Lipid-encapsulated dual-cutting endonucleases for DNA and gene editing

Abstract

A method for editing genes by administering chimeric nucleases to cells or organisms without the use of viral vectors.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on September 17, 2018 is Sequence Listing ST25.txt and is 77KB in size. Background of the Invention

[0004] There are an estimated 5,000-10,000 monogenic diseases, defined as genetic conditions caused by mutations in a single gene. These diseases typically present in childhood and result in multiple symptoms and sometimes even premature death. In total, it is estimated that approximately 6% of the population will be affected by these diseases at some point in their lifetime. Diagnosis and treatment of these diseases remain largely inadequate, and care is primarily palliative, focusing on disease management but not addressing the underlying genetic defect. There are many more diseases in which genetic mutations contribute to the pathogenesis of these diseases.

[0005] Gene editing is a gene therapy approach that relies on designing nucleases to recognize and cut specific DNA sequences, and then utilizes innate cellular DNA repair pathways, namely non-homologous end joining (NHEJ) and homology-directed repair (HDR), to introduce targeted modifications in the genome. Four families of nucleases have been used in this context: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-associated RNA-guided Cas9 (CRISPR-Cas9) nucleases. These can be designed to precisely introduce double-strand breaks at the target locus of interest. Gene editing opens the possibility of permanently modifying genomic sequences of interest by achieving targeted destruction, insertion, excision, and correction in both in vitro and in vivo settings. While these advances are expected to revolutionize this field, current gene editing methods are limited by the effectiveness of the modification, safety issues associated with nuclease specificity, and delivery of gene editing tools to target cell types.

[0006] As a component of the type II CRISPR system that constitutes the bacterial innate immune system, the Cas9 (CRISPR-associated) protein has caused a paradigm shift in the field of genome editing due to its ease of use. Programming Cas9 to cut a desired sequence is a simple matter of changing the sequence of the Cas9-associated guide RNA to be complementary to the target site. The ease of Cas9 targeted programming contrasts with the more intensive protein engineering required for other agents (zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs)). Cas9, as well as proteins from the type III CRISPR system, have been used in countless genome editing applications in a variety of organisms and are now entering the field of human therapeutic applications.

[0007] Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, which encodes an epithelial anion channel. The CFTR protein, cystic fibrosis transmembrane transport regulator, is found in a wide range of organs including the pancreas, kidneys, liver, lungs, gastrointestinal tract, and reproductive tract, making CF a multi-organ disease. Mutations in CFTR lead to suboptimal ion transport and fluid retention, resulting in the prominent clinical manifestations of abnormally thickened lung mucus and pancreatic insufficiency. In the lungs, dysfunctional CFTR impedes mucociliary clearance, making the organ susceptible to bacterial infection and inflammation, ultimately leading to airway occlusion, respiratory failure, and premature death. CF remains the most common and fatal genetic disease in the Caucasian population, with an estimated 70,000-100,000 patients worldwide, highlighting the real need to develop better treatments.

[0008] A major challenge in developing therapeutic strategies for CF is the wide diversity of mutation types. The deltaF508 (phenylalanine deletion at codon 508) mutation, with a prevalence of >80% in CF patients, is by far the most common, but more than 1,990 deleterious CFTR mutations have been described. These mutations result in premature stop codons, aberrant splicing, improper protein folding or trafficking to the cell surface, and dysfunctional CFTR with limited channel opening capacity. Pharmacological interventions have targeted several of these processes, and while drug administration has therapeutic effects in certain gating mutation types, more effective treatments are needed for the common deltaF508. However, advances in drugs to treat CF have not addressed mutations caused by aberrant splicing or premature stop codons; in these cases, gene editing may be most beneficial.

[0009] Similarly, in Western populations, approximately 15% of non-small cell lung cancer (NSCLC) patients carry activating mutations in the EGF receptor (EGFR) gene in their tumors.

[0010] Existing gene editing technologies, such as CRISPR-Cas9 (and Cas9 fusions), meganucleases, zinc finger proteins, type IIS restriction endonucleases (FokI and FokI fusions), and TALENS, are limited in their ability to introduce gene deletions of specific lengths or accurately repair target genes in a sufficient number of cells, which are of interest as therapeutic agents for treating many genetic diseases. In addition, for highly programmable RNA-guided nucleases, such as monomeric Cas9, studies have shown that the specificity of predictably binding, cutting, and repairing only its target site is limited, raising concerns about potential harmful changes to cellular genomic DNA that could inadvertently cause secondary diseases in patients. Finally, most nucleases are delivered in viral vectors. Viral vectors have the following potentials: immunity in many populations; immunogenicity after treatment; and genotoxicity. There is currently no non-viral delivery method that can safely deliver nucleases to target cells and allow controlled administration of nucleases in vivo.

[0011] There is still an unmet need to improve the existing gene editing technology to solve the above-mentioned problems so as to make the gene editing technology more efficient and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of a lipid-encapsulated double-cutting nuclease (TevCas9) after its preparation [components are not to scale]. The I-TevI ​​domain 10 is connected to the RNA-guided nuclease (Cas9) domain 12 via a linker domain 11. In a preferred embodiment, the formed particles also contain guide RNA 13 and donor DNA 14. The aforementioned nuclease is contained in a lipid particle 15, which has been formed into a sphere using an extrusion process.

[0013] Figure 2 Figure 2 shows lipid-encapsulated TevCas9 being internalized into the cell and nucleus to reach its target DNA. Figure 2 As shown in A, a cell 20 or a plurality of cells 20 are exposed to a novel lipid-encapsulated nuclease particle 21 containing TevCas9 25 via in vivo or ex vivo administration. Figure 2 As shown in B, the lipid-encapsulated nuclease particle 21 is endocytosed into the cell 20. The endosome 22 undergoes a maturation process in the cytosol and is targeted for degradation ( Figure 2 C) Under certain circumstances, TevCas9 25 can escape from the endosome 22 and enter the cytosol ( Figure 2 D). In eukaryotes, the nuclease (TevCas9) 25 is targeted to the nucleus 23 of the cell 20 via one or more nuclear localization sequences ("NLS"). Figure 2As depicted in E, through its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and once in the nucleus 23, the TevCas9 nuclease 25 binds and cleaves 26 the target genomic DNA 24 sequence.

[0014] Figure 3 is a diagram of the mechanism by which lipid-encapsulated TevCas9 modifies target DNA. The I-TevI ​​domain 27 targets the I-TevI ​​target sequence 29. The linker domain 30 connects the I-TevI ​​domain 27 to the Cas9 domain 28 that targets the Cas9 target sequence 31. The gene mutation 32 is surrounded by or close to the I-TevI ​​target sequence 29 and the Cas9 target sequence 31. Figure 3 As shown in B, TevCas9 25 cleaves the target sequence, leaving a deletion product 34 of predictable size with non-complementary DNA ends 35, 36. Figure 3 C illustrates that in the presence of single-stranded donor DNA with homology arms 37, cell 20 can insert donor DNA 37 sequence near the cleavage site through homology directed repair (HDR) pathway 38. Figure 3 D illustrates that in the presence of donor DNA 39 having DNA ends compatible with the DNA ends cleaved by TevCas9 25, cell 20 can insert donor DNA sequence 39 between cleavage sites by directional ligation using non-homologous end joining (NHEJ) pathway 40. In the absence of donor DNA, cell 20 can join DNA ends by NHEJ pathway 40 ( Figure 3 E).

[0015] FIG. 4A demonstrates in vitro cleavage of a CFTR DNA substrate using TevCas9 targeting the CFTR gene with the guide in SEQ ID 15. Figure 4B Cells transfected with a plasmid DNA version of TevCas9 fused to a cleavable GFP tag were imaged on a Cytation 5 (Biotek Instruments Inc, VT, USA) using phase contrast and GFP imaging 48 hours after treatment. Genomic DNA was extracted from the collected cells and editing at the CFTR gene was detected by PCR amplification and T7 endonuclease I cleavage assay.

[0016] Figure 5 It was demonstrated that TevCas9 targeting the CFTRδF508 mutation using the guide in SEQ ID 21 cleaved a DNA substrate containing the CFTRδF508 mutation in vitro, but did not cleave a substrate containing the wild-type CFTR sequence.

[0017] FIG6A illustrates that the saCas9 D10E mutation slows down the conversion of nicked supercoiled DNA to linear DNA. Figure 6BWe demonstrate that on a linear EMX1 DNA substrate, the saCas9D10E (D10E) ribonucleoprotein complex (RNP) cleaves the target substrate to a level similar to that of saCas9 wild-type (WT). The editing level of SaCas9D10E on computationally predicted off-targets is lower than that of wild-type saCas9 on the same off-targets.

[0018] Figure 7A is a schematic diagram of the spacing of I-TevI ​​sites in EGFR exon 19 deletion and wild-type (WT) EGFR. Figures 7B and 7C demonstrate that TevCas9 containing a nick mutation in Cas9 (H557A) targeting EGFR using a guide RNA in SEQ ID 16 cuts EGFR exon 19 deletion DNA substrate at a rate 4 times faster than wild-type EGFR. Figure 7D is an image of HCC827 cells carrying EGFR exon 19 deletion mutations treated with TevCas9 targeting EGFR being selectively killed compared to NuLi-1 cells carrying wild-type EGFR (WT). Summary of the invention

[0019] The present invention relates to a chimeric nuclease comprising a modified I-TevI ​​nuclease domain, preferably a modified I-TevI ​​nuclease domain. 1 Missing and with Lys 26 (which is the Lys in the untruncated version of I-TevI 27 ) and / or Cys 39 (which is the Cys in the untruncated version of I-TevI 40 ) modification; linker, in particular SEQ ID NO: 7-12 or a fragment thereof and / or containing one or more of the following mutations: Thr 95 (Refer to full length I-TevI), Val 117 , Lys 135 , Gln 158 or Asn 140 and a modified RNA-guided nuclease Staphylococcus aureus Cas9, which may be a wild-type or modified version, preferably containing its Glu 10 or Ala 557 mutation, wherein the I-TevI ​​polypeptide comprises the complete amino acid sequence of SEQ ID NO: 6 or a fragment thereof; and a guide RNA, in particular SEQ ID NO: 15, 16 or 21 or a fragment thereof, which targets the Cas9 domain, and a pharmaceutically acceptable formulation comprising a chimeric nuclease, cationic and / or neutral lipid nanoparticles, optionally a DNA binding compound, in particular GL67 (N 4 -cholesteryl-spermine), and a pharmaceutically acceptable carrier thereof.

[0020] In a further embodiment of the present invention, the lipid nanoparticles may contain exogenous donor DNA in the formulation.

[0021] Another embodiment of the present invention relates to a method of editing genes in vivo by administering chimeric nucleases to cells or organisms without the use of viral vectors using controlled dosages.

[0022] Another embodiment of the invention relates to a method for deleting or replacing selected sequences in a DNA molecule of defined length by delivering the chimeric nuclease to a whole organism in vivo or to isolated cells in culture ex vivo, wherein the cells are mammalian cells, bacteria, insect cells or plant cells.

[0023] In yet another embodiment, the novel chimeric nuclease targets two independent target sites on a selected DNA molecule, cuts at one target site or at both target sites, and forms fragments of 30 to 36 nucleotides in length.

[0024] In a further example, the novel purified chimeric nuclease further comprises a guide RNA.

[0025] Another aspect of the present invention is to use an extrusion process to form particles having a diameter of about 100 nM comprising an excipient, wherein the excipient is selected from the group consisting of polysorbate, polyphosphate, calcium chloride, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium edetate, potassium chloride, potassium phosphate and starch or a mixture of these substances, so that the novel chimeric nuclease can be administered to a patient using a nebulizer containing the formulation.

[0026] In a preferred embodiment, the present invention relates to a method for treating lung-related diseases in a patient in need thereof by administering a novel chimeric nuclease that modifies the DNA of lung epithelial cells, wherein the chimeric nuclease replaces the CFTRδF508 mutation in the CFTR gene to treat cystic fibrosis, or cleaves the EGFR exon 19 deletion to treat non-small cell lung cancer.

[0027] In yet another embodiment, the present invention relates to a chimeric nuclease comprising a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, wherein the RNA-guided nuclease Staphylococcus aureus Cas9 contains Ala 10 , Ala 557 or Ala 580 mutations, and deletions of EGFR exon 19 targeting the EGFR gene.

[0028] In a further embodiment, the guide RNA targets a specific CTFR gene sequence to cut out a specific EGFR gene sequence containing a CFTRδF508 mutation or an EGFR exon 19 deletion mutation.

[0029] The present invention also encompasses linkers comprising SEQ ID NOs:7–12 or fragments thereof, and modified donor DNA molecules selected from the group consisting of: a linear single strand of DNA comprising regions of homology flanking a site targeted and / or cleaved by a chimeric nuclease; a linear double-stranded DNA comprising regions of homology flanking a site targeted and / or cleaved by a chimeric nuclease; double-stranded DNA of the same length comprising DNA ends complementary to the ends of the DNA cleaved by the chimeric nuclease; a circular double-stranded DNA comprising regions of homology flanking a site targeted and / or cleaved by a chimeric nuclease; and a circular double-stranded DNA comprising an I-TevI ​​target site and a Cas9 target site, wherein the product cleaved from the double-stranded DNA contains an end complementary to the end cleaved by the chimeric nuclease.

[0030] In a further example consisting of a chimeric nuclease, the chimeric nuclease comprises a modified GIY-YIG nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 or Streptococcus pyogenes Cas9 or an EQR Streptococcus pyogenes Cas9 variant containing Glu 10 Mutation (SEQ ID NO: 19) and / or Ala 840 mutation and / or a mutation that cleaves the sugar-phosphate backbone of the target DNA on one strand of the target DNA, wherein the GIY-YIG nuclease domain is selected from a gene family consisting of I-Bmol and Eco29kI.

[0031] In yet a further embodiment, the invention comprises a chimeric nuclease comprising a modified I-TevI ​​nuclease domain, a linker and a modified nuclease or DNA targeting domain, wherein the modified nuclease or DNA targeting domain is selected from the group consisting of LAGLIDADG, His-Cys Box, HNH, PD-(D / E)xK and Vsr-like meganucleases, zinc finger nucleases, CRISPR proteins and DNA binding domains, wherein the CRISPR proteins are selected from scCas9 (Streptococcus canis), fnCas9 (Francisella novicida), cjCas9 (Campylobacter jejuni), Cpf1 (Lachnospiraceae bacterium), Cas12a (Acidaminococcus Sp), Cas13a (Leptorichia shahii)) and Cas3 (Streptococcus thermophilus), and the DNA binding domain is selected from the group consisting of a zinc finger motif and a TALE activation domain.

[0032] In a further example, the present invention encompasses a modified RNA-guided nuclease Staphylococcus aureus Cas9 and a guide RNA, wherein the guide RNA contains a sequence targeting a genetic polymorphism, a different sequence in the CFTR or EGFR gene, a sequence of a retargeting nuclease, a bridging nucleic acid, and / or a mixture of guide RNAs. DETAILED DESCRIPTION

[0033] Definitions and Acronyms

[0034] For convenience, certain terms used in the specification, examples, and appended claims are collected here. These definitions should be read in light of the disclosure and understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0035] The article "a / an" is used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. The term "and / or" used in this article is defined as the possibility of having one or the other or both. For example, "A and / or B" provides a situation with only A or only B or a combination of A and B. If the claim is written as A and / or B and / or C, then the composition can include only A, only B, only C, including A and B but not C, including B and C but not A, including A and C but not B, or including all three A, B and C as components.

[0036] The term "bioavailable" is art-recognized and refers to a form of the disclosure that allows it, or a portion of an administered amount, to be absorbed, incorporated, or otherwise physiologically available to a subject or patient to which it is administered.

[0037] As used herein, the term "exogenous donor DNA" refers to any sequence of DNA that differs in whole or in part from the original target DNA sequence.

[0038] As used herein, the term "flexible linker" refers to the situation when the RNA-guided nuclease domain (Cas9) binds to the target DNA sequence, and the amino acid linker domain ensures the mobility of the I-TevI ​​domain to allow recognition, binding and cleavage of its target sequence under cell physiological conditions (usually: pH ~ 7.2, temperature: ~ 37 ° C, [K +] ~ 140mM, [Na +] ~ 5–15mM, [Cl-] ~ 4mM, [Ca ++] ~ 0.0001mM). The length of the amino acid linker can affect the preferred number of nucleotides between the Cas9 target site and the I-TevI ​​target site. Certain amino acids in the linker can also make specific contacts with the DNA sequence targeted by TevCas9. These linker-DNA contacts can affect the flexibility of the I-TevI ​​domain. Replacing amino acids in the linker domain can affect the ability of the linker domain to contact DNA.

