Treatment systems and methods for symptoms and diseases using an alternating electric field and a CRISPR-CAS system

Abstract

Disclosed are systems and methods for inducing apoptosis and treating or reducing the occurrence of at least one symptom, disease, disorder, or infection in a subject. The systems and methods rely on the application of an alternating electric field and the administration of a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system. Also provided is a kit for performing the methods disclosed herein.

Description

Background Art

[0001] [Cross - Reference to Related Applications / Incorporation - by - Reference Statement] Not applicable

[0002] [Statement Regarding Federally Sponsored Research or Development] Not applicable

[0003] A Tumor Treating Field (TT field) is a low - intensity (e.g., 1 - 3 V / cm) alternating electric field within the intermediate frequency range (100 - 500 kHz) that targets solid tumors by inhibiting mitosis. This non - invasive treatment is for solid tumors and is described, for example, in U.S. Patent Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, and 10,441,776. The TT field is typically delivered through two pairs of transducer arrays that generate a magnetic field perpendicular to the tumor being treated, and the electrode arrays that make up each pair are placed on opposite sides of the body part being treated. Specifically, in the OPTUNE® system, one pair of electrodes is placed on the left - right (LR) of the tumor and the other pair of electrodes is placed on the anterior - posterior (AP) of the tumor. The TT field is approved for the treatment of glioblastoma multiforme (GBM) and can be delivered, for example, via the OPTUNE® system (Novocure of Jersey, Channel Islands of Helius) that includes transducer arrays placed on the shaved head of the patient.

[0004] Each transducer array used for delivery of TT fields with the OPTUNE® device consists of a series of ceramic disk electrodes coupled to the patient's skin (such as the shaved head of the patient for GBM treatment) via a layer of conductive medical gel. The purpose of the medical gel is to deform to fit the body's contours and provide good electrical contact between the array and the skin, and thus the gel interface bridges the skin and reduces interference. This device is intended for the patient to wear continuously for 2 - 4 days for hygiene care and re-shaving (if necessary), and then re-wear a new array set. Thus, the medical gel remains in substantial continuous contact with the area of the patient's skin for 2 - 4 days at a time, and the area of the skin is only briefly exposed to the environment without further application of the medical gel until the medical gel is reapplied.

[0005] TT fields have been shown to slow DNA damage repair mechanisms, particularly those responsible for the repair of double-strand DNA breaks. In particular, BRCA1 and Fanconi anemia proteins have been shown to have their gene and protein expression downregulated after application of an alternating electric field (Giladi et al., (2017) Radiation Oncology, 12(1):206; Karanam et al. (2017) Cell Death Dis, 8(3):e2711; Mumblat et al. (2021) International Journal of Radiation Oncology, Biology, Physics, 111(3):e463).

[0006] Clustered regularly interspaced short palindromic repeat - CRISPR associated nuclease protein (known as CRISPR - Cas9), a gene editing system derived from prokaryotes, is an important component of prokaryotic adaptive immunity. This gene editing mechanism of the CRISPR - Cas9 system has been adapted and used in mammalian cells to suppress or promote gene expression at precise sequences within DNA. The mechanism by which CRISPR - Cas9 functions is to recognize DNA elements with single - guide RNA for specific target sequences, followed by cleavage of double - stranded DNA by the Cas9 enzyme (Zhang et al. (2021) Mol. Cancer, 20:126; Afolabi et al. (2019) Immunology, 158(2):63 - 69). In this regard, DNA repair mechanisms are employed in the current applications of the CRISPR - Cas9 system (Richardson et al. (2018) Nature Genetics, 50(8):1132 - 1139).

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, there is a need in the art for new and improved systems and methods for treating cancer and other conditions and diseases or reducing their occurrence. The present disclosure is directed to such new and improved systems and methods.

Modes for Carrying Out the Invention

[0008] Before explaining in detail at least one embodiment of the concept(s) of the present invention by way of illustrative language and results, it should be understood that the application of the concept(s) of the present invention is not limited to the details of the configuration and arrangement of components described in the following explanation. The concept(s) of the present invention can have other embodiments or can be practiced or implemented in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning, and the embodiments are illustrative and not exhaustive. Also, it should be understood that the expressions and terms used herein are for explanatory purposes and should not be regarded as limiting.

[0009] Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed invention concept(s) shall have the meanings commonly understood by those of ordinary skill in the art, and furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and are as described in various general and more specific references cited and described throughout this specification. The nomenclature used in connection with the analytical chemistry, organic synthetic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0010] All patents, published patent applications, and non-patent publications described herein demonstrate the skill level of those of ordinary skill in the art in the technical fields related to the presently disclosed invention concept(s). Also, it should be understood that the expressions and terms used herein are for explanatory purposes and should not be regarded as limiting.

