcga caggaggatt tctacccctt cctcaaggac 1500 aatagggaga aaatcgaaaa gattctgacc ttcaggatcc cctactacgt gggccctctt 1560 gccaggggca acagccgatt cgcttggatg acaagaaaga gcgaggagac catcaccccc 1620 tggaacttcg aggaagtggt ggacaaagga gcaagcgcgc agtctttcat cgaacggatg 1680 accaatttcg acaaaaacct gcctaacgag aaggtgctgc ccaagcacag cctgctttac 1740 gagtacttca ccgtgtacaa cgagctcacc aaggtgaaat atgtgaccga gggcatgcga 1800 aaacccgctt tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1860 aacaggaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttt 1920 gatagcgtgg aaataagcgg cgtggaggac aggttcaacg ccagcctggg cacctaccac 1980 gacttgttga agataatcaa agacaaggat ttcctggata atgaggagaa cgaggatata 2040 ctcgaggaca tcgtgctgac tttgaccctg tttgaggacc gagagatgat tgaagaaagg 2100 ctcaaaacct acgcccacct gttcgacgac aaagtgatga aacaactgaa gagacgaaga 2160 tacaccggct ggggcagact gtccaggaag ctcatcaacg gcattaggga caagcagagc 2220 ggcaagacca tcctggattt cctgaagtcc gacggcttcg ccaaccgaaa cttcatgcag 2280 ctgattcacg atgacagctt gaccttcaag gaggacatcc agaaggccca ggttagcggc 2340 cagggcgact ccctgcacga acatattgca aacctggcag gctcccctgc gatcaagaag 2400 ggcatactgc agaccgttaa ggttgtggac gaattggtca aggtcatggg caggcacaag 2460 cccgaaaaca tagttataga gatggccaga gagaaccaga ccacccaaaa gggccagaag 2520 aacagccggg agcgcatgaa aaggatcgag gagggtatca aggaactcgg aagccagatc 2580 ctcaaagagc accccgtgga gaatacccag ctccagaacg agaagctgta cctgtactac 2640 ctgcagaacg gcagggacat gtacgttgac caggagttgg acatcaacag gctttcagac 2700 tatgacgtgg atcacatagt gccccagagc tttcttaaag acgatagcat cgacaacaag 2760 gtcctgaccc gctccgacaa aaacaggggc aaaagcgaca acgtgccaag cgaagaggtg 2820 gttaaaaaga tgaagaacta ctggaggcaa ctgctcaacg cgaaattgat cacccagaga 2880 aagttcgata acctgaccaa ggccgagagg ggcggactct ccgaacttga caaagcgggc 2940 ttcataaaga ggcagctggt cgagacccga cagatcacga agcacgtggc ccaaatcctc 3000 gacagcagaa tgaataccaa gtacgatgag aatgacaaac tcatcaggga agtgaaagtg 3060 attaccctga agagcaagtt ggtgtccgac tttcgcaaag atttccagtt ctacaaggtg 3120 agggagatca acaactacca ccatgcccac gacgcatacc tgaacgccgt ggtcggcacc 3180 gccctgatta agaagtatcc aaagctggag tccgaatttg tctacggcga ctacaaagtt 3240 tacgatgtga ggaagatgat cgctaagagc gaacaggaga tcggcaaggc caccgctaag 3300 tatttcttct acagcaacat catgaacttt ttcaagaccg agatcacact tgccaacggc 3360 gaaatcagga agaggccgct tatcgagacc aacggtgaga ccggcgagat cgtgtgggac 3420 aagggcaggg acttcgccac cgtgaggaaa gtcctgagca tgccccaggt gaatattgtg 3480 aaaaaaactg aggtgcagac aggcggcttt agcaaggaat ccatcctgcc caagaggaac 3540 agcgacaagc tgatcgcccg gaagaaggac tgggacccta agaagtatgg aggcttcgac 3600 agccccaccg tagcctacag cgtgctggtg gtcgcgaagg tagagaaggg gaagagcaag 3660 aaactgaaga gcgtgaagga gctgctcggc ataaccatca tggagaggtc cagctttgag 3720 aagaacccca ttgacttttt ggaagccaag ggctacaaag aggtcaaaaa ggacctgatc 3780 atcaaactcc ccaagtactc cctgtttgaa ttggagaacg gcagaaagag gatgctggcg 3840 agcgctgggg aactgcaaaa gggcaacgaa ctggcgctgc ccagcaagta cgtgaatttt 3900 ctgtacctgg cgtcccacta cgaaaagctg aaaggcagcc ccgaggacaa cgagcagaag 3960 cagctgttcg tggagcagca caagcattac ctggacgaga taatcgagca aatcagcgag 4020 ttcagcaaga gggtgattct ggccgacgcg aacctggata aggtcctcag cgcctacaac 4080 aagcaccgag acaaacccat cagggagcag gccgagaata tcatacacct gttcaccctg 4140 acaaatctgg gcgcacctgc ggcattcaaa tacttcgata ccaccatcga caggaaaagg 4200 tacactagca ctaaggaggt gctggatgcc accttgatcc accagtccat taccggcctg 4260 tatgagacca ggatcgacct gagccagctt ggaggcgact ctagggcgga cccaaaaaag 4320 aaaaggaagg tggaattcta cccatacgat gttccagatt acgct 4365 <210> 44 <211> 4371 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE8-Cas9 <400> 44 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacatg aacacgagca cgagcatgag 120 cacgaacacg aacacgaagc tagcctggac agcaccgctc ccaaaaagaa aaggaaggtg 180 ggcattcacg gcgtgcctgc ggccgacaaa aagtacagca tcggccttga tatcggcacc 240 aatagcgtgg gctgggccgt tatcacagac gaatacaagg tacccagcaa gaagttcaag 300 gtgctgggga atacagacag gcactctatc aagaaaaacc ttatcggggc tctgctgttt 360 gactcaggcg agaccgccga ggccaccagg ttgaagagga ccgcaaggcg aaggtacacc 420 cggaggaaga acaggatctg ctatctgcag gagatcttca gcaacgagat ggccaaggtg 480 gacgacagct tcttccacag gctggaggag agcttccttg tcgaggagga taagaagcac 540 gaacgacacc ccatcttcgg caacatagtc gacgaggtcg cttatcacga gaagtacccc 600 accatctacc acctgcgaaa gaaattggtg gatagcaccg ataaagccga cttgcgactt 660 atctacttgg ctctggcgca catgattaag ttcaggggcc acttcctgat cgagggcgac 720 cttaaccccg acaacagtga cgtagacaaa ttgttcatcc agcttgtaca gacctataac 780 cagctgttcg aggaaaaccc tattaacgcc agcggggtgg atgcgaaggc catacttagc 840 gccaggctga gcaaaagcag gcgcttggag aacctgatag cccagctgcc cggtgaaaag 900 aagaacggcc tcttcggtaa tctgattgcc ctgagcctgg gcctgacccc caacttcaag 960 agcaacttcg acctggcaga agatgccaag ctgcagttga gtaaggacac ctatgacgac 1020 gacttggaca atctgctcgc ccaaatcggc gaccagtacg ctgacctgtt cctcgccgcc 1080 aagaaccttt ctgacgcaat cctgcttagc gatatcctta gggtgaacac agagatcacc 1140 aaggcccccc tgagcgccag catgatcaag aggtacgacg agcaccatca ggacctgacc 1200 cttctgaagg ccctggtgag gcagcaactg cccgagaagt acaaggagat ctttttcgac 1260 cagagcaaga acggctacgc cggctacatc gacggcggag ccagccaaga ggagttctac 1320 aagttcatca agcccatcct ggagaagatg gatggcaccg aggagctgct ggtgaagctg 1380 aacagggaag atttgctccg gaagcagagg acctttgaca acggtagcat cccccaccag 1440 atccacctgg gcgagctgca cgcaatactg aggcgacagg aggatttcta ccccttcctc 1500 aaggacaata gggagaaaat cgaaaagatt ctgaccttca ggatccccta ctacgtgggc 1560 cctcttgcca ggggcaacag ccgattcgct tggatgacaa gaaagagcga ggagaccatc 1620 accccctgga acttcgagga agtggtggac aaaggagcaa gcgcgcagtc tttcatcgaa 1680 cggatgacca atttcgacaa aaacctgcct aacgagaagg tgctgcccaa gcacagcctg 1740 ctttacgagt acttcaccgt gtacaacgag ctcaccaagg tgaaatatgt gaccgagggc 1800 atgcgaaaac ccgctttcct gagcggcgag cagaagaagg ccatcgtgga cctgctgttc 1860 aagaccaaca ggaaggtgac cgtgaagcag ctgaaggagg actacttcaa gaagatcgag 1920 tgctttgata gcgtggaaat aagcggcgtg gaggacaggt tcaacgccag cctgggcacc 1980 taccacgact tgttgaagat aatcaaagac aaggatttcc tggataatga ggagaacgag 2040 gatatactcg aggacatcgt gctgactttg accctgtttg aggaccgaga gatgattgaa 2100 gaaaggctca aaacctacgc ccacctgttc gacgacaaag tgatgaaaca actgaagaga 2160 cgaagataca ccggctgggg cagactgtcc aggaagctca tcaacggcat tagggacaag 2220 cagagcggca agaccatcct ggatttcctg aagtccgacg gcttcgccaa ccgaaacttc 2280 atgcagctga ttcacgatga cagcttgacc ttcaaggagg acatccagaa ggcccaggtt 2340 agcggccagg gcgactccct gcacgaacat attgcaaacc tggcaggctc ccctgcgatc 2400 aagaagggca tactgcagac cgttaaggtt gtggacgaat tggtcaaggt catgggcagg 2460 cacaagcccg aaaacatagt tatagagatg gccagagaga accagaccac ccaaaagggc 2520 cagaagaaca gccgggagcg catgaaaagg atcgaggagg gtatcaagga actcggaagc 2580 cagatcctca aagagcaccc cgtggagaat acccagctcc agaacgagaa gctgtacctg 2640 tactacctgc agaacggcag ggacatgtac gttgaccagg agttggacat caacaggctt 2700 tcagactatg acgtggatca catagtgccc cagagctttc ttaaagacga tagcatcgac 2760 aacaaggtcc tgacccgctc cgacaaaaac aggggcaaaa gcgacaacgt gccaagcgaa 2820 gaggtggtta aaaagatgaa gaactactgg aggcaactgc tcaacgcgaa attgatcacc 2880 cagagaaagt tcgataacct gaccaaggcc gagaggggcg gactctccga acttgacaaa 2940 gcgggcttca taaagaggca gctggtcgag acccgacaga tcacgaagca cgtggcccaa 3000 atcctcgaca gcagaatgaa taccaagtac gatgagaatg acaaactcat cagggaagtg 3060 aaagtgatta ccctgaagag caagttggtg tccgactttc gcaaagattt ccagttctac 3120 aaggtgaggg agatcaacaa ctaccaccat gcccacgacg catacctgaa cgccgtggtc 3180 ggcaccgccc tgattaagaa gtatccaaag ctggagtccg aatttgtcta cggcgactac 3240 aaagtttacg atgtgaggaa gatgatcgct aagagcgaac aggagatcgg caaggccacc 3300 gctaagtatt tcttctacag caacatcatg aactttttca agaccgagat cacacttgcc 3360 aacggcgaaa tcaggaagag gccgcttatc gagaccaacg gtgagaccgg cgagatcgtg 3420 tgggacaagg gcagggactt cgccaccgtg aggaaagtcc tgagcatgcc ccaggtgaat 3480 attgtgaaaa aaactgaggt gcagacaggc ggctttagca aggaatccat cctgcccaag 3540 aggaacagcg acaagctgat cgcccggaag aaggactggg accctaagaa gtatggaggc 3600 ttcgacagcc ccaccgtagc ctacagcgtg ctggtggtcg cgaaggtaga gaaggggaag 3660 agcaagaaac tgaagagcgt gaaggagctg ctcggcataa ccatcatgga gaggtccagc 3720 tttgagaaga accccattga ctttttggaa gccaagggct acaaagaggt caaaaaggac 3780 ctgatcatca aactccccaa gtactccctg tttgaattgg agaacggcag aaagaggatg 3840 ctggcgagcg ctggggaact gcaaaagggc aacgaactgg cgctgcccag caagtacgtg 3900 aattttctgt acctggcgtc ccactacgaa aagctgaaag gcagccccga ggacaacgag 3960 cagaagcagc tgttcgtgga gcagcacaag cattacctgg acgagataat cgagcaaatc 4020 agcgagttca gcaagagggt gattctggcc gacgcgaacc tggataaggt cctcagcgcc 4080 tacaacaagc accgagacaa acccatcagg gagcaggccg agaatatcat acacctgttc 4140 accctgacaa atctgggcgc acctgcggca ttcaaatact tcgataccac catcgacagg 4200 aaaaggtaca ctagcactaa ggaggtgctg gatgccacct tgatccacca gtccattacc 4260 ggcctgtatg agaccaggat cgacctgagc cagcttggag gcgactctag ggcggaccca 4320 aaaaagaaaa ggaaggtgga attctaccca tacgatgttc cagattacgc t 4371 <210> 45 <211> 4377 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE9-Cas9 <400> 45 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacatg aacatgaaca cgagcacgag 120 catgagcacg aacacgaaca cgaagctagc ctggacagca ccgctcccaa aaagaaaagg 180 aaggtgggca ttcacggcgt gcctgcggcc gacaaaaagt acagcatcgg ccttgatatc 240 ggcaccaata gcgtgggctg ggccgttatc acagacgaat acaaggtacc cagcaagaag 300 ttcaaggtgc tggggaatac agacaggcac tctatcaaga aaaaccttat cggggctctg 360 ctgtttgact caggcgagac cgccgaggcc accaggttga agaggaccgc aaggcgaagg 420 tacacccgga ggaagaacag gatctgctat ctgcaggaga tcttcagcaa cgagatggcc 480 aaggtggacg acagcttctt ccacaggctg gaggagagct tccttgtcga ggaggataag 540 aagcacgaac gacaccccat cttcggcaac atagtcgacg aggtcgctta tcacgagaag 600 taccccacca tctaccacct gcgaaagaaa ttggtggata gcaccgataa agccgacttg 660 cgacttatct acttggctct ggcgcacatg attaagttca ggggccactt cctgatcgag 720 ggcgacctta accccgacaa cagtgacgta gacaaattgt tcatccagct tgtacagacc 780 tataaccagc tgttcgagga aaaccctatt aacgccagcg gggtggatgc gaaggccata 840 cttagcgcca ggctgagcaa aagcaggcgc ttggagaacc tgatagccca gctgcccggt 900 gaaaagaaga acggcctctt cggtaatctg attgccctga gcctgggcct gacccccaac 960 ttcaagagca acttcgacct ggcagaagat gccaagctgc agttgagtaa ggacacctat 1020 gacgacgact tggacaatct gctcgcccaa atcggcgacc agtacgctga cctgttcctc 1080 gccgccaaga acctttctga cgcaatcctg cttagcgata tccttagggt gaacacagag 1140 atcaccaagg cccccctgag cgccagcatg atcaagaggt acgacgagca ccatcaggac 1200 ctgacccttc tgaaggccct ggtgaggcag caactgcccg agaagtacaa ggagatcttt 1260 ttcgaccaga gcaagaacgg ctacgccggc tacatcgacg gcggagccag ccaagaggag 1320 ttctacaagt tcatcaagcc catcctggag aagatggatg gcaccgagga gctgctggtg 1380 aagctgaaca gggaagattt gctccggaag cagaggacct ttgacaacgg tagcatcccc 1440 caccagatcc acctgggcga gctgcacgca atactgaggc gacaggagga tttctacccc 1500 ttcctcaagg acaataggga gaaaatcgaa aagattctga ccttcaggat cccctactac 1560 gtgggccctc ttgccagggg caacagccga ttcgcttgga tgacaagaaa gagcgaggag 1620 accatcaccc cctggaactt cgaggaagtg gtggacaaag gagcaagcgc gcagtctttc 1680 atcgaacgga tgaccaattt cgacaaaaac ctgcctaacg agaaggtgct gcccaagcac 1740 agcctgcttt acgagtactt caccgtgtac aacgagctca ccaaggtgaa atatgtgacc 1800 gagggcatgc gaaaacccgc tttcctgagc ggcgagcaga agaaggccat cgtggacctg 1860 ctgttcaaga ccaacaggaa ggtgaccgtg aagcagctga aggaggacta cttcaagaag 1920 atcgagtgct ttgatagcgt ggaaataagc ggcgtggagg acaggttcaa cgccagcctg 1980 ggcacctacc acgacttgtt gaagataatc aaagacaagg atttcctgga taatgaggag 2040 aacgaggata tactcgagga catcgtgctg actttgaccc tgtttgagga ccgagagatg 2100 attgaagaaa ggctcaaaac ctacgcccac ctgttcgacg acaaagtgat gaaacaactg 2160 aagagacgaa gatacaccgg ctggggcaga ctgtccagga agctcatcaa cggcattagg 2220 gacaagcaga gcggcaagac catcctggat ttcctgaagt ccgacggctt cgccaaccga 2280 aacttcatgc agctgattca cgatgacagc ttgaccttca aggaggacat ccagaaggcc 2340 caggttagcg gccagggcga ctccctgcac gaacatattg caaacctggc aggctcccct 2400 gcgatcaaga agggcatact gcagaccgtt aaggttgtgg acgaattggt caaggtcatg 2460 ggcaggcaca agcccgaaaa catagttata gagatggcca gagagaacca gaccacccaa 2520 aagggccaga agaacagccg ggagcgcatg aaaaggatcg aggagggtat caaggaactc 2580 ggaagccaga tcctcaaaga gcaccccgtg gagaataccc agctccagaa cgagaagctg 2640 tacctgtact acctgcagaa cggcagggac atgtacgttg accaggagtt ggacatcaac 2700 aggctttcag actatgacgt ggatcacata gtgccccaga gctttcttaa agacgatagc 2760 atcgacaaca aggtcctgac ccgctccgac aaaaacaggg gcaaaagcga caacgtgcca 2820 agcgaagagg tggttaaaaa gatgaagaac tactggaggc aactgctcaa cgcgaaattg 2880 atcacccaga gaaagttcga taacctgacc aaggccgaga ggggcggact ctccgaactt 2940 gacaaagcgg gcttcataaa gaggcagctg gtcgagaccc gacagatcac gaagcacgtg 3000 gcccaaatcc tcgacagcag aatgaatacc aagtacgatg agaatgacaa actcatcagg 3060 gaagtgaaag tgattaccct gaagagcaag ttggtgtccg actttcgcaa agatttccag 3120 ttctacaagg tgagggagat caacaactac caccatgccc acgacgcata cctgaacgcc 3180 gtggtcggca ccgccctgat taagaagtat ccaaagctgg agtccgaatt tgtctacggc 3240 gactacaaag tttacgatgt gaggaagatg atcgctaaga gcgaacagga gatcggcaag 3300 gccaccgcta agtatttctt ctacagcaac atcatgaact ttttcaagac cgagatcaca 3360 cttgccaacg gcgaaatcag gaagaggccg cttatcgaga ccaacggtga gaccggcgag 3420 atcgtgtggg acaagggcag ggacttcgcc accgtgagga aagtcctgag catgccccag 3480 gtgaatattg tgaaaaaaac tgaggtgcag acaggcggct ttagcaagga atccatcctg 3540 cccaagagga acagcgacaa gctgatcgcc cggaagaagg actgggaccc taagaagtat 3600 ggaggcttcg acagccccac cgtagcctac agcgtgctgg tggtcgcgaa ggtagagaag 3660 gggaagagca agaaactgaa gagcgtgaag gagctgctcg gcataaccat catggagagg 3720 tccagctttg agaagaaccc cattgacttt ttggaagcca agggctacaa agaggtcaaa 3780 aaggacctga tcatcaaact ccccaagtac tccctgtttg aattggagaa cggcagaaag 3840 aggatgctgg cgagcgctgg ggaactgcaa aagggcaacg aactggcgct gcccagcaag 3900 tacgtgaatt ttctgtacct ggcgtcccac tacgaaaagc tgaaaggcag ccccgaggac 3960 aacgagcaga agcagctgtt cgtggagcag cacaagcatt acctggacga gataatcgag 4020 caaatcagcg agttcagcaa gagggtgatt ctggccgacg cgaacctgga taaggtcctc 4080 agcgcctaca acaagcaccg agacaaaccc atcagggagc aggccgagaa tatcatacac 4140 ctgttcaccc tgacaaatct gggcgcacct gcggcattca aatacttcga taccaccatc 4200 gacaggaaaa ggtacactag cactaaggag gtgctggatg ccaccttgat ccaccagtcc 4260 attaccggcc tgtatgagac caggatcgac ctgagccagc ttggaggcga ctctagggcg 4320 gacccaaaaa agaaaaggaa ggtggaattc tacccatacg atgttccaga tacgct 4377 <210> 46 <211> 4383 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE10-Cas9 <400> 46 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgaacatg aacatgaaca tgaacacgag 120 cacgagcatg agcacgaaca cgaacacgaa gctagcctgg acagcaccgc tcccaaaaag 180 aaaaggaagg tgggcattca cggcgtgcct gcggccgaca aaaagtacag catcggcctt 240 gatatcggca ccaatagcgt gggctgggcc gttatcacag acgaatacaa ggtacccagc 300 aagaagttca aggtgctggg gaatacagac aggcactcta tcaagaaaaa ccttatcggg 360 gctctgctgt ttgactcagg cgagaccgcc gaggccacca ggttgaagag gaccgcaagg 420 cgaaggtaca cccggaggaa gaacaggatc tgctatctgc aggagatctt cagcaacgag 480 atggccaagg tggacgacag cttcttccac aggctggagg agagcttcct tgtcgaggag 540 gataagaagc acgaacgaca ccccatcttc ggcaacatag tcgacgaggt cgcttatcac 600 gagaagtacc ccaccatcta ccacctgcga aagaaattgg tggatagcac cgataaagcc 660 gacttgcgac ttatctactt ggctctggcg cacatgatta agttcagggg ccacttcctg 720 atcgagggcg accttaaccc cgacaacagt gacgtagaca aattgttcat ccagcttgta 780 cagacctata accagctgtt cgaggaaaac cctattaacg ccagcggggt ggatgcgaag 840 gccatactta gcgccaggct gagcaaaagc aggcgcttgg agaacctgat agcccagctg 900 cccggtgaaa agaagaacgg cctcttcggt aatctgattg ccctgagcct gggcctgacc 960 cccaacttca agagcaactt cgacctggca gaagatgcca agctgcagtt gagtaaggac 1020 acctatgacg acgacttgga caatctgctc gcccaaatcg gcgaccagta cgctgacctg 1080 ttcctcgccg ccaagaacct ttctgacgca atcctgctta gcgatatcct tagggtgaac 1140 acagagatca ccaaggcccc cctgagcgcc agcatgatca agaggtacga cgagcaccat 1200 caggacctga cccttctgaa ggccctggtg aggcagcaac tgcccgagaa gtacaaggag 1260 atctttttcg accagagcaa gaacggctac gccggctaca tcgacggcgg agccagccaa 1320 gaggagttct acaagttcat caagcccatc ctggagaaga tggatggcac cgaggagctg 1380 ctggtgaagc tgaacaggga agatttgctc cggaagcaga ggacctttga caacggtagc 1440 atcccccacc agatccacct gggcgagctg cacgcaatac tgaggcgaca ggaggatttc 1500 taccccttcc tcaaggacaa tagggagaaa atcgaaaaga ttctgacctt caggatcccc 1560 tactacgtgg gccctcttgc caggggcaac agccgattcg cttggatgac aagaaagagc 1620 gaggagacca tcaccccctg gaacttcgag gaagtggtgg acaaaggagc aagcgcgcag 1680 tctttcatcg aacggatgac caatttcgac aaaaacctgc ctaacgagaa ggtgctgccc 1740 aagcacagcc tgctttacga gtacttcacc gtgtacaacg agctcaccaa ggtgaaatat 1800 gtgaccgagg gcatgcgaaa acccgctttc ctgagcggcg agcagaagaa ggccatcgtg 1860 gacctgctgt tcaagaccaa caggaaggtg accgtgaagc agctgaagga ggactacttc 1920 aagaagatcg agtgctttga tagcgtggaa ataagcggcg tggaggacag gttcaacgcc 1980 agcctgggca cctaccacga cttgttgaag ataatcaaag acaaggattt cctggataat 2040 gaggagaacg aggatatact cgaggacatc gtgctgactt tgaccctgtt tgaggaccga 2100 gagatgattg aagaaaggct caaaacctac gcccacctgt tcgacgacaa agtgatgaaa 2160 caactgaaga gacgaagata caccggctgg ggcagactgt ccaggaagct catcaacggc 2220 attagggaca agcagagcgg caagaccatc ctggatttcc tgaagtccga cggcttcgcc 2280 aaccgaaact tcatgcagct gattcacgat gacagcttga ccttcaagga ggacatccag 2340 aaggcccagg ttagcggcca gggcgactcc ctgcacgaac atattgcaaa cctggcaggc 2400 tcccctgcga tcaagaaggg catactgcag accgttaagg ttgtggacga attggtcaag 2460 gtcatgggca ggcacaagcc cgaaaacata gttatagaga tggccagaga gaaccagacc 2520 acccaaaagg gccagaagaa cagccgggag cgcatgaaaa ggatcgagga gggtatcaag 2580 gaactcggaa gccagatcct caaagagcac cccgtggaga atacccagct ccagaacgag 2640 aagctgtacc tgtactacct gcagaacggc agggacatgt acgttgacca ggagttggac 2700 atcaacaggc tttcagacta tgacgtggat cacatagtgc cccagagctt tcttaaagac 2760 gatagcatcg acaacaaggt cctgacccgc tccgacaaaa acaggggcaa aagcgacaac 2820 gtgccaagcg aagaggtggt taaaaagatg aagaactact ggaggcaact gctcaacgcg 2880 aaattgatca cccagagaaa gttcgataac ctgaccaagg ccgagagggg cggactctcc 2940 gaacttgaca aagcgggctt cataaagagg cagctggtcg agacccgaca gatcacgaag 3000 cacgtggccc aaatcctcga cagcagaatg aataccaagt acgatgagaa tgacaaactc 3060 atcagggaag tgaaagtgat taccctgaag agcaagttgg tgtccgactt tcgcaaagat 3120 ttccagttct acaaggtgag ggagatcaac aactaccacc atgcccacga cgcatacctg 3180 aacgccgtgg tcggcaccgc cctgattaag aagtatccaa agctggagtc cgaatttgtc 3240 tacggcgact acaaagttta cgatgtgagg aagatgatcg ctaagagcga acaggagatc 3300 ggcaaggcca ccgctaagta tttcttctac agcaacatca tgaacttttt caagaccgag 3360 atcacacttg ccaacggcga aatcaggaag aggccgctta tcgagaccaa cggtgagacc 3420 ggcgagatcg tgtgggacaa gggcagggac ttcgccaccg tgaggaaagt cctgagcatg 3480 ccccaggtga atattgtgaa aaaaactgag gtgcagacag gcggctttag caaggaatcc 3540 atcctgccca agaggaacag cgacaagctg atcgcccgga agaaggactg ggaccctaag 3600 aagtatggag gcttcgacag ccccaccgta gcctacagcg tgctggtggt cgcgaaggta 3660 gagaagggga agagcaagaa actgaagagc gtgaaggagc tgctcggcat aaccatcatg 3720 gagaggtcca gctttgagaa gaaccccatt gactttttgg aagccaaggg ctacaaagag 3780 gtcaaaaagg acctgatcat caaactcccc aagtactccc tgtttgaatt ggagaacggc 3840 agaaagagga tgctggcgag cgctggggaa ctgcaaaagg gcaacgaact ggcgctgccc 3900 agcaagtacg tgaattttct gtacctggcg tcccactacg aaaagctgaa aggcagcccc 3960 gaggacaacg agcagaagca gctgttcgtg gagcagcaca agcattacct ggacgagata 4020 atcgagcaaa tcagcgagtt cagcaagagg gtgattctgg ccgacgcgaa cctggataag 4080 gtcctcagcg cctacaacaa gcaccgagac aaacccatca gggagcaggc cgagaatatc 4140 atacacctgt tcaccctgac aaatctgggc gcacctgcgg cattcaaata cttcgatacc 4200 accatcgaca ggaaaaggta cactagcact aaggaggtgc tggatgccac cttgatccac 4260 cagtccatta ccggcctgta tgagaccagg atcgacctga gccagcttgg aggcgactct 4320 agggcggacc caaaaaagaa aaggaaggtg gaattctacc catacgatgt tccagattac 4380 gct 4383 <210> 47 <211> 4389 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE11-Cas9 <400> 47 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt catgaacacg aacatgaaca tgaacatgaa 120 cacgagcacg agcatgagca cgaacacgaa cacgaagcta gcctggacag caccgctccc 180 aaaaagaaaa ggaaggtggg cattcacggc gtgcctgcgg ccgacaaaaa gtacagcatc 240 ggccttgata tcggcaccaa tagcgtgggc tgggccgtta tcacagacga atacaaggta 300 cccagcaaga agttcaaggt gctggggaat acagacaggc actctatcaa gaaaaacctt 360 atcggggctc tgctgtttga ctcaggcgag accgccgagg ccaccaggtt gaagaggacc 420 gcaaggcgaa ggtacacccg gaggaagaac aggatctgct atctgcagga gatcttcagc 480 aacgagatgg ccaaggtgga cgacagcttc ttccacaggc tggaggagag cttccttgtc 540 gaggaggata agaagcacga acgacacccc atcttcggca acatagtcga cgaggtcgct 600 tatcacgaga agtaccccac catctaccac ctgcgaaaga aattggtgga tagcaccgat 660 aaagccgact tgcgacttat ctacttggct ctggcgcaca tgattaagtt caggggccac 720 ttcctgatcg agggcgacct taaccccgac aacagtgacg tagacaaatt gttcatccag 780 cttgtacaga cctataacca gctgttcgag gaaaacccta ttaacgccag cggggtggat 840 gcgaaggcca tacttagcgc caggctgagc aaaagcaggc gcttggagaa cctgatagcc 900 cagctgcccg gtgaaaagaa gaacggcctc ttcggtaatc tgattgccct gagcctgggc 960 ctgaccccca acttcaagag caacttcgac ctggcagaag atgccaagct gcagttgagt 1020 aaggacacct atgacgacga cttggacaat ctgctcgccc aaatcggcga ccagtacgct 1080 gacctgttcc tcgccgccaa gaacctttct gacgcaatcc tgcttagcga tatccttagg 1140 gtgaacacag agatcaccaa ggcccccctg agcgccagca tgatcaagag gtacgacgag 1200 caccatcagg acctgaccct tctgaaggcc ctggtgaggc agcaactgcc cgagaagtac 1260 aaggagatct ttttcgacca gagcaagaac ggctacgccg gctacatcga cggcggagcc 1320 agccaagagg agttctacaa gttcatcaag cccatcctgg agaagatgga tggcaccgag 1380 gagctgctgg tgaagctgaa cagggaagat ttgctccgga agcagaggac ctttgacaac 1440 ggtagcatcc cccaccagat ccacctgggc gagctgcacg caatactgag gcgacaggag 1500 gatttctacc ccttcctcaa ggacaatagg gagaaaatcg aaaagattct gaccttcagg 1560 atcccctact acgtgggccc tcttgccagg ggcaacagcc gattcgcttg gatgacaaga 1620 aagagcgagg agaccatcac cccctggaac ttcgaggaag tggtggacaa aggagcaagc 1680 gcgcagtctt tcatcgaacg gatgaccaat ttcgacaaaa acctgcctaa cgagaaggtg 1740 ctgcccaagc acagcctgct ttacgagtac ttcaccgtgt acaacgagct caccaaggtg 1800 aaatatgtga ccgagggcat gcgaaaaccc gctttcctga gcggcgagca gaagaaggcc 1860 atcgtggacc tgctgttcaa gaccaacagg aaggtgaccg tgaagcagct gaaggaggac 1920 tacttcaaga agatcgagtg ctttgatagc gtggaaataa gcggcgtgga ggacaggttc 1980 aacgccagcc tgggcaccta ccacgacttg ttgaagataa tcaaagacaa ggatttcctg 2040 gataatgagg agaacgagga tatactcgag gacatcgtgc tgactttgac cctgtttgag 2100 gaccgagaga tgattgaaga aaggctcaaa acctacgccc acctgttcga cgacaaagtg 2160 atgaaacaac tgaagagacg aagatacacc ggctggggca gactgtccag gaagctcatc 2220 aacggcatta gggacaagca gagcggcaag accatcctgg atttcctgaa gtccgacggc 2280 ttcgccaacc gaaacttcat gcagctgatt cacgatgaca gcttgacctt caaggaggac 2340 atccagaagg cccaggttag cggccagggc gactccctgc acgaacatat tgcaaacctg 2400 gcaggctccc ctgcgatcaa gaagggcata ctgcagaccg ttaaggttgt ggacgaattg 2460 gtcaaggtca tgggcaggca caagcccgaa aacatagtta tagagatggc cagagagaac 2520 cagaccaccc aaaagggcca gaagaacagc cgggagcgca tgaaaaggat cgaggagggt 2580 atcaaggaac tcggaagcca gatcctcaaa gagcaccccg tggagaatac ccagctccag 2640 aacgagaagc tgtacctgta ctacctgcag aacggcaggg acatgtacgt tgaccaggag 2700 ttggacatca acaggctttc agactatgac gtggatcaca tagtgcccca gagctttctt 2760 aaagacgata gcatcgacaa caaggtcctg acccgctccg acaaaaacag gggcaaaagc 2820 gacaacgtgc caagcgaaga ggtggttaaa aagatgaaga actactggag gcaactgctc 2880 aacgcgaaat tgatcaccca gagaaagttc gataacctga ccaaggccga gaggggcgga 2940 ctctccgaac ttgacaaagc gggcttcata aagaggcagc tggtcgagac ccgacagatc 3000 acgaagcacg tggcccaaat cctcgacagc agaatgaata ccaagtacga tgagaatgac 3060 aaactcatca gggaagtgaa agtgattacc ctgaagagca agttggtgtc cgactttcgc 3120 aaagatttcc agttctacaa ggtgagggag atcaacaact accaccatgc ccacgacgca 3180 tacctgaacg ccgtggtcgg caccgccctg attaagaagt atccaaagct ggagtccgaa 3240 tttgtctacg gcgactacaa agtttacgat gtgaggaaga tgatcgctaa gagcgaacag 3300 gagatcggca aggccaccgc taagtatttc ttctacagca acatcatgaa ctttttcaag 3360 accgagatca cacttgccaa cggcgaaatc aggaagaggc cgcttatcga gaccaacggt 3420 gagaccggcg agatcgtgtg ggacaagggc agggacttcg ccaccgtgag gaaagtcctg 3480 agcatgcccc aggtgaatat tgtgaaaaaa actgaggtgc agacaggcgg ctttagcaag 3540 gaatccatcc tgcccaagag gaacagcgac aagctgatcg cccggaagaa ggactgggac 3600 cctaagaagt atggaggctt cgacagcccc accgtagcct acagcgtgct ggtggtcgcg 3660 aaggtagaga aggggaagag caagaaactg aagagcgtga aggagctgct cggcataacc 3720 atcatggaga ggtccagctt tgagaagaac cccattgact ttttggaagc caagggctac 3780 aaagaggtca aaaaggacct gatcatcaaa ctccccaagt actccctgtt tgaattggag 3840 aacggcagaa agaggatgct ggcgagcgct ggggaactgc aaaagggcaa cgaactggcg 3900 