[0039] As used herein, the term "including" is used to mean "including, but not limited to." "Including" and "including, but not limited to" are used interchangeably.

[0040] The terms "inhalation administration", "inhale / inhaled / inhalation" or "inhalation therapy" are used interchangeably and, as used herein, include administering a substantially uniform distribution of appropriately sized particles to the respiratory epithelium of the nose, the central airways, the peripheral portions of the lungs and / or the alveolar regions of the lungs or by intratracheal instillation. Such particles may be introduced into the patient and / or generated using an appropriate device, preferably a nebulizer.

[0041] The term "patient", "subject" or "host" to be treated by the present method can refer to humans or non-human animals. Non-human animals include companion animals (e.g., cats, dogs) and animals raised for food (i.e., food animals), such as cows, pigs and chickens.

[0042] The term "pharmaceutically acceptable carrier" is well known in the art and refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in delivering or transporting any of the present compositions or their components from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the present compositions and their components and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as dextrose, lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as microcrystalline cellulose, sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), and cellulose acetate; (4) glycols such as propylene glycol; (5) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol. Alcohols; (6) esters such as ethyl oleate, glyceryl behenate and ethyl laurate; (7) buffers such as monohydrogen phosphate and dihydrogen phosphate, Tris / boric acid / EDTA and Tris / acetic acid / EDTA; (8) pyrogen-free water; (9) isotonic saline; (10) Ringer's solution; (11) ethanol; (12) phosphate buffer solution; (13) polysorbates; (14) polyphosphate esters; and (15) other non-toxic compatible substances used in pharmaceutical formulations. The disclosed excipients can serve more than one function. For example, a solubilizer can also be a suspending aid, an emulsifier, a preservative, etc.

[0043] In certain preferred embodiments, the pharmaceutically acceptable excipient is a crystal expansion excipient. As used herein, the term "crystallization expansion excipient" or "crystallization expansion agent" means an excipient that provides volume and structure to the lyophilized cake. These crystal expansion agents are inert and do not react with proteins or nucleic acids. In addition, the crystal expansion agent is able to crystallize under lyophilization conditions. Examples of suitable crystal expansion agents include hydrophilic excipients such as water-soluble polymers; sugars such as mannitol, sorbitol, xylitol, glucitol, dulcitol, inositol, arabitol, arabitol, galactitol, iditol, allitol, maltitol, fructose, sorbitol, glucose, xylose, trehalose, allose, dextrose, altrose, lactose, glucose, fructose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose, lactulose, fucose, rhamnose, sucrose, maltose, lactose, lactulose, fucose, rhamnose, sucrose, sucrose, maltose, lactose, lactulose, sucrose ... Sugar, maltotriose, raffinose, atritol, its optional active form (D or L) and the corresponding racemate; inorganic salts, mineral salts and mineral organic salts, such as calcium salts, such as lactate, gluconate, glycerophosphate, citrate, monohydrogen phosphate and dihydrogen phosphate, succinate, sulfate and tartrate, and the above-mentioned salts of aluminum and magnesium; carbohydrates, such as traditional monosaccharides and disaccharides and corresponding polyhydroxy alcohols; proteins, such as albumin; amino acids, such as glycine; emulsifiable fats and polyvinylpyrrolidone. Preferred crystalline bulking agents are selected from the group consisting of glycine, mannitol, dextran, dextrose, lactose, sucrose, polyvinylpyrrolidone, trehalose, glucose and combinations thereof. Particularly useful bulking agents include dextran.

[0044] As used herein, the term "pharmaceutically acceptable salt" is well known in the art and refers to relatively non-toxic inorganic and organic acid addition salts, or inorganic or organic base addition salts of compounds, including, for example, salts contained in the compositions of the present invention. Some examples of pharmaceutically acceptable salts include: (1) calcium chloride; (2) sodium chloride; (3) sodium citrate; (4) sodium hydroxide; (5) sodium phosphate; (6) sodium ethylenediaminetetraacetate; (7) potassium chloride; (8) potassium phosphate; and (9) other non-toxic compatible substances used in pharmaceutical formulations.

[0045] As used herein, the term "replacement" refers to replacing an amino acid in a sequence with a different amino acid. As used herein, the abbreviation X10Y indicates that the amino acid Y has "substituted" the amino acid X present at position 10 in the sequence. For example, W26C indicates that the amino acid tryptophan-26 (Trp, W) has been changed to cysteine ​​(Cys). Similarly, the symbol AA X means that AA is the amino acid that replaces the amino acid present at position X. For example, Lys 26Indicates the replacement of the amino acid at position 26 in the sequence with lysine. The use of the two abbreviations is interchangeable. In addition, the use of the one or three letter abbreviations for the amino acids is also interchangeable.

[0046] As used herein, the term "therapeutic agent" is well known in the art and refers to any chemical or biochemical moiety that acts locally or systemically in a subject as a biologically, physiologically or pharmacologically active substance. Examples of therapeutic agents, also known as "drugs," are described in well-known references such as the Merck Index, the Physician's Desk Reference, and The Pharmacological Basis of Therapeutics, and include, but are not limited to, pharmaceutical agents; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or alleviate disease or condition; substances that affect the structure or function of the body; or prodrugs, which become biologically active or more active after being placed in a physiological environment.

[0047] As used herein, the term "therapeutic effect" is well known in the art and refers to a local or systemic effect in an animal, especially a mammal, and more particularly in a human being, caused by a pharmacologically active substance. Thus, the term means any substance intended to be used for diagnosing, curing, alleviating, treating or preventing a disease or for enhancing the desired physical or mental progress and / or condition in an animal or human being. The phrase "therapeutically effective amount" means the amount of such a substance that produces a certain desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutically effective amount of such a substance will vary according to the subject and the disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., which can be easily determined by a person of ordinary skill in the art. For example, certain compositions of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0048] As used herein, the term "treatment" includes any effect that improves a condition, disease, illness, etc., such as alleviation, reduction, regulation, or elimination. As used herein, "treatment" may include both prophylactic and therapeutic treatments. For example, therapeutic treatments may include delaying, inhibiting, or preventing the progression of cystic fibrosis or non-small cell lung cancer, reducing or eliminating symptoms associated with cystic fibrosis or non-small cell lung cancer. Prophylactic treatments may include preventing, inhibiting, or delaying the onset of cystic fibrosis or non-small cell lung cancer.

[0049] As used herein, "effective amount" refers to an amount sufficient to elicit a desired response. In the present invention, the desired biological response is that cystic fibrosis and / or non-small cell lung cancer (NSCLC) is treated.

[0050] As used herein, "buffer" is any acid or salt combination that is pharmaceutically acceptable and capable of maintaining the composition of the present invention within a desired pH range. The buffer in the disclosed composition maintains the pH at about 2 to about 8.5, about 5.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 7.5, or about 6.5. Suitable buffers include any pharmaceutically acceptable buffer capable of maintaining the above pH ranges, such as, for example, acetate, tartrate, phosphate, or citrate buffers. In one embodiment, the buffer is a phosphate buffer. In another embodiment, the buffer is an acetate buffer. In one embodiment, the buffer is disodium hydrogen phosphate, sodium chloride, potassium chloride, and potassium dihydrogen phosphate.

[0051] In the disclosed compositions, the concentration of the buffer is typically about 0.1 mM to about 1000 mM, about 0.2 mM to about 200 mM, about 0.5 mM to about 50 mM, about 1 mM to about 10 mM, or about 6.0 mM.

[0052] As used herein, "antimicrobial" is a pharmaceutically acceptable preservative, suitable for application to a subject, which inhibits, prevents or delays the growth of microorganisms in the compositions of the present invention, including, for example, bacteria, viruses and fungi. Suitable antimicrobial agents for the compositions and methods of the present invention include, but are not limited to, cresols, benzyl alcohol, phenol, benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol, methylparaben, propylparaben, thimerosal and phenylmercuric nitrate and phenylmercuric acetate. In one embodiment, the antimicrobial is metacresol, chlorocresol or phenol. In another embodiment, the antimicrobial is chlorocresol or phenol. In another embodiment, the antimicrobial is phenol. As used herein, the effective amount of an antimicrobial is an amount that effectively inhibits, prevents or delays the growth of microorganisms in the compositions of the present invention, including, for example, bacteria, viruses and fungi. In the compositions of the present invention, the amount of antimicrobial agent is generally from about 0.1 to about 20 mg / ml, from about 0.2 to about 30 mg / ml, from about 0.2 to about 10 mg / ml, from about 0.25 to about 5 mg / ml, from about 0.5 to about 50 mg / ml, from about 1 to about 10 mg / ml, about 3 mg / ml or about 5 mg / ml.

[0053] The compositions of the present invention may also be freeze-dried using freeze-drying techniques known in the art and stored in powder form, which may be reconstituted prior to administration. As used herein, the term "freeze-drying" is a freeze-drying or dehydration technique that involves removing a solvent, preferably a water-miscible solvent, more preferably water, from the compositions of the present invention, typically by sublimation under high vacuum while the composition is in a frozen state. Typically, freeze-drying is performed in a freeze-drying apparatus (lyophilizer) that includes a drying chamber with variable temperature control, a condenser to collect water, and a vacuum system for reducing the pressure in the drying chamber.

[0054] As used herein, the term "lyophilized composition" means the solid residue or powder that is produced or remains after the lyophilization procedure as defined above. The lyophilized composition of the present invention generally also comprises a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" refers to a substance that is added to the solution before lyophilization to enhance the characteristics of the lyophilized cake, such as color, texture, strength and volume. Pharmaceutically acceptable excipients can be, for example, buffers and pH regulators, crystal expansion excipients, stabilizers and tonicity enhancing agents.

[0055] As used herein, a stabilizer is a composition that maintains the chemical, biological or stability of the chimeric nuclease. Examples of stabilizers include polyols, including sugars, preferably monosaccharides or disaccharides, such as glucose, trehalose, raffinose or sucrose; sugar alcohols, such as, for example, mannitol, sorbitol or inositol, polyhydroxy alcohols, such as glycerol or propylene glycol or mixtures thereof, and albumin.

[0056] Pharmaceutically acceptable salts are salts suitable for administration to subjects such as humans. The chimeric nuclease of the present invention may have one or more sufficiently acidic protons that can react with suitable organic or inorganic bases to form base addition salts. Base addition salts include base addition salts derived from inorganic bases such as ammonium or alkali metal or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.; and organic bases such as alkoxides, alkylamides, alkylamines, and arylamines, etc. Therefore, such bases that can be used to prepare the salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc. The chimeric nuclease of the present invention having a sufficiently basic group such as an amine can react with an organic or inorganic acid to form an acid addition salt. Acids commonly used to form acid addition salts from compounds having a basic group are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of such salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioate, hexyne-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, and the like.

[0057] Although specific embodiments of the present invention have been discussed, the above description is illustrative rather than restrictive. After reading this specification, many variations of the present invention will be apparent to those skilled in the art. The full scope of the present invention should be determined by reference to the full scope of the claims and their equivalents and the specification and these variations.

[0058] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0059] The above discussion is intended to illustrate the principles and various embodiments of the present invention. Once the above disclosure is fully understood, many changes and modifications will become apparent to those skilled in the art. It is intended that the appended claims be interpreted as covering all such changes and modifications.

[0060] abbreviation

[0061] The abbreviations used herein are defined as follows:

[0062] AA Amino Acid

[0063] Cas9 CRISPR-associated protein 9

[0064] CF Cystic Fibrosis

[0065] CFTR cystic fibrosis transmembrane transport regulator

[0066] cjCas9 Campylobacter jejuni Cas9

[0067] Cpf1 CRISPR 1 from Prevotella and Francisella

[0068] CRISPR Clustered Regularly Interspaced Short Palindromic Repeats

[0069] DLS Dynamic Light Scattering

[0070] DMEM Dulbecco's modified Eagle's medium

[0071] DMPE 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine

[0072] DNA deoxyribonucleic acid

[0073] DOAB Dioctadecyl dimethyl ammonium bromide

[0074] DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

[0075] DPPC Dipalmitoylphosphatidylcholine

[0076] E.Coli Escherichia coli

[0077] EDTA Ethylenediaminetetraacetic acid

[0078] EGFR Epidermal Growth Factor Receptor

[0079] ELISA enzyme-linked immunosorbent assay

[0080] fnCas9, the new killer Francisella Cas9

[0081] HDR Homology-Directed Repair

[0082] IMAC Immobilized Metal Affinity Chromatography

[0083] IPTG Isopropyl β-D-1-thiogalactopyranoside

[0084] MPEG-5000-DMPE N-(Carbonyl-methoxypolyethylene glycol 5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine

[0085] NSCLC Non-small cell lung cancer

[0086] NHEJ Non-homologous end joining

[0087] NLS Nuclear Localization Signal

[0088] PC Phosphatidylcholine

[0089] PCR polymerase chain reaction

[0090] PE Phosphoethanolamine

[0091] RNA Ribonucleic acid

[0092] saCas9 Staphylococcus aureus Cas9

[0093] scCas9 Streptococcus canis Cas9

[0094] SDS Sodium dodecyl sulfate

[0095] spCas9 Streptococcus pyogenes Cas9

[0096] TALEN transcription activator-like effector nuclease

[0097] TEV Tobacco etch virus

[0098] TevCas9 Modified I-TevI ​​domain, linker peptide, and modified RNA-guided nuclease Staphylococcus aureus Cas9

[0099] ZFN Zinc Finger Nuclease

[0100] The inventors have discovered a chimeric nuclease comprising a modified version of the I-TevI ​​domain, a linker peptide, and a modified version of the RNA-guided nuclease Staphylococcus aureus Cas9 ("saCas9") (hereinafter referred to as "TevCas9"), which, when mixed with lipid nanoparticles with or without exogenous donor DNA, replaces a DNA sequence in the presence of exogenous donor DNA or deletes a defined length of DNA in the absence of exogenous donor DNA when delivered to cells. This novel chimeric nuclease has been shown to edit genes in human cells as well as cells of other organisms such as bacteria, yeast, insects, plants or other mammals, both in whole organisms (in vivo) and in isolated cell cultures (ex vivo).

[0101] The novel chimeric nuclease discovered by the inventors has the following advantages in particular compared to existing gene editing technologies and methods.

[0102] a. The nuclease is a modified version of the TevCas9 nuclease that can target two independent target sites as a single protein and cut DNA at one or both of these sites. It can be reprogrammed to many different target DNA sequences by modifying one or more of the I-TevI ​​domain, the linker domain, the Cas9 domain, or the guide RNA (which targets the Cas9 domain to its target sequence);

[0103] b. If the nuclease cuts at two sites, it cuts a precise length of DNA (~30–36 bases, depending on the sites targeted by I-TevI ​​and Cas9);

[0104] c. The Cas9 domain contains a mutation (D10E) that is rationally designed to modify the Cas9 nuclease activity and / or increase the specificity of the Cas9 domain to its target binding site;

[0105] d. In the presence of exogenous donor DNA, the present invention is designed to replace a higher percentage of target DNA sequences in cells than prior art or practice;

[0106] e. The nuclease can be purified as a single continuous protein bound to the guide RNA, thus simplifying manufacturing;

[0107] f. Lipid nanoparticles allow non-viral delivery to target cells with high efficiency and low toxicity, allowing controlled administration of nucleases. Although other lipid-based nuclease delivery technologies exist, none are suitable compositions for in vivo use;

[0108] g. Lipid nanoparticles have also been designed to deliver nucleases via aerosolization (inhalation);

[0109] h. A version of the nuclease targets and cleaves the CFTR gene to correct the CFTRδF508 mutation for the treatment of cystic fibrosis (SEQ ID NO 1); and

[0110] i. Another version of the nuclease was designed to target and cleave clinically relevant EGFR exon 19 deletion mutations (SEQ ID NOs 2–4), which are present in a variety of cancers, including non-small cell lung cancer (NSCLC).