[0011] All of the compositions, assemblies, systems, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions, assemblies, systems, kits, and methods of the inventive concept(s) are described with respect to specific embodiments, it will be apparent to those skilled in the art that changes can be made in the compositions and / or methods and in the steps or the order of the steps of the methods described herein without departing from the spirit, concept, and scope of the inventive concept(s). All such similar alternatives and modifications apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the inventive concept(s) as defined in the appended claims.

[0012] The following terms used in accordance with this disclosure are to be understood to have the following meanings unless otherwise specified.

[0013] The use of the terms "a" or "an" when used in combination with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," "one or two or more." Accordingly, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the expression "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plural" refers to "two or more."

[0014] The use of the term "at least one" is understood to include quantities greater than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or more than 1000 depending on the term to which it is added, and the quantities 100 / 1000 are not considered limiting and higher limits may also yield satisfactory results. Additionally, the use of the term "at least one of X, Y, Z" is understood to include only X, only Y, only Z, and any combination of X, Y, Z. The use of ordinal terms ("first", "second", "third", "fourth", etc.) is only for the purpose of distinguishing two or more items and does not imply any order or importance of one item over another, or any additional order, etc.

[0015] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly indicated otherwise to refer only to alternative cases or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by either A being true (or present) and B being false (or present), A being false (or present) and B being true (or present), or both A and B being true (or present).

[0016] As used herein, references to "one embodiment", "an embodiment", "some embodiments", "an example", "for example", or "an illustration" mean that a particular element, function, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. For example, the phrases "in some embodiments" or "in an example" may appear in various places in the specification, but not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples are to be construed as not limiting the claims.

[0017] Throughout this application, the term "about" is used to indicate that a value includes variations due to the inherent error of a composition / device / device, the method used to determine the value, or the variations that exist between the subjects of study. For example, but not limited to, when the term "about" is used, the specified value is appropriate for performing the disclosed method and can vary plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent from the specified value, as understood by those skilled in the art.

[0018] As used herein and in the claims (if any), the terms "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unlisted elements or method steps.

[0019] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed prior to that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC. Also, if order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB may also be used. Continuing with this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. One of ordinary skill in the art will understand that, unless otherwise apparent from the context, there is usually no limit to the number of items or terms being combined.

[0020] As used herein, the term "substantially" means that the event or situation described below occurs completely, or that the event or situation described below occurs to a significant degree or extent. For example, when associated with a particular event or situation, the term "substantially" means that the event or situation described thereafter occurs at least 80% of the time, at least 85% of the time, at least 90% of the time, or at least 95% of the time. For example, the term "substantially adjacent" can mean that two items are 100% adjacent to each other, that two items are very close to each other but not 100% adjacent, or that a portion of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0021] The term "single guide RNA" or "sgRNA" refers to a DNA-targeted RNA that contains a guide sequence that targets a CRISPR-associated endonuclease to a target genomic DNA and a scaffold sequence (e.g., tracrRNA) that interacts with the endonuclease.

[0022] The terms "CRISPR-associated endonuclease", "Cas polypeptide", and "Cas nuclease" refer to a family of clustered regularly interspaced short palindromic repeat (CRISPR)-associated polypeptides or nucleases that cleave DNA at a site specified by a nucleotide guide sequence contained within a crRNA transcript to generate blunt ends upon double-strand cleavage. CRISPR-associated endonucleases require both crRNA and tracrRNA for site-specific DNA recognition and cleavage. The crRNA binds to the tracrRNA via a region of partial complementarity and directs the endonuclease to a region of the target DNA that is homologous to the crRNA, referred to as the "protospacer".

[0023] The term "ribonucleoprotein complex" or "RNP complex" refers to a complex comprising an sgRNA and a CRISPR-associated endonuclease polypeptide.

[0024] The term "pharmaceutically acceptable" refers to compounds and compositions suitable for administration to humans and / or animals without excessive side effects such as toxicity, irritation, and / or allergic reactions, commensurate with a reasonable benefit / risk ratio.

[0025] As used herein, the terms "patient" or "subject" include human and veterinary subjects. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including but not limited to humans, domestic animals, farm animals, non-human primates, and other animals having mammary tissue.

[0026] The term "treatment" refers to both therapeutic treatment and prophylactic measures. Persons in need of treatment include not only those already suffering from a particular symptom / disease / infection but also those who are at risk of developing a particular symptom / disease / infection (such as those in need of prophylactic measures), among others. The term "treating" refers to administering a drug / agent / method to a patient for therapeutic and / or prophylactic purposes.

[0027] As used herein, the terms "therapeutic composition" or "pharmaceutical composition" refer to agents that can be administered in vivo to provide a therapeutic and / or prophylactic effect.

[0028] Administering a therapeutically effective amount or a prophylactically effective amount is intended to provide a therapeutic benefit in the treatment, prevention, and / or management of a disease, condition, and / or infection. The specific therapeutically effective amount can be readily determined by a general physician and can vary depending on factors known in the art such as, but not limited to, the type of symptom / disease / infection, the patient's medical history and age, the stage of the symptom / disease / infection, and the co-administration of other agents.