ctgcccagca agtacgtgaa ttttctgtac ctggcgtccc actacgaaaa gctgaaaggc 3960 agccccgagg acaacgagca gaagcagctg ttcgtggagc agcacaagca ttacctggac 4020 gagataatcg agcaaatcag cgagttcagc aagagggtga ttctggccga cgcgaacctg 4080 gataaggtcc tcagcgccta caacaagcac cgagacaaac ccatcaggga gcaggccgag 4140 aatatcatac acctgttcac cctgacaaat ctgggcgcac ctgcggcatt caaatacttc 4200 gataccacca tcgacaggaa aaggtacact agcactaagg aggtgctgga tgccaccttg 4260 atccaccagt ccattaccgg cctgtatgag accaggatcg acctgagcca gcttggaggc 4320 gactctaggg cggacccaaa aaagaaaagg aaggtggaat tctacccata cgatgttcca 4380 gattacgct 4389 <210> 48 <211> 4395 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-HE12-Cas9 <400> 48 atgggcggtt ctcatcatca tcatcatcat ggtatggcct cccgccggcg ctggtgcaaa 60 cgccgccggg gttctggtgg tggtggaggt cacgaacatg aacacgaaca tgaacatgaa 120 catgaacacg agcacgagca tgagcacgaa cacgaacacg aagctagcct ggacagcacc 180 gctcccaaaa agaaaaggaa ggtgggcatt cacggcgtgc ctgcggccga caaaaagtac 240 agcatcggcc ttgatatcgg caccaatagc gtgggctggg ccgttatcac agacgaatac 300 aaggtaccca gcaagaagtt caaggtgctg gggaatacag acaggcactc tatcaagaaa 360 aaccttatcg gggctctgct gtttgactca ggcgagaccg ccgaggccac caggttgaag 420 aggaccgcaa ggcgaaggta cacccggagg aagaacagga tctgctatct gcaggagatc 480 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acaggctgga ggagagcttc 540 cttgtcgagg aggataagaa gcacgaacga caccccatct tcggcaacat agtcgacgag 600 gtcgcttatc acgagaagta ccccaccatc taccacctgc gaaagaaatt ggtggatagc 660 accgataaag ccgacttgcg acttatctac ttggctctgg cgcacatgat taagttcagg 720 ggccacttcc tgatcgaggg cgaccttaac cccgacaaca gtgacgtaga caaattgttc 780 atccagcttg tacagaccta taaccagctg ttcgaggaaa accctattaa cgccagcggg 840 gtggatgcga aggccatact tagcgccagg ctgagcaaaa gcaggcgctt ggagaacctg 900 atagcccagc tgcccggtga aaagaagaac ggcctcttcg gtaatctgat tgccctgagc 960 ctgggcctga cccccaactt caagagcaac ttcgacctgg cagaagatgc caagctgcag 1020 ttgagtaagg acacctatga cgacgacttg gacaatctgc tcgcccaaat cggcgaccag 1080 tacgctgacc tgttcctcgc cgccaagaac ctttctgacg caatcctgct tagcgatatc 1140 cttagggtga acacagagat caccaaggcc cccctgagcg ccagcatgat caagaggtac 1200 gacgagcacc atcaggacct gacccttctg aaggccctgg tgaggcagca actgcccgag 1260 aagtacaagg agatcttttt cgaccagagc aagaacggct acgccggcta catcgacggc 1320 ggagccagcc aagaggagtt ctacaagttc atcaagccca tcctggagaa gatggatggc 1380 accgaggagc tgctggtgaa gctgaacagg gaagatttgc tccggaagca gaggaccttt 1440 gacaacggta gcatccccca ccagatccac ctgggcgagc tgcacgcaat actgaggcga 1500 caggaggatt tctacccctt cctcaaggac aatagggaga aaatcgaaaa gattctgacc 1560 ttcaggatcc cctactacgt gggccctctt gccaggggca acagccgatt cgcttggatg 1620 acaagaaaga gcgaggagac catcaccccc tggaacttcg aggaagtggt ggacaaagga 1680 gcaagcgcgc agtctttcat cgaacggatg accaatttcg acaaaaacct gcctaacgag 1740 aaggtgctgc ccaagcacag cctgctttac gagtacttca ccgtgtacaa cgagctcacc 1800 aaggtgaaat atgtgaccga gggcatgcga aaacccgctt tcctgagcgg cgagcagaag 1860 aaggccatcg tggacctgct gttcaagacc aacaggaagg tgaccgtgaa gcagctgaag 1920 gaggactact tcaagaagat cgagtgcttt gatagcgtgg aaataagcgg cgtggaggac 1980 aggttcaacg ccagcctggg cacctaccac gacttgttga agataatcaa agacaaggat 2040 ttcctggata atgaggagaa cgaggatata ctcgaggaca tcgtgctgac tttgaccctg 2100 tttgaggacc gagagatgat tgaagaaagg ctcaaaacct acgcccacct gttcgacgac 2160 aaagtgatga aacaactgaa gagacgaaga tacaccggct ggggcagact gtccaggaag 2220 ctcatcaacg gcattaggga caagcagagc ggcaagacca tcctggattt cctgaagtcc 2280 gacggcttcg ccaaccgaaa cttcatgcag ctgattcacg atgacagctt gaccttcaag 2340 gaggacatcc agaaggccca ggttagcggc cagggcgact ccctgcacga acatattgca 2400 aacctggcag gctcccctgc gatcaagaag ggcatactgc agaccgttaa ggttgtggac 2460 gaattggtca aggtcatggg caggcacaag cccgaaaaca tagttataga gatggccaga 2520 gagaaccaga ccacccaaaa gggccagaag aacagccggg agcgcatgaa aaggatcgag 2580 gagggtatca aggaactcgg aagccagatc ctcaaagagc accccgtgga gaatacccag 2640 ctccagaacg agaagctgta cctgtactac ctgcagaacg gcagggacat gtacgttgac 2700 caggagttgg acatcaacag gctttcagac tatgacgtgg atcacatagt gccccagagc 2760 tttcttaaag acgatagcat cgacaacaag gtcctgaccc gctccgacaa aaacaggggc 2820 aaaagcgaca acgtgccaag cgaagaggtg gttaaaaaga tgaagaacta ctggaggcaa 2880 ctgctcaacg cgaaattgat cacccagaga aagttcgata acctgaccaa ggccgagagg 2940 ggcggactct ccgaacttga caaagcgggc ttcataaaga ggcagctggt cgagacccga 3000 cagatcacga agcacgtggc ccaaatcctc gacagcagaa tgaataccaa gtacgatgag 3060 aatgacaaac tcatcaggga agtgaaagtg attaccctga agagcaagtt ggtgtccgac 3120 tttcgcaaag atttccagtt ctacaaggtg agggagatca acaactacca ccatgcccac 3180 gacgcatacc tgaacgccgt ggtcggcacc gccctgatta agaagtatcc aaagctggag 3240 tccgaatttg tctacggcga ctacaaagtt tacgatgtga ggaagatgat cgctaagagc 3300 gaacaggaga tcggcaaggc caccgctaag tatttcttct acagcaacat catgaacttt 3360 ttcaagaccg agatcacact tgccaacggc gaaatcagga agaggccgct tatcgagacc 3420 aacggtgaga ccggcgagat cgtgtgggac aagggcaggg acttcgccac cgtgaggaaa 3480 gtcctgagca tgccccaggt gaatattgtg aaaaaaactg aggtgcagac aggcggcttt 3540 agcaaggaat ccatcctgcc caagaggaac agcgacaagc tgatcgcccg gaagaaggac 3600 tgggacccta agaagtatgg aggcttcgac agccccaccg tagcctacag cgtgctggtg 3660 gtcgcgaagg tagagaaggg gaagagcaag aaactgaaga gcgtgaagga gctgctcggc 3720 ataaccatca tggagaggtc cagctttgag aagaacccca ttgacttttt ggaagccaag 3780 ggctacaaag aggtcaaaaa ggacctgatc atcaaactcc ccaagtactc cctgtttgaa 3840 ttggagaacg gcagaaagag gatgctggcg agcgctgggg aactgcaaaa gggcaacgaa 3900 ctggcgctgc ccagcaagta cgtgaatttt ctgtacctgg cgtcccacta cgaaaagctg 3960 aaaggcagcc ccgaggacaa cgagcagaag cagctgttcg tggagcagca caagcattac 4020 ctggacgaga taatcgagca aatcagcgag ttcagcaaga gggtgattct ggccgacgcg 4080 aacctggata aggtcctcag cgcctacaac aagcaccgag acaaacccat cagggagcag 4140 gccgagaata tcatacacct gttcaccctg acaaatctgg gcgcacctgc ggcattcaaa 4200 tacttcgata ccaccatcga caggaaaagg tacactagca ctaaggaggt gctggatgcc 4260 accttgatcc accagtccat taccggcctg tatgagacca ggatcgacct gagccagctt 4320 ggaggcgact ctagggcgga cccaaaaaag aaaaggaagg tggaattcta cccatacgat 4380 gttccagatt acgct 4395 <210> 49 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; AP <400> 49 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgcgccggc gctggtgcaa acgccgccgg 90 <210> 50 <211> 4404 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; AP-Cas9 <400> 50 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgcgccggc gctggtgcaa acgccgccgg ggttctggtg gtggtggagg tcacgaacat 120 gaacatgaac atgaacacga gcacgagcat gagcacgaac acgaacacga agctagcctg 180 gacagcaccg ctcccaaaaa gaaaaggaag gtgggcattc acggcgtgcc tgcggccgac 240 aaaaagtaca gcatcggcct tgatatcggc accaatagcg tgggctgggc cgttatcaca 300 gacgaataca aggtacccag caagaagttc aaggtgctgg ggaatacaga caggcactct 360 atcaagaaaa accttatcgg ggctctgctg tttgactcag gcgagaccgc cgaggccacc 420 aggttgaaga ggaccgcaag gcgaaggtac acccggagga agaacaggat ctgctatctg 480 caggagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca caggctggag 540 gagagcttcc ttgtcgagga ggataagaag cacgaacgac accccatctt cggcaacata 600 gtcgacgagg tcgcttatca cgagaagtac cccaccatct accacctgcg aaagaaattg 660 gtggatagca ccgataaagc cgacttgcga cttatctact tggctctggc gcacatgatt 720 aagttcaggg gccacttcct gatcgagggc gaccttaacc ccgacaacag tgacgtagac 780 aaattgttca tccagcttgt acagacctat aaccagctgt tcgaggaaaa ccctattaac 840 gccagcgggg tggatgcgaa ggccatactt agcgccaggc tgagcaaaag caggcgcttg 900 gagaacctga tagcccagct gcccggtgaa aagaagaacg gcctcttcgg taatctgatt 960 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc agaagatgcc 1020 aagctgcagt tgagtaagga cacctatgac gacgacttgg acaatctgct cgcccaaatc 1080 ggcgaccagt acgctgacct gttcctcgcc gccaagaacc tttctgacgc aatcctgctt 1140 agcgatatcc ttagggtgaa cacagagatc accaaggccc ccctgagcgc cagcatgatc 1200 aagaggtacg acgagcacca tcaggacctg acccttctga aggccctggt gaggcagcaa 1260 ctgcccgaga agtacaagga gatctttttc gaccagagca agaacggcta cgccggctac 1320 atcgacggcg gagccagcca agaggagttc tacaagttca tcaagcccat cctggagaag 1380 atggatggca ccgaggagct gctggtgaag ctgaacaggg aagatttgct ccggaagcag 1440 aggacctttg acaacggtag catcccccac cagatccacc tgggcgagct gcacgcaata 1500 ctgaggcgac aggaggattt ctaccccttc ctcaaggaca atagggagaa aatcgaaaag 1560 attctgacct tcaggatccc ctactacgtg ggccctcttg ccaggggcaa cagccgattc 1620 gcttggatga caagaaagag cgaggagacc atcaccccct ggaacttcga ggaagtggtg 1680 gacaaaggag caagcgcgca gtctttcatc gaacggatga ccaatttcga caaaaacctg 1740 cctaacgaga aggtgctgcc caagcacagc ctgctttacg agtacttcac cgtgtacaac 1800 gagctcacca aggtgaaata tgtgaccgag ggcatgcgaa aacccgcttt cctgagcggc 1860 gagcagaaga aggccatcgt ggacctgctg ttcaagacca acaggaaggt gaccgtgaag 1920 cagctgaagg aggactactt caagaagatc gagtgctttg atagcgtgga aataagcggc 1980 gtggaggaca ggttcaacgc cagcctgggc acctaccacg acttgttgaa gataatcaaa 2040 gacaaggatt tcctggataa tgaggagaac gaggatatac tcgaggacat cgtgctgact 2100 ttgaccctgt ttgaggaccg agagatgatt gaagaaaggc tcaaaaccta cgcccacctg 2160 ttcgacgaca aagtgatgaa acaactgaag agacgaagat acaccggctg gggcagactg 2220 tccaggaagc tcatcaacgg cattagggac aagcagagcg gcaagaccat cctggatttc 2280 ctgaagtccg acggcttcgc caaccgaaac ttcatgcagc tgattcacga tgacagcttg 2340 accttcaagg aggacatcca gaaggcccag gttagcggcc agggcgactc cctgcacgaa 2400 catattgcaa acctggcagg ctcccctgcg atcaagaagg gcatactgca gaccgttaag 2460 gttgtggacg aattggtcaa ggtcatgggc aggcacaagc ccgaaaacat agttatagag 2520 atggccagag agaaccagac cacccaaaag ggccagaaga acagccggga gcgcatgaaa 2580 aggatcgagg agggtatcaa ggaactcgga agccagatcc tcaaagagca ccccgtggag 2640 aatacccagc tccagaacga gaagctgtac ctgtactacc tgcagaacgg cagggacatg 2700 tacgttgacc aggagttgga catcaacagg ctttcagact atgacgtgga tcacatagtg 2760 ccccagagct ttcttaaaga cgatagcatc gacaacaagg tcctgacccg ctccgacaaa 2820 aacaggggca aaagcgacaa cgtgccaagc gaagaggtgg ttaaaaagat gaagaactac 2880 tggaggcaac tgctcaacgc gaaattgatc acccagagaa agttcgataa cctgaccaag 2940 gccgagaggg gcggactctc cgaacttgac aaagcgggct tcataaagag gcagctggtc 3000 gagacccgac agatcacgaa gcacgtggcc caaatcctcg acagcagaat gaataccaag 3060 tacgatgaga atgacaaact catcagggaa gtgaaagtga ttaccctgaa gagcaagttg 3120 gtgtccgact ttcgcaaaga tttccagttc tacaaggtga gggagatcaa caactaccac 3180 catgcccacg acgcatacct gaacgccgtg gtcggcaccg ccctgattaa gaagtatcca 3240 aagctggagt ccgaatttgt ctacggcgac tacaaagttt acgatgtgag gaagatgatc 3300 gctaagagcg aacaggagat cggcaaggcc accgctaagt atttcttcta cagcaacatc 3360 atgaactttt tcaagaccga gatcacactt gccaacggcg aaatcaggaa gaggccgctt 3420 atcgagacca acggtgagac cggcgagatc gtgtgggaca agggcaggga cttcgccacc 3480 gtgaggaaag tcctgagcat gccccaggtg aatattgtga aaaaaactga ggtgcagaca 3540 ggcggcttta gcaaggaatc catcctgccc aagaggaaca gcgacaagct gatcgcccgg 3600 aagaaggact gggaccctaa gaagtatgga ggcttcgaca gccccaccgt agcctacagc 3660 gtgctggtgg tcgcgaaggt agagaagggg aagagcaaga aactgaagag cgtgaaggag 3720 ctgctcggca taaccatcat ggagaggtcc agctttgaga agaaccccat tgactttttg 3780 gaagccaagg gctacaaaga ggtcaaaaag gacctgatca tcaaactccc caagtactcc 3840 ctgtttgaat tggagaacgg cagaaagagg atgctggcga gcgctgggga actgcaaaag 3900 ggcaacgaac tggcgctgcc cagcaagtac gtgaattttc tgtacctggc gtcccactac 3960 gaaaagctga aaggcagccc cgaggacaac gagcagaagc agctgttcgt ggagcagcac 4020 aagcattacc tggacgagat aatcgagcaa atcagcgagt tcagcaagag ggtgattctg 4080 gccgacgcga acctggataa ggtcctcagc gcctacaaca agcaccgaga caaacccatc 4140 agggagcagg ccgagaatat catacacctg ttcaccctga caaatctggg cgcacctgcg 4200 gcattcaaat acttcgatac caccatcgac aggaaaaggt acactagcac taaggaggtg 4260 ctggatgcca ccttgatcca ccagtccatt accggcctgt atgagaccag gatcgacctg 4320 agccagcttg gaggcgactc tagggcggac ccaaaaaaga aaaggaaggt ggaattctac 4380 ccatacgatg ttccagatta cgct 4404 <210> 51 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; dNP2 <400> 51 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaagatca agaaggttaa aaaaaagggt cgcaagggct ctaaaattaa aaaagtcaag 120 aagaaaggaa gaaaa 135 <210> 52 <211> 4368 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; dNP2-Cas9 <400> 52 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaagatca agaaggttaa aaaaaagggt cgcaagggct ctaaaattaa aaaagtcaag 120 aagaaaggaa gaaaagctag cctggacagc accgctccca aaaagaaaag gaaggtgggc 180 attcacggcg tgcctgcggc cgacaaaaag tacagcatcg gccttgatat cggcaccaat 240 agcgtgggct gggccgttat cacagacgaa tacaaggtac ccagcaagaa gttcaaggtg 300 ctggggaata cagacaggca ctctatcaag aaaaacctta tcggggctct gctgtttgac 360 tcaggcgaga ccgccgaggc caccaggttg aagaggaccg caaggcgaag gtacacccgg 420 aggaagaaca ggatctgcta tctgcaggag atcttcagca acgagatggc caaggtggac 480 gacagcttct tccacaggct ggaggagagc ttccttgtcg aggaggataa gaagcacgaa 540 cgacacccca tcttcggcaa catagtcgac gaggtcgctt atcacgagaa gtaccccacc 600 atctaccacc tgcgaaagaa attggtggat agcaccgata aagccgactt gcgacttatc 660 tacttggctc tggcgcacat gattaagttc aggggccact tcctgatcga gggcgacctt 720 aaccccgaca acagtgacgt agacaaattg ttcatccagc ttgtacagac ctataaccag 780 ctgttcgagg aaaaccctat taacgccagc ggggtggatg cgaaggccat acttagcgcc 840 aggctgagca aaagcaggcg cttggagaac ctgatagccc agctgcccgg tgaaaagaag 900 aacggcctct tcggtaatct gattgccctg agcctgggcc tgacccccaa cttcaagagc 960 aacttcgacc tggcagaaga tgccaagctg cagttgagta aggacaccta tgacgacgac 1020 ttggacaatc tgctcgccca aatcggcgac cagtacgctg acctgttcct cgccgccaag 1080 aacctttctg acgcaatcct gcttagcgat atccttaggg tgaacacaga gatcaccaag 1140 gcccccctga gcgccagcat gatcaagagg tacgacgagc accatcagga cctgaccctt 1200 ctgaaggccc tggtgaggca gcaactgccc gagaagtaca aggagatctt tttcgaccag 1260 agcaagaacg gctacgccgg ctacatcgac ggcggagcca gccaagagga gttctacaag 1320 ttcatcaagc ccatcctgga gaagatggat ggcaccgagg agctgctggt gaagctgaac 1380 agggaagatt tgctccggaa gcagaggacc tttgacaacg gtagcatccc ccaccagatc 1440 cacctgggcg agctgcacgc aatactgagg cgacaggagg atttctaccc cttcctcaag 1500 gacaataggg agaaaatcga aaagattctg accttcagga tcccctacta cgtgggccct 1560 cttgccaggg gcaacagccg attcgcttgg atgacaagaa agagcgagga gaccatcacc 1620 ccctggaact tcgaggaagt ggtggacaaa ggagcaagcg cgcagtcttt catcgaacgg 1680 atgaccaatt tcgacaaaaa cctgcctaac gagaaggtgc tgcccaagca cagcctgctt 1740 tacgagtact tcaccgtgta caacgagctc accaaggtga aatatgtgac cgagggcatg 1800 cgaaaacccg ctttcctgag cggcgagcag aagaaggcca tcgtggacct gctgttcaag 1860 accaacagga aggtgaccgt gaagcagctg aaggaggact acttcaagaa gatcgagtgc 1920 tttgatagcg tggaaataag cggcgtggag gacaggttca acgccagcct gggcacctac 1980 cacgacttgt tgaagataat caaagacaag gatttcctgg ataatgagga gaacgaggat 2040 atactcgagg acatcgtgct gactttgacc ctgtttgagg accgagagat gattgaagaa 2100 aggctcaaaa cctacgccca cctgttcgac gacaaagtga tgaaacaact gaagagacga 2160 agatacaccg gctggggcag actgtccagg aagctcatca acggcattag ggacaagcag 2220 agcggcaaga ccatcctgga tttcctgaag tccgacggct tcgccaaccg aaacttcatg 2280 cagctgattc acgatgacag cttgaccttc aaggaggaca tccagaaggc ccaggttagc 2340 ggccagggcg actccctgca cgaacatatt gcaaacctgg caggctcccc tgcgatcaag 2400 aagggcatac tgcagaccgt taaggttgtg gacgaattgg tcaaggtcat gggcaggcac 2460 aagcccgaaa acatagttat agagatggcc agagagaacc agaccaccca aaagggccag 2520 aagaacagcc gggagcgcat gaaaaggatc gaggagggta tcaaggaact cggaagccag 2580 atcctcaaag agcaccccgt ggagaatacc cagctccaga acgagaagct gtacctgtac 2640 tacctgcaga acggcaggga catgtacgtt gaccaggagt tggacatcaa caggctttca 2700 gactatgacg tggatcacat agtgccccag agctttctta aagacgatag catcgacaac 2760 aaggtcctga cccgctccga caaaaacagg ggcaaaagcg acaacgtgcc aagcgaagag 2820 gtggttaaaa agatgaagaa ctactggagg caactgctca acgcgaaatt gatcacccag 2880 agaaagttcg ataacctgac caaggccgag aggggcggac tctccgaact tgacaaagcg 2940 ggcttcataa agaggcagct ggtcgagacc cgacagatca cgaagcacgt ggcccaaatc 3000 ctcgacagca gaatgaatac caagtacgat gagaatgaca aactcatcag ggaagtgaaa 3060 gtgattaccc tgaagagcaa gttggtgtcc gactttcgca aagatttcca gttctacaag 3120 gtgagggaga tcaacaacta ccaccatgcc cacgacgcat acctgaacgc cgtggtcggc 3180 accgccctga ttaagaagta tccaaagctg gagtccgaat ttgtctacgg cgactacaaa 3240 gtttacgatg tgaggaagat gatcgctaag agcgaacagg agatcggcaa ggccaccgct 3300 aagtatttct tctacagcaa catcatgaac tttttcaaga ccgagatcac acttgccaac 3360 ggcgaaatca ggaagaggcc gcttatcgag accaacggtg agaccggcga gatcgtgtgg 3420 gacaagggca gggacttcgc caccgtgagg aaagtcctga gcatgcccca ggtgaatatt 3480 gtgaaaaaaa ctgaggtgca gacaggcggc tttagcaagg aatccatcct gcccaagagg 3540 aacagcgaca agctgatcgc ccggaagaag gactgggacc ctaagaagta tggaggcttc 3600 gacagcccca ccgtagccta cagcgtgctg gtggtcgcga aggtagagaa ggggaagagc 3660 aagaaactga agagcgtgaa ggagctgctc ggcataacca tcatggagag gtccagcttt 3720 gagaagaacc ccattgactt tttggaagcc aagggctaca aagaggtcaa aaaggacctg 3780 atcatcaaac tccccaagta ctccctgttt gaattggaga acggcagaaa gaggatgctg 3840 gcgagcgctg gggaactgca aaagggcaac gaactggcgc tgcccagcaa gtacgtgaat 3900 tttctgtacc tggcgtccca ctacgaaaag ctgaaaggca gccccgagga caacgagcag 3960 aagcagctgt tcgtggagca gcacaagcat tacctggacg agataatcga gcaaatcagc 4020 gagttcagca agagggtgat tctggccgac gcgaacctgg ataaggtcct cagcgcctac 4080 aacaagcacc gagacaaacc catcagggag caggccgaga atatcataca cctgttcacc 4140 ctgacaaatc tgggcgcacc tgcggcattc aaatacttcg ataccaccat cgacaggaaa 4200 aggtacacta gcactaagga ggtgctggat gccaccttga tccaccagtc cattaccggc 4260 ctgtatgaga ccaggatcga cctgagccag cttggaggcg actctagggc ggacccaaaa 4320 aagaaaagga aggtggaatt ctacccatac gatgttccag attacgct 4368 <210> 53 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; dNP2-HE10 <400> 53 atgggcggtt ctcatcatca tcatcatcat catatgaaga tcaagaaggt taaaaaaaag 60 ggtcgcaagg gctctaaaat taaaaaagtc aagaagaaag gaagaaaagc tagcggtggt 120 ggtggaggtc acgaacatga acatgaacat gaacacgagc acgagcatga gcacgaacac 180 gaacacgaa 189 <210> 54 <211> 4416 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; dNP2-HE10-Cas9 <400> 54 atgggcggtt ctcatcatca tcatcatcat catatgaaga tcaagaaggt taaaaaaaag 60 ggtcgcaagg gctctaaaat taaaaaagtc aagaagaaag gaagaaaagc tagcggtggt 120 ggtggaggtc acgaacatga acatgaacat gaacacgagc acgagcatga gcacgaacac 180 gaacacgaac tggacagcac cgctcccaaa aagaaaagga aggtgggcat tcacggcgtg 240 cctgcggccg acaaaaagta cagcatcggc cttgatatcg gcaccaatag cgtgggctgg 300 gccgttatca cagacgaata caaggtaccc agcaagaagt tcaaggtgct ggggaataca 360 gacaggcact ctatcaagaa aaaccttatc ggggctctgc tgtttgactc aggcgagacc 420 gccgaggcca ccaggttgaa gaggaccgca aggcgaaggt acacccggag gaagaacagg 480 atctgctatc tgcaggagat cttcagcaac gagatggcca aggtggacga cagcttcttc 540 cacaggctgg aggagagctt ccttgtcgag gaggataaga agcacgaacg acaccccatc 600 ttcggcaaca tagtcgacga ggtcgcttat cacgagaagt accccaccat ctaccacctg 660 cgaaagaaat tggtggatag caccgataaa gccgacttgc gacttatcta cttggctctg 720 gcgcacatga ttaagttcag gggccacttc ctgatcgagg gcgaccttaa ccccgacaac 780 agtgacgtag acaaattgtt catccagctt gtacagacct ataaccagct gttcgaggaa 840 aaccctatta acgccagcgg ggtggatgcg aaggccatac ttagcgccag gctgagcaaa 900 agcaggcgct tggagaacct gatagcccag ctgcccggtg aaaagaagaa cggcctcttc 960 ggtaatctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg 1020 gcagaagatg ccaagctgca gttgagtaag gacacctatg acgacgactt ggacaatctg 1080 ctcgcccaaa tcggcgacca gtacgctgac ctgttcctcg ccgccaagaa cctttctgac 1140 gcaatcctgc ttagcgatat ccttagggtg aacacagaga tcaccaaggc ccccctgagc 1200 gccagcatga tcaagaggta cgacgagcac catcaggacc tgacccttct gaaggccctg 1260 gtgaggcagc aactgcccga gaagtacaag gagatctttt tcgaccagag caagaacggc 1320 tacgccggct acatcgacgg cggagccagc caagaggagt tctacaagtt catcaagccc 1380 atcctggaga agatggatgg caccgaggag ctgctggtga agctgaacag ggaagatttg 1440 ctccggaagc agaggacctt tgacaacggt agcatccccc accagatcca cctgggcgag 1500 ctgcacgcaa tactgaggcg acaggaggat ttctacccct tcctcaagga caatagggag 1560 aaaatcgaaa agattctgac cttcaggatc ccctactacg tgggccctct tgccaggggc 1620 aacagccgat tcgcttggat gacaagaaag agcgaggaga ccatcacccc ctggaacttc 1680 gaggaagtgg tggacaaagg agcaagcgcg cagtctttca tcgaacggat gaccaatttc 1740 gacaaaaacc tgcctaacga gaaggtgctg cccaagcaca gcctgcttta cgagtacttc 1800 accgtgtaca acgagctcac caaggtgaaa tatgtgaccg agggcatgcg aaaacccgct 1860 ttcctgagcg gcgagcagaa gaaggccatc gtggacctgc tgttcaagac caacaggaag 1920 gtgaccgtga agcagctgaa ggaggactac ttcaagaaga tcgagtgctt tgatagcgtg 1980 gaaataagcg gcgtggagga caggttcaac gccagcctgg gcacctacca cgacttgttg 2040 aagataatca aagacaagga tttcctggat aatgaggaga acgaggatat actcgaggac 2100 atcgtgctga ctttgaccct gtttgaggac cgagagatga ttgaagaaag gctcaaaacc 2160 tacgcccacc tgttcgacga caaagtgatg aaacaactga agagacgaag atacaccggc 2220 tggggcagac tgtccaggaa gctcatcaac ggcattaggg acaagcagag cggcaagacc 2280 atcctggatt tcctgaagtc cgacggcttc gccaaccgaa acttcatgca gctgattcac 2340 gatgacagct tgaccttcaa ggaggacatc cagaaggccc aggttagcgg ccagggcgac 2400 tccctgcacg aacatattgc aaacctggca ggctcccctg cgatcaagaa gggcatactg 2460 cagaccgtta aggttgtgga cgaattggtc aaggtcatgg gcaggcacaa gcccgaaaac 2520 atagttatag agatggccag agagaaccag accacccaaa agggccagaa gaacagccgg 2580 gagcgcatga aaaggatcga ggagggtatc aaggaactcg gaagccagat cctcaaagag 2640 caccccgtgg agaataccca gctccagaac gagaagctgt acctgtacta cctgcagaac 2700 ggcagggaca tgtacgttga ccaggagttg gacatcaaca ggctttcaga ctatgacgtg 2760 gatcacatag tgccccagag ctttcttaaa gacgatagca tcgacaacaa ggtcctgacc 2820 cgctccgaca aaaacagggg caaaagcgac aacgtgccaa gcgaagaggt ggttaaaaag 2880 atgaagaact actggaggca actgctcaac gcgaaattga tcacccagag aaagttcgat 2940 aacctgacca aggccgagag gggcggactc tccgaacttg acaaagcggg cttcataaag 3000 aggcagctgg tcgagacccg acagatcacg aagcacgtgg cccaaatcct cgacagcaga 3060 atgaatacca agtacgatga gaatgacaaa ctcatcaggg aagtgaaagt gattaccctg 3120 aagagcaagt tggtgtccga ctttcgcaaa gatttccagt tctacaaggt gagggagatc 3180 aacaactacc accatgccca cgacgcatac ctgaacgccg tggtcggcac cgccctgatt 3240 aagaagtatc caaagctgga gtccgaattt gtctacggcg actacaaagt ttacgatgtg 3300 aggaagatga tcgctaagag cgaacaggag atcggcaagg ccaccgctaa gtatttcttc 3360 tacagcaaca tcatgaactt tttcaagacc gagatcacac ttgccaacgg cgaaatcagg 3420 aagaggccgc ttatcgagac caacggtgag accggcgaga tcgtgtggga caagggcagg 3480 gacttcgcca ccgtgaggaa agtcctgagc atgccccagg tgaatattgt gaaaaaaact 3540 gaggtgcaga caggcggctt tagcaaggaa tccatcctgc ccaagaggaa cagcgacaag 3600 ctgatcgccc ggaagaagga ctgggaccct aagaagtatg gaggcttcga cagccccacc 3660 gtagcctaca gcgtgctggt ggtcgcgaag gtagagaagg ggaagagcaa gaaactgaag 3720 agcgtgaagg agctgctcgg cataaccatc atggagaggt ccagctttga gaagaacccc 3780 attgactttt tggaagccaa gggctacaaa gaggtcaaaa aggacctgat catcaaactc 3840 cccaagtact ccctgtttga attggagaac ggcagaaaga ggatgctggc gagcgctggg 3900 gaactgcaaa agggcaacga actggcgctg cccagcaagt acgtgaattt tctgtacctg 3960 gcgtcccact acgaaaagct gaaaggcagc cccgaggaca acgagcagaa gcagctgttc 4020 gtggagcagc acaagcatta cctggacgag ataatcgagc aaatcagcga gttcagcaag 4080 agggtgattc tggccgacgc gaacctggat aaggtcctca gcgcctacaa caagcaccga 4140 gacaaaccca tcagggagca ggccgagaat atcatacacc tgttcaccct gacaaatctg 4200 ggcgcacctg cggcattcaa atacttcgat accaccatcg acaggaaaag gtacactagc 4260 actaaggagg tgctggatgc caccttgatc caccagtcca ttaccggcct gtatgagacc 4320 aggatcgacc tgagccagct tggaggcgac tctagggcgg acccaaaaaa gaaaaggaag 4380 gtggaattct acccatacga tgttccagat tacgct 4416 <210> 55 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; R9 <400> 55 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgagacgaa gacgaagacg tagacgtaga 90 <210> 56 <211> 4323 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; R9-Cas9 <400> 56 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgagacgaa gacgaagacg tagacgtaga gctagcctgg acagcaccgc tcccaaaaag 120 aaaaggaagg tgggcattca cggcgtgcct gcggccgaca aaaagtacag catcggcctt 180 gatatcggca ccaatagcgt gggctgggcc gttatcacag acgaatacaa ggtacccagc 240 aagaagttca aggtgctggg gaatacagac aggcactcta tcaagaaaaa ccttatcggg 300 gctctgctgt ttgactcagg cgagaccgcc gaggccacca ggttgaagag gaccgcaagg 360 cgaaggtaca cccggaggaa gaacaggatc tgctatctgc aggagatctt cagcaacgag 420 atggccaagg tggacgacag cttcttccac aggctggagg agagcttcct tgtcgaggag 480 gataagaagc acgaacgaca ccccatcttc ggcaacatag tcgacgaggt cgcttatcac 540 gagaagtacc ccaccatcta ccacctgcga aagaaattgg tggatagcac cgataaagcc 600 gacttgcgac ttatctactt ggctctggcg cacatgatta agttcagggg ccacttcctg 660 atcgagggcg accttaaccc cgacaacagt gacgtagaca aattgttcat ccagcttgta 720 cagacctata accagctgtt cgaggaaaac cctattaacg ccagcggggt ggatgcgaag 780 gccatactta gcgccaggct gagcaaaagc aggcgcttgg agaacctgat agcccagctg 840 cccggtgaaa agaagaacgg cctcttcggt aatctgattg ccctgagcct gggcctgacc 900 cccaacttca agagcaactt