[0111] It is known that GIY-YIG nucleases such as I-TevI ​​are fused to a DNA binding domain via a flexible linker (WO2014 / 121222). Previous versions of double-cut TevCas9 have been described, comprising amino acids 1-92 of the wild-type I-TevI ​​nuclease domain, a linker region containing amino acids 93-169 of the I-TevI ​​linker region, and Streptococcus pyogenes Cas9 ("spCas9") (Wolfs JM et al., (2016), 'Biasing Genome-Editing Events Toward Precise Length Deletions with an RNA-Guided TevCas9 Dual Nuclease, 'Proc Natl Acad Sci USA, 113 (52): 14988-93). The chimeric nuclease of the present invention comprises the following:

[0112] The iI-TevI ​​nuclease domain binds novel target sequences, allowing targeting of clinically relevant gene sequences, such as the CFTR gene;

[0113] ii. Various flexible linker regions designed to confer different DNA binding or nuclease activities to TevCas9;

[0114] iii. saCas9 nuclease domain (US-1988 / 065406B2). Compared to spCas9, the use of saCas9 produces a smaller DNA coding sequence (~3.7 kilobases for Tev-saCas9 vs. ~4.6 kilobases for Tev-spCas9) and a lower molecular weight TevCas9 protein (~144 kilodaltons for Tev-saCas9 vs. ~179 kilodaltons for Tev-spCas9), which is more suitable for a variety of delivery technologies; as found by the inventors of the claimed technology, compared to spCas9, the cleavage by the saCas9 domain between the 3rd and 4th nucleotides is predictable, which is more suitable for defined length deletions.

[0115] iv. a version in which the guide RNA is targeted to a specific CFTR gene sequence near the CFTRδF508 mutation; and

[0116] v. A second version in which the guide RNA is targeted to a specific EGFR gene sequence and is designed to cleave only DNA with appropriately spaced I-TevI ​​sites and Cas9 target sites. Such appropriately spaced sites are present in certain EGFR exon 19 deletion mutations (SEQ ID NOs 2-4) but not in wild-type EGFR (SEQ ID NO 5);

[0117] a. The present invention includes lipid nanoparticles of certain compositions that are selectively sized to an average diameter of about 100 nM. These lipid nanoparticles are capable of delivering nucleases to cells with high efficiency and low toxicity;

[0118] b. Pharmaceutical preparations of lipids, nucleases and exogenous donor DNA;

[0119] c. a pharmaceutical formulation of lipids, nucleases, and exogenous donor DNA suitable for aerosolization (inhalation); and

[0120] d. One version of the invention contains exogenous donor DNA that, when delivered with the TevCas9 nuclease in lipid nanoparticles, is capable of integrating into the region between or around the two sites targeted by the nuclease.

[0121] The novel chimeric nuclease composition of the present application contains different combinations of I-TevI ​​domain, linker domain, Cas9 domain and guide RNA.

[0122] Versions that target the CFTR gene include:

[0123] i. an I-TevI ​​domain according to the amino acid sequence of SEQ ID NO: 6;

[0124] ii. a linker domain according to any one of SEQ ID NOs: 7-12;

[0125] iii. a saCas9 domain according to the amino acid sequence of SEQ ID NO: 13; and

[0126] iv. a guide RNA according to the RNA sequence of SEQ ID NO: 15 or 21.

[0127] Versions that target the EGFR gene include:

[0128] i. an I-TevI ​​domain according to the amino acid sequence of SEQ ID NO: 6;

[0129] ii. a linker domain having an amino acid sequence according to any one of SEQ ID NOs: 7-12;

[0130] iii. a saCas9 domain according to the amino acid sequence of SEQ ID NO: 13; and

[0131] iv. Guide RNA of the RNA sequence in SEQ ID NO:16.

[0132] The I-TevI ​​domain of a preferred embodiment is the 93-amino acid I-TevI ​​domain of Enterobacteriaceae phage T4 according to the following sequence:

[0133] MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIA(SEQ ID NO:6)

[0134] The saCas9 of a preferred embodiment is a polypeptide comprising 1,053 amino acids according to the following sequence:

[0135] MKRNYILGLLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRR

[0136] (SEQ IDNO:13)

[0137] The preferred embodiment has Glu 10 The mutated saCas9 is a polypeptide comprising 1,053 amino acids according to the following sequence (mutations are underlined):

[0138] MKRNYILGL E IGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRR

[0139] (SEQ IDNO:14)

[0140] The version of the guide RNA that targets the CFTR gene contains 101 ribonucleotides according to the following sequence:

[0141] GCGUCAUCAAAGCAUGCCAACGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU(SEQ ID NO:15)

[0142] AUAUCAUUGGUGUUUCCUAUGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU(SEQ ID NO:21)

[0143] The version of the guide RNA that targets the EGFR gene is 101 ribonucleotides in length according to the following sequence:

[0144] AAUUUUAACUUUCUCACCUUCGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU (SEQ ID NO: 16).

[0145] The linker used in any of the above constructs may be selected from the group consisting of:

[0146]

[0147]

[0148] synthesis

[0149] Example 1: Method for making TevCas9 nuclease:

[0150] The DNA coding sequences of the I-TevI ​​domain, the linker domain, and the Cas9 domain mentioned above were synthesized into a continuous DNA sequence using techniques known in the art. Gene synthesis was performed by Bio Basic (Markham, Ontario, Canada). In short, short oligonucleotides (~50–60 base pairs) were synthesized, which contained overlapping regions to cover the entire sequence of the I-TevI ​​domain, the linker domain, and the Cas9 domain. These oligonucleotides were mixed together with blocks of approximately 1 kilobase of the sequence to be synthesized, and these ~1 kilobase blocks were synthesized using polymerase chain reaction (PCR). The ~1 kilobase blocks were then mixed and PCR was performed to synthesize the I-TevI ​​domain, the linker domain, and the Cas9 domain. In addition, in order to enhance the expression of TevCas9 in Escherichia coli and simplify restriction endonuclease digestion, the DNA sequence of TevCas9 was optimized before synthesis. First, three base pair DNA codons that are not commonly used by Escherichia coli ("E. coli") were replaced with more frequently occurring codons (e.g., among the six codons encoding the amino acid arginine, the relative abundance of the codon AGG is 0.03, while the relative abundance of the codon CGT is 0.42). A total of 37% of the codons were changed to codons preferred by E. coli. Second, the content of the nucleotides cytosine and guanine was increased from 39.6% to 48.6%. Third, two E. coli ribosome binding sites were removed from the sequence. Fourth, the NdeI restriction endonuclease site was removed from the internal sequence. The continuous DNA was digested with restriction endonucleases NdeI and BamHI (New England Biolabs, Ipswich, Massachusetts, USA), whose target site only appeared once in the DNA sequence, and then inserted into a similarly digested pET-11a expression vector (EMD Millipore, Burlington, Massachusetts, USA) suitable for expressing TevCas9 in Escherichia coli using DNA ligase (New England Biolabs, Ipswich, Massachusetts, USA). The pET-11a vector containing TevCas9 was transformed into an Escherichia coli expression strain T7 Express (New England Biolabs#C2566, Ipswich, Massachusetts, USA), which has been optimized for the expression of proteins including nucleases. Alternatively, the Escherichia coli expression strain BL-21 (DE3) (New England Biolabs#C2527, Ipswich, Massachusetts, USA) was used. Successful transformation was confirmed by the resistance of E. coli to ampicillin or tetracycline, and the coding sequence of TevCas9 was verified by DNA sequencing of the expression vector derived from the transformed E. coli.As measured by spectrophotometry at a wavelength of 600nM, the transformed Escherichia coli grows to an optical density of 0.4 to 0.6 at 37°C, and the expression of the TevCas9 protein from the pET-11a vector in the Escherichia coli expression strain transformed using IPTG induction at 16°C is 10-12 hours. Compared with the uninduced samples, the successful expression of TevCas9 was verified by the presence of a band of about 150kDa on the SDS-polyacrylamide gel stained by Coomassie in the sample of the induced material. Escherichia coli cells were harvested by centrifugation and resuspended in a lysis buffer comprising 10mM imidazole (Sigma, St.Louis, Missouri, USA), 300-500mM sodium chloride (Sigma-Aldrich, St.Louis, Missouri, USA) and 50mM sodium phosphate (binary) (Sigma, St.Louis, Missouri, USA), pH 8.0 [buffer 1]. Alternatively, the sodium phosphate (binary) in buffer 1 is replaced with 10 mM Tris hydrochloride (Sigma, St. Louis, Missouri, USA) at pH 8. Escherichia coli is lysed by homogenization, using a high pressure liquid homogenizer (Avestin, Ottawa, Ontario, Canada) at 600-1000 bar, or using any other suitable lysis method known in the art, such as using an ultrasonic instrument (Branson Ultrasonics, Danbury, Connecticut, USA) sonication, lysozyme treatment, using a French pressure cell (Glen Mills, Clifton, New Jersey, USA) homogenization or using a Dounce homogenizer (Corning, Corning, New York, USA) homogenization. The cracked material is centrifuged at 12,000 rpm for 20-30 minutes at 4 ° C, and the supernatant containing soluble TevCas9 is used for subsequent purification steps. The precipitation contains cell debris, insoluble intracellular material, and any insoluble TevCas9. Successful lysis and solubilization was verified by the presence of an approximately 150 kDa band on a Coomassie-stained SDS-polyacrylamide gel in the supernatant samples compared to the pelleted resuspended samples.

[0151] TevCas9 nuclease was purified as follows:

[0152] 1. The nuclease-containing lysate was applied to a nuclease-binding immobilized metal affinity chromatography (IMAC) column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

[0153] 2. Wash the IMAC column with buffer 1.

[0154] 3. TevCas9 that remained bound to the column was eluted with a solution containing: 250 mM imidazole (Sigma, St. Louis, Missouri, USA), 300 mM-500 mM sodium chloride (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 mM sodium phosphate (dibasic) (Sigma, St. Louis, Missouri, USA), pH 7.6-8.0 [buffer 2]. Alternatively, sodium phosphate (dibasic) in buffer 2 was replaced with 10 mM Tris hydrochloride (Sigma, St. Louis, Missouri, USA) at pH 7.6-8.

[0155] 4. The eluate was treated with tobacco etch virus (TEV) protease (New England Biolabs, Ipswich, MA, USA) and incubated with appropriate guide RNA. The guide RNA was synthesized by Integrated DNA Technology (Coralville, IA, USA).

[0156] 5. Re-apply the treated eluate to the IMAC column and collect the flow-through containing TevCas9 nuclease and guide RNA.

[0157] 6. Successful purification of the TevCas9 nuclease was confirmed by the presence of a 150 kilodalton protein band on a Coomassie-stained SDS-polyacrylamide gel. Successful co-purification of TevCas9 with guide RNA was confirmed by treating the eluate sample with proteinase K (New England Biolabs, Ipswich, MA, USA), then splitting the sample into two and further treating one subsample with RNase A (New England Biolabs, Ipswich, MA, USA), while the other was in a control buffer without RNase A. An RNA band of ~100 nucleotides was visible on the urea-polyacrylamide gel in the control sample, but not in the RNase A-treated sample.

[0158] 7. The solution containing TevCas9 nuclease and guide RNA was dialyzed into a solution containing phosphate buffered saline at pH 7.4.

[0159] Example 2: Method for making lipid nanoparticles:

[0160] The lipid nanoparticles of the preferred embodiment comprise one of the following mixtures:

[0161] I. Lipid nanoparticles No. 1 contain DOPE (Avanti Polar Lipids, Alabaster, AL, United States) and MPEG-5000-DMPE (Avanti Polar Lipids, Alabaster, AL, United States) in a molar ratio of 2:0.05;

[0162] II. Lipid nanoparticles No. 2 comprise DPPC (Avanti Polar Lipids, Alabaster, Alabama, USA), cholesterol (SUPELCO, Bellefonte, PA, United States) and DOBA (Sigma, St. Louis, Missouri, USA) in a molar ratio of 7:2:1, respectively; and

[0163] III.3 lipid nanoparticles contain DPPC, cholesterol and MPEG-5000-DMPE (Avanti Polar Lipids, Alabaster, Alabama, USA) in a molar ratio of 4:1:0.125, respectively.

[0164] Lipid nanoparticles were fabricated with an average diameter of approximately 100 nM.

[0165] Select one of No. 1-3 lipid mixtures. For example, DOPE and MPEG-5000-DMPE are mixed together in an organic solvent such as chloroform with an appropriate molar ratio. The organic solvent is then evaporated, and the dried lipid mixture is resuspended in a solution of phosphate buffered saline containing a pH of 7.4 using a vigorous vortex. The resuspended lipid mixture is then extruded through a 100nM polycarbonate membrane (T&T Scientific Corporation, Knoxville, Tennessee, USA) balanced in phosphate buffered saline to produce lipid nanoparticles with an average diameter of about 100nM. The solution is sterilized by filtration through a 0.2 μM sterile filter (VWR Scientific, Radnor, Pennsylvania, USA). The average diameter and size distribution of lipid nanoparticles are determined by dynamic light scattering (DLS) using Zetasizer (Malvern Panalytical Co., Ltd., Malvern, UK) or other suitable techniques known in the art.

[0166] Example 3: Composition of donor DNA

[0167] The donor DNA contains DNA sequences intended to repair the gene defect. It also contains DNA sequences not found in the target genomic DNA; these sequences do not interfere with normal gene function, but are intended to knock out I-TevI ​​and / or Cas9 sites and / or introduce one or more DNA sequences for tracking the successful repair of the target gene. Examples of donor DNA include, but are not limited to, the following:

[0168] I. Linear single-stranded DNA of varying lengths containing homology regions flanking the site targeted / cleaved by TevCas9;

[0169] II. Linear double-stranded DNA of varying lengths containing homology regions flanking the site targeted / cleaved by TevCas9;

[0170] III. double-stranded DNA of the same length, which is cut by the nuclease and further comprises a DNA end complementary to the DNA end cut by TevCas9;

[0171] IV. a circular double-stranded DNA comprising homology regions flanking the site targeted / cleaved by TevCas9; and

[0172] V. A circular double-stranded DNA comprising an I-TevI ​​target site and a Cas9 target site, wherein the product cleaved from the double-stranded DNA comprises an end complementary to the end cleaved by TevCas9.

[0173] Example 4: Methods for assembling lipid-encapsulated TevCas9 and transfecting cells

[0174] For ex vivo cell transfection: To assemble lipid-encapsulated TevCas9, lipid nanoparticles were mixed with TevCas9 at a molar ratio of 2000:1 in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, Missouri, USA) and incubated at room temperature for 10 minutes. Cells were transfected with 8.7 x 10E-17 to 3.1 x 10E-17 moles of lipid-encapsulated TevCas9 per cell.

[0175] For in vivo cell transfection: To assemble lipid-encapsulated TevCas9, lipid nanoparticles were mixed with TevCas9 at a molar ratio of 2000:1 in phosphate-buffered saline and incubated for 10 min at room temperature. The molar ratio of lipid-encapsulated TevCas9 per cell for in vivo transfection was determined.

[0176] Other Implementations

[0177] Nucleases may contain different combinations of I-TevI ​​domains, linker domains, Cas9 domains, or guide RNAs, as highlighted below.

[0178] Modification of the I-TevI ​​domain: Other versions of the I-TevI ​​nuclease domain may contain different combinations of mutations to change the site targeted by the I-TevI ​​domain or the activity of the I-TevI ​​domain, including mutations that change the sequence recognized by I-TevI, such as K26 and / or C39. Other versions of the nuclease may replace the I-TevI ​​domain with other GIY-YIG nuclease domains such as I-BmoI, Eco29kI, etc. Other versions do not contain Met when expressed in E. coli due to processing 1 .

[0179] Modification of the linker domain: The linker domain may contain one or more of the following to change the binding specificity or activity of TevCas9, including: a). I-TevI ​​linker domain, which contains one or more mutations of amino acids T95, V117, K135, Q158 or N140; b). The linker may contain various combinations of amino acids shown in SEQ ID NO: 9-12.

[0180] Modifications of the Cas9 domain: Other versions of the Cas9 domain may contain the following: a). a version of the saCas9 domain that includes the D10E mutation (SEQ ID NO: 14); b). a version of the saCas9 domain that cleaves the target DNA on one strand of the target DNA, such as the H557A mutation (SEQ ID NO: 17); c). a version of the saCas9 domain that binds to the target DNA but does not cleave it, such as mutations at both the D10A and H557A mutations (SEQ ID NO: 18); d). a version of the previously described spCas9 EQR mutant that includes mutations D1135E, R1335Q, and T1337R combined with the D10E mutation (SEQ ID NO: 19); and e). a previously described spCas9 A version of the EQR mutant that contains mutations D1135E, R1335Q, and T1337R in combination with the D10E mutation, and a mutation that cleaves the target DNA on one strand of the target DNA, such as the H840A mutation (SEQ ID NO: 20). Other versions of the saCas9 domain do not contain Met 1 .

[0181] Other versions can replace the Cas9 domain with other nucleases or DNA binding domains, such as: a). Meganucleases, such as family LAGLIDADG, His-Cys Box, HNH, PD-(D / E)xK, Vsr-like, etc.; b). Zinc finger nucleases; c). Other CRISPR proteins, such as scCas9, fnCas9, cjCas9, Cpf1, Cas12a, Cas13a, Cas3, etc.; and d). Other DNA binding domains, such as zinc finger motifs, TALE activation domains, etc.

[0182] Modifications of guide RNA: a). Other versions of guide RNA can target the same DNA region in the CFTR gene or EGFR gene, but contain different sequences to account for genetic polymorphisms in the population; b). Other versions of guide RNA can target different sequences in the CFTR gene or EGFR gene; c). Other versions of guide RNA can target other sequences in the genome to retarget the nuclease to other clinically relevant targets; d). Other versions of guide RNA can contain bridging nucleic acids ("BNA") to enhance target site specificity; and e). Other versions can contain mixtures of guide RNAs to target multiple sequences within the same gene.

[0183] Modifications of lipid nanoparticles: a) Other versions of lipid nanoparticles 1, 2 or 3 may have different ratios of each lipid component; b) Other versions of lipid nanoparticles may have different average diameters; c) Other versions of lipid nanoparticles may include different cationic or neutral lipids; d) Other versions of lipid nanoparticles may include peptides that target specific cell types; e) Other versions of lipid nanoparticles may include compounds that bind DNA, such as GL67 (N 4 -cholesteryl-spermine); f). The lipid nanoparticles can be lyophilized to improve stability; and g). The lipid nanoparticles can be resuspended in solutions other than phosphate-buffered saline, such as sterile isotonic saline, water for injection, etc.

[0184] Modification of the composition of donor DNA: a). Other versions of linear double-stranded donor DNA may contain longer single-stranded DNA regions complementary to the target sequence; and b). Other versions of circular double-stranded DNA may contain additional DNA sequences intended to increase the rate of homology-directed repair.

[0185] Variants of the method of assembling lipid-encapsulated nucleases and transfecting cells: a). Other versions of lipid-encapsulated nucleases can contain different molar ratios of lipid nanoparticles to nucleases; b). Media other than DMEM or phosphate-buffered saline can be used for the incubation step; c). Nucleases and lipid nanoparticles can be incubated for less than or more than 10 minutes; and d). Other molar amounts of nuclease per cell can be used in the transfection reaction.

[0186] Variants of the method for making nucleases: a). Other E. coli expression strains can be used, such as LS5218 (Escherichia coli Genetic Stock Center - Yale University, New Haven, CT, USA) or BL21-DE3 (New England Biolabs, Ipswich, MA, USA). b). Buffer 1 or 2 can contain different concentrations of imidazole, sodium chloride, sodium phosphate (dibasic) or tris hydrochloride and buffered to different pH; c). Other processing steps can be used, such as cation or anion exchange chromatography; d). The nuclease can be dialyzed into solutions other than phosphate-buffered saline, such as sterile isotonic saline, water for injection, etc.; e). The nuclease can be lyophilized to improve stability; f). The guide RNA can be co-expressed from the pACYC-Duet1 expression vector (EMD Millipore, Burlington, MA, USA). The DNA coding sequence for the synthetic guide RNA (Integrated DNA Technology, Coralville, Iowa, USA) was digested with restriction endonucleases and inserted into a similarly digested second expression site in the pACYC-Duet1 expression vector; and g). Guide RNA can be synthesized from double-stranded DNA by transcribing the guide using the T7 RNA polymerase HiScribe kit (New England Biolabs #E2040S, Ipswich, Massachusetts, USA) and purifying the guide using the RNA Cleanup kit (New England Biolabs #T2030L, Ipswich, Massachusetts, USA).

[0187] test

[0188] Example 1: Demonstration of correction of CFTRδF508 and CFTR protein in model cell lines

[0189] Functional approach

[0190] Cultures of immortalized epithelial cells homozygous for the CFTRδF508 mutation, such as the CuFi-1 cell line ( CRL-4013 TMThe cells were then treated with a series of concentrations of lipid-encapsulated TevCas9 and donor DNA (Specific Biologics, Toronto, Ontario, Canada) in a pharmaceutical formulation targeting the CFTRδF508 mutation. Appropriate control cell lines, such as NuLi-1 ( CRL-4011 TM , American Type Culture Collection, Manassas, VA, USA) immortalized epithelial cells.

[0191] By T7 endonuclease I assay ( Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, MA, USA), deep gene sequencing using the Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, USA), or other suitable methods to measure the proportion of cells with CFTRδF508 corrected relative to uncorrected cells. CRL-4011 TM , American Type Culture Collection, Manassas, VA, USA). CRL-4013 TM , American Type Culture Collection, Manassas, VA, USA) and mock-treated CuFi-1 cultures ( CRL-4013 TM CFTR functionality was measured using short-circuit current measurements in a UssingCamber (Warner Instruments, Hamden, Connecticut, USA) in the presence of a chloride gradient in a 1:1 ratio (A.T.C., American Type Culture Collection, Manassas, VA, USA). The effect of TevCas9 treatment in control cell lines (e.g., NuLi-1) was also measured.

[0192] To demonstrate disruption of EGFR expression and activity in a model cell line by inducing one or more EGFR exon 19 deletion mutations, cultures of immortalized epithelial cells expressing one or more EGFR exon 19 deletion mutations, such as the HCC827 cell line ( CRL-2868 TM , American Type Culture Collection, Manassas, VA, USA) were treated with a range of concentrations of lipid-encapsulated TevCas9 (Specific Biologics, Toronto, Ontario, Canada) targeting EGFR exon 19 deletion in phosphate-buffered saline, sterile isotonic saline, or water for injection. Appropriate control cell lines were used, such as NuLi-1 ( CRL-4011 TM , American Type Culture Collection, Manassas, VA) or immortalized epithelial cell lines.

[0193] By T7 endonuclease I assay ( Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, MA, USA), deep gene sequencing using the Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, USA), or other suitable methods to measure the proportion of cells disrupted by EGFR exon 19 deletions relative to uncorrected cells. The effect of TevCas9 treatment on control cell lines (e.g., NuLi-1 ( CRL-4011 TM , American Type Culture Collection, Manassas, VA, USA). EGFR protein expression and activity were measured using enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, USA), which detected the effects of treated HCC827 cultures ( CRL-2868 TM , American Type Culture Collection, Manassas, VA, USA) versus mock-treated HCC827 cultures. The expression of phosphorylated (i.e., activated), unphosphorylated, and total EGFR proteins in control cell lines (e.g., NuLi-1 ( CRL-4011 TM, American Type Culture Collection, Manassas, VA, USA).

[0194] Example 1: Animal model testing planned to demonstrate efficacy and determine dose-limiting toxicity

[0195] In an exemplary method demonstrating correction of CFTRδF508 and / or cystic fibrosis symptoms treated with lipid-encapsulated TevCas9 in an animal model (e.g., mouse, rat, minipig, or ferret), lipid-encapsulated TevCas9 targeting CFTRδF508 in a pharmaceutical formulation of phosphate-buffered saline, sterile isotonic saline, or water for injection is delivered directly to the lungs by intubation or intranasal administration. The procedure time for each treatment is approximately 30-6000 seconds, depending on the animal model used.

[0196] In another method, lipid-encapsulated TevCas9 in a drug formulation targeting CFTRδF508 was nebulized using a commercial nebulizer ( (Trudell Medical, London, Ontario, Canada) or PARI-LC (PARI USA, Midlothian, Virginia, USA)) atomization. After atomization, the average size of the lipid nanoparticles was confirmed to be about 100nM by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Co., Ltd., Malvern, UK) or other suitable techniques known in the art. The composition and concentration of lipid-encapsulated TevCas9 were confirmed using a MicroGram lipid assay kit (ProFoldin, Hudson, Massachusetts, USA) and a band of about 150kDa on a Coomassie-stained SDS-polyacrylamide gel after atomization. For the measurement of gene correction rate, a representative sheep (minipig) animal model homozygous for the CFTRδF508 mutation (Exemplar Genetics, Sioux City, Iowa, USA) was exposed to lipid-encapsulated TevCas9 targeting CFTRδF508 and appropriate controls through the mouth, nose or directly to the lungs. General maintenance of these animals includes breeding and littering; age-appropriate biosecure housing; good nutrition; basic vaccinations and veterinary care; and documentation consistent with animal welfare guidelines. Maintenance of these CFTRδF508-specific animals may include one or more of the following: surgery to treat intestinal obstruction; pancreatic enzyme replacement therapy; vitamins and H2 blockers; and / or proton pump inhibitors to improve gastric acid control. Minipigs were treated with a range of concentrations of lipid-encapsulated TevCas9 that were predicted to be effective for 2 days to 4 weeks for acute toxicity studies based on the model cell line studies described above, and for up to 24 months for chronic toxicity studies.

[0197] Monitor the general health of the animals after treatment to assess any adverse events associated with treatment, such as changes in behavior, body weight, or food consumption; immune response; changes in cardiovascular health; death, etc. Other post-treatment efficacy measures may include:

[0198] I. Forced expiratory volume, such as the forced expiratory volume (or other appropriate method) for each animal after treatment;

[0199] II. Overall survival rate of each animal relative to controls;

[0200] III. Other lung function measurements (e.g., use of a mechanical ventilator that allows for general lung function assessment); and

[0201] IV. Measuring mutations in vivo by tissue sampling and mutation detection methods such as polymerase chain reaction.

[0202] After treatment with lipid-encapsulated TevCas9, animals were sacrificed and lung and tracheal tissues were harvested.

[0203] By T7 endonuclease I assay ( Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, MA, USA), deep gene sequencing using the Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, USA), or other suitable methods to measure the proportion of CFTRδF508 corrected cells relative to uncorrected cells.

[0204] In a method demonstrating disruption of one or more EGFR exon 19 deletion mutations and / or symptoms of non-small cell lung cancer (NSCLC) treated with TevCas9 in an animal model, a lipid-encapsulated TevCas9 targeting an EGFR exon 19 deletion mutation in a pharmaceutical formulation of phosphate-buffered saline, sterile isotonic saline, or water for injection is delivered directly to the lungs by mouth, nose, or directly to the lungs. The procedure time for each treatment is approximately 30-6000 seconds, depending on the animal model used.

[0205] In another approach, lipid-encapsulated TevCas9 targeting EGFR exon 19 deletion mutations in a pharmaceutical formulation was nebulized using a commercial nebulizer ( (Trudell Medical, London, Ontario, Canada) or PARI-LC (PARI USA, Midlothian, Virginia, USA)) atomization. After atomization, the average size of the lipid nanoparticles was confirmed to be about 100nM by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Co., Ltd., Malvern, UK) or other suitable techniques known in the art. The composition and concentration of lipid-encapsulated TevCas9 nanoparticles were confirmed using a MicroGram lipid assay kit (ProFoldin, Hudson, Massachusetts, USA) and a band of about 150kDa on a Coomassie-stained SDS-polyacrylamide gel after atomization. In order to measure the gene disruption rate, a representative mouse (mouse) animal model homozygous for one or more EGFR exon 19 deletion mutations was exposed to lipid-encapsulated TevCas9 targeting EGFR exon 19 deletions through the nose, mouth or directly to the lungs. Mice were treated with a range of concentrations of TevCas9, which was predicted to be effective for acute toxicity studies for 2 days to 4 weeks based on model cell line studies, and effective for chronic toxicity studies for up to 24 months.

[0206] Monitor the general health of the animals after treatment to assess any adverse events associated with treatment, such as changes in behavior, weight, or food consumption; immune response; changes in cardiovascular health; death, etc. Other post-treatment efficacy measures may include:

[0207] I. Quantification of EGFR activated protein by positron emission tomography (PET) using EGFR mutation tracers;

[0208] II. Overall survival rate of each animal relative to controls; and

[0209] III. Measurement of tumor formation / reduction in each animal over time.

[0210] Through tissue sampling and mutation detection methods such as The EGFR mutation test version 2 (Roche Diagnostics, Risch-Rotkreuz, Switzerland) measured in vivo mutations. After treatment with aerosolized lipid-encapsulated TevCas9, animals were sacrificed and lung and tracheal tissues were harvested.

[0211] By T7 endonuclease I assay ( Mutation Detection Kit, New England Biolabs # E3321, Ipswich, Massachusetts, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, Massachusetts, USA), deep gene sequencing using the Illumina MiSeq system and barcode primers flanking the target sites (Illumina, San Diego, California, USA) or other suitable methods to measure the ratio of cells destroyed by EGFR exon 19 deletion mutations relative to undestroyed cells. EGFR protein expression and activity in harvested tissue cells were measured using enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, Missouri, USA), which detects phosphorylated (i.e., activated), unphosphorylated and total EGFR protein. The dose-limiting toxicity that can be determined for the first human clinical study is based on the predicted effective dose from the above-mentioned animal model studies. A series of concentrations (e.g., milligrams per kilogram of body weight) of lipid-encapsulated TevCas9 are atomized and delivered to suitable animal models for toxicology studies, such as crab-eating monkeys or other non-human primates. The animals' general health is monitored for any adverse events related to treatment, such as changes in behavior, body weight, or food consumption; immune response; changes in cardiovascular health; death, etc. Other measures of efficacy that may be measured in the study include those listed above.

[0212] Treatment Effects

[0213] The novel chimeric nucleases of the present invention have been intentionally designed to modify the DNA of lung epithelial cells to treat monogenic diseases, although they can function in vivo or ex vivo in other cell types or cells of other organisms such as bacteria, yeast, insects, plants or other mammals to treat monogenic or polygenic and infectious diseases.

[0214] Example 1: Method for targeted insertion or replacement of DNA sequences in all or part of the human cell genome

[0215] Figure 2 The mechanism of action of the cellular uptake of the novel chimeric nuclease of the present invention is described. Figure 2 As shown in A, a cell 20 or a plurality of cells 20 are exposed to a novel lipid-encapsulated nuclease particle 21 containing TevCas9 25 by in vivo or ex vivo administration. Figure 2 As shown in Figure B, the lipid-encapsulated nuclease particle 21 is internalized into the cell 20, and the endosome 22 undergoes a maturation process in the cytosol and is targeted for degradation ( Figure 2 C) Under certain circumstances, TevCas9 25 can escape from the endosome 22 and enter the cytosol ( Figure 2D). In eukaryotes, the nuclease (TevCas9) 25 is targeted to the nucleus 23 of the cell 20 via one or more nuclear localization sequences ("NLS"). Figure 2 As depicted in E, through its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and once in the nucleus 23, the TevCas9 nuclease 25 binds and cleaves 26 the target genomic DNA 24 sequence.

[0216] Figure 3 The mechanism by which the TevCas9 nuclease cleaves DNA is illustrated. Figure 3 A shows key features of TevCas9 shown bound to its target genomic DNA sequence 24 prior to the cleavage reaction. The I-TevI ​​domain 27 targets the I-TevI ​​target sequence 29. The linker domain 30 connects the I-TevI ​​domain 27 to the Cas9 domain 28 targeting the Cas9 target sequence 31. The genetic mutation 32 is surrounded by or close to the I-TevI ​​target sequence 29 and the Cas9 target sequence 31. Figure 3 As shown in B, TevCas9 25 cleaves the target sequence, leaving a deletion product 34 of predictable size with non-complementary DNA ends 35, 36. Figure 3 C illustrates that in the presence of single-stranded donor DNA with homology arms 37, cell 20 can insert donor DNA 37 sequence near the cleavage site through homology directed repair (HDR) pathway 38. Figure 3 D illustrates that in the presence of donor DNA 39 having DNA ends compatible with the DNA ends cleaved by TevCas9 25, cell 20 can insert donor DNA sequence 39 between cleavage sites by directional ligation using non-homologous end joining (NHEJ) pathway 40. In the absence of donor DNA, cell 20 can join DNA ends by NHEJ pathway 40 ( Figure 3 E).

[0217] Example 2: Treatment of Cystic Fibrosis

[0218] For the treatment of cystic fibrosis, the exogenous donor DNA comprises a DNA sequence that repairs the CFTRδF508 mutation, and involves a method of using the exogenous donor DNA as a template to target and delete a DNA sequence of a defined length in human somatic cells to stimulate homology-directed repair ( Figure 3 C).

[0219] Example 3: Treatment of non-small cell lung cancer

[0220] For the application of treating non-small cell lung cancer, a Cas9 domain version that cuts only one strand of DNA (D10A or H557A mutation) or a nuclease-deficient version (D10A+H557A mutation) is used, and the sequence targeted is the EGFR exon 19 deletion mutation (SEQ ID NO: 2-4). However, in the present application, the nuclease does not contain exogenous donor DNA. In the absence of exogenous donor DNA, the cell can remove the DNA sequence between the two sites targeted by the nuclease by non-homologous end joining ( Figure 3 E).