cgacctggca gaagatgcca agctgcagtt gagtaaggac 960 acctatgacg acgacttgga caatctgctc gcccaaatcg gcgaccagta cgctgacctg 1020 ttcctcgccg ccaagaacct ttctgacgca atcctgctta gcgatatcct tagggtgaac 1080 acagagatca ccaaggcccc cctgagcgcc agcatgatca agaggtacga cgagcaccat 1140 caggacctga cccttctgaa ggccctggtg aggcagcaac tgcccgagaa gtacaaggag 1200 atctttttcg accagagcaa gaacggctac gccggctaca tcgacggcgg agccagccaa 1260 gaggagttct acaagttcat caagcccatc ctggagaaga tggatggcac cgaggagctg 1320 ctggtgaagc tgaacaggga agatttgctc cggaagcaga ggacctttga caacggtagc 1380 atcccccacc agatccacct gggcgagctg cacgcaatac tgaggcgaca ggaggatttc 1440 taccccttcc tcaaggacaa tagggagaaa atcgaaaaga ttctgacctt caggatcccc 1500 tactacgtgg gccctcttgc caggggcaac agccgattcg cttggatgac aagaaagagc 1560 gaggagacca tcaccccctg gaacttcgag gaagtggtgg acaaaggagc aagcgcgcag 1620 tctttcatcg aacggatgac caatttcgac aaaaacctgc ctaacgagaa ggtgctgccc 1680 aagcacagcc tgctttacga gtacttcacc gtgtacaacg agctcaccaa ggtgaaatat 1740 gtgaccgagg gcatgcgaaa acccgctttc ctgagcggcg agcagaagaa ggccatcgtg 1800 gacctgctgt tcaagaccaa caggaaggtg accgtgaagc agctgaagga ggactacttc 1860 aagaagatcg agtgctttga tagcgtggaa ataagcggcg tggaggacag gttcaacgcc 1920 agcctgggca cctaccacga cttgttgaag ataatcaaag acaaggattt cctggataat 1980 gaggagaacg aggatatact cgaggacatc gtgctgactt tgaccctgtt tgaggaccga 2040 gagatgattg aagaaaggct caaaacctac gcccacctgt tcgacgacaa agtgatgaaa 2100 caactgaaga gacgaagata caccggctgg ggcagactgt ccaggaagct catcaacggc 2160 attagggaca agcagagcgg caagaccatc ctggatttcc tgaagtccga cggcttcgcc 2220 aaccgaaact tcatgcagct gattcacgat gacagcttga ccttcaagga ggacatccag 2280 aaggcccagg ttagcggcca gggcgactcc ctgcacgaac atattgcaaa cctggcaggc 2340 tcccctgcga tcaagaaggg catactgcag accgttaagg ttgtggacga attggtcaag 2400 gtcatgggca ggcacaagcc cgaaaacata gttatagaga tggccagaga gaaccagacc 2460 acccaaaagg gccagaagaa cagccgggag cgcatgaaaa ggatcgagga gggtatcaag 2520 gaactcggaa gccagatcct caaagagcac cccgtggaga atacccagct ccagaacgag 2580 aagctgtacc tgtactacct gcagaacggc agggacatgt acgttgacca ggagttggac 2640 atcaacaggc tttcagacta tgacgtggat cacatagtgc cccagagctt tcttaaagac 2700 gatagcatcg acaacaaggt cctgacccgc tccgacaaaa acaggggcaa aagcgacaac 2760 gtgccaagcg aagaggtggt taaaaagatg aagaactact ggaggcaact gctcaacgcg 2820 aaattgatca cccagagaaa gttcgataac ctgaccaagg ccgagagggg cggactctcc 2880 gaacttgaca aagcgggctt cataaagagg cagctggtcg agacccgaca gatcacgaag 2940 cacgtggccc aaatcctcga cagcagaatg aataccaagt acgatgagaa tgacaaactc 3000 atcagggaag tgaaagtgat taccctgaag agcaagttgg tgtccgactt tcgcaaagat 3060 ttccagttct acaaggtgag ggagatcaac aactaccacc atgcccacga cgcatacctg 3120 aacgccgtgg tcggcaccgc cctgattaag aagtatccaa agctggagtc cgaatttgtc 3180 tacggcgact acaaagttta cgatgtgagg aagatgatcg ctaagagcga acaggagatc 3240 ggcaaggcca ccgctaagta tttcttctac agcaacatca tgaacttttt caagaccgag 3300 atcacacttg ccaacggcga aatcaggaag aggccgctta tcgagaccaa cggtgagacc 3360 ggcgagatcg tgtgggacaa gggcagggac ttcgccaccg tgaggaaagt cctgagcatg 3420 ccccaggtga atattgtgaa aaaaactgag gtgcagacag gcggctttag caaggaatcc 3480 atcctgccca agaggaacag cgacaagctg atcgcccgga agaaggactg ggaccctaag 3540 aagtatggag gcttcgacag ccccaccgta gcctacagcg tgctggtggt cgcgaaggta 3600 gagaagggga agagcaagaa actgaagagc gtgaaggagc tgctcggcat aaccatcatg 3660 gagaggtcca gctttgagaa gaaccccatt gactttttgg aagccaaggg ctacaaagag 3720 gtcaaaaagg acctgatcat caaactcccc aagtactccc tgtttgaatt ggagaacggc 3780 agaaagagga tgctggcgag cgctggggaa ctgcaaaagg gcaacgaact ggcgctgccc 3840 agcaagtacg tgaattttct gtacctggcg tcccactacg aaaagctgaa aggcagcccc 3900 gaggacaacg agcagaagca gctgttcgtg gagcagcaca agcattacct ggacgagata 3960 atcgagcaaa tcagcgagtt cagcaagagg gtgattctgg ccgacgcgaa cctggataag 4020 gtcctcagcg cctacaacaa gcaccgagac aaacccatca gggagcaggc cgagaatatc 4080 atacacctgt tcaccctgac aaatctgggc gcacctgcgg cattcaaata cttcgatacc 4140 accatcgaca ggaaaaggta cactagcact aaggaggtgc tggatgccac cttgatccac 4200 cagtccatta ccggcctgta tgagaccagg atcgacctga gccagcttgg aggcgactct 4260 agggcggacc caaaaaagaa aaggaaggtg gaattctacc catacgatgt tccagattac 4320 gct 4323 <210> 57 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; R9-HE10 <400> 57 atgggcggtt ctcatcatca tcatcatcat catatgagac gaagacgaag acgtagacgt 60 agagctagcg gtggtggtgg aggtcacgaa catgaacatg aacatgaaca cgagcacgag 120 catgagcacg aacacgaaca cgaa 144 <210> 58 <211> 4371 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; R9-HE10-Cas9 <400> 58 atgggcggtt ctcatcatca tcatcatcat catatgagac gaagacgaag acgtagacgt 60 agagctagcg gtggtggtgg aggtcacgaa catgaacatg aacatgaaca cgagcacgag 120 catgagcacg aacacgaaca cgaactggac agcaccgctc ccaaaaagaa aaggaaggtg 180 ggcattcacg gcgtgcctgc ggccgacaaa aagtacagca tcggccttga tatcggcacc 240 aatagcgtgg gctgggccgt tatcacagac gaatacaagg tacccagcaa gaagttcaag 300 gtgctgggga atacagacag gcactctatc aagaaaaacc ttatcggggc tctgctgttt 360 gactcaggcg agaccgccga ggccaccagg ttgaagagga ccgcaaggcg aaggtacacc 420 cggaggaaga acaggatctg ctatctgcag gagatcttca gcaacgagat ggccaaggtg 480 gacgacagct tcttccacag gctggaggag agcttccttg tcgaggagga taagaagcac 540 gaacgacacc ccatcttcgg caacatagtc gacgaggtcg cttatcacga gaagtacccc 600 accatctacc acctgcgaaa gaaattggtg gatagcaccg ataaagccga cttgcgactt 660 atctacttgg ctctggcgca catgattaag ttcaggggcc acttcctgat cgagggcgac 720 cttaaccccg acaacagtga cgtagacaaa ttgttcatcc agcttgtaca gacctataac 780 cagctgttcg aggaaaaccc tattaacgcc agcggggtgg atgcgaaggc catacttagc 840 gccaggctga gcaaaagcag gcgcttggag aacctgatag cccagctgcc cggtgaaaag 900 aagaacggcc tcttcggtaa tctgattgcc ctgagcctgg gcctgacccc caacttcaag 960 agcaacttcg acctggcaga agatgccaag ctgcagttga gtaaggacac ctatgacgac 1020 gacttggaca atctgctcgc ccaaatcggc gaccagtacg ctgacctgtt cctcgccgcc 1080 aagaaccttt ctgacgcaat cctgcttagc gatatcctta gggtgaacac agagatcacc 1140 aaggcccccc tgagcgccag catgatcaag aggtacgacg agcaccatca ggacctgacc 1200 cttctgaagg ccctggtgag gcagcaactg cccgagaagt acaaggagat ctttttcgac 1260 cagagcaaga acggctacgc cggctacatc gacggcggag ccagccaaga ggagttctac 1320 aagttcatca agcccatcct ggagaagatg gatggcaccg aggagctgct ggtgaagctg 1380 aacagggaag atttgctccg gaagcagagg acctttgaca acggtagcat cccccaccag 1440 atccacctgg gcgagctgca cgcaatactg aggcgacagg aggatttcta ccccttcctc 1500 aaggacaata gggagaaaat cgaaaagatt ctgaccttca ggatccccta ctacgtgggc 1560 cctcttgcca ggggcaacag ccgattcgct tggatgacaa gaaagagcga ggagaccatc 1620 accccctgga acttcgagga agtggtggac aaaggagcaa gcgcgcagtc tttcatcgaa 1680 cggatgacca atttcgacaa aaacctgcct aacgagaagg tgctgcccaa gcacagcctg 1740 ctttacgagt acttcaccgt gtacaacgag ctcaccaagg tgaaatatgt gaccgagggc 1800 atgcgaaaac ccgctttcct gagcggcgag cagaagaagg ccatcgtgga cctgctgttc 1860 aagaccaaca ggaaggtgac cgtgaagcag ctgaaggagg actacttcaa gaagatcgag 1920 tgctttgata gcgtggaaat aagcggcgtg gaggacaggt tcaacgccag cctgggcacc 1980 taccacgact tgttgaagat aatcaaagac aaggatttcc tggataatga ggagaacgag 2040 gatatactcg aggacatcgt gctgactttg accctgtttg aggaccgaga gatgattgaa 2100 gaaaggctca aaacctacgc ccacctgttc gacgacaaag tgatgaaaca actgaagaga 2160 cgaagataca ccggctgggg cagactgtcc aggaagctca tcaacggcat tagggacaag 2220 cagagcggca agaccatcct ggatttcctg aagtccgacg gcttcgccaa ccgaaacttc 2280 atgcagctga ttcacgatga cagcttgacc ttcaaggagg acatccagaa ggcccaggtt 2340 agcggccagg gcgactccct gcacgaacat attgcaaacc tggcaggctc ccctgcgatc 2400 aagaagggca tactgcagac cgttaaggtt gtggacgaat tggtcaaggt catgggcagg 2460 cacaagcccg aaaacatagt tatagagatg gccagagaga accagaccac ccaaaagggc 2520 cagaagaaca gccgggagcg catgaaaagg atcgaggagg gtatcaagga actcggaagc 2580 cagatcctca aagagcaccc cgtggagaat acccagctcc agaacgagaa gctgtacctg 2640 tactacctgc agaacggcag ggacatgtac gttgaccagg agttggacat caacaggctt 2700 tcagactatg acgtggatca catagtgccc cagagctttc ttaaagacga tagcatcgac 2760 aacaaggtcc tgacccgctc cgacaaaaac aggggcaaaa gcgacaacgt gccaagcgaa 2820 gaggtggtta aaaagatgaa gaactactgg aggcaactgc tcaacgcgaa attgatcacc 2880 cagagaaagt tcgataacct gaccaaggcc gagaggggcg gactctccga acttgacaaa 2940 gcgggcttca taaagaggca gctggtcgag acccgacaga tcacgaagca cgtggcccaa 3000 atcctcgaca gcagaatgaa taccaagtac gatgagaatg acaaactcat cagggaagtg 3060 aaagtgatta ccctgaagag caagttggtg tccgactttc gcaaagattt ccagttctac 3120 aaggtgaggg agatcaacaa ctaccaccat gcccacgacg catacctgaa cgccgtggtc 3180 ggcaccgccc tgattaagaa gtatccaaag ctggagtccg aatttgtcta cggcgactac 3240 aaagtttacg atgtgaggaa gatgatcgct aagagcgaac aggagatcgg caaggccacc 3300 gctaagtatt tcttctacag caacatcatg aactttttca agaccgagat cacacttgcc 3360 aacggcgaaa tcaggaagag gccgcttatc gagaccaacg gtgagaccgg cgagatcgtg 3420 tgggacaagg gcagggactt cgccaccgtg aggaaagtcc tgagcatgcc ccaggtgaat 3480 attgtgaaaa aaactgaggt gcagacaggc ggctttagca aggaatccat cctgcccaag 3540 aggaacagcg acaagctgat cgcccggaag aaggactggg accctaagaa gtatggaggc 3600 ttcgacagcc ccaccgtagc ctacagcgtg ctggtggtcg cgaaggtaga gaaggggaag 3660 agcaagaaac tgaagagcgt gaaggagctg ctcggcataa ccatcatgga gaggtccagc 3720 tttgagaaga accccattga ctttttggaa gccaagggct acaaagaggt caaaaaggac 3780 ctgatcatca aactccccaa gtactccctg tttgaattgg agaacggcag aaagaggatg 3840 ctggcgagcg ctggggaact gcaaaagggc aacgaactgg cgctgcccag caagtacgtg 3900 aattttctgt acctggcgtc ccactacgaa aagctgaaag gcagccccga ggacaacgag 3960 cagaagcagc tgttcgtgga gcagcacaag cattacctgg acgagataat cgagcaaatc 4020 agcgagttca gcaagagggt gattctggcc gacgcgaacc tggataaggt cctcagcgcc 4080 tacaacaagc accgagacaa acccatcagg gagcaggccg agaatatcat acacctgttc 4140 accctgacaa atctgggcgc acctgcggca ttcaaatact tcgataccac catcgacagg 4200 aaaaggtaca ctagcactaa ggaggtgctg gatgccacct tgatccacca gtccattacc 4260 ggcctgtatg agaccaggat cgacctgagc cagcttggag gcgactctag ggcggaccca 4320 aaaaagaaaa ggaaggtgga attctaccca tacgatgttc cagattacgc t 4371 <210> 59 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; TAT <400> 59 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgtatggac gcaagaagcg ccgccagcgc cgccgc 96 <210> 60 <211> 4329 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; TAT-Cas9 <400> 60 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgtatggac gcaagaagcg ccgccagcgc cgccgcgcta gcctggacag caccgctccc 120 aaaaagaaaa ggaaggtggg cattcacggc gtgcctgcgg ccgacaaaaa gtacagcatc 180 ggccttgata tcggcaccaa tagcgtgggc tgggccgtta tcacagacga atacaaggta 240 cccagcaaga agttcaaggt gctggggaat acagacaggc actctatcaa gaaaaacctt 300 atcggggctc tgctgtttga ctcaggcgag accgccgagg ccaccaggtt gaagaggacc 360 gcaaggcgaa ggtacacccg gaggaagaac aggatctgct atctgcagga gatcttcagc 420 aacgagatgg ccaaggtgga cgacagcttc ttccacaggc tggaggagag cttccttgtc 480 gaggaggata agaagcacga acgacacccc atcttcggca acatagtcga cgaggtcgct 540 tatcacgaga agtaccccac catctaccac ctgcgaaaga aattggtgga tagcaccgat 600 aaagccgact tgcgacttat ctacttggct ctggcgcaca tgattaagtt caggggccac 660 ttcctgatcg agggcgacct taaccccgac aacagtgacg tagacaaatt gttcatccag 720 cttgtacaga cctataacca gctgttcgag gaaaacccta ttaacgccag cggggtggat 780 gcgaaggcca tacttagcgc caggctgagc aaaagcaggc gcttggagaa cctgatagcc 840 cagctgcccg gtgaaaagaa gaacggcctc ttcggtaatc tgattgccct gagcctgggc 900 ctgaccccca acttcaagag caacttcgac ctggcagaag atgccaagct gcagttgagt 960 aaggacacct atgacgacga cttggacaat ctgctcgccc aaatcggcga ccagtacgct 1020 gacctgttcc tcgccgccaa gaacctttct gacgcaatcc tgcttagcga tatccttagg 1080 gtgaacacag agatcaccaa ggcccccctg agcgccagca tgatcaagag gtacgacgag 1140 caccatcagg acctgaccct tctgaaggcc ctggtgaggc agcaactgcc cgagaagtac 1200 aaggagatct ttttcgacca gagcaagaac ggctacgccg gctacatcga cggcggagcc 1260 agccaagagg agttctacaa gttcatcaag cccatcctgg agaagatgga tggcaccgag 1320 gagctgctgg tgaagctgaa cagggaagat ttgctccgga agcagaggac ctttgacaac 1380 ggtagcatcc cccaccagat ccacctgggc gagctgcacg caatactgag gcgacaggag 1440 gatttctacc ccttcctcaa ggacaatagg gagaaaatcg aaaagattct gaccttcagg 1500 atcccctact acgtgggccc tcttgccagg ggcaacagcc gattcgcttg gatgacaaga 1560 aagagcgagg agaccatcac cccctggaac ttcgaggaag tggtggacaa aggagcaagc 1620 gcgcagtctt tcatcgaacg gatgaccaat ttcgacaaaa acctgcctaa cgagaaggtg 1680 ctgcccaagc acagcctgct ttacgagtac ttcaccgtgt acaacgagct caccaaggtg 1740 aaatatgtga ccgagggcat gcgaaaaccc gctttcctga gcggcgagca gaagaaggcc 1800 atcgtggacc tgctgttcaa gaccaacagg aaggtgaccg tgaagcagct gaaggaggac 1860 tacttcaaga agatcgagtg ctttgatagc gtggaaataa gcggcgtgga ggacaggttc 1920 aacgccagcc tgggcaccta ccacgacttg ttgaagataa tcaaagacaa ggatttcctg 1980 gataatgagg agaacgagga tatactcgag gacatcgtgc tgactttgac cctgtttgag 2040 gaccgagaga tgattgaaga aaggctcaaa acctacgccc acctgttcga cgacaaagtg 2100 atgaaacaac tgaagagacg aagatacacc ggctggggca gactgtccag gaagctcatc 2160 aacggcatta gggacaagca gagcggcaag accatcctgg atttcctgaa gtccgacggc 2220 ttcgccaacc gaaacttcat gcagctgatt cacgatgaca gcttgacctt caaggaggac 2280 atccagaagg cccaggttag cggccagggc gactccctgc acgaacatat tgcaaacctg 2340 gcaggctccc ctgcgatcaa gaagggcata ctgcagaccg ttaaggttgt ggacgaattg 2400 gtcaaggtca tgggcaggca caagcccgaa aacatagtta tagagatggc cagagagaac 2460 cagaccaccc aaaagggcca gaagaacagc cgggagcgca tgaaaaggat cgaggagggt 2520 atcaaggaac tcggaagcca gatcctcaaa gagcaccccg tggagaatac ccagctccag 2580 aacgagaagc tgtacctgta ctacctgcag aacggcaggg acatgtacgt tgaccaggag 2640 ttggacatca acaggctttc agactatgac gtggatcaca tagtgcccca gagctttctt 2700 aaagacgata gcatcgacaa caaggtcctg acccgctccg acaaaaacag gggcaaaagc 2760 gacaacgtgc caagcgaaga ggtggttaaa aagatgaaga actactggag gcaactgctc 2820 aacgcgaaat tgatcaccca gagaaagttc gataacctga ccaaggccga gaggggcgga 2880 ctctccgaac ttgacaaagc gggcttcata aagaggcagc tggtcgagac ccgacagatc 2940 acgaagcacg tggcccaaat cctcgacagc agaatgaata ccaagtacga tgagaatgac 3000 aaactcatca gggaagtgaa agtgattacc ctgaagagca agttggtgtc cgactttcgc 3060 aaagatttcc agttctacaa ggtgagggag atcaacaact accaccatgc ccacgacgca 3120 tacctgaacg ccgtggtcgg caccgccctg attaagaagt atccaaagct ggagtccgaa 3180 tttgtctacg gcgactacaa agtttacgat gtgaggaaga tgatcgctaa gagcgaacag 3240 gagatcggca aggccaccgc taagtatttc ttctacagca acatcatgaa ctttttcaag 3300 accgagatca cacttgccaa cggcgaaatc aggaagaggc cgcttatcga gaccaacggt 3360 gagaccggcg agatcgtgtg ggacaagggc agggacttcg ccaccgtgag gaaagtcctg 3420 agcatgcccc aggtgaatat tgtgaaaaaa actgaggtgc agacaggcgg ctttagcaag 3480 gaatccatcc tgcccaagag gaacagcgac aagctgatcg cccggaagaa ggactgggac 3540 cctaagaagt atggaggctt cgacagcccc accgtagcct acagcgtgct ggtggtcgcg 3600 aaggtagaga aggggaagag caagaaactg aagagcgtga aggagctgct cggcataacc 3660 atcatggaga ggtccagctt tgagaagaac cccattgact ttttggaagc caagggctac 3720 aaagaggtca aaaaggacct gatcatcaaa ctccccaagt actccctgtt tgaattggag 3780 aacggcagaa agaggatgct ggcgagcgct ggggaactgc aaaagggcaa cgaactggcg 3840 ctgcccagca agtacgtgaa ttttctgtac ctggcgtccc actacgaaaa gctgaaaggc 3900 agccccgagg acaacgagca gaagcagctg ttcgtggagc agcacaagca ttacctggac 3960 gagataatcg agcaaatcag cgagttcagc aagagggtga ttctggccga cgcgaacctg 4020 gataaggtcc tcagcgccta caacaagcac cgagacaaac ccatcaggga gcaggccgag 4080 aatatcatac acctgttcac cctgacaaat ctgggcgcac ctgcggcatt caaatacttc 4140 gataccacca tcgacaggaa aaggtacact agcactaagg aggtgctgga tgccaccttg 4200 atccaccagt ccattaccgg cctgtatgag accaggatcg acctgagcca gcttggaggc 4260 gactctaggg cggacccaaa aaagaaaagg aaggtggaat tctacccata cgatgttcca 4320 gattacgct 4329 <210> 61 <211> 150 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide; TAT-HE10 <400> 61 atgggcggtt ctcatcatca tcatcatcat catatgtatg gacgcaagaa gcgccgccag 60 cgccgccgcg ctagcggtgg tggtggaggt cacgaacatg aacatgaaca tgaacacgag 120 cacgagcatg agcacgaaca cgaacacgaa 150 <210> 62 <211> 4377 <212> DNA <213> Artificial Sequence <220> <223> gene sequence of penetrating peptide-linkage-Cas protein; TAT-HE10-Cas9 <400> 62 atgggcggtt ctcatcatca tcatcatcat catatgtatg gacgcaagaa gcgccgccag 60 cgccgccgcg ctagcggtgg tggtggaggt cacgaacatg aacatgaaca tgaacacgag 120 cacgagcatg agcacgaaca cgaacacgaa ctggacagca ccgctcccaa aaagaaaagg 180 aaggtgggca ttcacggcgt gcctgcggcc gacaaaaagt acagcatcgg ccttgatatc 240 ggcaccaata gcgtgggctg ggccgttatc acagacgaat acaaggtacc cagcaagaag 300 ttcaaggtgc tggggaatac agacaggcac tctatcaaga aaaaccttat cggggctctg 360 ctgtttgact caggcgagac cgccgaggcc accaggttga agaggaccgc aaggcgaagg 420 tacacccgga ggaagaacag gatctgctat ctgcaggaga tcttcagcaa cgagatggcc 480 aaggtggacg acagcttctt ccacaggctg gaggagagct tccttgtcga ggaggataag 540 aagcacgaac gacaccccat cttcggcaac atagtcgacg aggtcgctta tcacgagaag 600 taccccacca tctaccacct gcgaaagaaa ttggtggata gcaccgataa agccgacttg 660 cgacttatct acttggctct ggcgcacatg attaagttca ggggccactt cctgatcgag 720 ggcgacctta accccgacaa cagtgacgta gacaaattgt tcatccagct tgtacagacc 780 tataaccagc tgttcgagga aaaccctatt aacgccagcg gggtggatgc gaaggccata 840 cttagcgcca ggctgagcaa aagcaggcgc ttggagaacc tgatagccca gctgcccggt 900 gaaaagaaga acggcctctt cggtaatctg attgccctga gcctgggcct gacccccaac 960 ttcaagagca acttcgacct ggcagaagat gccaagctgc agttgagtaa ggacacctat 1020 gacgacgact tggacaatct gctcgcccaa atcggcgacc agtacgctga cctgttcctc 1080 gccgccaaga acctttctga cgcaatcctg cttagcgata tccttagggt gaacacagag 1140 atcaccaagg cccccctgag cgccagcatg atcaagaggt acgacgagca ccatcaggac 1200 ctgacccttc tgaaggccct ggtgaggcag caactgcccg agaagtacaa ggagatcttt 1260 ttcgaccaga gcaagaacgg ctacgccggc tacatcgacg gcggagccag ccaagaggag 1320 ttctacaagt tcatcaagcc catcctggag aagatggatg gcaccgagga gctgctggtg 1380 aagctgaaca gggaagattt gctccggaag cagaggacct ttgacaacgg tagcatcccc 1440 caccagatcc acctgggcga gctgcacgca atactgaggc gacaggagga tttctacccc 1500 ttcctcaagg acaataggga gaaaatcgaa aagattctga ccttcaggat cccctactac 1560 gtgggccctc ttgccagggg caacagccga ttcgcttgga tgacaagaaa gagcgaggag 1620 accatcaccc cctggaactt cgaggaagtg gtggacaaag gagcaagcgc gcagtctttc 1680 atcgaacgga tgaccaattt cgacaaaaac ctgcctaacg agaaggtgct gcccaagcac 1740 agcctgcttt acgagtactt caccgtgtac aacgagctca ccaaggtgaa atatgtgacc 1800 gagggcatgc gaaaacccgc tttcctgagc ggcgagcaga agaaggccat cgtggacctg 1860 ctgttcaaga ccaacaggaa ggtgaccgtg aagcagctga aggaggacta cttcaagaag 1920 atcgagtgct ttgatagcgt ggaaataagc ggcgtggagg acaggttcaa cgccagcctg 1980 ggcacctacc acgacttgtt gaagataatc aaagacaagg atttcctgga taatgaggag 2040 aacgaggata tactcgagga catcgtgctg actttgaccc tgtttgagga ccgagagatg 2100 attgaagaaa ggctcaaaac ctacgcccac ctgttcgacg acaaagtgat gaaacaactg 2160 aagagacgaa gatacaccgg ctggggcaga ctgtccagga agctcatcaa cggcattagg 2220 gacaagcaga gcggcaagac catcctggat ttcctgaagt ccgacggctt cgccaaccga 2280 aacttcatgc agctgattca cgatgacagc ttgaccttca aggaggacat ccagaaggcc 2340 caggttagcg gccagggcga ctccctgcac gaacatattg caaacctggc aggctcccct 2400 gcgatcaaga agggcatact gcagaccgtt aaggttgtgg acgaattggt caaggtcatg 2460 ggcaggcaca agcccgaaaa catagttata gagatggcca gagagaacca gaccacccaa 2520 aagggccaga agaacagccg ggagcgcatg aaaaggatcg aggagggtat caaggaactc 2580 ggaagccaga tcctcaaaga gcaccccgtg gagaataccc agctccagaa cgagaagctg 2640 tacctgtact acctgcagaa cggcagggac atgtacgttg accaggagtt ggacatcaac 2700 aggctttcag actatgacgt ggatcacata gtgccccaga gctttcttaa agacgatagc 2760 atcgacaaca aggtcctgac ccgctccgac aaaaacaggg gcaaaagcga caacgtgcca 2820 agcgaagagg tggttaaaaa gatgaagaac tactggaggc aactgctcaa cgcgaaattg 2880 atcacccaga gaaagttcga taacctgacc aaggccgaga ggggcggact ctccgaactt 2940 gacaaagcgg gcttcataaa gaggcagctg gtcgagaccc gacagatcac gaagcacgtg 3000 gcccaaatcc tcgacagcag aatgaatacc aagtacgatg agaatgacaa actcatcagg 3060 gaagtgaaag tgattaccct gaagagcaag ttggtgtccg actttcgcaa agatttccag 3120 ttctacaagg tgagggagat caacaactac caccatgccc acgacgcata cctgaacgcc 3180 gtggtcggca ccgccctgat taagaagtat ccaaagctgg agtccgaatt tgtctacggc 3240 gactacaaag tttacgatgt gaggaagatg atcgctaaga gcgaacagga gatcggcaag 3300 gccaccgcta agtatttctt ctacagcaac atcatgaact ttttcaagac cgagatcaca 3360 cttgccaacg gcgaaatcag gaagaggccg cttatcgaga ccaacggtga gaccggcgag 3420 atcgtgtggg acaagggcag ggacttcgcc accgtgagga aagtcctgag catgccccag 3480 gtgaatattg tgaaaaaaac tgaggtgcag acaggcggct ttagcaagga atccatcctg 3540 cccaagagga acagcgacaa gctgatcgcc cggaagaagg actgggaccc taagaagtat 3600 ggaggcttcg acagccccac cgtagcctac agcgtgctgg tggtcgcgaa ggtagagaag 3660 gggaagagca agaaactgaa gagcgtgaag gagctgctcg gcataaccat catggagagg 3720 tccagctttg agaagaaccc cattgacttt ttggaagcca agggctacaa agaggtcaaa 3780 aaggacctga tcatcaaact ccccaagtac tccctgtttg aattggagaa cggcagaaag 3840 aggatgctgg cgagcgctgg ggaactgcaa aagggcaacg aactggcgct gcccagcaag 3900 tacgtgaatt ttctgtacct ggcgtcccac tacgaaaagc tgaaaggcag ccccgaggac 3960 aacgagcaga agcagctgtt cgtggagcag cacaagcatt acctggacga gataatcgag 4020 caaatcagcg agttcagcaa gagggtgatt ctggccgacg cgaacctgga taaggtcctc 4080 agcgcctaca acaagcaccg agacaaaccc atcagggagc aggccgagaa tatcatacac 4140 ctgttcaccc tgacaaatct gggcgcacct gcggcattca aatacttcga taccaccatc 4200 gacaggaaaa ggtacactag cactaaggag gtgctggatg ccaccttgat ccaccagtcc 4260 attaccggcc tgtatgagac caggatcgac ctgagccagc ttggaggcga ctctagggcg 4320 gacccaaaaa agaaaaggaa ggtggaattc tacccatacg atgttccaga tacgct 4377 <210> 63 <211> 4050 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; AsCas12a <400> 63 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctgctg ctgctagcac acagttcgag ggctttacca acctgtatca ggtgagcaag 120 acactgcggt ttgagctgat cccacagggc aagaccctga agcacatcca ggagcagggc 180 ttcatcgagg aggacaaggc ccgcaatgat cactacaagg agctgaagcc catcatcgat 240 cggatctaca agacctatgc cgaccagtgc ctgcagctgg tgcagctgga ttgggagaac 300 ctgagcgccg ccatcgactc ctatagaaag gagaaaaccg aggagacaag gaacgccctg 360 atcgaggagc aggccacata tcgcaatgcc atccacgact acttcatcgg ccggacagac 420 aacctgaccg atgccatcaa taagagacac gccgagatct acaagggcct gttcaaggcc 480 gagctgttta atggcaaggt gctgaagcag ctgggcaccg tgaccacaac cgagcacgag 540 aacgccctgc tgcggagctt cgacaagttt acaacctact tctccggctt ttatgagaac 600 aggaagaacg tgttcagcgc cgaggatatc agcacagcca tcccacaccg catcgtgcag 660 gacaacttcc ccaagtttaa ggagaattgt cacatcttca cacgcctgat caccgccgtg 720 cccagcctgc gggagcactt tgagaacgtg aagaaggcca tcggcatctt cgtgagcacc 780 tccatcgagg aggtgttttc cttccctttt tataaccagc tgctgacaca gacccagatc 840 gacctgtata accagctgct gggaggaatc tctcgggagg caggcaccga gaagatcaag 900 ggcctgaacg aggtgctgaa tctggccatc cagaagaatg atgagacagc ccacatcatc 960 gcctccctgc cacacagatt catccccctg tttaagcaga tcctgtccga taggaacacc 1020 ctgtctttca tcctggagga gtttaagagc gacgaggaag tgatccagtc cttctgcaag 1080 tacaagacac tgctgagaaa cgagaacgtg ctggagacag ccgaggccct gtttaacgag 1140 ctgaacagca tcgacctgac acacatcttc atcagccaca agaagctgga gacaatcagc 1200 agcgccctgt gcgaccactg ggatacactg aggaatgccc tgtatgagcg gagaatctcc 1260 gagctgacag gcaagatcac caagtctgcc aaggagaagg tgcagcgcag cctgaagcac 1320 gaggatatca acctgcagga gatcatctct gccgcaggca aggagctgag cgaggccttc 1380 aagcagaaaa ccagcgagat cctgtcccac gcacacgccg ccctggatca gccactgcct 1440 acaaccctga agaagcagga ggagaaggag atcctgaagt ctcagctgga cagcctgctg 1500 ggcctgtacc acctgctgga ctggtttgcc gtggatgagt ccaacgaggt ggaccccgag 1560 ttctctgccc ggctgaccgg catcaagctg gagatggagc cttctctgag cttctacaac 1620 aaggccagaa attatgccac caagaagccc tactccgtgg agaagttcaa gctgaacttt 1680 cagatgccta cactggcctc tggctgggac gtgaataagg agaagaacaa tggcgccatc 1740 ctgtttgtga agaacggcct gtactatctg ggcatcatgc caaagcagaa gggcaggtat 1800 aaggccctga gcttcgagcc cacagagaaa accagcgagg gctttgataa gatgtactat 1860 gactacttcc ctgatgccgc caagatgatc ccaaagtgca gcacccagct gaaggccgtg 1920 acagcccact ttcagaccca cacaaccccc atcctgctgt ccaacaattt catcgagcct 1980 ctggagatca caaaggagat ctacgacctg aacaatcctg agaaggagcc aaagaagttt 2040 cagacagcct acgccaagaa aaccggcgac cagaagggct acagagaggc cctgtgcaag 2100 tggatcgact tcacaaggga ttttctgtcc aagtatacca agacaacctc tatcgatctg 2160 tctagcctgc ggccatcctc tcagtataag gacctgggcg agtactatgc cgagctgaat 2220 cccctgctgt accacatcag cttccagaga atcgccgaga aggagatcat ggatgccgtg 2280 gagacaggca agctgtacct gttccagatc tataacaagg actttgccaa gggccaccac 2340 ggcaagccta atctgcacac actgtattgg accggcctgt tttctccaga gaacctggcc 2400 aagacaagca tcaagctgaa tggccaggcc gagctgttct accgccctaa gtccaggatg 2460 aagaggatgg cacaccggct gggagagaag atgctgaaca agaagctgaa ggatcagaaa 2520 accccaatcc ccgacaccct gtaccaggag ctgtacgact atgtgaatca cagactgtcc 2580 cacgacctgt ctgatgaggc cagggccctg ctgcccaacg tgatcaccaa ggaggtgtct 2640 cacgagatca tcaaggatag