[0221] Alternatively, the inhalation route is a rapid and effective way to deliver drugs locally to the lungs and for systemic administration of certain agents. Inhaled drug therapy is widely used to treat respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD). Research is ongoing to develop an inhalation system for the treatment of cystic fibrosis.

[0222] The following examples are not intended to limit the scope of the present disclosure, but are provided to illustrate how to make and use the compounds disclosed herein.Many other embodiments of the present disclosure will be apparent to those skilled in the art.

[0223] A nebulizer is a device that delivers medication to the lungs in the form of an aerosolized vapor. Nebulizers are commonly used to treat respiratory diseases such as asthma and COPD, for example, the nebulization of corticosteroids, although nebulization has also been used to treat and prevent lung infections such as (Insmed Incorporated, Bridgewater, New Jersey, USA).

[0224] Nebulizers may require some procedures to prepare the liquid for nebulization. The drug is usually placed in a cup in the nebulizer chamber in liquid form. After loading, the device is turned on and compressed air is generated to convert the liquid into vapor in the nebulizer chamber. The patient puts the mouthpiece of the nebulizer chamber into his mouth and takes a deep breath, holding his breath for 5-10 seconds to ensure that the drug reaches the lower part of the lungs. There are many such devices. Many modern nebulizers are breath-activated and rely on the force of the patient's inhalation to bring the nebulized liquid out of the device, thereby ensuring that the drug is delivered only to the patient and not the surrounding environment. This also ensures the consistency of delivering a full dose of drug to the patient.

[0225] The use of nebulizers is well known and nebulizers are commercially available from a number of sources, such as (Trudell Medical, London, Ontario, Canada) or PARI-LC (PARI USA, Midlothian, Virginia, USA). In an example of the present invention, a nebulizer is used to deliver the lipid-encapsulated novel chimeric nuclease of the present application comprising a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 to lung epithelial tissue. A sterile liquid version of the therapeutic agent of interest is loaded into the nebulizer chamber and then atomized and inhaled into the lungs by the patient through deep breathing.

[0226] Some advantages of using a nebulizer over oral or intravenous administration are that: less medication may be required than with oral or intravenous administration; onset of action may be faster via inhalation than with the oral route; adverse effects may be less severe because the medication is delivered locally to the lung tissue where the disease itself occurs; and inhaled medication therapy is painless and relatively comfortable for the patient, which encourages compliance.

[0227] No non-invasive route of administration can provide the speed of action that inhaled drugs can provide. One of the advantages of inhaled drugs is that they are absorbed faster than subcutaneously injected molecules and provide a more immediate physiological response. Small or large molecules, especially hydrophobic molecules, can be absorbed within seconds after inhalation and can therefore be used to treat a variety of symptoms that occur suddenly or require long-term administration. Pain, panic, anxiety, nausea, cardiovascular crisis, bronchoconstriction, sleep induction, spasms, Parkinson's lock-up and hot flashes are some of the rapid onset conditions that inhaled drugs can address.

[0228] Most protein-based drug products have a certain water solubility and can be quickly and effectively absorbed from the lungs. Those that are more hydrophobic are even absorbed faster within seconds to minutes. Those that are more hydrophilic are absorbed within minutes to tens of minutes. In an example of the present invention, a vial is aseptically filled with a therapeutic dose of hydrophobic lipid nanoparticles, and another vial is aseptically filled with a therapeutic dose of chimeric nuclease, the chimeric nuclease comprising a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, which is water-soluble and hydrophilic for delivery to the lungs at a therapeutic dose. The dosage range of lipid nanoparticles and chimeric nucleases can be 1 to 1000 mg, preferably about 5 to 200 mg. The claimed lipid-encapsulated chimeric nuclease comprising a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 can be absorbed by lung cells within a few hours, and complete cutting on an in vitro DNA substrate has been observed within 2 hours. Other aerosol therapy has been delivered every day. Therefore, the aerosol application of lipid-encapsulated chimeric nuclease can be used once a day or less frequently, depending on its efficacy based on each patient. Chimeric nuclease is produced by BioVectra (Charlottetown, PE, Canada), and vials are aseptically filled by Dalton Pharma Services (Mississauga, Ontario, Canada). Lipid nanoparticles are manufactured and aseptically filled vials by TransferraNanosciences (Burnaby, BC, Canada).

[0229] The dosage of any disclosed composition will vary according to the patient's symptoms, age and weight, the nature and severity of the illness to be treated or prevented, the route of administration and the form of the composition. Any of the present preparations can be administered in a single dose or divided doses. The dosage of the composition can be easily determined by techniques known to those skilled in the art or as taught herein.

[0230] In certain embodiments, the dosage of the present compound is generally in the range of about 1 to 1000 mg, particularly in the range of about 5 to 200 mg, depending on the patient's body weight.

[0231] For any particular composition of the present disclosure, it may be necessary to determine an effective dose or amount and any possible effects on the time of administration of the formulation. This can be accomplished by routine experimentation as described herein, using one or more groups of animals (preferably at least 5 animals per group), or if appropriate, in human trials. The effectiveness of any of the present compositions and methods of treatment or prevention can be assessed by administering the composition and evaluating the effect of the administration by measuring one or more applicable indices and comparing the post-treatment values ​​of these indices with the pre-treatment values ​​of the same indices.

[0232] The precise time and amount of administration of any particular present composition that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the present composition, the patient's physiological condition (including age, sex, disease type and stage, general physical condition, response to a given dose and drug type), route of administration, etc. The guide proposed herein can be used to optimize treatment, such as determining the best time and amount of administration, which will only require routine experiments consisting of monitoring the subject and adjusting the dosage and / or time. When the subject is receiving treatment, the health of the patient can be monitored by measuring one or more related indicators at a predetermined time during treatment. Treatments including compositions, amounts, time of administration, and preparations can be optimized according to the results of this monitoring. By measuring the same parameters, the patient can be reassessed regularly to determine the degree of improvement. The amount of the present composition used and possibly the time of administration can be adjusted based on these reassessments.

[0233] Treatment may be initiated with a smaller dose which is less than the optimum dose of the compound. Thereafter, the dose may be increased by small increments until the optimum therapeutic effect is achieved.

[0234] The use of the present combination can reduce the required dosage of any single agent contained in the combination, because the onset and duration of action of the different agents can be complementary. The therapeutic efficacy of the present combination can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining LD 50 and ED 50 .

[0235] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of any present composition is preferably in the range of ED 50 The dosage may vary within this range, depending on the dosage form employed and the route of administration utilized. For the compositions of the present disclosure, the therapeutically effective dose can be initially estimated from cell culture assays.

[0236] preparation

[0237] Pharmaceutical compositions of the present disclosure can be applied in various ways, depending on their intended use, as is well known in the art. For example, compositions of the present disclosure will be applied by atomization. Alternatively, preparations disclosed herein can be applied intravenously, subcutaneously or intramuscularly. These preparations can be prepared in a conventional manner, and if desired, compositions can be mixed with any conventional additives such as excipients, solubilizers, suspension aids, emulsifiers or preservatives. Disclosed excipients can be used for more than one function. For example, solubilizers can also be suspension aids, emulsifiers, preservatives, etc.

[0238] The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.The amount of the composition that may be combined with the carrier materials to produce a single dose will vary depending upon the subject being treated and the particular mode of administration.

[0239] Methods for preparing these formulations include the step of bringing into association a composition of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agent with a liquid carrier.

[0240] It should be understood that the disclosed compositions can include lyophilized or freeze-dried compounds disclosed herein. For example, disclosed herein are compositions in the form of crystalline and / or amorphous powders of the disclosed compounds. Such forms can be reconstituted for use as, for example, aqueous compositions.

[0241] Liquid dosage forms for injection include pharmaceutically acceptable solutions, emulsions, microemulsions, solutions and suspensions. In addition to the present composition, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, glycerol, tetrahydrofuran alcohol and fatty acid esters of sorbitan, cyclodextrin, albumin, hyaluronic acid, chitosan and mixtures thereof. Polyethylene glycol (PEG) may be used to obtain desired properties in solubility, stability, half-life and other pharmaceutically favorable properties. Representative examples of stabilizing components include polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose and combinations thereof. Other excipients such as solvent binders or antioxidants that can be used include, but are not limited to, butylhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C and xylitol. Typically, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the present composition with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary with the requirements of the particular composition, but typically include nonionic surfactants (Tweens, pluronics or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are usually prepared from isotonic solutions.

[0242] It should be noted that an excipient given as an example may have more than one function. For example, a solubilizer may also be a suspending aid, an emulsifier, a preservative, etc.

[0243] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate and cyclodextrins. For example, appropriate fluidity can be maintained by the use of coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0244] The compositions of the present invention are generally sterile, storage-stable and pharmaceutically acceptable aqueous solutions that are easy to administer and do not require reconstitution prior to administration. The compositions of the present invention are suitable for administration to a subject, which means that they are pharmaceutically acceptable, non-toxic, do not contain any components that would adversely affect the biological effects of the chimeric nuclease, and have a pH close to physiological conditions, which avoids aspiration and / or injection site reactions. For example, the compositions of the present invention do not contain any cells.

[0245] The composition is typically stored in a sealed container, vial or cartridge that is generally suitable for long-term storage. "Suitable for long-term storage" means that the vial, container or cartridge will not allow the components of the composition of the present invention to escape or flow into foreign components, such as microorganisms, when kept at 25°C for at least 3 months. The composition of the present invention is preferably administered by nebulization, typically breath-actuated nebulization.

[0246] The compositions of the invention may also be administered by injection as described herein.

[0247] The compositions of the invention can be administered alone or in combination with additional therapeutic agents, such as antiviral agents, antimicrobial agents, chemotherapeutic agents, and immunotherapies.

[0248] As used herein, a vial may also comprise two containers, one of which contains a chimeric nuclease or lipid particle as described herein in the form of a lyophilized powder as described below, and a second container containing a liquid for reconstitution of the lyophilized powder. The contents of the two containers may be mixed before administration.

[0249] As described above, the compositions of the invention may be administered by nebulization. Suitable volumes of the compositions of the invention for nebulization include about 0.5 to about 1 ml, about 1 to about 2 ml, about 2 to about 10 ml, or about 10 to about 20 ml.

[0250] In the compositions of the invention, the concentration of the chimeric nuclease is about 0.1 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 100.0 mg / ml, about 30.0 mg / ml to about 300.0 mg / ml, about 500 mg / ml to about 2000 mg / ml and about 2.0 mg / ml.

[0251] In the compositions of the present invention, the concentration of lipid nanoparticles is about 0.1 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 100.0 mg / ml, about 30.0 mg / ml to about 300.0 mg / ml, about 500 mg / ml to about 2000 mg / ml and about 2.0 mg / ml.

[0252] SEQ ID NO 1

[0253]

[0254]

[0255] Length: 4,443

[0256] Type: DNA

[0257] Organism: Homo sapiens

[0258] Characterization: Cystic fibrosis transmembrane transport regulator (CFTR) delta F508 DNA coding sequence

[0259] Additional information: Nucleotides missing compared to wild-type CFTR are shown as ("-"). Sequences targeted by TevCas9 are highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double underlined, and the PAM sequence is in lowercase.

[0260] SEQ ID NO 2

[0261]

[0262] Length: 132

[0263] Type: DNA

[0264] Organism: Homo sapiens

[0265] Features: Epidermal growth factor receptor (EGFR) exon 19C.2235-2249 deletion sequence

[0266] Additional information: Nucleotides missing compared to wild-type EGFR are shown as ("-"). Sequences targeted by TevCas9 are highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double underlined, and the PAM sequence is in lowercase. Intron sequences are in bold italics.

[0267] SEQ ID NO 3

[0268]

[0269] Length: 132

[0270] Type: DNA

[0271] Organism: Homo sapiens

[0272] Features: Epidermal growth factor receptor (EGFR) exon 19C.2236_2250 deletion sequence

[0273] Additional information: Nucleotides missing compared to wild-type EGFR are shown as ("-"). Sequences targeted by TevCas9 are highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double underlined, and the PAM sequence is in lowercase. Intron sequences are in bold italics.

[0274] SEQ ID NO 4

[0275]

[0276] Length: 132

[0277] Type: DNA

[0278] Organism: Homo sapiens

[0279] Features: Epidermal growth factor receptor (EGFR) exon 19C.22372251 deletion sequence

[0280] Additional information: Nucleotides missing compared to wild-type EGFR are shown as ("-"). Sequences targeted by TevCas9 are highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double underlined, and the PAM sequence is in lowercase. Intron sequences are in bold italics.

[0281] SEQ ID NO 5

[0282]

[0283] Length: 147

[0284] Type: DNA

[0285] Organism: Homo sapiens

[0286] FEATURE: Epidermal growth factor receptor (EGFR) exon 19 wild-type sequence

[0287] Additional information: Sequences targeted by TevCas9 are highlighted. I-Tevl sites are underlined, Cas9 guide RNA sequences are double underlined, and PAM sequences are in lowercase. Note: I-Tevl and Cas9 target sites are not properly spaced, so I-TevI ​​should not bind or cleave its target site. Intron sequences are in bold italics.

[0288] SEQ ID NO 6

[0289]

[0290] Length: 93

[0291] Type: Amino Acid

[0292] Organism: Enterobacteriaceae phage T4

[0293] Features: I-TEVI domain

[0294] Additional Information:

[0295] SEQ ID NO 7

[0296]

[0297] Length: 83

[0298] Type: Amino Acid

[0299] Organism: Artificial

[0300] Features: Linker domain

[0301] Additional information: V117F; mutation is underlined

[0302] SEQ ID NO 8

[0303]

[0304] Length: 83

[0305] Type: Amino Acid

[0306] Organism: Artificial

[0307] Features: Linker domain

[0308] Additional information: K135R / N140S; mutations are underlined

[0309] SEQ ID NO 9

[0310]

[0311] Length: 83

[0312] Type: Amino Acid

[0313] Organism: Artificial

[0314] Features: Linker domain

[0315] Additional information: V117F / K135R / N140S; mutations are underlined

[0316] SEQ ID NO 10

[0317]

[0318] Length: 86

[0319] Type: Amino Acid

[0320] Organism: Artificial

[0321] Features: Linker domain variants

[0322] Additional Information:

[0323] SEQ ID NO 11

[0324]

[0325] Length: 87

[0326] Type: Amino Acid

[0327] Organism: Artificial

[0328] Features: Linker domain variants

[0329] Additional Information:

[0330] SEQ ID NO 12

[0331]

[0332] Length: 95

[0333] Type: Amino Acid

[0334] Organism: Artificial

[0335] Features: Linker domain variants

[0336] Additional Information:

[0337] SEQ ID NO 13

[0338]

[0339] Length: 1,053

[0340] Type: Amino Acid

[0341] Organism: Staphylococcus aureus

[0342] feature:

[0343] Additional Information:

[0344] SEQ ID NO 14

[0345]

[0346] Length: 1,053

[0347] Type: Amino Acid

[0348] Organism: Artificial

[0349] Features: SACAS9 D10E mutation

[0350] Additional information: mutations are underlined

[0351] SEQ ID NO 15

[0352]

[0353] Length: 101

[0354] Type: RNA

[0355] Organism: Artificial

[0356] Features: Target CFTR gene

[0357] Additional Information:

[0358] SEQ ID NO 16

[0359]

[0360] Length: 101

[0361] Type: RNA

[0362] Organism: Artificial

[0363] Features: Target EGFR gene

[0364] Additional Information:

[0365] SEQ ID NO 17

[0366]

[0367] Length: 1,053

[0368] Type: Amino Acid

[0369] Organism: Artificial

[0370] Features: SACAS9 D10E+H557A mutation

[0371] Additional information: mutations are underlined

[0372] SEQ ID NO 18

[0373]

[0374]

[0375] Length: 1,053

[0376] Type: Amino Acid

[0377] Organism: Artificial

[0378] Features: SACAS9 D10A+H557A mutation

[0379] Additional information: mutations are underlined

[0380] SEQ ID NO 19

[0381]

[0382] Length: 1,368

[0383] Type: Amino Acid

[0384] Organism: Artificial

[0385] Characterization: SpCas9 D10E, D1135E, R1335Q, T1337R mutations

[0386] Additional information: mutations are underlined

[0387] SEQ ID NO 20

[0388]

[0389] Length: 1,368

[0390] Type: Amino Acid

[0391] Organism: Artificial

[0392] Characterization: SpCas9 D10E, H840A, D1135E, R1335Q, T1337R mutations

[0393] Additional information: mutations are underlined

[0394] SEQ ID NO 21

[0395]

[0396] Length: 101

[0397] Type: RNA

[0398] Organism: Artificial

[0399] Features: Target CFTR delta F508 gene

[0400] Additional Information:. Sequence Listing <110> Specific Biologics THE UNIVERSITY OF WESTERN ONTARIO (GENOMICS) <120> Lipid-encapsulated dual-cutting endonucleases for DNA and gene editing <130> PPI21172454CA <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 4440 <212> DNA <213> Homo sapiens <400> 1 atgcagaggt cgcctctgga aaaggccagc gttgtctcca aacttttttt cagctggacc 60 agaccaattt tgaggaaagg atacagacag cgcctggaat tgtcagacat ataccaaatc 120 ccttctgttg attctgctga caatctatct gaaaaattgg aaagagaatg ggatagagag 180 ctggcttcaa agaaaaatcc taaactcatt aatgcccttc ggcgatgttt tttctggaga 240 tttatgttct atggaatctt tttattta ggggaagtca ccaaagcagt acagcctctc 300 ttactgggaa gaatcatagc ttcctatgac ccggataaca aggaggaacg ctctatcgcg 360 attatctag gcataggctt atgccttctc tttattgtga ggacactgct cctacaccca 420 gccattttg gccttcatca cattggaatg cagatgagaa tagctatgtt tagttgatt 480 tataagaaga ctttaaagct gtcaagccgt gttctagata aaataagtat tggacaactt 540 gttagtctcc tttccaacaa cctgaacaaa tttgatgaag gacttgcatt ggcacatttc 600 gtgtggatcg ctcctttgca agtggcactc ctcatggggc taatctggga gttgttacag 660 gcgtctgcct tctgtggact tggtttcctg atagtccttg ccctttttca ggctgggcta 720 gggagaatga tgatgaagta cagagatcag agagctggga agatcagtga aagacttgtg 780 attacctcag aaatgattga aaattccaa tctgttaagg catactgctg ggaagaagca 840 atggaaaaaa tgattgaaaa cttaagacaa acagaactga aactgactcg gaaggcagcc 900 tatgtgagat acttcaatag ctcagccttc ttcttctcag ggttctttgt ggtgttttta 960 tctgtgcttc cctatgcact aatcaaagga atcatcctcc ggaaaatatt caccaccatc 1020 tcattctgca ttgttctgcg catggcggtc actcggcaat ttccctgggc tgtacaaaca 1080 tggtatgact ctcttggagc aataaacaaa atacaggatt tcttacaaaa gcaagaatat 1140 aagacattgg aatataactt aacgactaca gaagtagtga tggagaatgt aacagccttc 1200 tgggaggagg gatttgggga attatttgag aaagcaaaac aaaacaataa caatagaaaa 1260 acttctaatg gtgatgacag cctcttctc agtaatttct cacttcttgg tactcctgtc 1320 ctgaaagata ttaatttcaa gatagaaaga ggacagttgt tggcggttgc tggatccact 1380 ggagcaggca agacttcact tctaatggtg attatgggag aactggagcc ttcagagggt 1440 aaaattaagc acagtggaag aatttcattc tgttctcagt tttcctggat tatgcctggc 1500 accattaaag aaaatatcat tggtgtttcc tatgatgaat atagatacag aagcgtcatc 1560 aaagcatgcc aactagaaga ggacatctcc aagttttgcag agaaagacaa tatagttctt 1620 ggagaaggtg gaatcacact gagtggaggt caacgagcaa gaatttcttt agcaagagca 1680 gtatacaaag atgctgattt gtatttatta gactctcctt ttggatacct agatgtttta 1740 acagaaaaag aaatatttga aagctgtgtc tgtaactga tggctaaca aactaggatt 1800 ttggtcactt ctaaaatgga acatttaaag aaagctgaca aaatattaat ttgcatgaa 1860 ggtagcagct atttttgg gatattca gaacccaaa atctacagcc agactttagc 1920 tcaaactca tgggatgtga ttctttcgac caatttagtg cagaagaag aattcaatc 1980 ctaactgaga ccttacaccg tttctcatta gaaggagatg ctcctgtctc ctggacagaa 2040 aaaaaaac aatcttta acagactgga gagtttgggg aaaaaggaa gattctatt 2100 ctcaatccaa tcaacctat acgaaattt tccattgtgc aaagactcc cttacaaatg 2160 aatggcatcg aagaggattc tgatgagcct ttagagagaa ggctgtcctt agtaccagat 2220 tctgagcagg gagaggcgat actgcctcgc atcagcgtga tcagcactgg ccccacgctt 2280 caggcacgaa ggaggcagtc tgtcctgaac ctgatgacac actcagttaa ccaggtcag 2340 aacattcacc gaagacaac agcatccaca cgaaagtgt cactggcccc tcaggcaac 2400 ttgactgaac tggatatata ttcaagaagg ttatctcaag aaactggctt ggaaataagt 2460 gaagaaatta acgaagaaga cttaaaggag tgctttttg atgatatgga gagcatacca 2520 gcagtgacta catggaacac ataccttcga tatattactg tccacaagag cttaattttt 2580 gtgctaattt ggtgcttagt aatttttctg gcagaggtgg ctgcttcttt ggttgtgctg 2640 tggctccttg gaaacactcc tcttcaagac aaagggaata gtactcatag tagaaataac 2700 agctatgcag tgattatcac cagcaccagt tcgtattatg tgttttacat ttacgtggga 2760 gtagccgaca ctttgcttgc tatgggattc ttcagaggtc taccactggt gcataccta 2820 atcacagtgt cgaaaatttt acaccacaaa atgttacatt ctgttcttca agcacctatg 2880 tcaaccctca acacgttgaa agcaggtggg attcttaata gattctccaa agatatagca 2940 atttggatg accttctgcc tcttaccata tttgacttca tccagttgtt attaattgtg 3000 attggagcta tagcagttgt cgcagtttta caaccctaca tctttgttgc aacagtgcca 3060 gtgatagtgg cttttattat gttgagagca tatttcctcc aaacctcaca gcaactcaaa 3120 3180 ctatggacac ttcgtgcctt cggacggcag ccttactttg aaactctgtt ccacaaagct 3240 ctgaatttac atactgccaa ctggttcttg tacctgtcaa cactgcgctg gttccaaatg 3300 agaataaa tgatttttgt catcttcttc attgctgtta ccttcatttc cattttaaca 3360 agggaag gaggaag agttggtatt atcctgactt tagccatgaa tatcatgagt 3420 acattgcagt gggctgtaaa ctccagcata gatgtggata gcttgatgcg atctgtgagc 3480 cgagtcttta agttcattga catgccaaca gaaggtaac ctaccaagtc aaccaaacca 3540 tacaagaatg gccaactctc gaaagttatg attattgaga attcacacgt gaaaagat 3600 gacatctggc cctcaggggg ccaaatgact gtcaaagatc tcacagcaaa atacacagaa 3660 ggtggaaatg ccatattaga gaacatttcc ttctcaataa gtcctggcca gagggtgggc 3720 ctcttgggaa gaactggatc agggaagagt actttgttat cagctttttt gagactactg 3780 3840 tggaggaaag cctttggagt gataccacag aaagtattta ttttttctgg aacatttaga 3900 aaaaacttgg atccctatga acagtggagt gatcaagaaa tatggaaagt tgcagatgag 3960 gttgggctca gatctgtgat agaacagttt cctgggaagc ttgactttgt ccttgtggat 4020 gggggctgtg tcctaagcca tggccacaag cagttgatgt gcttggctag atctgttctc 4080 agtaaggcga agatcttgct gcttgatgaa cccagtgctc atttggatcc agtaacatac 4140 caaataatta gaagaactct aaaacaagca tttgctgatt gcacagtaat tctctgtgaa 4200 cacaggatag aagcaatgct ggaatgccaa caatttttgg tcatagaaga gaacaaagtg 4260 cggcagtacg attccatcca gaaactgctg aacgagagga gcctcttccg gcaagccatc 4320 agcccctccg acagggtgaa gctctttccc caccggaact caagcaagtg caagtctaag 4380 ccccagattg ctgctctgaa agaggagaca gaagaagagg tgcaagatac aaggctttag 4440 <210> 2 <211> 3261 <212> DNA <213> Homo sapiens <400> 2 atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60 gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120 ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180 gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240 accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300 ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360 gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420 cagggacaaa agtgtgatcc aagctgtccc aatgggagct gctggggtgc aggagaggag 480 aactgccaga aactgaccaa aatcatctgt gcccagcagt gctccgggcg ctgccgtggc 540 aagtccccca gtgactgctg ccacaaccag tgtgctgcag gctgcacagg cccccgggag 600 agcgactgcc tggtctgccg caaattccga gacgaagcca cgtgcaagga cacctgcccc 660 ccactcatgc tctacaaccc caccacgtac cagatggatg tgaaccccga gggcaaatac 720 agctttggtg ccacctgcgt gaagaagtgt ccccgtaatt atgtggtgac agatcacggc 780 tcgtgcgtcc gagcctgtgg ggccgacagc tatgagatgg aggaagacgg cgtccgcaag 840 tgtaagaagt gcgaagggcc ttgccgcaaa gtgtgtaacg gaataggtat tggtgaattt 900 aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 960 agtggcgatc tccacatcct gccggtggca tttaggggtg actctcttcac acatactcct 1020 cctctggatc caaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1080 ctgattcagg cttggcctga aaacggacg gacctccatg cctttgagaa cctagaaatc 1140 atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1200 acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1260 aacaaaaatt tgtgctatgc aaataacaata aactggaaaa aactgtttgg gacctccggt 1320 cagaaaacca aaattataag caacagaggt gaaaacagct ccaaggccac aggccaggtc 1380 tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 1440 tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 1500 ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 1560 gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 1620 attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 1680 ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 1740 tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 1800 atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 1860 ctcttcatgc gaaggcgcca catcgttcgg aagcgcacgc tgcggaggct gctgcaggag 1920 agggagcttg tggagcctct tacacccagt ggagaagctc ccaaccaagc tctcttgagg 1980 atcttgaagg aaactgaatt caaaaagatc aaagtgctgg gctccggtgc gttcggcacg 2040 gtgtataagg gactctggat cccagaaggt gagaaagtta aaattcccgt cgctatcaaa 2100 acatctccga aagccaacaa ggaaatcctc gatgaagcct acgtgatggc cagcgtggac 2160 aaccccacg tgtgccgcct gctgggcatc tgcctcacct ccaccgtgca gctcatcacg 2220 cagctcatgc ccttcggctg cctcctggac tatgtccggg aacacaaaga caatattggc 2280 tcccagtacc tgctcaactg gtgtgtgcag atcgcaaagg gcatgaacta cttggaggac 2340 cgtcgcttgg tgcaccgcga cctggcagcc aggaacgtac tggtgaaaac accgcagcat 2400 gtcaagatca cagatttgg gctggccaaa ctgctgggtg cggaagagaa agaataccat 2460 gcagaaggag gcaaagtgcc tatcaagtgg atggcattgg aatcaatttt acacagaatc 2520 tatacccacc agagtgatgt ctggagctac ggggtgaccg tttgggagtt gatgaccttt 2580 ggatccaagc catatgacgg aatccctgcc agcgagatct cctccatcct ggagaaagga 2640 gaacgcctcc ctcagccacc catatgtacc atcgatgtct acatgatcat ggtcaagtgc 2700 tggatgatag acgcagatag tcgcccaaag ttccgtgagt tgatcatcga attctccaaa 2760 atggcccgag acccccagcg ctaccttgtc attcaggggg atgaaagaat gcatttgcca 2820 agtcctacag actccaactt ctaccgtgcc ctgatggatg aagaagacat ggacgacgtg 2880 gtggatgccg acgagtacct catcccacag cagggcttct tcagcagccc ctccacgtca 2940 cggactcccc tcctgagctc tctgagtgca accagcaaca attccaccgt ggcttgcatt 3000 gatagaaatg ggctgcaaag ctgtcccatc aaggaagaca gcttcttgca gcgatacagc 3060 tcagacccca caggcgcctt gactgaggac agcatagacg acaccttcct cccagtgcct 3120 ggtgagtggc ttgtctggaa acagtcctgc tcctcaacct cctcgaccca ctcagcagca 3180 gccagtctcc agtgtccaag ccaggtgctc cctccagcat ctccagaggg ggaaacagtg 3240 gcagatttgc agacacagtg a 3261 <210> 3 <211> 3260 <212> DNA <213> Homo sapiens <400> 3 atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60 gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120 ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180 gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240 accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300 ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360 gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420 cagggacaaa agtgtgatcc aagctgtccc aatgggagct gctggggtgc aggagaggag 480 aactgccaga aactgaccaa aatcatctgt gcccagcagt gctccgggcg ctgccgtggc 540 aagtccccca gtgactgctg ccacaaccag tgtgctgcag gctgcacagg cccccgggag 600 agcgactgcc tggtctgccg caaattccga gacgaagcca cgtgcaagga cacctgcccc 660 ccactcatgc tctacaaccc caccacgtac cagatggatg tgaaccccga gggcaaatac 720 agctttggtg ccacctgcgt gaagaagtgt ccccgtaatt atgtggtgac agatcacggc 780 tcgtgcgtcc gagcctgtgg ggccgacagc tatgagatgg aggaagacgg cgtccgcaag 840 tgtaagaagt gcgaagggcc ttgccgcaaa gtgtgtaacg gaataggtat tggtgaattt 900 aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 960 agtggcgatc tccacatcct gccggtggca tttaggggtg actccttcac acatactcct 1020 cctctggatc cacaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1080 ctgattcagg cttggcctga aaacaggacg gacctccatg cctttgagaa cctagaaatc 1140 atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1200 acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1260 aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 1320 cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 1380 tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 1440 tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 1500 ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 1560 gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 1620 attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 1680 ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 1740 tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 1800 atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 1860 ctcttcatgc gaaggcgcca catcgttcgg aagcgcacgc tgcggaggct gctgcaggag 1920 agggagcttg tggagcctct tacacccagt ggagaagctc ccaaccaagc tctcttgagg 1980 atcttgaagg aaactgaatt caaaaagatc aaagtgctgg gctccggtgc gttcggcacg 2040 gtgtataagg gactctggat cccagaaggt gagaaagtta aaattcccgt cgctatcaag 2100 acatctccga aagccaacaa ggaaatcctc gatgagccta cgtgatggcc agcgtggaca 2160 acccccacgt gtgccgcctg ctgggcatct gcctcacctc caccgtgcag ctcatcacgc 2220 agctcatgcc cttcggctgc ctcctggact atgtccggga acacaaagac aatattggct 2280 cccagtacct gctcaactgg tgtgtgcaga tcgcaaaggg catgaactac ttggaggacc 2340 gtcgcttggt gcaccgcgac ctggcagcca ggaacgtact ggtgaaaaca ccgcagcatg 2400 tcaagatcac agattttggg ctggccaaac tgctgggtgc ggaagagaaa gaataccatg 2460 cagaaggagg caaagtgcct atcaagtgga tggcattgga atcaatttta cacagaatct 2520 atacccacca gagtgatgtc tggagctacg gggtgaccgt ttgggagttg atgacctttg 2580 gatccaagcc atatgacgga atccctgcca gcgagatctc ctccatcctg gagaaaggag 2640 aacgcctccc tcagccaccc atatgtacca tcgatgcta catgatcatg gtcaagtgct 2700 ggatgataga cgcagatagt cgcccaaagt tccgtgagtt gatcatcgaa ttctccaaaa 2760 tggcccgaga cccccagcgc taccttgtca ttcaggggga tgaaagaatg catttgccaa 2820 gtcctacaga ctccaacttc taccgtgccc tgatggatga agaagacatg gacgacgtgg 2880 tggatgccga cgagtacctc atcccacagc agggcttctt cagcagcccc tccacgtcac 2940 ggactcccct cctgagctct ctgagtgcaa ccagcaacaa ttccaccgtg gcttgcattg 3000 atagaaatgg gctgcaaagc tgtcccatca aggaagacag cttcttgcag cgatacagct 3060 cagaccccac aggcgccttg actgaggaca gcatagacga caccttcctc ccagtgcctg 3120 gtgagtggct tgtctggaaa cagtcctgct cctcaacctc ctcgacccac tcagcagcag 3180 ccagtctcca gtgtccaagc caggtgctcc ctccagcatc tccagagggg gaaacagtgg 3240 cagatttgca gacacagtga 3260 <210> 4 <211> 3259 <212> DNA <213> Homo sapiens <400> 4 atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60 gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120 ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180 gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240 accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300 ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360 gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420 cagggacaaa agtgtgatcc aagctgtccc aatgggagct gctggggtgc aggagaggag 480 aactgccaga aactgaccaa aatcatctgt gcccagcagt gctccgggcg ctgccgtggc 540 aagtccccca gtgactgctg ccacaaccag tgtgctgcag gctgcacagg cccccgggag 600 agcgactgcc tggtctgccg caaattccga gacgaagcca cgtgcaagga cacctgcccc 660 ccactcatgc tctacaaccc caccacgtac cagatggatg tgaaccccga gggcaaatac 720 agctttggtg ccacctgcgt gaagaagtgt ccccgtaatt atgtggtgac agatcacggc 780 tcgtgcgtcc gagcctgtgg ggccgacagc tatgagatgg aggaagacgg cgtccgcaag 840 tgtaagaagt gcgaagggcc ttgccgcaaa gtgtgtaacg gaataggtat tggtgaattt 900 aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 960 agtggcgatc tccacatcct gccggtggca tttaggggtg actctcttcac acatactcct 1020 cctctggatc caaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1080 ctgattcagg cttggcctga aaacggacg gacctccatg cctttgagaa cctagaaatc 1140 atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1200 acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1260 aacaaaaatt tgtgctatgc aaatacaata aactggaaaa aactgtttgg gacctccggt 1320 cagaaaacca aaattataag caacagaggt gaaaacagct gcaaggccac aggccaggtc 1380 tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 1440 tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 1500 ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 1560 gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 1620 attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 1680 ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 1740 tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 1800 atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 1860 ctcttcatgc gaaggcgcca catcgttcgg aagcgcacgc tgcggaggct gctgcaggag 1920 agggagcttg tggagcctct tacacccagt ggagaagctc ccaaccaagc tctcttgagg 1980 atcttgaagg aaactgaatt caaaaagatc aaagtgctgg gctccggtgc gttcggcacg 2040 gtgtataagg gactctggat cccagaaggt gagaaagtta aaattcccgt cgctatcaag 2100 catctccgaa agccaacaag gaaatcctcg atgagcctac gtgatggcca gcgtggacaa 2160 cccccacgtg tgccgcctgc tgggcatctg cctcacctcc accgtgcagc tcatcacgca 2220 gctcatgccc ttcggctgcc tcctggacta tgtccgggaa cacaaagaca atattggctc 2280 ccagtacctg ctcaactggt gtgtgcagat cgcaaagggc atgaactact tggaggaccg 2340 tcgcttggtg caccgcgacc tggcagccag gaacgtactg gtgaaaacac cgcagcatgt 2400 caagatcaca gattttgggc tggccaaact gctgggtgcg gaagagaaag aataccatgc 2460 agaaggaggc aaagtgccta tcaagtggat ggcattggaa tcaattttac acagaatcta 2520 tacccaccag agtgatgtct ggagctacgg ggtgaccgtt tgggagttga tgacctttgg 2580 atccaagcca tatgacggaa tccctgccag cgagatctcc tccatcctgg agaaggaga 2640 acgcctccct cagccaccca tatgtaccat cgatgtctac atgatcatgg tcaagtgctg 2700 gatgatagac gcagatagtc gcccaaagtt ccgtgagttg atcatcgaat tctccaaaat 2760 ggcccgagac ccccagcgct accttgtcat tcagggggat gaaagaatgc atttgccaag 2820 tcctacagac tccaacttct accgtgccct gatggatgaa gaagacatgg acgacgtggt 2880 ggatgccgac gagtacctca tcccacagca gggcttcttc agcagcccct ccacgtcacg 2940 gactcccctc ctgagctctc tgagtgcaac cagcaacaat tccaccgtgg cttgcattga 3000 tagaaatggg ctgcaaagct gtcccatcaa ggaagacagc ttcttgcagc gatacagctc 3060 agaccccaca ggcgccttga ctgaggacag catagacgac accttcctcc cagtgcctgg 3120 tgagtggctt gtctggaaac agtcctgctc ctcaacctcc tcgacccact cagcagcagc 3180 cagtctccag tgtccaagcc aggtgctccc tccagcatct ccagaggggg aaacagtggc 3240 agatttgcag acacagtga 3259 <210> 5 <211> 3276 <212> DNA <213> Homo sapiens <400> 5 atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60 gcgagtcggg ctctggagga aaagaaagtt tgccaaggca cgagtaacaa gctcacgcag 120 ttgggcactt ttgaagatca ttttctcagc ctccagagga tgttcaataa ctgtgaggtg 180 gtccttggga atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag 240 accatccagg aggtggctgg ttatgtcctc attgccctca acacagtgga gcgaattcct 300 ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta tgccttagca 360 gtcttatcta actatgatgc aaataaaacc ggactgaagg agctgcccat gagaaattta 420 cagggacaaa agtgtgatcc aagctgtccc aatgggagct gctggggtgc aggagaggag 480 aactgccaga aactgaccaa aatcatctgt gcccagcagt gctccgggcg ctgccgtggc 540 aagtccccca gtgactgctg ccacaaccag tgtgctgcag gctgcacagg cccccgggag 600 agcgactgcc tggtctgccg caaattccga gacgaagcca cgtgcaagga cacctgcccc 660 ccactcatgc tctacaaccc caccacgtac cagatggatg tgaaccccga gggcaaatac 720 agctttggtg ccacctgcgt gaagaagtgt ccccgtaatt atgtggtgac agatcacggc 780 tcgtgcgtcc gagcctgtgg ggccgacagc tatgagatgg aggaagacgg cgtccgcaag 840 tgtaagaagt gcgaagggcc ttgccgcaaa gtgtgtaacg gaataggtat tggtgaattt 900 aaagactcac tctccataaa tgctacgaat attaaacact tcaaaaactg cacctccatc 960 agtggcgatc tccacatcct gccggtggca tttaggggtg actctcttcac acatactcct 1020 cctctggatc caaggaact ggatattctg aaaaccgtaa aggaaatcac agggtttttg 1080 ctgattcagg cttggcctga aaacggacg gacctccatg cctttgagaa cctagaaatc 1140 atacgcggca ggaccaagca acatggtcag ttttctcttg cagtcgtcag cctgaacata 1200 acatccttgg gattacgctc cctcaaggag ataagtgatg gagatgtgat aatttcagga 1260 aacaaaaatt tgtgctatgc aaataacaata aactggaaaa aactgtttgg gacctccggt 1320 cagaaaacca aaattataag caacagaggt gaaaacagct ccaaggccac aggccaggtc 1380 tgccatgcct tgtgctcccc cgagggctgc tggggcccgg agcccaggga ctgcgtctct 1440 tgccggaatg tcagccgagg cagggaatgc gtggacaagt gcaaccttct ggagggtgag 1500 ccaagggagt ttgtggagaa ctctgagtgc atacagtgcc acccagagtg cctgcctcag 1560 gccatgaaca tcacctgcac aggacgggga ccagacaact gtatccagtg tgcccactac 1620 attgacggcc cccactgcgt caagacctgc ccggcaggag tcatgggaga aaacaacacc 1680 ctggtctgga agtacgcaga cgccggccat gtgtgccacc tgtgccatcc aaactgcacc 1740 tacggatgca ctgggccagg tcttgaaggc tgtccaacga atgggcctaa gatcccgtcc 1800 atcgccactg ggatggtggg ggccctcctc ttgctgctgg tggtggccct ggggatcggc 1860 ctcttcatgc gaaggcgcca catcgttcgg aagcgcacgc tgcggaggct gctgcaggag 1920 agggagcttg tggagcctct tacacccagt ggagaagctc ccaaccaagc tctcttgagg 1980 atcttgaagg aaactgaatt caaaaagatc aaagtgctgg gctccggtgc gttcggcacg 2040 gtgtataagg gactctggat cccagaaggt gagaaagtta aaattcccgt cgctatcaag 2100 gaattaagag aagcaacatc tccgaaagcc aacaaggaaa tcctcgatga agcctacgtg 2160 atggccagcg tggacaaccc cccacgtgc cgcctgctg gcatctgcct cacctccacc 2220 gtgcagctca tcaccagct catgccctc ggctgcctcc tggactgt ccgggacac 2280 aaagacaata ttggctccca gtacctgctc aactgtgtg tgcagatcgc aaagggcatg 2340 aactacttgg aggaccgtcg cttggtgcac cgcgacctgg cagccaggaa cgtactggtg 2400 aaaaacaccgc agcatgtca gatcacagat ttgggctgg ccaactgct gggtgcggaa 2460 gagaaagaat accatgcaga aggaggcaa gtgcctatca agtggatggc attggaatca 2520 attttacaca gatctatac ccaccagt gatgtctgga gctacggggt gaccgttttgg 2580 gagttgatga cctttggatc caagccatat gacggaatcc ctgccagcga gatctcctcc 2640 atcctggaga aaggagaacg cctccctcag ccaccatat gtaccatcga tgtctacatg 2700 atcatggtca agtgctggat gatagacgca gatagtcgcc caaagttccg tgagttgatc 2760 atcgaatttct ccaaaatgc ccgagacccc cagcgctacc ttgtcattca gggggatgaa 2820 agaatgcatt tgccaagtcc tacagactcc aacttctacc gtgccctgat ggatgaagaa 2880 gacatggacg acgtggtgga tgccgacgag tacctcatcc cacagcaggg cttcttcagc 2940 agcccctcca cgtcacggac tcccctctg agctctctga gtgcaaccag caacaattcc 3000 accgtggctt gcattgatag aaatgggctg caaagctgtc ccatcaagga agacagcttc 3060 ttgcagcgat acagctcaga ccccacaggc gccttgactg aggacagcat agacgacacc 3120 ttcctcccag tgcctggtga gtggcttgtc tggaaacagt cctgctcctc aacctcctcg 3180 acccactcag cagcagccag tctccagtgt ccaagccagg tgctccctcc agcatctcca 3240 gagggggaaa cagtggcaga tttgcagaca cagtga 3276 <210> 6 <211> 93 <212> PRT <213> T4 <400> 6 Met Gly Lys Ser Gly Ile Tyr Gln Ile Lys Asn Thr Leu Asn Asn Lys 1 5 10 15 Val Tyr Val Gly Ser Ala Lys Asp Phe Glu Lys Arg Trp Lys Arg His 20 25 30 Phe Lys Asp Leu Glu Lys Gly Cys His Ser Ser Ile Lys Leu Gln Arg 35 40 45 Ser Phe Asn Lys His Gly Asn Val Phe Glu Cys Ser Ile Leu Glu Glu 50 55 60 Ile Pro Tyr Glu Lys Asp Leu Ile Ile Glu Arg Glu Asn Phe Trp Ile 65 70 75 80 Lys Glu Leu Asn Ser Lys Ile Asn Gly Tyr Asn Ile Ala 85 90 <210> 7 <211> 83 <212> PRT <213> Artificial Sequence <220> <223> V117F Mutation Linker <400> 7 Asp Ala Thr Phe Gly Asp Thr Cys Ser Thr His Pro Leu Lys Glu Glu 1 5 10 15 Ile Ile Lys Lys Arg Ser Glu Thr Phe Lys Ala Lys Met Leu Lys Leu 20 25 30 Gly Pro Asp Gly Arg Lys Ala Leu Tyr Ser Lys Pro Gly Ser Lys Asn 35 40 45 Gly Arg Trp Asn Pro Glu Thr His Lys Phe Cys Lys Cys Gly Val Arg 50 55 60 Ile Gln Thr Ser Ala Tyr Thr Cys Ser Lys Cys Arg Asn Gly Gly Ser 65 70 75 80 Gly Gly Ser <210> 8 <211> 83 <212> PRT <213> Artificial sequence <220> <223> V117F K135R N140S mutant linker <400> 8 Asp Ala Thr Phe Gly Asp Thr Cys Ser Thr His Pro Leu Lys Glu Glu 1 5 10 15 Ile Ile Lys Lys Arg Ser Glu Thr Val Lys Ala Lys Met Leu Lys Leu 20 25 30 Gly Pro Asp Gly Arg Lys Ala Leu Tyr Ser Arg Pro Gly Ser Lys Ser 