gcgctttacc agcgacaagt tctttttcca cgtgcctatc 2700 acactgaact atcaggccgc caattcccca tctaagttca accagagggt gaatgcctac 2760 ctgaaggagc accccgagac acctatcatc ggcatcgatc ggggcgagag aaacctgatc 2820 tatatcacag tgatcgactc caccggcaag atcctggagc agcggagcct gaacaccatc 2880 cagcagtttg attaccagaa gaagctggac aacagggaga aggagagggt ggcagcaagg 2940 caggcctggt ctgtggtggg cacaatcaag gatctgaagc agggctatct gagccaggtc 3000 atccacgaga tcgtggacct gatgatccac taccaggccg tggtggtgct ggagaacctg 3060 aatttcggct ttaagagcaa gaggaccggc atcgccgaga aggccgtgta ccagcagttc 3120 gagaagatgc tgatcgataa gctgaattgc ctggtgctga aggactatcc agcagagaaa 3180 gtgggaggcg tgctgaaccc ataccagctg acagaccagt tcacctcctt tgccaagatg 3240 ggcacccagt ctggcttcct gttttacgtg cctgccccat atacatctaa gatcgatccc 3300 ctgaccggct tcgtggaccc cttcgtgtgg aaaaccatca agaatcacga gagccgcaag 3360 cacttcctgg agggcttcga ctttctgcac tacgacgtga aaaccggcga cttcatcctg 3420 cactttaaga tgaacagaaa tctgtccttc cagaggggcc tgcccggctt tatgcctgca 3480 tgggatatcg tgttcgagaa gaacgagaca cagtttgacg ccaagggcac ccctttcatc 3540 gccggcaaga gaatcgtgcc agtgatcgag aatcacagat tcaccggcag ataccgggac 3600 ctgtatcctg ccaacgagct gatcgccctg ctggaggaga agggcatcgt gttcagggat 3660 ggctccaaca tcctgccaaa gctgctggag aatgacgatt ctcacgccat cgacaccatg 3720 gtggccctga tccgcagcgt gctgcagatg cggaactcca atgccgccac aggcgaggac 3780 tatatcaaca gccccgtgcg cgatctgaat ggcgtgtgct tcgactcccg gtttcagaac 3840 ccagagtggc ccatggacgc cgatgccaat ggcgcctacc acatcgccct gaagggccag 3900 ctgctgctga atcacctgaa ggagagcaag gatctgaagc tgcagaacgg catctccaat 3960 caggactggc tggcctacat ccaggagctg cgcaacaaaa ggccggcggc cacgaaaaag 4020 gccggccagg caaaaaagaa aaaggaattc 4050 <210> 64 <211> 3813 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; LbCas12a <400> 64 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctgctg ctgctagcag caagctggag aagtttacaa actgctactc cctgtctaag 120 accctgaggt tcaaggccat ccctgtgggc aagacccagg agaacatcga caataagcgg 180 ctgctggtgg aggacgagaa gagagccgag gattataagg gcgtgaagaa gctgctggat 240 cgctactatc tgtcttttat caacgacgtg ctgcacagca tcaagctgaa gaatctgaac 300 aattacatca gcctgttccg gaagaaaacc agaaccgaga aggagaataa ggagctggag 360 aacctggaga tcaatctgcg gaaggagatc gccaaggcct tcaagggcaa cgagggctac 420 aagtccctgt ttaagaagga tatcatcgag acaatcctgc cagagttcct ggacgataag 480 gacgagatcg ccctggtgaa cagcttcaat ggctttacca cagccttcac cggcttcttt 540 gataacagag agaatatgtt ttccgaggag gccaagagca catccatcgc cttcaggtgt 600 atcaacgaga atctgacccg ctacatctct aatatggaca tcttcgagaa ggtggacgcc 660 atctttgata agcacgaggt gcaggagatc aaggagaaga tcctgaacag cgactatgat 720 gtggaggatt tctttgaggg cgagttcttt aactttgtgc tgacacagga gggcatcgac 780 gtgtataacg ccatcatcgg cggcttcgtg accgagagcg gcgagaagat caagggcctg 840 aacgagtaca tcaacctgta taatcagaaa accaagcaga agctgcctaa gtttaagcca 900 ctgtataagc aggtgctgag cgatcgggag tctctgagct tctacggcga gggctataca 960 tccgatgagg aggtgctgga ggtgtttaga aacaccctga acaagaacag cgagatcttc 1020 agctccatca agaagctgga gaagctgttc aagaattttg acgagtactc tagcgccggc 1080 atctttgtga agaacggccc cgccatcagc acaatctcca aggatatctt cggcgagtgg 1140 aacgtgatcc gggacaagtg gaatgccgag tatgacgata tccacctgaa gaagaaggcc 1200 gtggtgaccg agaagtacga ggacgatcgg agaaagtcct tcaagaagat cggctccttt 1260 tctctggagc agctgcagga gtacgccgac gccgatctgt ctgtggtgga gaagctgaag 1320 gagatcatca tccagaaggt ggatgagatc tacaaggtgt atggctcctc tgagaagctg 1380 ttcgacgccg attttgtgct ggagaagagc ctgaagaaga acgacgccgt ggtggccatc 1440 atgaaggacc tgctggattc tgtgaagagc ttcgagaatt acatcaaggc cttctttggc 1500 gagggcaagg agacaaacag ggacgagtcc ttctatggcg attttgtgct ggcctacgac 1560 atcctgctga aggtggacca catctacgat gccatccgca attatgtgac ccagaagccc 1620 tactctaagg ataagttcaa gctgtatttt cagaaccctc agttcatggg cggctgggac 1680 aaggataagg agacagacta tcgggccacc atcctgagat acggctccaa gtactatctg 1740 gccatcatgg ataagaagta cgccaagtgc ctgcagaaga tcgacaagga cgatgtgaac 1800 ggcaattacg agaagatcaa ctataagctg ctgcccggcc ctaataagat gctgccaaag 1860 gtgttctttt ctaagaagtg gatggcctac tataacccca gcgaggacat ccagaagatc 1920 tacaagaatg gcacattcaa gaagggcgat atgtttaacc tgaatgactg tcacaagctg 1980 atcgacttct ttaaggatag catctcccgg tatccaaagt ggtccaatgc ctacgatttc 2040 aacttttctg agacagagaa gtataaggac atcgccggct tttacagaga ggtggaggag 2100 cagggctata aggtgagctt cgagtctgcc agcaagaagg aggtggataa gctggtggag 2160 gagggcaagc tgtatatgtt ccagatctat aacaaggact tttccgataa gtctcacggc 2220 acacccaatc tgcacaccat gtacttcaag ctgctgtttg acgagaacaa tcacggacag 2280 atcaggctga gcggaggagc agagctgttc atgaggcgcg cctccctgaa gaaggaggag 2340 ctggtggtgc acccagccaa ctcccctatc gccaacaaga atccagataa tcccaagaaa 2400 accacaaccc tgtcctacga cgtgtataag gataagaggt tttctgagga ccagtacgag 2460 ctgcacatcc caatcgccat caataagtgc cccaagaaca tcttcaagat caatacagag 2520 gtgcgcgtgc tgctgaagca cgacgataac ccctatgtga tcggcatcga taggggcgag 2580 cgcaatctgc tgtatatcgt ggtggtggac ggcaagggca acatcgtgga gcagtattcc 2640 ctgaacgaga tcatcaacaa cttcaacggc atcaggatca agacagatta ccactctctg 2700 ctggacaaga aggagaagga gaggttcgag gcccgccaga actggacctc catcgagaat 2760 atcaaggagc tgaaggccgg ctatatctct caggtggtgc acaagatctg cgagctggtg 2820 gagaagtacg atgccgtgat cgccctggag gacctgaact ctggctttaa gaatagccgc 2880 gtgaaggtgg agaagcaggt gtatcagaag ttcgagaaga tgctgatcga taagctgaac 2940 tacatggtgg acaagaagtc taatccttgt gcaacaggcg gcgccctgaa gggctatcag 3000 atcaccaata agttcgagag ctttaagtcc atgtctaccc agaacggctt catcttttac 3060 atccctgcct ggctgacatc caagatcgat ccatctaccg gctttgtgaa cctgctgaaa 3120 accaagtata ccagcatcgc cgattccaag aagttcatca gctcctttga caggatcatg 3180 tacgtgcccg aggaggatct gttcgagttt gccctggact ataagaactt ctctcgcaca 3240 gacgccgatt acatcaagaa gtggaagctg tactcctacg gcaaccggat cagaatcttc 3300 cggaatccta agaagaacaa cgtgttcgac tgggaggagg tgtgcctgac cagcgcctat 3360 aaggagctgt tcaacaagta cggcatcaat tatcagcagg gcgatatcag agccctgctg 3420 tgcgagcagt ccgacaaggc cttctactct agctttatgg ccctgatgag cctgatgctg 3480 cagatgcgga acagcatcac aggccgcacc gacgtggatt ttctgatcag ccctgtgaag 3540 aactccgacg gcatcttcta cgatagccgg aactatgagg cccaggagaa tgccatcctg 3600 ccaaagaacg ccgacgccaa tggcgcctat aacatcgcca gaaaggtgct gtgggccatc 3660 ggccagttca agaaggccga ggacgagaag ctggataagg tgaagatcgc catctctaac 3720 aaggagtggc tggagtacgc ccagaccagc gtgaagcaca aaaggccggc ggccacgaaa 3780 aaggccggcc aggcaaaaaa gaaaaaggaa ttc 3813 <210> 65 <211> 4305 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; SpCas9 <400> 65 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctgctg ctgctagcct ggacagcacc gctcccaaaa agaaaaggaa ggtgggcatt 120 cacggcgtgc ctgcggccga caaaaagtac agcatcggcc ttgatatcgg caccaatagc 180 gtgggctggg ccgttatcac agacgaatac aaggtaccca gcaagaagtt caaggtgctg 240 gggaatacag acaggcactc tatcaagaaa aaccttatcg gggctctgct gtttgactca 300 ggcgagaccg ccgaggccac caggttgaag aggaccgcaa ggcgaaggta cacccggagg 360 aagaacagga tctgctatct gcaggagatc ttcagcaacg agatggccaa ggtggacgac 420 agcttcttcc acaggctgga ggagagcttc cttgtcgagg aggataagaa gcacgaacga 480 caccccatct tcggcaacat agtcgacgag gtcgcttatc acgagaagta ccccaccatc 540 taccacctgc gaaagaaatt ggtggatagc accgataaag ccgacttgcg acttatctac 600 ttggctctgg cgcacatgat taagttcagg ggccacttcc tgatcgaggg cgaccttaac 660 cccgacaaca gtgacgtaga caaattgttc atccagcttg tacagaccta taaccagctg 720 ttcgaggaaa accctattaa cgccagcggg gtggatgcga aggccatact tagcgccagg 780 ctgagcaaaa gcaggcgctt ggagaacctg atagcccagc tgcccggtga aaagaagaac 840 ggcctcttcg gtaatctgat tgccctgagc ctgggcctga cccccaactt caagagcaac 900 ttcgacctgg cagaagatgc caagctgcag ttgagtaagg acacctatga cgacgacttg 960 gacaatctgc tcgcccaaat cggcgaccag tacgctgacc tgttcctcgc cgccaagaac 1020 ctttctgacg caatcctgct tagcgatatc cttagggtga acacagagat caccaaggcc 1080 cccctgagcg ccagcatgat caagaggtac gacgagcacc atcaggacct gacccttctg 1140 aaggccctgg tgaggcagca actgcccgag aagtacaagg agatcttttt cgaccagagc 1200 aagaacggct acgccggcta catcgacggc ggagccagcc aagaggagtt ctacaagttc 1260 atcaagccca tcctggagaa gatggatggc accgaggagc tgctggtgaa gctgaacagg 1320 gaagattgc tccggaagca gaggaccttt gacaacggta gcatccccca ccagatccac 1380 ctgggcgagc tgcacgcaat actgaggcga caggaggatt tctacccctt cctcaaggac 1440 aatagggaga aaatcgaaaa gattctgacc ttcaggatcc cctactacgt gggccctctt 1500 gccaggggca acagccgatt cgcttggatg acaagaaaga gcgaggagac catcaccccc 1560 tggaacttcg aggaagtggt ggacaaagga gcaagcgcgc agtctttcat cgaacggatg 1620 accaatttcg acaaaaacct gcctaacgag aaggtgctgc ccaagcacag cctgctttac 1680 gagtacttca ccgtgtacaa cgagctcacc aaggtgaaat atgtgaccga gggcatgcga 1740 aaacccgctt tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc 1800 aacaggaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttt 1860 gatagcgtgg aaataagcgg cgtggaggac aggttcaacg ccagcctggg cacctaccac 1920 gacttgttga agataatcaa agacaaggat ttcctggata atgaggagaa cgaggatata 1980 ctcgaggaca tcgtgctgac tttgaccctg tttgaggacc gagagatgat tgaagaaagg 2040 ctcaaaacct acgcccacct gttcgacgac aaagtgatga aacaactgaa gagacgaaga 2100 tacaccggct ggggcagact gtccaggaag ctcatcaacg gcattaggga caagcagagc 2160 ggcaagacca tcctggattt cctgaagtcc gacggcttcg ccaaccgaaa cttcatgcag 2220 ctgattcacg atgacagctt gaccttcaag gaggacatcc agaaggccca ggttagcggc 2280 cagggcgact ccctgcacga acatattgca aacctggcag gctcccctgc gatcaagaag 2340 ggcatactgc agaccgttaa ggttgtggac gaattggtca aggtcatggg caggcacaag 2400 cccgaaaaca tagttataga gatggccaga gagaaccaga ccacccaaaa gggccagaag 2460 aacagccggg agcgcatgaa aaggatcgag gagggtatca aggaactcgg aagccagatc 2520 ctcaaagagc accccgtgga gaatacccag ctccagaacg agaagctgta cctgtactac 2580 ctgcagaacg gcagggacat gtacgttgac caggagttgg acatcaacag gctttcagac 2640 tatgacgtgg atcacatagt gccccagagc tttcttaaag acgatagcat cgacaacaag 2700 gtcctgaccc gctccgacaa aaacaggggc aaaagcgaca acgtgccaag cgaagaggtg 2760 gttaaaaaga tgaagaacta ctggaggcaa ctgctcaacg cgaaattgat cacccagaga 2820 aagttcgata acctgaccaa ggccgagagg ggcggactct ccgaacttga caaagcgggc 2880 ttcataaaga ggcagctggt cgagacccga cagatcacga agcacgtggc ccaaatcctc 2940 gacagcagaa tgaataccaa gtacgatgag aatgacaaac tcatcaggga agtgaaagtg 3000 attaccctga agagcaagtt ggtgtccgac tttcgcaaag atttccagtt ctacaaggtg 3060 agggagatca acaactacca ccatgcccac gacgcatacc tgaacgccgt ggtcggcacc 3120 gccctgatta agaagtatcc aaagctggag tccgaatttg tctacggcga ctacaaagtt 3180 tacgatgtga ggaagatgat cgctaagagc gaacaggaga tcggcaaggc caccgctaag 3240 tatttcttct acagcaacat catgaacttt ttcaagaccg agatcacact tgccaacggc 3300 gaaatcagga agaggccgct tatcgagacc aacggtgaga ccggcgagat cgtgtgggac 3360 aagggcaggg acttcgccac cgtgaggaaa gtcctgagca tgccccaggt gaatattgtg 3420 aaaaaaactg aggtgcagac aggcggcttt agcaaggaat ccatcctgcc caagaggaac 3480 agcgacaagc tgatcgcccg gaagaaggac tgggacccta agaagtatgg aggcttcgac 3540 agccccaccg tagcctacag cgtgctggtg gtcgcgaagg tagagaaggg gaagagcaag 3600 aaactgaaga gcgtgaagga gctgctcggc ataaccatca tggagaggtc cagctttgag 3660 aagaacccca ttgacttttt ggaagccaag ggctacaaag aggtcaaaaa ggacctgatc 3720 atcaaactcc ccaagtactc cctgtttgaa ttggagaacg gcagaaagag gatgctggcg 3780 agcgctgggg aactgcaaaa gggcaacgaa ctggcgctgc ccagcaagta cgtgaatttt 3840 ctgtacctgg cgtcccacta cgaaaagctg aaaggcagcc ccgaggacaa cgagcagaag 3900 cagctgttcg tggagcagca caagcattac ctggacgaga taatcgagca aatcagcgag 3960 ttcagcaaga gggtgattct ggccgacgcg aacctggata aggtcctcag cgcctacaac 4020 aagcaccgag acaaacccat cagggagcag gccgagaata tcatacacct gttcaccctg 4080 acaaatctgg gcgcacctgc ggcattcaaa tacttcgata ccaccatcga caggaaaagg 4140 tacactagca ctaaggaggt gctggatgcc accttgatcc accagtccat taccggcctg 4200 tatgagacca ggatcgacct gagccagctt ggaggcgact ctagggcgga cccaaaaaag 4260 aaaaggaagg tggaattcta cccatacgat gttccagatt acgct 4305 <210> 66 <211> 1350 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; AsCas12a <400> 66 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ala Ala Ala Ser Thr Gln Phe Glu Gly Phe 20 25 30 Thr Asn Leu Tyr Gln Val Ser Lys Thr Leu Arg Phe Glu Leu Ile Pro 35 40 45 Gln Gly Lys Thr Leu Lys His Ile Gln Glu Gin Gly Phe Ile Glu Glu 50 55 60 Asp Lys Ala Arg Asn Asp His Tyr Lys Glu Leu Lys Pro Ile Ile Asp 65 70 75 80 Arg Ile Tyr Lys Thr Tyr Ala Asp Gln Cys Leu Gln Leu Val Gln Leu 85 90 95 Asp Trp Glu Asn Leu Ser Ala Ala Ile Asp Ser Tyr Arg Lys Glu Lys 100 105 110 Thr Glu Glu Thr Arg Asn Ala Leu Ile Glu Glu Gln Ala Thr Tyr Arg 115 120 125 Asn Ala Ile His Asp Tyr Phe Ile Gly Arg Thr Asp Asn Leu Thr Asp 130 135 140 Ala Ile Asn Lys Arg His Ala Glu Ile Tyr Lys Gly Leu Phe Lys Ala 145 150 155 160 Glu Leu Phe Asn Gly Lys Val Leu Lys Gln Leu Gly Thr Val Thr Thr 165 170 175 Thr Glu His Glu Asn Ala Leu Leu Arg Ser Phe Asp Lys Phe Thr Thr 180 185 190 Tyr Phe Ser Gly Phe Tyr Glu Asn Arg Lys Asn Val Phe Ser Ala Glu 195 200 205 Asp Ile Ser Thr Ala Ile Pro His Arg Ile Val Gln Asp Asn Phe Pro 210 215 220 Lys Phe Lys Glu Asn Cys His Ile Phe Thr Arg Leu Ile Thr Ala Val 225 230 235 240 Pro Ser Leu Arg Glu His Phe Glu Asn Val Lys Lys Ala Ile Gly Ile 245 250 255 Phe Val Ser Thr Ser Ile Glu Glu Val Phe Ser Phe Pro Phe Tyr Asn 260 265 270 Gln Leu Leu Thr Gln Thr Gln Ile Asp Leu Tyr Asn Gln Leu Leu Gly 275 280 285 Gly Ile Ser Arg Glu Ala Gly Thr Glu Lys Ile Lys Gly Leu Asn Glu 290 295 300 Val Leu Asn Leu Ala Ile Gln Lys Asn Asp Glu Thr Ala His Ile Ile 305 310 315 320 Ala Ser Leu Pro His Arg Phe Ile Pro Leu Phe Lys Gln Ile Leu Ser 325 330 335 Asp Arg Asn Thr Leu Ser Phe Ile Leu Glu Glu Phe Lys Ser Asp Glu 340 345 350 Glu Val Ile Gln Ser Phe Cys Lys Tyr Lys Thr Leu Leu Arg Asn Glu 355 360 365 Asn Val Leu Glu Thr Ala Glu Ala Leu Phe Asn Glu Leu Asn Ser Ile 370 375 380 Asp Leu Thr His Ile Phe Ile Ser His Lys Lys Leu Glu Thr Ile Ser 385 390 395 400 Ser Ala Leu Cys Asp His Trp Asp Thr Leu Arg Asn Ala Leu Tyr Glu 405 410 415 Arg Arg Ile Ser Glu Leu Thr Gly Lys Ile Thr Lys Ser Ala Lys Glu 420 425 430 Lys Val Gln Arg Ser Leu Lys His Glu Asp Ile Asn Leu Gln Glu Ile 435 440 445 Ile Ser Ala Ala Gly Lys Glu Leu Ser Glu Ala Phe Lys Gln Lys Thr 450 455 460 Ser Glu Ile Leu Ser His Ala His Ala Ala Leu Asp Gln Pro Leu Pro 465 470 475 480 Thr Thr Leu Lys Lys Gln Glu Glu Lys Glu Ile Leu Lys Ser Gln Leu 485 490 495 Asp Ser Leu Leu Gly Leu Tyr His Leu Leu Asp Trp Phe Ala Val Asp 500 505 510 Glu Ser Asn Glu Val Asp Pro Glu Phe Ser Ala Arg Leu Thr Gly Ile 515 520 525 Lys Leu Glu Met Glu Pro Ser Leu Ser Phe Tyr Asn Lys Ala Arg Asn 530 535 540 Tyr Ala Thr Lys Lys Pro Tyr Ser Val Glu Lys Phe Lys Leu Asn Phe 545 550 555 560 Gln Met Pro Thr Leu Ala Ser Gly Trp Asp Val Asn Lys Glu Lys Asn 565 570 575 Asn Gly Ala Ile Leu Phe Val Lys Asn Gly Leu Tyr Tyr Leu Gly Ile 580 585 590 Met Pro Lys Gln Lys Gly Arg Tyr Lys Ala Leu Ser Phe Glu Pro Thr 595 600 605 Glu Lys Thr Ser Glu Gly Phe Asp Lys Met Tyr Tyr Asp Tyr Phe Pro 610 615 620 Asp Ala Ala Lys Met Ile Pro Lys Cys Ser Thr Gln Leu Lys Ala Val 625 630 635 640 Thr Ala His Phe Gln Thr His Thr Thr Pro Ile Leu Leu Ser Asn Asn 645 650 655 Phe Ile Glu Pro Leu Glu Ile Thr Lys Glu Ile Tyr Asp Leu Asn Asn 660 665 670 Pro Glu Lys Glu Pro Lys Lys Phe Gln Thr Ala Tyr Ala Lys Lys Thr 675 680 685 Gly Asp Gln Lys Gly Tyr Arg Glu Ala Leu Cys Lys Trp Ile Asp Phe 690 695 700 Thr Arg Asp Phe Leu Ser Lys Tyr Thr Lys Thr Thr Ser Ile Asp Leu 705 710 715 720 Ser Ser Leu Arg Pro Ser Ser Gln Tyr Lys Asp Leu Gly Glu Tyr Tyr 725 730 735 Ala Glu Leu Asn Pro Leu Leu Tyr His Ile Ser Phe Gln Arg Ile Ala 740 745 750 Glu Lys Glu Ile Met Asp Ala Val Glu Thr Gly Lys Leu Tyr Leu Phe 755 760 765 Gln Ile Tyr Asn Lys Asp Phe Ala Lys Gly His His Gly Lys Pro Asn 770 775 780 Leu His Thr Leu Tyr Trp Thr Gly Leu Phe Ser Pro Glu Asn Leu Ala 785 790 795 800 Lys Thr Ser Ile Lys Leu Asn Gly Gln Ala Glu Leu Phe Tyr Arg Pro 805 810 815 Lys Ser Arg Met Lys Arg Met Ala His Arg Leu Gly Glu Lys Met Leu 820 825 830 Asn Lys Lys Leu Lys Asp Gln Lys Thr Pro Ile Pro Asp Thr Leu Tyr 835 840 845 Gln Glu Leu Tyr Asp Tyr Val Asn His Arg Leu Ser His Asp Leu Ser 850 855 860 Asp Glu Ala Arg Ala Leu Leu Pro Asn Val Ile Thr Lys Glu Val Ser 865 870 875 880 His Glu Ile Ile Lys Asp Arg Arg Phe Thr Ser Asp Lys Phe Phe Phe 885 890 895 His Val Pro Ile Thr Leu Asn Tyr Gln Ala Ala Asn Ser Pro Ser Lys 900 905 910 Phe Asn Gln Arg Val Asn Ala Tyr Leu Lys Glu His Pro Glu Thr Pro 915 920 925 Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr Ile Thr Val 930 935 940 Ile Asp Ser Thr Gly Lys Ile Leu Glu Gln Arg Ser Leu Asn Thr Ile 945 950 955 960 Gln Gln Phe Asp Tyr Gln Lys Lys Leu Asp Asn Arg Glu Lys Glu Arg 965 970 975 Val Ala Ala Arg Gln Ala Trp Ser Val Val Gly Thr Ile Lys Asp Leu 980 985 990 Lys Gln Gly Tyr Leu Ser Gln Val Ile His Glu Ile Val Asp Leu Met 995 1000 1005 Ile His Tyr Gln Ala Val Val Val Leu Glu Asn Leu Asn Phe Gly Phe 1010 1015 1020 Lys Ser Lys Arg Thr Gly Ile Ala Glu Lys Ala Val Tyr Gln Gln Phe 1025 1030 1035 1040 Glu Lys Met Leu Ile Asp Lys Leu Asn Cys Leu Val Leu Lys Asp Tyr 1045 1050 1055 Pro Ala Glu Lys Val Gly Gly Val Leu Asn Pro Tyr Gln Leu Thr Asp 1060 1065 1070 Gln Phe Thr Ser Phe Ala Lys Met Gly Thr Gln Ser Gly Phe Leu Phe 1075 1080 1085 Tyr Val Pro Ala Pro Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe 1090 1095 1100 Val Asp Pro Phe Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys 1105 1110 1115 1120 His Phe Leu Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly 1125 1130 1135 Asp Phe Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg 1140 1145 1150 Gly Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn 1155 1160 1165 Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys Arg 1170 1175 1180 Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr Arg Asp 1185 1190 1195 1200 Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu Lys Gly Ile 1205 1210 1215 Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu Leu Glu Asn Asp 1220 1225 1230 Asp Ser His Ala Ile Asp Thr Met Val Ala Leu Ile Arg Ser Val Leu 1235 1240 1245 Gln Met Arg Asn Ser Asn Ala Ala Thr Gly Glu Asp Tyr Ile Asn Ser 1250 1255 1260 Pro Val Arg Asp Leu Asn Gly Val Cys Phe Asp Ser Arg Phe Gln Asn 1265 1270 1275 1280 Pro Glu Trp Pro Met Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Ala 1285 1290 1295 Leu Lys Gly Gln Leu Leu Leu Asn His Leu Lys Glu Ser Lys Asp Leu 1300 1305 1310 Lys Leu Gln Asn Gly Ile Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln 1315 1320 1325 Glu Leu Arg Asn Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala 1330 1335 1340 Lys Lys Lys Lys Glu Phe 1345 1350 <210> 67 <211> 1271 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; LbCas12a <400> 67 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ala Ala Ala Ser Ser Lys Leu Glu Lys Phe 20 25 30 Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro 35 40 45 Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu 50 55 60 Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp 65 70 75 80 Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu 85 90 95 Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr 100 105 110 Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys 115 120 125 Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe 130 135 140 Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys 145 150 155 160 Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe 165 170 175 Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys 180 185 190 Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr 195 200 205 Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys 210 215 220 His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp 225 230 235 240 Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln 245 250 255 Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu 260 265 270 Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn 275 280 285 Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln 290 295 300 Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr 305 310 315 320 Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn 325 330 335 Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn 340 345 350 Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala 355 360 365 Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg 370 375 380 Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala 385 390 395 400 Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys 405 410 415 Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp 420 425 430 Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp 435 440 445 Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp 450 455 460 Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile 465 470 475 480 Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys 485 490 495 Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr 500 505 510 Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile 515 520 525 Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp 530 535 540 Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp 545 550 555 560 Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser 565 570 575 Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln 580 585 590 Lys Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr 595 600 605 Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser 610 615 620 Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile 625 630 635 640 Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp 645 650 655 Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro 660 665 670 Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr 675 680 685 Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys 690 695 700 Val Ser Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu 705 710 715 720 Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp 725 730 735 Lys Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu 740 745 750 Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu 755 760 765 Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His 770 775 780 Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys 785 790 795 800 Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu 805 810 815 Asp Gln Tyr Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys 820 825 830 Asn Ile Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp 835 840 845 Asp Asn Pro Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu 850 855 860 Tyr Ile Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser 865 870 875 880 Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp 885 890 895 Tyr His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg 900 905 910 Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr 915 920 925 Ile Ser Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp 930 935 940 Ala Val Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg 945 950 955 960 Val Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile 965 970 975 Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr 980 985 990 Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe 995 1000 1005 Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp 1010 1015 1020 Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys 1025 1030 1035 1040 Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe 1045 1050 1055 Asp Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu 1060 1065 1070 Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp 1075 1080 1085 Lys Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys 1090 1095 1100 Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr 1105 1110 1115 1120 Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile 1125 1130 1135 Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe 1140 1145 1150 Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly 1155 1160 1165 Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly 1170 1175 1180 Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu 1185 1190 1195 1200 Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val 1205 1210 1215 Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp 1220 1225 1230 Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln 1235 1240 1245 Thr Ser Val Lys His Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln 1250 1255 1260 Ala Lys Lys Lys Lys Glu Phe 1265 1270 <210> 68 <211> 1435 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; SpCas9 <400> 68 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ala Ala Ala Ser Leu Asp Ser Thr Ala Pro 20 25 30 Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala Ala Asp Lys 35 40 45 Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala 50 55 60 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 65 70 75 80 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 85 90 95 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 100 105 110 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 115 120 125 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 130 135 140 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 145 150 155 160 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 165 170 175 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 180 185 190 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 195 200 205 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 210 215 220 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 225 230 235 240 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 245 250 255 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 260 265 270 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 275 280 285 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 290 295 300 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 305 310 315 320 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 325 330 335 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 340 345 350 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 355 360 365 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 370 375 380 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 385 390 395 400 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 405 410 415 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 420 425 430 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 435 440 445 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 450 455 460 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 465 470 475 480 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 485 490 495 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 500 505 510 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 515 520 525 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 530 535 540 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 545 550 555 560 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 565 570 575 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 580 585 590 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 595 600 605 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 610 615 620 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 625 630 635 640 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 645 650 655 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 660 665 670 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 675 680 685 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 690 695 700 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 705 710 715 720 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 725 730 735 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 740 745 750 Ile Gln Lys Ala Gln Val Ser Gly Gly Gly Asp Ser Leu His Glu His 755 760 765 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 770 775 780 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 785 790 795 800 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 805 810 815 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 820 825 830 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn 835 840 845 Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly 850 855 860 Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp 865 870 875 880 Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser 885 890 895 Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser 900 905 910 Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp 915 920 925 Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn 930 935 940 Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly 945 950 955 960 Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val 965 970 975 Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp 980 985 990 Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val 995 1000 1005 Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn 1010 1015 1020 Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr 1025 1030 1035 1040 Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1045 1050 1055 Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1060 1065 1070 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1075 1080 1085 Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys 1090 1095 1100 Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1105 1110 1115 1120 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1125 1130 1135 Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys 1140 1145 1150 Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1155 1160 1165 Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1170 1175 1180 Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1185 1190 1195 1200 Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg 1205 1210 1215 Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1220 1225 1230 Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1235 1240 1245 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu 1250 1255 1260 Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe 1265 1270 1275 1280 Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1285 1290 1295 Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1300 1305 1310 Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1315 1320 1325 Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp 1330 1335 1340 Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1345 1350 1355 1360 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1365 1370 1375 Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu 1380 1385 1390 Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser 1395 1400 1405 Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1410 1415 1420 Glu Phe Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1425 1430 1435 <210> 69 <211> 1356 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; AP-AsCas12a <400> 69 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser 20 25 30 Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr Leu 35 40 45 Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln Glu 50 55 60 Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys Glu 65 70 75 80 Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln Cys 85 90 95 Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile Asp 100 105 110 Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile Glu 115 120 125 Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly Arg 130 135 140 Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile Tyr 145 150 155 160 Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys Gln 165 170 175 Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg Ser 180 185 190 Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg Lys 195 200 205 Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg Ile 210 215 220 Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe Thr 225 230 235 240 Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn Val 245 250 255 Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val Phe 260 265 270 Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp Leu 275 280 285 Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu Lys 290 295 300 Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn Asp 305 310 315 320 Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro Leu 325 330 335 Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu Glu 340 345 350 Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr Lys 355 360 365 Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu Phe 370 375 380 Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His Lys 385 390 395 400 Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr Leu 405 410 415 Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys Ile 420 425 430 Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu Asp 435 440 445 Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser Glu 450 455 460 Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala Ala 465 470 475 480 Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys Glu 485 490 495 Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu Leu 500 505 510 Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe Ser 515 520 525 Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser Phe 530 535 540 Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val Glu 545 550 555 560 Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp Asp 565 570 575 Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn Gly 580 585 590 Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys Ala 595 600 605 Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys Met 610 615 620 Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys Ser 625 630 635 640 Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr Pro 645 650 655 Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys Glu 660 665 670 Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln Thr 675 680 685 Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala Leu 690 695 700 Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr Lys 705 710 715 720 Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr Lys 725 730 735 Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His Ile 740 745 750 Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu Thr 755 760 765 Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys Gly 770 775 780 His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu Phe 785 790 795 800 Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln Ala 805 810 815 Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His Arg 820 825 830 Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr Pro 835 840 845 Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His Arg 850 855 860 Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn Val 865 870 875 880 Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe Thr 885 890 895 Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln Ala 900 905 910 Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu Lys 915 920 925 Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn 930 935 940 Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu Gln 945 950 955 960 Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu Asp 965 970 975 Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val Val 980 985 990 Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile His 995 1000 1005 Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu Glu 1010 1015 1020 Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu Lys 1025 1030 1035 1040 Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Cys 1045 1050 1055 Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val Leu Asn 1060 1065 1070 Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly Thr 1075 1080 1085 Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys Ile 1090 1095 1100 Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile Lys 1105 1110 1115 1120 Asn His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe Leu His 1125 1130 1135 Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn Arg 1140 1145 1150 Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp Asp 1155 1160 1165 Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro 1170 1175 1180 Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg Phe 1185 1190 1195 1200 Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu 1205 1210 1215 Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro 1220 1225 1230 Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala 1235 1240 1245 Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly 1250 1255 1260 Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys Phe 1265 1270 1275 1280 Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala Asn 1285 1290 1295 Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His Leu 1300 1305 1310 Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn Gln Asp 1315 1320 1325 Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys Arg Pro Ala Ala Thr 1330 1335 1340 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1345 1350 1355 <210> 70 <211> 1371 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; dNP2-AsCas12a <400> 70 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys 20 25 30 Gly Ser Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Ala Ser Thr 35 40 45 Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr Leu Arg 50 55 60 Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln Glu Gln 65 70 75 80 Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys Glu Leu 85 90 95 Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln Cys Leu 100 105 110 Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile Asp Ser 115 120 125 Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile Glu Glu 130 135 140 Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly Arg Thr 145 150 155 160 Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile Tyr Lys 165 170 175 Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys Gln Leu 180 185 190 Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg Ser Phe 195 200 205 Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg Lys Asn 210 215 220 Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg Ile Val 225 230 235 240 Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe Thr Arg 245 250 255 Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn Val Lys 260 265 270 Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val Phe Ser 275 280 285 Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp Leu Tyr 290 295 300 Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu Lys Ile 305 310 315 320 Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn Asp Glu 325 330 335 Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro Leu Phe 340 345 350 Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu Glu Glu 355 360 365 Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr Lys Thr 370 375 380 Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu Phe Asn 385 390 395 400 Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His Lys Lys 405 410 415 Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr Leu Arg 420 425 430 Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys Ile Thr 435 440 445 Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu Asp Ile 450 455 460 Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser Glu Ala 465 470 475 480 Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala Ala Leu 485 490 495 Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys Glu Ile 500 505 510 Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu Leu Asp 515 520 525 Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe Ser Ala 530 535 540 Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser Phe Tyr 545 550 555 560 Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val Glu Lys 565 570 575 Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp Asp Val 580 585 590 Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn Gly Leu 595 600 605 Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys Ala Leu 610 615 620 Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys Met Tyr 625 630 635 640 Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys Ser Thr 645 650 655 Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr Pro Ile 660 665 670 Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys Glu Ile 675 680 685 Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln Thr Ala 690 695 700 Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala Leu Cys 705 710 715 720 Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr Lys Thr 725 730 735 Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr Lys Asp 740 745 750 Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His Ile Ser 755 760 765 Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu Thr Gly 770 775 780 Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys Gly His 785 790 795 800 His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu Phe Ser 805 810 815 Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln Ala Glu 820 825 830 Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His Arg Leu 835 840 845 Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr Pro Ile 850 855 860 Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His Arg Leu 865 870 875 880 Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn Val Ile 885 890 895 Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe Thr Ser 900 905 910 Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln Ala Ala 915 920 925 Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu Lys Glu 930 935 940 His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu 945 950 955 960 Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu Gln Arg 965 970 975 Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu Asp Asn 980 985 990 Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val Val Gly 995 1000 1005 Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile His Glu 1010 1015 1020 Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu Glu Asn 1025 1030 1035 1040 Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu Lys Ala 1045 1050 1055 Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Cys Leu 1060 1065 1070 Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val Leu Asn Pro 1075 1080 1085 Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly Thr Gln 1090 1095 1100 Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys Ile Asp 1105 1110 1115 1120 Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile Lys Asn 1125 1130 1135 His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe Leu His Tyr 1140 1145 1150 Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn Arg Asn 1155 1160 1165 Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp Asp Ile 1170 1175 1180 Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe 1185 1190 1195 1200 Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr 1205 1210 1215 Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu 1220 1225 1230 Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys 1235 1240 1245 Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu 1250 1255 1260 Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly Glu 1265 1270 1275 1280 Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys Phe Asp 1285 1290 1295 Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala Asn Gly 1300 1305 1310 Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His Leu Lys 1315 1320 1325 Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn Gln Asp Trp 1330 1335 1340 Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys Arg Pro Ala Ala Thr Lys 1345 1350 1355 1360 Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1365 1370 <210> 71 <211> 1356 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; R9-AsCas12a <400> 71 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Ala Ser 20 25 30 Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr Leu 35 40 45 Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln Glu 50 55 60 Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys Glu 65 70 75 80 Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln Cys 85 90 95 Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile Asp 100 105 110 Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile Glu 115 120 125 Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly Arg 130 135 140 Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile Tyr 145 150 155 160 Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys Gln 165 170 175 Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg Ser 180 185 190 Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg Lys 195 200 205 Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg Ile 210 215 220 Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe Thr 225 230 235 240 Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn Val 245 250 255 Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val Phe 260 265 270 Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp Leu 275 280 285 Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu Lys 290 295 300 Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn Asp 305 310 315 320 Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro Leu 325 330 335 Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu Glu 340 345 350 Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr Lys 355 360 365 Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu Phe 370 375 380 Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His Lys 385 390 395 400 Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr Leu 405 410 415 Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys Ile 420 425 430 Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu Asp 435 440 445 Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser Glu 450 455 460 Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala Ala 465 470 475 480 Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys Glu 485 490 495 Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu Leu 500 505 510 Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe Ser 515 520 525 Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser Phe 530 535 540 Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val Glu 545 550 555 560 Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp Asp 565 570 575 Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn Gly 580 585 590 Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys Ala 595 600 605 Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys Met 610 615 620 Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys Ser 625 630 635 640 Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr Pro 645 650 655 Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys Glu 660 665 670 Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln Thr 675 680 685 Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala Leu 690 695 700 Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr Lys 705 710 715 720 Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr Lys 725 730 735 Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His Ile 740 745 750 Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu Thr 755 760 765 Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys Gly 770 775 780 His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu Phe 785 790 795 800 Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln Ala 805 810 815 Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His Arg 820 825 830 Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr Pro 835 840 845 Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His Arg 850 855 860 Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn Val 865 870 875 880 Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe Thr 885 890 895 Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln Ala 900 905 910 Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu Lys 915 920 925 Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn 930 935 940 Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu Gln 945 950 955 960 Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu Asp 965 970 975 Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val Val 980 985 990 Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile His 995 1000 1005 Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu Glu 1010 