35 40 45 Gly Arg Trp Asn Pro Glu Thr His Lys Phe Cys Lys Cys Gly Val Arg 50 55 60 Ile Gln Thr Ser Ala Tyr Thr Cys Ser Lys Cys Arg Asn Gly Gly Ser 65 70 75 80 Gly Gly Ser <210> 9 <211> 83 <212> PRT <213> Artificial sequence <220> <223> Linker domain variants <400> 9 Asp Ala Thr Phe Gly Asp Thr Cys Ser Thr His Pro Leu Lys Glu Glu 1 5 10 15 Ile Ile Lys Lys Arg Ser Glu Thr Phe Lys Ala Lys Met Leu Lys Leu 20 25 30 Gly Pro Asp Gly Arg Lys Ala Leu Tyr Ser Arg Pro Gly Ser Lys Ser 35 40 45 Gly Arg Trp Asn Pro Glu Thr His Lys Phe Cys Lys Cys Gly Val Arg 50 55 60 Ile Gln Thr Ser Ala Tyr Thr Cys Ser Lys Cys Arg Asn Gly Gly Ser 65 70 75 80 Gly Gly Ser <210> 10 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> Adaptor Domain Variant <400> 10 Asp Ala Thr Phe Gly Asp Thr Cys Ser Thr His Pro Leu Lys Glu Glu 1 5 10 15 Ile Ile Lys Lys Arg Ser Glu Thr Val Lys Ala Lys Met Leu Lys Leu 20 25 30 Gly Pro Asp Gly Arg Lys Ala Leu Tyr Ser Arg Pro Gly Ser Lys Ser 35 40 45 Gly Arg Trp Asn Pro Glu Thr His Lys Phe Cys Lys Cys Gly Val Arg 50 55 60 Ile Gln Thr Ser Ala Tyr Thr Cys Ser Lys Cys Arg Asn Gly Gly Ser 65 70 75 80 Gly Gly Thr Gly Gly Ser 85 <210> 11 <211> 87 <212> PRT <213> Artificial sequence <220> <223> Linker domain variants <400> 11 Asp Ala Thr Phe Gly Asp Thr Cys Ser Thr His Pro Leu Lys Glu Glu 1 5 10 15 Ile Ile Lys Lys Arg Ser Glu Thr Val Lys Ala Lys Met Leu Lys Leu 20 25 30 Gly Pro Asp Gly Arg Lys Ala Leu Tyr Ser Arg Pro Gly Ser Lys Ser 35 40 45 Gly Arg Trp Asn Pro Glu Thr His Lys Phe Cys Lys Cys Gly Val Arg 50 55 60 Ile Gln Thr Ser Ala Tyr Thr Cys Ser Lys Cys Arg Asn Gly Gly Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser 85 <210> 12 <211> 95 <212> PRT <213> Artificial sequence <220> <223> Linker domain variants <400> 12 Asp Ala Thr Phe Gly Asp Thr Cys Ser Thr His Pro Leu Lys Glu Glu 1 5 10 15 Ile Ile Lys Lys Arg Ser Glu Thr Val Lys Ala Lys Met Leu Lys Leu 20 25 30 Gly Pro Asp Gly Arg Lys Ala Leu Tyr Ser Arg Pro Gly Ser Lys Ser 35 40 45 Gly Arg Trp Asn Pro Glu Thr His Lys Phe Cys Lys Cys Gly Val Arg 50 55 60 Ile Gln Thr Ser Ala Tyr Thr Cys Ser Lys Cys Arg Asn Lys Glu Ser 65 70 75 80 Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser Leu Asp 85 90 95 <210> 13 <211> 1053 <212> PRT <213> Staphylococcus aureus <400> 13 Met Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val 1 5 10 15 Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly 20 25 30 Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg 35 40 45 Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile 50 55 60 Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His 65 70 75 80 Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu 85 90 95 Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu 100 105 110 Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr 115 120 125 Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala 130 135 140 Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys 145 150 155 160 Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr 165 170 175 Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln 180 185 190 Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg 195 200 205 Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys 210 215 220 Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe 225 230 235 240 Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr 245 250 255 Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn 260 265 270 Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe 275 280 285 Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu 290 295 300 Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys 305 310 315 320 Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr 325 330 335 Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala 340 345 350 Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu 355 360 365 Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser 370 375 380 Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile 385 390 395 400 Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala 405 410 415 Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln 420 425 430 Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro 435 440 445 Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile 450 455 460 Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg 465 470 475 480 Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys 485 490 495 Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr 500 505 510 Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp 515 520 525 Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu 530 535 540 Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro 545 550 555 560 Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys 565 570 575 Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu 580 585 590 Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile 595 600 605 Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu 610 615 620 Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp 625 630 635 640 Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu 645 650 655 Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys 660 665 670 Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp 675 680 685 Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp 690 695 700 Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys 705 710 715 720 Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys 725 730 735 Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu 740 745 750 Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp 755 760 765 Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile 770 775 780 Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu 785 790 795 800 Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu 805 810 815 Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His 820 825 830 Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly 835 840 845 Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr 850 855 860 Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile 865 870 875 880 Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp 885 890 895 Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr 900 905 910 Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val 915 920 925 Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser 930 935 940 Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala 945 950 955 960 Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly 965 970 975 Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile 980 985 990 Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met 995 1000 1005 Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys 1010 1015 1020 Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu 1025 1030 1035 Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly 1040 1045 1050 <210> 14 <211> 1053 <212> PRT <213> Artificial Sequence <220> <223> SACAS9 D10E Mutation <400> 14 Met Lys Arg Asn Tyr Ile Leu Gly Leu Glu Ile Gly Ile Thr Ser Val 1 5 10 15 Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly 20 25 30 Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg 35 40 45 Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile 50 55 60 Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His 65 70 75 80 Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu 85 90 95 Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu 100 105 110 Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr 115 120 125 Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala 130 135 140 Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys 145 150 155 160 Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr 165 170 175 Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln 180 185 190 Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg 195 200 205 Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys 210 215 220 Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe 225 230 235 240 Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr 245 250 255 Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn 260 265 270 Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe 275 280 285 Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu 290 295 300 Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys 305 310 315 320 Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr 325 330 335 Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala 340 345 350 Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu 355 360 365 Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser 370 375 380 Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile 385 390 395 400 Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala 405 410 415 Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln 420 425 430 Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro 435 440 445 Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile 450 455 460 Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg 465 470 475 480 Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys 485 490 495 Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr 500 505 510 Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp 515 520 525 Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu 530 535 540 Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro 545 550 555 560 Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys 565 570 575 Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu 580 585 590 Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile 595 600 605 Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu 610 615 620 Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp 625 630 635 640 Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu 645 650 655 Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys 660 665 670 Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp 675 680 685 Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp 690 695 700 Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys 705 710 715 720 Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys 725 730 735 Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu 740 745 750 Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp 755 760 765 Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile 770 775 780 Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu 785 790 795 800 Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu 805 810 815 Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His 820 825 830 Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly 835 840 845 Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr 850 855 860 Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile 865 870 875 880 Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp 885 890 895 Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr 900 905 910 Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val 915 920 925 Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser 930 935 940 Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala 945 950 955 960 Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly 965 970 975 Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile 980 985 990 Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met 995 1000 1005 Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys 1010 1015 1020 Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu 1025 1030 1035 Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly 1040 1045 1050 <210> 15 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Target CFTR gene <400> 15 gcgucaucaa agcaugccaa c 21 <210> 16 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Target EGFR gene <400> 16 aagccaacaa ggaaauccuc ga 22 <210> 17 <211> 1053 <212> PRT <213> Artificial sequence <220> <223> SACAS9 D10E + H557A mutation <400> 17 Met Lys Arg Asn Tyr Ile Leu Gly Leu Glu Ile Gly Ile Thr Ser Val 1 5 10 15 Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly 20 25 30 Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg 35 40 45 Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile 50 55 60 Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His 65 70 75 80 Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu 85 90 95 Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu 100 105 110 Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr 115 120 125 Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala 130 135 140 Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys 145 150 155 160 Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr 165 170 175 Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln 180 185 190 Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg 195 200 205 Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys 210 215 220 Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe 225 230 235 240 Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr 245 250 255 Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn 260 265 270 Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe 275 280 285 Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu 290 295 300 Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys 305 310 315 320 Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr 325 330 335 Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala 340 345 350 Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu 355 360 365 Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser 370 375 380 Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile 385 390 395 400 Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala 405 410 415 Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln 420 425 430 Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro 435 440 445 Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile 450 455 460 Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg 465 470 475 480 Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys 485 490 495 Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr 500 505 510 Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp 515 520 525 Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu 530 535 540 Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp Ala Ile Ile Pro 545 550 555 560 Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys 565 570 575 Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu 580 585 590 Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile 595 600 605 Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu 610 615 620 Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp 625 630 635 640 Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu 645 650 655 Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys 660 665 670 Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp 675 680 685 Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp 690 695 700 Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys 705 710 715 720 Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys 725 730 735 Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu 740 745 750 Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp 755 760 765 Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile 770 775 780 Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu 785 790 795 800 Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu 805 810 815 Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His 820 825 830 Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly 835 840 845 Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr 850 855 860 Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile 865 870 875 880 Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp 885 890 895 Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr 900 905 910 Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val 915 920 925 Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser 930 935 940 Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala 945 950 955 960 Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly 965 970 975 Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile 980 985 990 Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met 995 1000 1005 Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys 1010 1015 1020 Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu 1025 1030 1035 Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly 1040 1045 1050 <210> 18 <211> 1053 <212> PRT <213> Artificial Sequence <220> <223> SACAS9 D10A + H557A Mutation <400> 18 Met Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val 1 5 10 15 Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly 20 25 30 Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg 35 40 45 Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile 50 55 60 Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His 65 70 75 80 Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu 85 90 95 Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu 100 105 110 Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr 115 120 125 Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala 130 135 140 Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys 145 150 155 160 Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr 165 170 175 Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln 180 185 190 Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg 195 200 205 Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys 210 215 220 Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe 225 230 235 240 Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr 245 250 255 Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn 260 265 270 Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe 275 280 285 Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu 290 295 300 Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys 305 310 315 320 Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr 325 330 335 Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala 340 345 350 Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu 355 360 365 Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser 370 375 380 Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile 385 390 395 400 Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala 405 410 415 Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln 420 425 430 Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro 435 440 445 Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile 450 455 460 Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg 465 470 475 480 Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys 485 490 495 Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr 500 505 510 Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp 515 520 525 Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu 530 535 540 Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp Ala Ile Ile Pro 545 550 555 560 Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys 565 570 575 Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu 580 585 590 Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile 595 600 605 Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu 610 615 620 Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp 625 630 635 640 Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu 645 650 655 Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys 660 665 670 Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp 675 680 685 Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp 690 695 700 Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys 705 710 715 720 Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys 725 730 735 Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu 740 745 750 Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp 755 760 765 Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile 770 775 780 Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu 785 790 795 800 Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu 805 810 815 Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His 820 825 830 Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly 835 840 845 Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr 850 855 860 Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile 865 870 875 880 Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp 885 890 895 Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr 900 905 910 Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val 915 920 925 Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser 930 935 940 Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala 945 950 955 960 Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly 965 970 975 Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile 980 985 990 Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met 995 1000 1005 Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys 1010 1015 1020 Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu 1025 1030 1035 Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly 1040 1045 1050 <210> 19 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> SPCAS9 D10E, D1135E, R1335Q, T1337R Mutations <400> 19 Met Asp Lys Lys Tyr Ser Ile Gly Leu Glu Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ser Arg Ser Arg Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Glu Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Gln Tyr Arg Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 20 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> SPCAS9 D10E, H840A, D1135E, R1335Q, T1337R Mutation <400> 20 Met Asp Lys Lys Tyr Ser Ile Gly Leu Glu Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ser Arg Ser Arg Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Glu Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Gln Tyr Arg Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365