1015 1020 Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu Lys 1025 1030 1035 1040 Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Cys 1045 1050 1055 Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val Leu Asn 1060 1065 1070 Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly Thr 1075 1080 1085 Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys Ile 1090 1095 1100 Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile Lys 1105 1110 1115 1120 Asn His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe Leu His 1125 1130 1135 Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn Arg 1140 1145 1150 Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp Asp 1155 1160 1165 Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro 1170 1175 1180 Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg Phe 1185 1190 1195 1200 Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu 1205 1210 1215 Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro 1220 1225 1230 Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala 1235 1240 1245 Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly 1250 1255 1260 Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys Phe 1265 1270 1275 1280 Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala Asn 1285 1290 1295 Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His Leu 1300 1305 1310 Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn Gln Asp 1315 1320 1325 Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys Arg Pro Ala Ala Thr 1330 1335 1340 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1345 1350 1355 <210> 72 <211> 1358 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; TAT-AsCas12a <400> 72 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 20 25 30 Ala Ser Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys 35 40 45 Thr Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile 50 55 60 Gln Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr 65 70 75 80 Lys Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp 85 90 95 Gln Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala 100 105 110 Ile Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu 115 120 125 Ile Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile 130 135 140 Gly Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu 145 150 155 160 Ile Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu 165 170 175 Lys Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu 180 185 190 Arg Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn 195 200 205 Arg Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His 210 215 220 Arg Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile 225 230 235 240 Phe Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu 245 250 255 Asn Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu 260 265 270 Val Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile 275 280 285 Asp Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr 290 295 300 Glu Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys 305 310 315 320 Asn Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile 325 330 335 Pro Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile 340 345 350 Leu Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys 355 360 365 Tyr Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala 370 375 380 Leu Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser 385 390 395 400 His Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp 405 410 415 Thr Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly 420 425 430 Lys Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His 435 440 445 Glu Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu 450 455 460 Ser Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His 465 470 475 480 Ala Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu 485 490 495 Lys Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His 500 505 510 Leu Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu 515 520 525 Phe Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu 530 535 540 Ser Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser 545 550 555 560 Val Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly 565 570 575 Trp Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys 580 585 590 Asn Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr 595 600 605 Lys Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp 610 615 620 Lys Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys 625 630 635 640 Cys Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr 645 650 655 Thr Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr 660 665 670 Lys Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe 675 680 685 Gln Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu 690 695 700 Ala Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr 705 710 715 720 Thr Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln 725 730 735 Tyr Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr 740 745 750 His Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val 755 760 765 Glu Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala 770 775 780 Lys Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly 785 790 795 800 Leu Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly 805 810 815 Gln Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala 820 825 830 His Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys 835 840 845 Thr Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn 850 855 860 His Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro 865 870 875 880 Asn Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg 885 890 895 Phe Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr 900 905 910 Gln Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr 915 920 925 Leu Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu 930 935 940 Arg Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu 945 950 955 960 Glu Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys 965 970 975 Leu Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser 980 985 990 Val Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val 995 1000 1005 Ile His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val 1010 1015 1020 Leu Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala 1025 1030 1035 1040 Glu Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu 1045 1050 1055 Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val 1060 1065 1070 Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met 1075 1080 1085 Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser 1090 1095 1100 Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr 1105 1110 1115 1120 Ile Lys Asn His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe 1125 1130 1135 Leu His Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met 1140 1145 1150 Asn Arg Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala 1155 1160 1165 Trp Asp Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly 1170 1175 1180 Thr Pro Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His 1185 1190 1195 1200 Arg Phe Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile 1205 1210 1215 Ala Leu Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile 1220 1225 1230 Leu Pro Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met 1235 1240 1245 Val Ala Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala 1250 1255 1260 Thr Gly Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val 1265 1270 1275 1280 Cys Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp 1285 1290 1295 Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn 1300 1305 1310 His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn 1315 1320 1325 Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys Arg Pro Ala 1330 1335 1340 Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1345 1350 1355 <210> 73 <211> 1277 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; AP-LbCas12a <400> 73 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Arg Arg Trp Cys Lys Arg Arg Arg Ala Ser 20 25 30 Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu 35 40 45 Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn 50 55 60 Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly 65 70 75 80 Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val 85 90 95 Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe 100 105 110 Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu 115 120 125 Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu 130 135 140 Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro 145 150 155 160 Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn 165 170 175 Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met 180 185 190 Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn 195 200 205 Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val 210 215 220 Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile 225 230 235 240 Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe 245 250 255 Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile 260 265 270 Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu 275 280 285 Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe 290 295 300 Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe 305 310 315 320 Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg 325 330 335 Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu 340 345 350 Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe 355 360 365 Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly 370 375 380 Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile 385 390 395 400 His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg 405 410 415 Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln 420 425 430 Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile 435 440 445 Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu 450 455 460 Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn 465 470 475 480 Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser 485 490 495 Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn 500 505 510 Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu 515 520 525 Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln 545 550 555 560 Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr 565 570 575 Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys 580 585 590 Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn 595 600 605 Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu 610 615 620 Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser 625 630 635 640 Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp 645 650 655 Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp 660 665 670 Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe 675 680 685 Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val 690 695 700 Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu 705 710 715 720 Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr 725 730 735 Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr 740 745 750 Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg 755 760 765 Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys 770 775 780 Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn 785 790 795 800 Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys 805 810 815 Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala 820 825 830 Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val Arg 835 840 845 Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp Arg 850 855 860 Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly Asn 865 870 875 880 Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly 885 890 895 Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys 900 905 910 Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys 915 920 925 Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu 930 935 940 Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser 945 950 955 960 Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln Lys 965 970 975 Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys 980 985 990 Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr 995 1000 1005 Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile 1010 1015 1020 Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly 1025 1030 1035 1040 Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys 1045 1050 1055 Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp 1060 1065 1070 Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala 1075 1080 1085 Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile Arg 1090 1095 1100 Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val 1105 1110 1115 1120 Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn 1125 1130 1135 Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys 1140 1145 1150 Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln Met 1155 1160 1165 Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro 1170 1175 1180 Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala 1185 1190 1195 1200 Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr 1205 1210 1215 Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala 1220 1225 1230 Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu 1235 1240 1245 Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Arg Pro Ala Ala 1250 1255 1260 Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1265 1270 1275 <210> 74 <211> 1292 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; dNP2-LbCas12a <400> 74 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys 20 25 30 Gly Ser Lys Ile Lys Lys Val Lys Lys Lys Gly Arg Lys Ala Ser Ser 35 40 45 Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg 50 55 60 Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys 65 70 75 80 Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val 85 90 95 Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu 100 105 110 His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg 115 120 125 Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu 130 135 140 Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly 145 150 155 160 Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu 165 170 175 Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn Gly 180 185 190 Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met Phe 195 200 205 Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu 210 215 220 Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val Asp 225 230 235 240 Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile Leu 245 250 255 Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn 260 265 270 Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly 275 280 285 Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr 290 295 300 Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys 305 310 315 320 Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr 325 330 335 Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg Asn 340 345 350 Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu 355 360 365 Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val 370 375 380 Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu 385 390 395 400 Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile His 405 410 415 Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg 420 425 430 Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu 435 440 445 Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile Ile 450 455 460 Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys 465 470 475 480 Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn Asp 485 490 495 Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser Phe 500 505 510 Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg 515 520 525 Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu 530 535 540 Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys 545 550 555 560 Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe 565 570 575 Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile 580 585 590 Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr 595 600 605 Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr 610 615 620 Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro 625 630 635 640 Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu 645 650 655 Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp Met 660 665 670 Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser 675 680 685 Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser 690 695 700 Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu 705 710 715 720 Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu Val 725 730 735 Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn 740 745 750 Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr Met 755 760 765 Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg Leu 770 775 780 Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys Glu 785 790 795 800 Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro 805 810 815 Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp 820 825 830 Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala Ile 835 840 845 Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val Arg Val 850 855 860 Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp Arg Gly 865 870 875 880 Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly Asn Ile 885 890 895 Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile 900 905 910 Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys Glu 915 920 925 Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu 930 935 940 Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu Leu 945 950 955 960 Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser Gly 965 970 975 Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln Lys Phe 980 985 990 Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser 995 1000 1005 Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn 1010 1015 1020 Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile Phe 1025 1030 1035 1040 Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe 1045 1050 1055 Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys 1060 1065 1070 Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp Leu 1075 1080 1085 Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp 1090 1095 1100 Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile 1105 1110 1115 1120 Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val Cys 1125 1130 1135 Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr 1140 1145 1150 Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala 1155 1160 1165 Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln Met Arg 1170 1175 1180 Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val 1185 1190 1195 1200 Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln 1205 1210 1215 Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn 1220 1225 1230 Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu 1235 1240 1245 Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp 1250 1255 1260 Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Arg Pro Ala Ala Thr 1265 1270 1275 1280 Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1285 1290 <210> 75 <211> 1277 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; R9-LbCas12a <400> 75 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Ala Ser 20 25 30 Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu 35 40 45 Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn 50 55 60 Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly 65 70 75 80 Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val 85 90 95 Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe 100 105 110 Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu 115 120 125 Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu 130 135 140 Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro 145 150 155 160 Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn 165 170 175 Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met 180 185 190 Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn 195 200 205 Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val 210 215 220 Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile 225 230 235 240 Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe 245 250 255 Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile 260 265 270 Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu 275 280 285 Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe 290 295 300 Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe 305 310 315 320 Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg 325 330 335 Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu 340 345 350 Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe 355 360 365 Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly 370 375 380 Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile 385 390 395 400 His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg 405 410 415 Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln 420 425 430 Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile 435 440 445 Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu 450 455 460 Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn 465 470 475 480 Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser 485 490 495 Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn 500 505 510 Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu 515 520 525 Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln 530 535 540 Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln 545 550 555 560 Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr 565 570 575 Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys 580 585 590 Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn 595 600 605 Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu 610 615 620 Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser 625 630 635 640 Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp 645 650 655 Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp 660 665 670 Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe 675 680 685 Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val 690 695 700 Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu 705 710 715 720 Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr 725 730 735 Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr 740 745 750 Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg 755 760 765 Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys 770 775 780 Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn 785 790 795 800 Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys 805 810 815 Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala 820 825 830 Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val Arg 835 840 845 Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp Arg 850 855 860 Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly Asn 865 870 875 880 Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly 885 890 895 Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys 900 905 910 Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys 915 920 925 Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu 930 935 940 Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser 945 950 955 960 Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln Lys 965 970 975 Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys 980 985 990 Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr 995 1000 1005 Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile 1010 1015 1020 Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly 1025 1030 1035 1040 Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser Lys 1045 1050 1055 Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu Asp 1060 1065 1070 Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp Ala 1075 1080 1085 Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile Arg 1090 1095 1100 Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu Val 1105 1110 1115 1120 Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile Asn 1125 1130 1135 Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp Lys 1140 1145 1150 Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln Met 1155 1160 1165 Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro 1170 1175 1180 Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala 1185 1190 1195 1200 Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr 1205 1210 1215 Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala 1220 1225 1230 Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu 1235 1240 1245 Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Arg Pro Ala Ala 1250 1255 1260 Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1265 1270 1275 <210> 76 <211> 1279 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence; TAT-LbCas12a <400> 76 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 20 25 30 Ala Ser Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys 35 40 45 Thr Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile 50 55 60 Asp Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr 65 70 75 80 Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn 85 90 95 Asp Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser 100 105 110 Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu 115 120 125 Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly 130 135 140 Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile 145 150 155 160 Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser 165 170 175 Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu 180 185 190 Asn Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys 195 200 205 Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu 210 215 220 Lys Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu 225 230 235 240 Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu 245 250 255 Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala 260 265 270 Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu 275 280 285 Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro 290 295 300 Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu 305 310 315 320 Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val 325 330 335 Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys 340 345 350 Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly 355 360 365 Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile 370 375 380 Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp 385 390 395 400 Asp Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp 405 410 415 Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln 420 425 430 Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys 435 440 445 Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser 450 455 460 Ser Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys 465 470 475 480 Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val 485 490 495 Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu 500 505 510 Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp 515 520 525 Ile Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val 530 535 540 Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn 545 550 555 560 Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg 565 570 575 Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp 580 585 590 Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn 595 600 605 Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys 610 615 620 Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn 625 630 635 640 Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys 645 650 655 Gly Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe 660 665 670 Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe 675 680 685 Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg 690 695 700 Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys 705 710 715 720 Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln 725 730 735 Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu 740 745 750 His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln 755 760 765 Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu 770 775 780 Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn 785 790 795 800 Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val 805 810 815 Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro 820 825 830 Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu 835 840 845 Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile 850 855 860 Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys 865 870 875 880 Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe 885 890 895 Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys 900 905 910 Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn 915 920 925 Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile 930 935 940 Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu 945 950 955 960 Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr 965 970 975 Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp 980 985 990 Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln 995 1000 1005 Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly 1010 1015 1020 Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser 1025 1030 1035 1040 Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp 1045 1050 1055 Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu 1060 1065 1070 Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr 1075 1080 1085 Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg 1090 1095 1100 Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu 1105 1110 1115 1120 Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly 1125 1130 1135 Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser 1140 1145 1150 Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu 1155 1160 1165 Gln Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile 1170 1175 1180 Ser Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr 1185 1190 1195 1200 Glu Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly 1205 1210 1215 Ala Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys 1220 1225 1230 Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn 1235 1240 1245 Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Lys Arg Pro 1250 1255 1260 Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys Glu Phe 1265 1270 1275 <210> 77 <211> 4068 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; AP-AsCas12a <400> 77 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgcgccggc gctggtgcaa acgccgccgg gctagcacac agttcgaggg ctttaccaac 120 ctgtatcagg tgagcaagac actgcggttt gagctgatcc cacagggcaa gaccctgaag 180 cacatccagg agcagggctt catcgaggag gacaaggccc gcaatgatca ctacaaggag 240 ctgaagccca tcatcgatcg gatctacaag acctatgccg accagtgcct gcagctggtg 300 cagctggatt gggagaacct gagcgccgcc atcgactcct atagaaagga gaaaaccgag 360 gagacaagga acgccctgat cgaggagcag gccacatatc gcaatgccat ccacgactac 420 ttcatcggcc ggacagacaa cctgaccgat gccatcaata agagacacgc cgagatctac 480 aagggcctgt tcaaggccga gctgtttaat ggcaaggtgc tgaagcagct gggcaccgtg 540 accacaaccg agcacgagaa cgccctgctg cggagcttcg acaagtttac aacctacttc 600 tccggctttt atgagaacag gaagaacgtg ttcagcgccg aggatatcag cacagccatc 660 ccacaccgca tcgtgcagga caacttcccc aagtttaagg agaattgtca catcttcaca 720 cgcctgatca ccgccgtgcc cagcctgcgg gagcactttg agaacgtgaa gaaggccatc 780 ggcatcttcg tgagcacctc catcgaggag gtgttttcct tcccttttta taaccagctg 840 ctgacacaga cccagatcga cctgtataac cagctgctgg gaggaatctc tcgggaggca 900 ggcaccgaga agatcaaggg cctgaacgag gtgctgaatc tggccatcca gaagaatgat 960 gagacagccc acatcatcgc ctccctgcca cacagattca tccccctgtt taagcagatc 1020 ctgtccgata ggaacaccct gtctttcatc ctggaggagt ttaagagcga cgaggaagtg 1080 atccagtcct tctgcaagta caagacactg ctgagaaacg agaacgtgct ggagacagcc 1140 gaggccctgt ttaacgagct gaacagcatc gacctgacac acatcttcat cagccacaag 1200 aagctggaga caatcagcag cgccctgtgc gaccactggg atacactgag gaatgccctg 1260 tatgagcgga gaatctccga gctgacaggc aagatcacca agtctgccaa ggagaaggtg 1320 cagcgcagcc tgaagcacga ggatatcaac ctgcaggaga tcatctctgc cgcaggcaag 1380 gagctgagcg aggccttcaa gcagaaaacc agcgagatcc tgtccccacgc acacgccgcc 1440 ctggatcagc cactgcctac aaccctgaag aagcaggagg agaaggagat cctgaagtct 1500 cagctggaca gcctgctggg cctgtaccac ctgctggact ggtttgccgt ggatgagtcc 1560 aacgaggtgg accccgagtt ctctgcccgg ctgaccggca tcaagctgga gatggagcct 1620 tctctgagct tctacaacaa ggccagaaat tatgccacca agaagcccta ctccgtggag 1680 aagttcaagc tgaactttca gatgcctaca ctggcctctg gctgggacgt gaataaggag 1740 aagaacaatg gcgccatcct gtttgtgaag aacggcctgt actatctggg catcatgcca 1800 aagcagaagg gcaggtataa ggccctgagc ttcgagccca cagagaaaac cagcgagggc 1860 tttgataaga tgtactatga ctacttccct gatgccgcca agatgatccc aaagtgcagc 1920 acccagctga aggccgtgac agcccacttt cagacccaca caacccccat cctgctgtcc 1980 aacaatttca tcgagcctct ggagatcaca aaggagatct acgacctgaa caatcctgag 2040 aaggagccaa agaagtttca gacagcctac gccaagaaaa ccggcgacca gaagggctac 2100 agagaggccc tgtgcaagtg gatcgacttc acaagggatt ttctgtccaa gtataccaag 2160 acaacctcta tcgatctgtc tagcctgcgg ccatcctctc agtataagga cctgggcgag 2220 tactatgccg agctgaatcc cctgctgtac cacatcagct tccagagaat cgccgagaag 2280 gagatcatgg atgccgtgga gacaggcaag ctgtacctgt tccagatcta taacaaggac 2340 tttgccaagg gccaccacgg caagcctaat ctgcacacac tgtattggac cggcctgttt 2400 tctccagaga acctggccaa gacaagcatc aagctgaatg gccaggccga gctgttctac 2460 cgccctaagt ccaggatgaa gaggatggca caccggctgg gagagaagat gctgaacaag 2520 aagctgaagg atcagaaaac cccaatcccc gacaccctgt accaggagct gtacgactat 2580 gtgaatcaca gactgtccca cgacctgtct gatgaggcca gggccctgct gcccaacgtg 2640 atcaccaagg aggtgtctca cgagatcatc aaggataggc gctttaccag cgacaagttc 2700 tttttccacg tgcctatcac actgaactat caggccgcca attccccatc taagttcaac 2760 cagagggtga atgcctacct gaaggagcac cccgagacac ctatcatcgg catcgatcgg 2820 ggcgagagaa acctgatcta tatcacagtg atcgactcca ccggcaagat cctggagcag 2880 cggagcctga acaccatcca gcagtttgat taccagaaga agctggacaa cagggagaag 2940 gagagggtgg cagcaaggca ggcctggtct gtggtgggca caatcaagga tctgaagcag 3000 ggctatctga gccaggtcat ccacgagatc gtggacctga tgatccacta ccaggccgtg 3060 gtggtgctgg agaacctgaa tttcggcttt aagagcaaga ggaccggcat cgccgagaag 3120 gccgtgtacc agcagttcga gaagatgctg atcgataagc tgaattgcct ggtgctgaag 3180 gactatccag cagagaaagt gggaggcgtg ctgaacccat accagctgac agaccagttc 3240 acctcctttg ccaagatggg cacccagtct ggcttcctgt tttacgtgcc tgccccatat 3300 acatctaaga tcgatcccct gaccggcttc gtggacccct tcgtgtggaa aaccatcaag 3360 aatcacgaga gccgcaagca cttcctggag ggcttcgact ttctgcacta cgacgtgaaa 3420 accggcgact tcatcctgca ctttaagatg aacagaaatc tgtccttcca gaggggcctg 3480 cccggcttta tgcctgcatg ggatatcgtg ttcgagaaga acgagacaca gtttgacgcc 3540 aagggcaccc ctttcatcgc cggcaagaga atcgtgccag tgatcgagaa tcacagattc 3600 accggcagat accgggacct gtatcctgcc aacgagctga tcgccctgct ggaggagaag 3660 ggcatcgtgt tcagggatgg ctccaacatc ctgccaaagc tgctggagaa tgacgattct 3720 cacgccatcg acaccatggt ggccctgatc cgcagcgtgc tgcagatgcg gaactccaat 3780 gccgccacag gcgaggacta tatcaacagc cccgtgcgcg atctgaatgg cgtgtgcttc 3840 gactcccggt ttcagaaccc agagtggccc atggacgccg atgccaatgg cgcctaccac 3900 atcgccctga agggccagct gctgctgaat cacctgaagg agagcaagga tctgaagctg 3960 cagaacggca tctccaatca ggactggctg gcctacatcc aggagctgcg caacaaaagg 4020 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggaattc 4068 <210> 78 <211> 4113 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; dNP2-AsCas12a <400> 78 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaagatca agaaggttaa aaaaaagggt cgcaagggct ctaaaattaa aaaagtcaag 120 aagaaaggaa gaaaagctag cacacagttc gagggcttta ccaacctgta tcaggtgagc 180 aagacactgc ggtttgagct gatcccacag ggcaagaccc tgaagcacat ccaggagcag 240 ggcttcatcg aggaggacaa ggcccgcaat gatcactaca aggagctgaa gcccatcatc 300 gatcggatct acaagaccta tgccgaccag tgcctgcagc tggtgcagct ggattgggag 360 aacctgagcg ccgccatcga ctcctataga aaggagaaaa ccgaggagac aaggaacgcc 420 ctgatcgagg agcaggccac atatcgcaat gccatccacg actacttcat cggccggaca 480 gacaacctga ccgatgccat caataagaga cacgccgaga tctacaaggg cctgttcaag 540 gccgagctgt ttaatggcaa ggtgctgaag cagctgggca ccgtgaccac aaccgagcac 600 gagaacgccc tgctgcggag cttcgacaag tttacaacct acttctccgg cttttatgag 660 aacaggaaga acgtgttcag cgccgaggat atcagcacag ccatcccaca ccgcatcgtg 720 caggacaact tccccaagtt taaggagaat tgtcacatct tcacacgcct gatcaccgcc 780 gtgcccagcc tgcgggagca ctttgagaac gtgaagaagg ccatcggcat cttcgtgagc 840 acctccatcg aggaggtgtt ttccttccct ttttataacc agctgctgac acagacccag 900 atcgacctgt ataaccagct gctgggagga atctctcggg aggcaggcac cgagaagatc 960 aagggcctga acgaggtgct gaatctggcc atccagaaga atgatgagac agcccacatc 1020 atcgcctccc tgccacacag attcatcccc ctgtttaagc agatcctgtc cgataggaac 1080 accctgtctt tcatcctgga ggagtttaag agcgacgagg aagtgatcca gtccttctgc 1140 aagtacaaga cactgctgag aaacgagaac gtgctggaga cagccgaggc cctgtttaac 1200 gagctgaaca gcatcgacct gacacacatc ttcatcagcc acaagaagct ggagacaatc 1260 agcagcgccc tgtgcgacca ctgggataca ctgaggaatg ccctgtatga gcggagaatc 1320 tccgagctga caggcaagat caccaagtct gccaaggaga aggtgcagcg cagcctgaag 1380 cacgaggata tcaacctgca ggagatcatc tctgccgcag gcaaggagct gagcgaggcc 1440 ttcaagcaga aaaccagcga gatcctgtcc cacgcacacg ccgccctgga tcagccactg 1500 cctacaaccc tgaagaagca ggaggagaag gagatcctga agtctcagct ggacagcctg 1560 ctgggcctgt accacctgct ggactggttt gccgtggatg agtccaacga ggtggacccc 1620 gagttctctg cccggctgac cggcatcaag ctggagatgg agccttctct gagcttctac 1680 aacaaggcca gaaattatgc caccaagaag ccctactccg tggagaagtt caagctgaac 1740 tttcagatgc ctacactggc ctctggctgg gacgtgaata aggagaagaa caatggcgcc 1800 atcctgtttg tgaagaacgg cctgtactat ctgggcatca tgccaaagca gaagggcagg 1860 tataaggccc tgagcttcga gcccacagag aaaaccagcg agggctttga taagatgtac 1920 tatgactact tccctgatgc cgccaagatg atcccaaagt gcagcaccca gctgaaggcc 1980 gtgacagccc actttcagac ccacacaacc cccatcctgc tgtccaacaa tttcatcgag 2040 cctctggaga tcacaaagga gatctacgac ctgaacaatc ctgagaagga gccaaagaag 2100 tttcagacag cctacgccaa gaaaaccggc gaccagaagg gctacagaga ggccctgtgc 2160 aagtggatcg acttcacaag ggattttctg tccaagtata ccaagacaac ctctatcgat 2220 ctgtctagcc tgcggccatc ctctcagtat aaggacctgg gcgagtacta tgccgagctg 2280 aatcccctgc tgtaccacat cagcttccag agaatcgccg agaaggagat catggatgcc 2340 gtggagacag gcaagctgta cctgttccag atctataaca aggactttgc caagggccac 2400 cacggcaagc ctaatctgca cacactgtat tggaccggcc tgttttctcc agagaacctg 2460 gccaagacaa gcatcaagct gaatggccag gccgagctgt tctaccgccc taagtccagg 2520 atgaagagga tggcacaccg gctgggagag aagatgctga acaagaagct gaaggatcag 2580 aaaaccccaa tccccgacac cctgtaccag gagctgtacg actatgtgaa tcacagactg 2640 tcccacgacc tgtctgatga ggccagggcc ctgctgccca acgtgatcac caaggaggtg 2700 tctcacgaga tcatcaagga taggcgcttt accagcgaca agttcttttt ccacgtgcct 2760 atcacactga actatcaggc cgccaattcc ccatctaagt tcaaccagag ggtgaatgcc 2820 tacctgaagg agcaccccga gacacctatc atcggcatcg atcggggcga gagaaacctg 2880 atctatatca cagtgatcga ctccaccggc aagatcctgg agcagcggag cctgaacacc 2940 atccagcagt ttgattacca gaagaagctg gacaacaggg agaaggagag ggtggcagca 3000 aggcaggcct ggtctgtggt gggcacaatc aaggatctga agcagggcta tctgagccag 3060 gtcatccacg agatcgtgga cctgatgatc cactaccagg ccgtggtggt gctggagaac 3120 ctgaatttcg gctttaagag caagaggacc ggcatcgccg agaaggccgt gtaccagcag 3180 ttcgagaaga tgctgatcga taagctgaat tgcctggtgc tgaaggacta tccagcagag 3240 aaagtgggag gcgtgctgaa cccataccag ctgacagacc agttcacctc ctttgccaag 3300 atgggcaccc agtctggctt cctgttttac gtgcctgccc catatacatc taagatcgat 3360 cccctgaccg gcttcgtgga ccccttcgtg tggaaaacca tcaagaatca cgagagccgc 3420 aagcacttcc tggagggctt cgactttctg cactacgacg tgaaaaccgg cgacttcatc 3480 ctgcacttta agatgaacag aaatctgtcc ttccagaggg gcctgcccgg ctttatgcct 3540 gcatgggata tcgtgttcga gaagaacgag acacagtttg acgccaaggg cacccctttc 3600 atcgccggca agagaatcgt gccagtgatc gagaatcaca gattcaccgg cagataccgg 3660 gacctgtatc ctgccaacga gctgatcgcc ctgctggagg agaagggcat cgtgttcagg 3720 gatggctcca acatcctgcc aaagctgctg gagaatgacg attctcacgc catcgacacc 3780 atggtggccc tgatccgcag cgtgctgcag atgcggaact ccaatgccgc cacaggcgag 3840 gactatatca acagccccgt gcgcgatctg aatggcgtgt gcttcgactc ccggtttcag 3900 aacccagagt ggcccatgga cgccgatgcc aatggcgcct accacatcgc cctgaagggc 3960 cagctgctgc tgaatcacct gaaggagagc aaggatctga agctgcagaa cggcatctcc 4020 aatcaggact ggctggccta catccaggag ctgcgcaaca aaaggccggc ggccacgaaa 4080 aaggccggcc aggcaaaaaa gaaaaaggaa ttc 4113 <210> 79 <211> 4068 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; R9-AsCas12a <400> 79 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgagacgaa gacgaagacg tagacgtaga gctagcacac agttcgaggg ctttaccaac 120 ctgtatcagg tgagcaagac actgcggttt gagctgatcc cacagggcaa gaccctgaag 180 cacatccagg agcagggctt catcgaggag gacaaggccc gcaatgatca ctacaaggag 240 ctgaagccca tcatcgatcg gatctacaag acctatgccg accagtgcct gcagctggtg 300 cagctggatt gggagaacct gagcgccgcc atcgactcct atagaaagga gaaaaccgag 360 gagacaagga acgccctgat cgaggagcag gccacatatc gcaatgccat ccacgactac 420 ttcatcggcc ggacagacaa cctgaccgat gccatcaata agagacacgc cgagatctac 480 aagggcctgt tcaaggccga gctgtttaat ggcaaggtgc tgaagcagct gggcaccgtg 540 accacaaccg agcacgagaa cgccctgctg cggagcttcg acaagtttac aacctacttc 600 tccggctttt atgagaacag gaagaacgtg ttcagcgccg aggatatcag cacagccatc 660 ccacaccgca tcgtgcagga caacttcccc aagtttaagg agaattgtca catcttcaca 720 cgcctgatca ccgccgtgcc cagcctgcgg gagcactttg agaacgtgaa gaaggccatc 780 ggcatcttcg tgagcacctc catcgaggag gtgttttcct tcccttttta taaccagctg 840 ctgacacaga cccagatcga cctgtataac cagctgctgg gaggaatctc tcgggaggca 900 ggcaccgaga agatcaaggg cctgaacgag gtgctgaatc tggccatcca gaagaatgat 960 gagacagccc acatcatcgc ctccctgcca cacagattca tccccctgtt taagcagatc 1020 ctgtccgata ggaacaccct gtctttcatc ctggaggagt ttaagagcga cgaggaagtg 1080 atccagtcct tctgcaagta caagacactg ctgagaaacg agaacgtgct ggagacagcc 1140 gaggccctgt ttaacgagct gaacagcatc gacctgacac acatcttcat cagccacaag 1200 aagctggaga caatcagcag cgccctgtgc gaccactggg atacactgag gaatgccctg 1260 tatgagcgga gaatctccga gctgacaggc aagatcacca agtctgccaa ggagaaggtg 1320 cagcgcagcc tgaagcacga ggatatcaac ctgcaggaga tcatctctgc cgcaggcaag 1380 gagctgagcg aggccttcaa gcagaaaacc agcgagatcc tgtccccacgc acacgccgcc 1440 ctggatcagc cactgcctac aaccctgaag aagcaggagg agaaggagat cctgaagtct 1500 cagctggaca gcctgctggg cctgtaccac ctgctggact ggtttgccgt ggatgagtcc 1560 aacgaggtgg accccgagtt ctctgcccgg ctgaccggca tcaagctgga gatggagcct 1620 tctctgagct tctacaacaa ggccagaaat tatgccacca agaagcccta ctccgtggag 1680 aagttcaagc tgaactttca gatgcctaca ctggcctctg gctgggacgt gaataaggag 1740 aagaacaatg gcgccatcct gtttgtgaag aacggcctgt actatctggg catcatgcca 1800 aagcagaagg gcaggtataa ggccctgagc ttcgagccca cagagaaaac cagcgagggc 1860 tttgataaga tgtactatga ctacttccct gatgccgcca agatgatccc aaagtgcagc 1920 acccagctga aggccgtgac agcccacttt cagacccaca caacccccat cctgctgtcc 1980 aacaatttca tcgagcctct ggagatcaca aaggagatct acgacctgaa caatcctgag 2040 aaggagccaa agaagtttca gacagcctac gccaagaaaa ccggcgacca gaagggctac 2100 agagaggccc tgtgcaagtg gatcgacttc acaagggatt ttctgtccaa gtataccaag 2160 acaacctcta tcgatctgtc tagcctgcgg ccatcctctc agtataagga cctgggcgag 2220 tactatgccg agctgaatcc cctgctgtac cacatcagct tccagagaat cgccgagaag 2280 gagatcatgg atgccgtgga gacaggcaag ctgtacctgt tccagatcta taacaaggac 2340 tttgccaagg gccaccacgg caagcctaat ctgcacacac tgtattggac cggcctgttt 2400 tctccagaga acctggccaa gacaagcatc aagctgaatg gccaggccga gctgttctac 2460 cgccctaagt ccaggatgaa gaggatggca caccggctgg gagagaagat gctgaacaag 2520 aagctgaagg atcagaaaac cccaatcccc gacaccctgt accaggagct gtacgactat 2580 gtgaatcaca gactgtccca cgacctgtct gatgaggcca gggccctgct gcccaacgtg 2640 atcaccaagg aggtgtctca cgagatcatc aaggataggc gctttaccag cgacaagttc 2700 tttttccacg tgcctatcac actgaactat caggccgcca attccccatc taagttcaac 2760 cagagggtga atgcctacct gaaggagcac cccgagacac ctatcatcgg catcgatcgg 2820 ggcgagagaa acctgatcta tatcacagtg atcgactcca ccggcaagat cctggagcag 2880 cggagcctga acaccatcca gcagtttgat taccagaaga agctggacaa cagggagaag 2940 gagagggtgg cagcaaggca ggcctggtct gtggtgggca caatcaagga tctgaagcag 3000 ggctatctga gccaggtcat ccacgagatc gtggacctga tgatccacta ccaggccgtg 3060 gtggtgctgg agaacctgaa tttcggcttt aagagcaaga ggaccggcat cgccgagaag 3120 gccgtgtacc agcagttcga gaagatgctg atcgataagc tgaattgcct ggtgctgaag 3180 gactatccag cagagaaagt gggaggcgtg ctgaacccat accagctgac agaccagttc 3240 acctcctttg ccaagatggg cacccagtct ggcttcctgt tttacgtgcc tgccccatat 3300 acatctaaga tcgatcccct gaccggcttc gtggacccct tcgtgtggaa aaccatcaag 3360 aatcacgaga gccgcaagca cttcctggag ggcttcgact ttctgcacta cgacgtgaaa 3420 accggcgact tcatcctgca ctttaagatg aacagaaatc tgtccttcca gaggggcctg 3480 cccggcttta tgcctgcatg ggatatcgtg ttcgagaaga acgagacaca gtttgacgcc 3540 aagggcaccc ctttcatcgc cggcaagaga atcgtgccag tgatcgagaa tcacagattc 3600 accggcagat accgggacct gtatcctgcc aacgagctga tcgccctgct ggaggagaag 3660 ggcatcgtgt tcagggatgg ctccaacatc ctgccaaagc tgctggagaa tgacgattct 3720 cacgccatcg acaccatggt ggccctgatc cgcagcgtgc tgcagatgcg gaactccaat 3780 gccgccacag gcgaggacta tatcaacagc cccgtgcgcg atctgaatgg cgtgtgcttc 3840 gactcccggt ttcagaaccc agagtggccc atggacgccg atgccaatgg cgcctaccac 3900 atcgccctga agggccagct gctgctgaat cacctgaagg agagcaagga tctgaagctg 3960 cagaacggca tctccaatca ggactggctg gcctacatcc aggagctgcg caacaaaagg 4020 ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggaattc 4068 <210> 80 <211> 4074 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; TAT-AsCas12a <400> 80 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgtatggac gcaagaagcg ccgccagcgc cgccgcgcta gcacacagtt cgagggcttt 120 accaacctgt atcaggtgag caagacactg cggtttgagc tgatcccaca gggcaagacc 180 ctgaagcaca tccaggagca gggcttcatc gaggaggaca aggcccgcaa tgatcactac 240 aaggagctga agcccatcat cgatcggatc tacaagacct atgccgacca gtgcctgcag 300 ctggtgcagc tggattggga gaacctgagc gccgccatcg actcctatag aaaggagaaa 360 accgaggaga caaggaacgc cctgatcgag gagcaggcca catatcgcaa tgccatccac 420 gactacttca tcggccggac agacaacctg accgatgcca tcaataagag acacgccgag 480 atctacaagg gcctgttcaa ggccgagctg tttaatggca aggtgctgaa gcagctgggc 540 accgtgacca caaccgagca cgagaacgcc ctgctgcgga gcttcgacaa gtttacaacc 600 tacttctccg gcttttatga gaacaggaag aacgtgttca gcgccgagga tatcagcaca 660 gccatccccac accgcatcgt gcaggacaac ttccccaagt ttaaggagaa ttgtcacatc 720 ttcacacgcc tgatcaccgc cgtgcccagc ctgcgggagc actttgagaa cgtgaagaag 780 gccatcggca tcttcgtgag cacctccatc gaggaggtgt tttccttccc tttttataac 840 cagctgctga cacagaccca gatcgacctg tataaccagc tgctgggagg aatctctcgg 900 gaggcaggca ccgagaagat caagggcctg aacgaggtgc tgaatctggc catccagaag 960 aatgatgaga cagcccacat catcgcctcc ctgccacaca gattcatccc cctgtttaag 1020 cagatcctgt ccgataggaa caccctgtct ttcatcctgg aggagtttaa gagcgacgag 1080 gaagtgatcc agtccttctg caagtacaag acactgctga gaaacgagaa cgtgctggag 1140 acagccgagg ccctgtttaa cgagctgaac agcatcgacc tgacacacat cttcatcagc 1200 cacaagaagc tggagacaat cagcagcgcc ctgtgcgacc actgggatac actgaggaat 1260 gccctgtatg agcggagaat ctccgagctg acaggcaaga tcaccaagtc tgccaaggag 1320 aaggtgcagc gcagcctgaa gcacgaggat atcaacctgc aggagatcat ctctgccgca 1380 ggcaaggagc tgagcgaggc cttcaagcag aaaaccagcg agatcctgtc ccacgcacac 1440 gccgccctgg atcagccact gcctacaacc ctgaagaagc aggaggagaa ggagatcctg 1500 aagtctcagc tggacagcct gctgggcctg taccacctgc tggactggtt tgccgtggat 1560 gagtccaacg aggtggaccc cgagttctct gcccggctga ccggcatcaa gctggagatg 1620 gagccttctc tgagcttcta caacaaggcc agaaattatg ccaccaagaa gccctactcc 1680 gtggagaagt tcaagctgaa ctttcagatg cctacactgg cctctggctg ggacgtgaat 1740 aaggagaaga acaatggcgc catcctgttt gtgaagaacg gcctgtacta tctgggcatc 1800 atgccaaagc agaagggcag gtataaggcc ctgagcttcg agcccacaga gaaaaccagc 1860 gagggctttg ataagatgta ctatgactac ttccctgatg ccgccaagat gatcccaaag 1920 tgcagcaccc agctgaaggc cgtgacagcc cactttcaga cccacacaac ccccatcctg 1980 ctgtccaaca atttcatcga gcctctggag atcacaaagg agatctacga cctgaacaat 2040 cctgagaagg agccaaagaa gtttcagaca gcctacgcca agaaaaccgg cgaccagaag 2100 ggctacagag aggccctgtg caagtggatc gacttcacaa gggattttct gtccaagtat 2160 accaagacaa cctctatcga tctgtctagc ctgcggccat cctctcagta taaggacctg 2220 ggcgagtact atgccgagct gaatcccctg ctgtaccaca tcagcttcca gagaatcgcc 2280 gagaaggaga tcatggatgc cgtggagaca ggcaagctgt acctgttcca gatctataac 2340 aaggactttg ccaagggcca ccacggcaag cctaatctgc acacactgta ttggaccggc 2400 ctgttttctc cagagaacct ggccaagaca agcatcaagc tgaatggcca ggccgagctg 2460 ttctaccgcc ctaagtccag gatgaagagg atggcacacc ggctgggaga gaagatgctg 2520 aacaagaagc tgaaggatca gaaaacccca atccccgaca ccctgtacca ggagctgtac 2580 gactatgtga atcacagact gtccccacgac ctgtctgatg aggccagggc cctgctgccc 2640 aacgtgatca ccaaggaggt gtctcacgag atcatcaagg ataggcgctt taccagcgac 2700 aagttctttt tccacgtgcc tatcacactg aactatcagg ccgccaattc cccatctaag 2760 ttcaaccaga gggtgaatgc ctacctgaag gagcaccccg agacacctat catcggcatc 2820 gatcggggcg agagaaacct gatctatatc acagtgatcg actccaccgg caagatcctg 2880 gagcagcgga gcctgaacac catccagcag tttgattacc agaagaagct ggacaacagg 2940 gagaaggaga gggtggcagc aaggcaggcc tggtctgtgg tgggcacaat caaggatctg 3000 aagcagggct atctgagcca ggtcatccac gagatcgtgg acctgatgat ccactaccag 3060 gccgtggtgg tgctggagaa cctgaatttc ggctttaaga gcaagaggac cggcatcgcc 3120 gagaaggccg tgtaccagca gttcgagaag atgctgatcg ataagctgaa ttgcctggtg 3180 ctgaaggact atccagcaga gaaagtggga ggcgtgctga acccatacca gctgacagac 3240 cagttcacct cctttgccaa gatgggcacc cagtctggct tcctgtttta cgtgcctgcc 3300 ccatatacat ctaagatcga tcccctgacc ggcttcgtgg accccttcgt gtggaaaacc 3360 atcaagaatc acgagagccg caagcacttc ctggagggct tcgactttct gcactacgac 3420 gtgaaaaccg gcgacttcat cctgcacttt aagatgaaca gaaatctgtc cttccagagg 3480 ggcctgcccg gctttatgcc tgcatgggat atcgtgttcg agaagaacga gacacagttt 3540 gacgccaagg gcaccccttt catcgccggc aagagaatcg tgccagtgat cgagaatcac 3600 agattcaccg gcagataccg ggacctgtat cctgccaacg agctgatcgc cctgctggag 3660 gagaagggca tcgtgttcag ggatggctcc aacatcctgc caaagctgct ggagaatgac 3720 gattctcacg ccatcgacac catggtggcc ctgatccgca gcgtgctgca gatgcggaac 3780 tccaatgccg ccacaggcga ggactatatc aacagccccg tgcgcgatct gaatggcgtg 3840 tgcttcgact cccggtttca gaacccagag tggcccatgg acgccgatgc caatggcgcc 3900 taccacatcg ccctgaaggg ccagctgctg ctgaatcacc tgaaggagag caaggatctg 3960 aagctgcaga acggcatctc caatcaggac tggctggcct acatccagga gctgcgcaac 4020 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga attc 4074 <210> 81 <211> 3831 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; AP-LbCas12a <400> 81 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgcgccggc gctggtgcaa acgccgccgg gctagcagca agctggagaa gtttacaaac 120 tgctactccc tgtctaagac cctgaggttc aaggccatcc ctgtgggcaa gacccaggag 180 aacatcgaca ataagcggct gctggtggag gacgagaaga gagccgagga ttataagggc 240 gtgaagaagc tgctggatcg ctactatctg tcttttatca acgacgtgct gcacagcatc 300 aagctgaaga atctgaacaa ttacatcagc ctgttccgga agaaaaccag aaccgagaag 360 gagaataagg agctggagaa cctggagatc aatctgcgga aggagatcgc caaggccttc 420 aagggcaacg agggctacaa gtccctgttt aagaaggata tcatcgagac aatcctgcca 480 gagttcctgg acgataagga cgagatcgcc ctggtgaaca gcttcaatgg ctttaccaca 540 gccttcaccg gcttctttga taacagagag aatatgtttt ccgaggaggc caagagcaca 600 tccatcgcct tcaggtgtat caacgagaat ctgacccgct acatctctaa tatggacatc 660 ttcgagaagg tggacgccat ctttgataag cacgaggtgc aggagatcaa ggagaagatc 720 ctgaacagcg actatgatgt ggaggatttc tttgagggcg agttctttaa ctttgtgctg 780 acacaggagg gcatcgacgt gtataacgcc atcatcggcg gcttcgtgac cgagagcggc 840 gagaagatca agggcctgaa cgagtacatc aacctgtata atcagaaaac caagcagaag 900 ctgcctaagt ttaagccact gtataagcag gtgctgagcg atcgggagtc tctgagcttc 960 tacggcgagg gctatacatc cgatgaggag gtgctggagg tgtttagaaa caccctgaac 1020 aagaacagcg agatcttcag ctccatcaag aagctggaga agctgttcaa gaattttgac 1080 gagtactcta gcgccggcat ctttgtgaag aacggccccg ccatcagcac aatctccaag 1140 gatatcttcg gcgagtggaa cgtgatccgg gacaagtgga atgccgagta tgacgatatc 1200 cacctgaaga agaaggccgt ggtgaccgag aagtacgagg acgatcggag aaagtccttc 1260 aagaagatcg gctccttttc tctggagcag ctgcaggagt acgccgacgc cgatctgtct 1320 gtggtggaga agctgaagga gatcatcatc cagaaggtgg atgagatcta caaggtgtat 1380 ggctcctctg agaagctgtt cgacgccgat tttgtgctgg agaagagcct gaagaagaac 1440 gacgccgtgg tggccatcat gaaggacctg ctggattctg tgaagagctt cgagaattac 1500 atcaaggcct tctttggcga gggcaaggag acaaacaggg acgagtcctt ctatggcgat 1560 tttgtgctgg cctacgacat cctgctgaag gtggaccaca tctacgatgc catccgcaat 1620 tatgtgaccc agaagcccta ctctaaggat aagttcaagc tgtattttca gaaccctcag 1680 ttcatgggcg gctgggacaa ggataaggag acagactatc gggccaccat cctgagatac 1740 ggctccaagt actatctggc catcatggat aagaagtacg ccaagtgcct gcagaagatc 1800 gacaaggacg atgtgaacgg caattacgag aagatcaact ataagctgct gcccggccct 1860 aataagatgc tgccaaaggt gttcttttct aagaagtgga tggcctacta taaccccagc 1920 gaggacatcc agaagatcta caagaatggc acatcaaga agggcgatat gtttaacctg 1980 aatgactgtc acaagctgat cgacttcttt aaggatagca tctcccggta tccaaagtgg 2040 tccaatgcct acgatttcaa cttttctgag acagagaagt ataaggacat cgccggcttt 2100 tacagagagg tggaggagca gggctataag gtgagcttcg agtctgccag caagaaggag 2160 gtggataagc tggtggagga gggcaagctg tatatgttcc agatctataa caaggacttt 2220 tccgataagt ctcacggcac acccaatctg cacaccatgt acttcaagct gctgtttgac 2280 gagaacaatc acggacagat caggctgagc ggaggagcag agctgttcat gaggcgcgcc 2340 tccctgaaga aggaggagct ggtggtgcac ccagccaact cccctatcgc caacaagaat 2400 ccagataatc ccaagaaaac cacaaccctg tcctacgacg tgtataagga taagaggttt 2460 tctgaggacc agtacgagct gcacatccca atcgccatca ataagtgccc caagaacatc 2520 ttcaagatca atacagaggt gcgcgtgctg ctgaagcacg acgataaccc ctatgtgatc 2580 ggcatcgata ggggcgagcg caatctgctg tatatcgtgg tggtggacgg caagggcaac 2640 atcgtggagc agtattccct gaacgagatc atcaacaact tcaacggcat caggatcaag 2700 acagattacc actctctgct ggacaagaag gagaaggaga ggttcgaggc ccgccagaac 2760 tggacctcca tcgagaatat caaggagctg aaggccggct atatctctca ggtggtgcac 2820 aagatctgcg agctggtgga gaagtacgat gccgtgatcg ccctggagga cctgaactct 2880 ggctttaaga atagccgcgt gaaggtggag aagcaggtgt atcagaagtt cgagaagatg 2940 ctgatcgata agctgaacta catggtggac aagaagtcta atccttgtgc aacaggcggc 3000 gccctgaagg gctatcagat caccaataag ttcgagagct ttaagtccat gtctacccag 3060 aacggcttca tcttttacat ccctgcctgg ctgacatcca agatcgatcc atctaccggc 3120 tttgtgaacc tgctgaaaac caagtatacc agcatcgccg attccaagaa gttcatcagc 3180 tcctttgaca ggatcatgta cgtgcccgag gaggatctgt tcgagtttgc cctggactat 3240 aagaacttct ctcgcacaga cgccgattac atcaagaagt ggaagctgta ctcctacggc 3300 aaccggatca gaatcttccg gaatcctaag aagaacaacg tgttcgactg ggaggaggtg 3360 tgcctgacca gcgcctataa ggagctgttc aacaagtacg gcatcaatta tcagcagggc 3420 gatatcagag ccctgctgtg cgagcagtcc gacaaggcct tctactctag ctttatggcc 3480 ctgatgagcc tgatgctgca gatgcggaac agcatcacag gccgcaccga cgtggatttt 3540 ctgatcagcc ctgtgaagaa ctccgacggc atcttctacg atagccggaa ctatgaggcc 3600 caggagaatg ccatcctgcc aaagaacgcc gacgccaatg gcgcctataa catcgccaga 3660 aaggtgctgt gggccatcgg ccagttcaag aaggccgagg acgagaagct ggataaggtg 3720 aagatcgcca tctctaacaa ggagtggctg gagtacgccc agaccagcgt gaagcacaaa 3780 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt c 3831 <210> 82 <211> 3876 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; dNP2-LbCas12a <400> 82 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgaagatca agaaggttaa aaaaaagggt cgcaagggct ctaaaattaa aaaagtcaag 120 aagaaaggaa gaaaagctag cagcaagctg gagaagttta caaactgcta ctccctgtct 180 aagaccctga ggttcaaggc catccctgtg ggcaagaccc aggagaacat cgacaataag 240 cggctgctgg tggaggacga gaagagagcc gaggattata agggcgtgaa gaagctgctg 300 gatcgctact atctgtcttt tatcaacgac gtgctgcaca gcatcaagct gaagaatctg 360 aacaattaca tcagcctgtt ccggaagaaa accagaaccg agaaggagaa taaggagctg 420 gagaacctgg agatcaatct gcggaaggag atcgccaagg ccttcaaggg caacgagggc 480 tacaagtccc tgtttaagaa ggatatcatc gagacaatcc tgccagagtt cctggacgat 540 aaggacgaga tcgccctggt gaacagcttc aatggcttta ccacagcctt caccggcttc 600 tttgataaca gagagaatat gttttccgag gaggccaaga gcacatccat cgccttcagg 660 tgtatcaacg agaatctgac ccgctacatc tctaatatgg acatcttcga gaaggtggac 720 gccatctttg ataagcacga ggtgcaggag atcaaggaga agatcctgaa cagcgactat 780 gatgtggagg atttctttga gggcgagttc tttaactttg tgctgacaca ggagggcatc 840 gacgtgtata acgccatcat cggcggcttc gtgaccgaga gcggcgagaa gatcaagggc 900 ctgaacgagt acatcaacct gtataatcag aaaaccaagc agaagctgcc taagtttaag 960 ccactgtata agcaggtgct gagcgatcgg gagtctctga gcttctacgg cgagggctat 1020 acatccgatg aggaggtgct ggaggtgttt agaaacaccc tgaacaagaa cagcgagatc 1080 ttcagctcca tcaagaagct ggagaagctg ttcaagaatt ttgacgagta ctctagcgcc 1140 ggcatctttg tgaagaacgg ccccgccatc agcacaatct ccaaggatat cttcggcgag 1200 tggaacgtga tccgggacaa gtggaatgcc gagtatgacg atatccacct gaagaagaag 1260 gccgtggtga ccgagaagta cgaggacgat cggagaaagt ccttcaagaa gatcggctcc 1320 ttttctctgg agcagctgca ggagtacgcc gacgccgatc tgtctgtggt ggagaagctg 1380 aaggagatca tcatccagaa ggtggatgag atctacaagg tgtatggctc ctctgagaag 1440 ctgttcgacg ccgattttgt gctggagaag agcctgaaga agaacgacgc cgtggtggcc 1500 atcatgaagg acctgctgga ttctgtgaag agcttcgaga attacatcaa ggccttcttt 1560 ggcgagggca aggagacaaa cagggacgag tccttctatg gcgattttgt gctggcctac 1620 gacatcctgc tgaaggtgga ccacatctac gatgccatcc gcaattatgt gacccagaag 1680 ccctactcta aggataagtt caagctgtat tttcagaacc ctcagttcat gggcggctgg 1740 gacaaggata aggagacaga ctatcgggcc accatcctga gatacggctc caagtactat 1800 ctggccatca tggataagaa gtacgccaag tgcctgcaga agatcgacaa ggacgatgtg 1860 aacggcaatt acgagaagat caactataag ctgctgcccg gccctaataa gatgctgcca 1920 aaggtgttct tttctaagaa gtggatggcc tactataacc ccagcgagga catccagaag 1980 atctacaaga atggcacatt caagaagggc gatatgttta acctgaatga ctgtcacaag 2040 ctgatcgact tctttaagga tagcatctcc cggtatccaa agtggtccaa tgcctacgat 2100 ttcaactttt ctgagacaga gaagtataag gacatcgccg gcttttacag agaggtggag 2160 gagcagggct ataaggtgag cttcgagtct gccagcaaga aggaggtgga taagctggtg 2220 gaggagggca agctgtatat gttccagatc tataacaagg acttttccga taagtctcac 2280 ggcacaccca atctgcacac catgtacttc aagctgctgt ttgacgagaa caatcacgga 2340 cagatcaggc tgagcggagg agcagagctg ttcatgaggc gcgcctccct gaagaaggag 2400 gagctggtgg tgcacccagc caactcccct atcgccaaca agaatccaga taatcccaag 2460 aaaaccacaa ccctgtccta cgacgtgtat aaggataaga ggttttctga ggaccagtac 2520 gagctgcaca tcccaatcgc catcaataag tgccccaaga acatcttcaa gatcaataca 2580 gaggtgcgcg tgctgctgaa gcacgacgat aacccctatg tgatcggcat cgataggggc 2640 gagcgcaatc tgctgtatat cgtggtggtg gacggcaagg gcaacatcgt ggagcagtat 2700 tccctgaacg agatcatcaa caacttcaac ggcatcagga tcaagacaga ttaccactct 2760 ctgctggaca agaaggagaa ggagaggttc gaggcccgcc agaactggac ctccatcgag 2820 aatatcaagg agctgaaggc cggctatatc tctcaggtgg tgcacaagat ctgcgagctg 2880 gtggagaagt acgatgccgt gatcgccctg gaggacctga actctggctt taagaatagc 2940 cgcgtgaagg tggagaagca ggtgtatcag aagttcgaga agatgctgat cgataagctg 3000 aactacatgg tggacaagaa gtctaatcct tgtgcaacag gcggcgccct gaagggctat 3060 cagatcacca ataagttcga gagctttaag tccatgtcta cccagaacgg cttcatcttt 3120 tacatccctg cctggctgac atccaagatc gatccatcta ccggctttgt gaacctgctg 3180 aaaaccaagt ataccagcat cgccgattcc aagaagttca tcagctcctt tgacaggatc 3240 atgtacgtgc ccgaggagga tctgttcgag tttgccctgg actataagaa cttctctcgc 3300 acagacgccg attacatcaa gaagtggaag ctgtactcct acggcaaccg gatcagaatc 3360 ttccggaatc ctaagaagaa caacgtgttc gactgggagg aggtgtgcct gaccagcgcc 3420 tataaggagc tgttcaacaa gtacggcatc aattatcagc agggcgatat cagagccctg 3480 ctgtgcgagc agtccgacaa ggccttctac tctagcttta tggccctgat gagcctgatg 3540 ctgcagatgc ggaacagcat cacaggccgc accgacgtgg attttctgat cagccctgtg 3600 aagaactccg acggcatctt ctacgatagc cggaactatg aggcccagga gaatgccatc 3660 ctgccaaaga acgccgacgc caatggcgcc tataacatcg ccagaaaggt gctgtgggcc 3720 atcggccagt tcaagaaggc cgaggacgag aagctggata aggtgaagat cgccatctct 3780 aacaaggagt ggctggagta cgcccagacc agcgtgaagc acaaaaggcc ggcggccacg 3840 aaaaaggccg gccaggcaaa aaagaaaaag gaattc 3876 <210> 83 <211> 3831 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; R9-LbCas12a <400> 83 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgagacgaa gacgaagacg tagacgtaga gctagcagca agctggagaa gtttacaaac 120 tgctactccc tgtctaagac cctgaggttc aaggccatcc ctgtgggcaa gacccaggag 180 aacatcgaca ataagcggct gctggtggag gacgagaaga gagccgagga ttataagggc 240 gtgaagaagc tgctggatcg ctactatctg tcttttatca acgacgtgct gcacagcatc 300 aagctgaaga atctgaacaa ttacatcagc ctgttccgga agaaaaccag aaccgagaag 360 gagaataagg agctggagaa cctggagatc aatctgcgga aggagatcgc caaggccttc 420 aagggcaacg agggctacaa gtccctgttt aagaaggata tcatcgagac aatcctgcca 480 gagttcctgg acgataagga cgagatcgcc ctggtgaaca gcttcaatgg ctttaccaca 540 gccttcaccg gcttctttga taacagagag aatatgtttt ccgaggaggc caagagcaca 600 tccatcgcct tcaggtgtat caacgagaat ctgacccgct acatctctaa tatggacatc 660 ttcgagaagg tggacgccat ctttgataag cacgaggtgc aggagatcaa ggagaagatc 720 ctgaacagcg actatgatgt ggaggatttc tttgagggcg agttctttaa ctttgtgctg 780 acacaggagg gcatcgacgt gtataacgcc atcatcggcg gcttcgtgac cgagagcggc 840 gagaagatca agggcctgaa cgagtacatc aacctgtata atcagaaaac caagcagaag 900 ctgcctaagt ttaagccact gtataagcag gtgctgagcg atcgggagtc tctgagcttc 960 tacggcgagg gctatacatc cgatgaggag gtgctggagg tgtttagaaa caccctgaac 1020 aagaacagcg agatcttcag ctccatcaag aagctggaga agctgttcaa gaattttgac 1080 gagtactcta gcgccggcat ctttgtgaag aacggccccg ccatcagcac aatctccaag 1140 gatatcttcg gcgagtggaa cgtgatccgg gacaagtgga atgccgagta tgacgatatc 1200 cacctgaaga agaaggccgt ggtgaccgag aagtacgagg acgatcggag aaagtccttc 1260 aagaagatcg gctccttttc tctggagcag ctgcaggagt acgccgacgc cgatctgtct 1320 gtggtggaga agctgaagga gatcatcatc cagaaggtgg atgagatcta caaggtgtat 1380 ggctcctctg agaagctgtt cgacgccgat tttgtgctgg agaagagcct gaagaagaac 1440 gacgccgtgg tggccatcat gaaggacctg ctggattctg tgaagagctt cgagaattac 1500 atcaaggcct tctttggcga gggcaaggag acaaacaggg acgagtcctt ctatggcgat 1560 tttgtgctgg cctacgacat cctgctgaag gtggaccaca tctacgatgc catccgcaat 1620 tatgtgaccc agaagcccta ctctaaggat aagttcaagc tgtattttca gaaccctcag 1680 ttcatgggcg gctgggacaa ggataaggag acagactatc gggccaccat cctgagatac 1740 ggctccaagt actatctggc catcatggat aagaagtacg ccaagtgcct gcagaagatc 1800 gacaaggacg atgtgaacgg caattacgag aagatcaact ataagctgct gcccggccct 1860 aataagatgc tgccaaaggt gttcttttct aagaagtgga tggcctacta taaccccagc 1920 gaggacatcc agaagatcta caagaatggc acatcaaga agggcgatat gtttaacctg 1980 aatgactgtc acaagctgat cgacttcttt aaggatagca tctcccggta tccaaagtgg 2040 tccaatgcct acgatttcaa cttttctgag acagagaagt ataaggacat cgccggcttt 2100 tacagagagg tggaggagca gggctataag gtgagcttcg agtctgccag caagaaggag 2160 gtggataagc tggtggagga gggcaagctg tatatgttcc agatctataa caaggacttt 2220 tccgataagt ctcacggcac acccaatctg cacaccatgt acttcaagct gctgtttgac 2280 gagaacaatc acggacagat caggctgagc ggaggagcag agctgttcat gaggcgcgcc 2340 tccctgaaga aggaggagct ggtggtgcac ccagccaact cccctatcgc caacaagaat 2400 ccagataatc ccaagaaaac cacaaccctg tcctacgacg tgtataagga taagaggttt 2460 tctgaggacc agtacgagct gcacatccca atcgccatca ataagtgccc caagaacatc 2520 ttcaagatca atacagaggt gcgcgtgctg ctgaagcacg acgataaccc ctatgtgatc 2580 ggcatcgata ggggcgagcg caatctgctg tatatcgtgg tggtggacgg caagggcaac 2640 atcgtggagc agtattccct gaacgagatc atcaacaact tcaacggcat caggatcaag 2700 acagattacc actctctgct ggacaagaag gagaaggaga ggttcgaggc ccgccagaac 2760 tggacctcca tcgagaatat caaggagctg aaggccggct atatctctca ggtggtgcac 2820 aagatctgcg agctggtgga gaagtacgat gccgtgatcg ccctggagga cctgaactct 2880 ggctttaaga atagccgcgt gaaggtggag aagcaggtgt atcagaagtt cgagaagatg 2940 ctgatcgata agctgaacta catggtggac aagaagtcta atccttgtgc aacaggcggc 3000 gccctgaagg gctatcagat caccaataag ttcgagagct ttaagtccat gtctacccag 3060 aacggcttca tcttttacat ccctgcctgg ctgacatcca agatcgatcc atctaccggc 3120 tttgtgaacc tgctgaaaac caagtatacc agcatcgccg attccaagaa gttcatcagc 3180 tcctttgaca ggatcatgta cgtgcccgag gaggatctgt tcgagtttgc cctggactat 3240 aagaacttct ctcgcacaga cgccgattac atcaagaagt ggaagctgta ctcctacggc 3300 aaccggatca gaatcttccg gaatcctaag aagaacaacg tgttcgactg ggaggaggtg 3360 tgcctgacca gcgcctataa ggagctgttc aacaagtacg gcatcaatta tcagcagggc 3420 gatatcagag ccctgctgtg cgagcagtcc gacaaggcct tctactctag ctttatggcc 3480 ctgatgagcc tgatgctgca gatgcggaac agcatcacag gccgcaccga cgtggatttt 3540 ctgatcagcc ctgtgaagaa ctccgacggc atcttctacg atagccggaa ctatgaggcc 3600 caggagaatg ccatcctgcc aaagaacgcc gacgccaatg gcgcctataa catcgccaga 3660 aaggtgctgt gggccatcgg ccagttcaag aaggccgagg acgagaagct ggataaggtg 3720 aagatcgcca tctctaacaa ggagtggctg gagtacgccc agaccagcgt gaagcacaaa 3780 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt c 3831 <210> 84 <211> 3837 <212> DNA <213> Artificial Sequence <220> <223> gene sequence; TAT-LbCas12a <400> 84 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atgtatggac gcaagaagcg ccgccagcgc cgccgcgcta gcagcaagct ggagaagttt 120 acaaactgct actccctgtc taagaccctg aggttcaagg ccatccctgt gggcaagacc 180 caggagaaca tcgacaataa gcggctgctg gtggaggacg agaagagagc cgaggattat 240 aagggcgtga agaagctgct ggatcgctac tatctgtctt ttatcaacga cgtgctgcac 300 agcatcaagc tgaagaatct gaacaattac atcagcctgt tccggaagaa aaccagaacc 360 gagaaggaga ataaggagct ggagaacctg gagatcaatc tgcggaagga gatcgccaag 420 gccttcaagg gcaacgaggg ctacaagtcc ctgtttaaga aggatatcat cgagacaatc 480 ctgccagagt tcctggacga taaggacgag atcgccctgg tgaacagctt caatggcttt 540 accacagcct tcaccggctt ctttgataac agagagaata tgttttccga ggaggccaag 600 agcacatcca tcgccttcag gtgtatcaac gagaatctga cccgctacat ctctaatatg 660 gacatcttcg agaaggtgga cgccatcttt gataagcacg aggtgcagga gatcaaggag 720 aagatcctga acagcgacta tgatgtggag gatttctttg agggcgagtt ctttaacttt 780 gtgctgacac aggagggcat cgacgtgtat aacgccatca tcggcggctt cgtgaccgag 840 agcggcgaga agatcaaggg cctgaacgag tacatcaacc tgtataatca gaaaaccaag 900 cagaagctgc ctaagtttaa gccactgtat aagcaggtgc tgagcgatcg ggagtctctg 960 agcttctacg gcgagggcta tacatccgat gaggaggtgc tggaggtgtt tagaaacacc 1020 ctgaacaaga acagcgagat cttcagctcc atcaagaagc tggagaagct gttcaagaat 1080 tttgacgagt actctagcgc cggcatcttt gtgaagaacg gccccgccat cagcacaatc 1140 tccaaggata tcttcggcga gtggaacgtg atccgggaca agtggaatgc cgagtatgac 1200 gatatccacc tgaagaagaa ggccgtggtg accgagaagt acgaggacga tcggagaaag 1260 tccttcaaga agatcggctc cttttctctg gagcagctgc aggagtacgc cgacgccgat 1320 ctgtctgtgg tggagaagct gaaggagatc atcatccaga aggtggatga gatctacaag 1380 gtgtatggct cctctgagaa gctgttcgac gccgattttg tgctggagaa gagcctgaag 1440 aagaacgacg ccgtggtggc catcatgaag gacctgctgg attctgtgaa gagcttcgag 1500 aattacatca aggccttctt tggcgagggc aaggagacaa acagggacga gtccttctat 1560 ggcgattttg tgctggccta cgacatcctg ctgaaggtgg accacatcta cgatgccatc 1620 cgcaattatg tgacccagaa gccctactct aaggataagt tcaagctgta ttttcagaac 1680 cctcagttca tgggcggctg ggacaaggat aaggagacag actatcgggc caccatcctg 1740 agatacggct ccaagtacta tctggccatc atggataaga agtacgccaa gtgcctgcag 1800 aagatcgaca aggacgatgt gaacggcaat tacgagaaga tcaactataa gctgctgccc 1860 ggccctaata agatgctgcc aaaggtgttc ttttctaaga agtggatggc ctactataac 1920 cccagcgagg acatccagaa gatctacaag aatggcacat tcaagaaggg cgatatgttt 1980 aacctgaatg actgtcacaa gctgatcgac ttctttaagg atagcatctc ccggtatcca 2040 aagtggtcca atgcctacga tttcaacttt tctgagacag agaagtataa ggacatcgcc 2100 ggcttttaca gagaggtgga ggagcagggc tataaggtga gcttcgagtc tgccagcaag 2160 aaggaggtgg ataagctggt ggaggagggc aagctgtata tgttccagat ctataacaag 2220 gacttttccg ataagtctca cggcacaccc aatctgcaca ccatgtactt caagctgctg 2280 tttgacgaga acaatcacgg acagatcagg ctgagcggag gagcagagct gttcatgagg 2340 cgcgcctccc tgaagaagga ggagctggtg gtgcacccag ccaactcccc tatcgccaac 2400 aagaatccag ataatcccaa gaaaaccaca accctgtcct acgacgtgta taaggataag 2460 aggttttctg aggaccagta cgagctgcac atcccaatcg ccatcaataa gtgccccaag 2520 aacatcttca agatcaatac agaggtgcgc gtgctgctga agcacgacga taacccctat 2580 gtgatcggca tcgatagggg cgagcgcaat ctgctgtata tcgtggtggt ggacggcaag 2640 ggcaacatcg tggagcagta ttccctgaac gagatcatca acaacttcaa cggcatcagg 2700 atcaagacag attaccactc tctgctggac aagaaggaga aggagaggtt cgaggcccgc 2760 cagaactgga cctccatcga gaatatcaag gagctgaagg ccggctatat ctctcaggtg 2820 gtgcacaaga tctgcgagct ggtggagaag tacgatgccg tgatcgccct ggaggacctg 2880 aactctggct ttaagaatag ccgcgtgaag gtggagaagc aggtgtatca gaagttcgag 2940 aagatgctga tcgataagct gaactacatg gtggacaaga agtctaatcc ttgtgcaaca 3000 ggcggcgccc tgaagggcta tcagatcacc aataagttcg agagctttaa gtccatgtct 3060 acccagaacg gcttcatctt ttacatccct gcctggctga catccaagat cgatccatct 3120 accggctttg tgaacctgct gaaaaccaag tataccagca tcgccgattc caagaagttc 3180 atcagctcct ttgacaggat catgtacgtg cccgaggagg atctgttcga gtttgccctg 3240 gactataaga acttctctcg cacagacgcc gattacatca agaagtggaa gctgtactcc 3300 tacggcaacc ggatcagaat cttccggaat cctaagaaga acaacgtgtt cgactgggag 3360 gaggtgtgcc tgaccagcgc ctataaggag ctgttcaaca agtacggcat caattatcag 3420 cagggcgata tcagagccct gctgtgcgag cagtccgaca aggccttcta ctctagcttt 3480 atggccctga tgagcctgat gctgcagatg cggaacagca tcacaggccg caccgacgtg 3540 gattttctga tcagccctgt gaagaactcc gacggcatct tctacgatag ccggaactat 3600 gaggcccagg agaatgccat cctgccaaag aacgccgacg ccaatggcgc ctataacatc 3660 gccagaaagg tgctgtgggc catcggccag ttcaagaagg ccgaggacga gaagctggat 3720 aaggtgaaga tcgccatctc taacaaggag tggctggagt acgcccagac cagcgtgaag 3780 cacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattc 3837

Claims (13)

A cell penetrating peptide for Cas protein-RNA complex (Ribonucleoprotein: RNP) comprising the following general formula (1).
[General formula 1]
Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly] _m -[His-Glu] _n
In the above formula, m is an integer of 3 to 7, and n is an integer of 5 to 15. According to claim 1,
In Formula 1, n is an integer of 9 to 15 Cas protein-RNA complex (Ribonucleoprotein: RNP) for cell penetrating peptide. According to claim 1,
In Formula 1, n is an integer of 10 to 12 Cas protein-RNA complex (Ribonucleoprotein: RNP) for cell penetrating peptide. According to claim 1,
The cell-penetrating peptide for the Cas protein-RNA complex (Ribonucleoprotein: RNP) is a cell-penetrating peptide for the Cas protein-RNA complex (Ribonucleoprotein: RNP), characterized in that it consists of SEQ ID NO: 6. a) Cas protein to which the cell-penetrating peptide of Formula 1 is bound; and
b) a guide RNA; a composition for gene editing comprising a complex (RNP) composed of.
[General formula 1]
Arg-Arg-Arg-Trp-Cys-Lys-Arg-Arg-Arg-Ala-Ser-[Gly] _m -[His-Glu] _n
In the above formula, m is an integer of 3 to 7, and n is an integer of 5 to 15. 6. The method of claim 5,
In the general formula 1, m is an integer of 4 to 6, characterized in that the gene editing composition. 6. The method of claim 5,
In the general formula 1, n is an integer of 9 to 15, a composition for gene editing, characterized in that. 6. The method of claim 5,
In the general formula 1, n is an integer of 10 to 12, a composition for gene editing, characterized in that. 6. The method of claim 5,
The Cas protein is a composition for gene editing, characterized in that consisting of SEQ ID NO: 9. 6. The method of claim 5,
The guide RNA is a gene editing composition, characterized in that it is in the form of a double RNA (dual RNA) or single-stranded guide RNA (sgRNA) including crRNA and tracrRNA. 6. The method of claim 5,
The composition is a prokaryotic cell, a eukaryotic cell or a gene editing composition for inducing a targeted mutation of a single- or multi-location gene in a eukaryotic organism other than a human. 1) The composition for gene editing of item 5 is locally injected into isolated prokaryotic cells, eukaryotic cells or eukaryotic organisms other than humans, microinjection, electroporation, and lipofection any selected from the group consisting of A method for producing a transformant other than a human comprising the step of introducing one. A transformant other than a human prepared by the method of claim 12.

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