Claims

1. A Staphylococcus aureus Cas9 nuclease, the nuclease comprising the amino acid sequence of SEQ ID NO:

14.

2. A chimeric nuclease comprising an I-Tev1 nuclease domain, a linker and an RNA-guided nuclease Staphylococcus aureus Cas9, wherein: The RNA-guided nuclease Staphylococcus aureus Cas9 is a Staphylococcus aureus Cas9 having glutamic acid at the position corresponding to position 10 of the amino acid sequence shown in SEQ ID NO:

13.

3. The chimeric nuclease according to claim 2, wherein The chimeric nuclease comprises 3.7 kilobases having a molecular weight of 144 kilodaltons.

4. The chimeric nuclease according to claim 2, wherein The chimeric nuclease comprises a modified I-Tevl nuclease domain, and the modified I-Tevl nuclease domain is shown in SEQ ID NO:

6.

5. The chimeric nuclease according to claim 4, wherein The linker is selected from the group consisting of SEQ ID NOs: 7-12.

6. The chimeric nuclease according to claim 2, wherein The linker comprises one or more selected from the group consisting of SEQ ID NOs: 9-12.

7. The chimeric nuclease according to claim 2, wherein The nuclease S. aureus Cas9 cleaves the sugar-phosphate backbone on one strand of the target DNA.

8. The chimeric nuclease according to claim 2, wherein The nuclease S. aureus Cas9 binds to target DNA but does not cleave the target DNA.

9. The chimeric nuclease according to claim 2, wherein The chimeric nuclease comprises a Met-free 1 The I-Tevl nuclease domain.

10. A pharmaceutically acceptable preparation comprising the chimeric nuclease according to any one of claims 2 to 9, lipid nanoparticles and a pharmaceutically acceptable carrier thereof.

11. The preparation according to claim 10, wherein The lipid nanoparticle further comprises exogenous donor DNA.

12. The formulation of claim 11, further comprising a guide RNA, wherein the guide RNA targets the Cas9 domain to genomic DNA.

13. The pharmaceutically acceptable formulation of claim 10, further comprising a DNA binding compound.

14. The pharmaceutically acceptable preparation according to claim 13, wherein The DNA binding compound is GL67 (N 4 -cholesteryl-spermine).

15. The pharmaceutically acceptable formulation of claim 10, further comprising a targeting polypeptide, nucleic acid or sugar.

16. The pharmaceutically acceptable preparation according to claim 10, wherein: The lipid nanoparticles contain cationic and / or neutral lipids.

17. The pharmaceutically acceptable preparation according to claim 10, wherein: The lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dioctadecyldimethylammonium bromide, N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylcholine, cholesterol, and any combination thereof.

18. The pharmaceutically acceptable preparation according to claim 10, wherein The lipid nanoparticles were prepared using an extrusion method.

19. The preparation according to claim 18, wherein The extrusion process produced particles with a diameter of 50-500 nM.

20. The preparation according to claim 19, wherein The formulation further comprises an excipient.

21. The preparation according to claim 20, wherein The excipient is selected from the group consisting of polysorbate, polyphosphate, calcium chloride, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium edetate, potassium chloride, potassium phosphate and starch or a mixture of these substances.

22. The formulation according to claim 20, further comprising polysorbate, polyphosphate, calcium chloride, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium edetate, potassium chloride, potassium phosphate and starch and mixtures of these substances.

23. The preparation according to claim 20, wherein The particles are formed using sonication.

24. The formulation of claim 10, further comprising a guide RNA, wherein the guide RNA targets a specific CFTR gene sequence.

25. The formulation of claim 10, further comprising a guide RNA, wherein the guide RNA targets a specific EGFR gene sequence.

26. The preparation according to claim 25, wherein The specific EGFR gene sequence contains an EGFR exon 19 deletion mutation.

27. A preparation containing the chimeric nuclease according to claim 5, further comprising a guide RNA, wherein the guide RNA comprises the complete RNA sequence of SEQ ID NO:

15.

28. The preparation according to claim 27, wherein The formulation targets the CFTR gene to cleave 10 to 20 nucleotides downstream of the CFTRδF508 mutation.

29. A preparation containing the chimeric nuclease according to claim 5, further comprising a guide RNA, wherein the guide RNA comprises the complete RNA sequence of SEQ ID NO: 21 to cut off the CFTRδF508 mutation.

30. A preparation containing the chimeric nuclease according to claim 5, further comprising a guide RNA, wherein the guide RNA comprises the complete RNA sequence of SEQ ID NO:

16.

31. The preparation according to claim 30, wherein The formulation targets the EGFR gene.

32. Use of the chimeric nuclease according to any one of claims 2 to 9 in the preparation of a medicament for editing genomic DNA.

33. The use according to claim 32, wherein The use described does not use viral vectors.

34. The use according to claim 32, wherein The chimeric nuclease targets two independent target sites on a selected DNA molecule.

35. The use according to claim 34, wherein The selected DNA molecule is cleaved at a target site.

36. The use according to claim 34, wherein The selected DNA molecule is cleaved at one target site and the sugar-phosphate backbone is cleaved at a second target site.

37. The use according to claim 34, wherein The selected DNA molecule is cleaved at two target sites.

38. The use according to claim 36, wherein Fragments of the selected DNA molecule are cleaved from the selected DNA molecule.

39. The use according to claim 38, wherein The fragments are 30 to 36 nucleotides in length.

40. Use of the chimeric nuclease according to any one of claims 2 to 9 in the preparation of a medicament for deleting a DNA molecule of a defined length.

41. Use of the chimeric nuclease of any one of claims 2 to 9 in the preparation of a medicament for replacing a selected sequence in a DNA molecule.

42. The use according to claim 40 or 41, wherein The formulations are administered ex vivo to isolated cells in culture.

43. The use according to claim 40 or 41, wherein The cell or organism is selected from the group consisting of: a mammalian cell, a bacteria, an insect cell and a plant cell.

44. The use according to claim 43, wherein The mammalian cells are somatic cells.

45. The use according to claim 40, wherein The chimeric nuclease removes selected sequences of targeted DNA molecules by non-homologous end joining.

46. ​​Use of the chimeric nuclease of any one of claims 2-9 in the preparation of a medicament for treating a lung-related disease in a patient in need thereof.

47. The use according to claim 46, wherein The disease is cystic fibrosis.

48. The use according to claim 46, wherein The chimeric nuclease targets and cleaves the CFTR gene.

49. The use according to claim 48, wherein The CFTRδF508 mutation is cleaved from the CFTR gene.

50. The use according to claim 49, wherein The chimeric nuclease targets and cleaves 10 to 20 nucleotides on either side of the CFTRδF508 mutation.

51. The use according to claim 46, wherein The disease is cancer.

52. The use according to claim 46, wherein The chimeric nuclease targets and cleaves the EGFR gene.

53. The use according to claim 52, wherein The chimeric nuclease deletes the fragment in which exon 19 of EGFR is deleted.

54. The use according to claim 53, wherein The disease is non-small cell lung cancer.

55. Use of the chimeric nuclease of any one of claims 2 to 9 in the preparation of a medicament for treating monogenic and infectious diseases.

56. The use according to claim 55, wherein The mutant RNA-guided nuclease Staphylococcus aureus Cas9 targets the EGFR exon 19 deletion of the EGFR gene.

57. A modified donor DNA molecule selected from the group consisting of a linear single strand of DNA comprising homology regions flanking a site targeted and / or cleaved by a chimeric nuclease according to any one of claims 2-9.

58. A chimeric nuclease comprising a Lys at position corresponding to the amino acid sequence shown in SEQ ID NO:

6. 26 and / or Cys 39 A modified I-Tevl nuclease domain mutated at a position corresponding to position 10 of the amino acid sequence shown in SEQ ID No: 13, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, wherein the RNA-guided nuclease Staphylococcus aureus Cas9 is a Staphylococcus aureus Cas9 having glutamic acid at a position corresponding to position 10 of the amino acid sequence shown in SEQ ID No:

13.

59. A chimeric nuclease comprising a GIY-YIG nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, wherein the RNA-guided nuclease Staphylococcus aureus Cas9 is a Staphylococcus aureus Cas9 having glutamic acid at the position corresponding to position 10 of the amino acid sequence shown in SEQ ID No:

13.

60. The chimeric nuclease of claim 58, wherein The GIY-YIG nuclease domain is selected from the group consisting of I-Bmol and Eco29kI.

61. A chimeric nuclease comprising an I-Tev1 nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 and a guide RNA, wherein the guide RNA contains a sequence targeting a CFTR or EGFR gene; wherein, The modified RNA-guided nuclease Staphylococcus aureus Cas9 is a Staphylococcus aureus Cas9 having glutamic acid at the position corresponding to position 10 of the amino acid sequence shown in SEQ ID NO:

13.

62. A chimeric nuclease comprising an I-Tev1 nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 and a guide RNA, wherein the guide RNA contains a bridging nucleic acid; wherein, The modified RNA-guided nuclease Staphylococcus aureus Cas9 is a Staphylococcus aureus Cas9 having glutamic acid at the position corresponding to position 10 of the amino acid sequence shown in SEQ ID NO:

13.

63. A modified donor DNA molecule comprising linear single-stranded DNA or linear double-stranded DNA, wherein the linear double-stranded DNA comprises homology regions flanking the site targeted and / or cleaved by the chimeric nuclease according to any one of claims 2-9.

64. A modified donor DNA molecule comprising double-stranded DNA of the same length, the double-stranded DNA comprising DNA ends complementary to the DNA ends cleaved by the chimeric nuclease according to any one of claims 2-9.

65. A modified donor DNA molecule comprising a circular double-stranded DNA comprising homology regions flanking a site targeted and / or cleaved by a chimeric nuclease according to any one of claims 2-9.

66. A modified donor DNA molecule, comprising a circular double-stranded DNA, wherein the circular double-stranded DNA comprises an I-TevI ​​target site and a Cas9 target site, wherein the product cut from the double-stranded DNA contains an end complementary to the end cut by the chimeric nuclease according to any one of claims 2-9.

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