[0029] The term "effective amount", when used in accordance with the concepts (s) of the present invention, refers to an amount of a biologically active molecule or its conjugate or derivative, or an amount of a treatment protocol (i.e., an alternating electric field), sufficient to exert a detectable therapeutic effect without undue side effects (such as, but not limited to, toxicity, irritation, allergic reactions, etc.) commensurate with a reasonable benefit / risk ratio. Therapeutic effects include, for example, but are not limited to, preventing, inhibiting, or reducing the occurrence of at least one symptom, disease, and / or infection. The effective amount for a subject varies depending on the type of subject, the subject's physique and health status, the nature and severity of the symptom / disease / infection to be treated, the method of administration, the treatment period, the nature of the combination therapy (if any), the specific formulation used, etc. Therefore, an exact effective amount cannot be specified in advance. However, the effective amount in a particular situation can be determined by one of ordinary skill in the art by performing routine experiments based on the information provided herein.

[0030] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjuvant therapy" and is understood to mean that a patient in need of treatment is treated or administered with another agent for a symptom / disease / infection in combination with the treatment of the present disclosure. This concurrent therapy can be a sequential therapy where the patient is first treated with one treatment protocol / pharmaceutical composition and then with another treatment protocol / pharmaceutical composition, or the two treatment protocols / pharmaceutical compositions are administered simultaneously.

[0031] Here, with respect to the concept(s) of the present invention, systems, kits, and methods are provided for inducing apoptosis and / or treating or reducing the occurrence of at least one symptom, disease, disorder, or infection in a subject. The systems, kits, and methods combine the application of an alternating electric field with a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system. The alternating electric field (e.g., TT field) delays DNA damage repair mechanisms, particularly those responsible for repairing double-strand DNA breaks. CRISPR-Cas is a gene editing system that has been adapted and used in mammalian cells to suppress or promote the expression of genes at precise sequences within DNA. The CRISPR-Cas9 system uses a single guide RNA (sgRNA) for a specific target sequence to recognize a DNA element, after which a double-strand DNA break is cleaved by the Cas9 enzyme. Currently, the CRISPR-Cas9 system employs DNA repair mechanisms. However, in the present disclosure, the application of an alternating electric field inhibits the DNA damage repair mechanism, so that the DNA breaks formed by exposure to the CRISPR-Cas system are not repaired, and thus the cell(s) exposed to the combined treatment of the alternating electric field and the CRISPR-Cas system undergo apoptosis.

[0032] This ability to efficiently induce apoptosis only in mutant cells (or other target cells that uniquely possess the specific sequence to which the sgRNA is directed) provides patients with a treatment option with fewer toxicities and / or adverse events compared to systemic therapies such as chemotherapy (but not limited to these).

[0033] Certain non-limiting embodiments of the present disclosure include a method of inducing apoptosis in at least one cell. In this method, an alternating current electric field is applied to the at least one cell for a certain period of time, and the application of the alternating current electric field down-regulates at least one DNA damage repair pathway in the at least one cell. The at least one cell is further exposed to a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system comprising (i) a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence within the at least one cell, and (ii) a CRISPR-associated endonuclease or a gene encoding the same.

[0034] Certain non-limiting embodiments of the present disclosure include a method of treating or reducing the occurrence of at least one symptom, disease, disorder, or infection in a subject. In this method, an alternating current electric field is applied to at least a part of the subject for a certain period of time, and the application of the alternating current electric field down-regulates at least one DNA damage repair pathway in at least a part of the subject. A CRISPR-Cas system is also administered to the subject, and the CRISPR-Cas system comprises (i) a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one symptom, disease, disorder, or infection, and (ii) a CRISPR-associated endonuclease or a gene encoding the same.

[0035] Any type of conductive and / or non-conductive electrode(s) and / or transducer array(s) that are known in the art or contemplated herein and can be used to generate an alternating electric field can be applied to cells / subjects to generate an alternating electric field in accordance with the present disclosure. Non-limiting examples of electrodes and transducer arrays that can be used to generate an alternating electric field in accordance with the present disclosure include, but are not limited to, those that function as part of the TTFields systems described in U.S. Pat. Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, 10,441,776, and 11,452,863, and those described in U.S. Patent Application Nos. 2018 / 0001078, 2018 / 0160933, 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016.

[0036] According to the present invention, the alternating electric field can be generated at any frequency that down-regulates at least one DNA damage repair pathway in at least a part of the cell(s) / subject. For example (but not limited to), the alternating electric field can be at about 50 kHz, about 75 kHz, about 100 kHz, about 125 kHz, about 150 kHz, about 175 kHz, about 200 kHz, about 225 kHz, about 250 kHz, about 275 kHz, about 300 kHz, about 325 kHz, about 350 kHz, about 375 kHz, about 400 kHz, about 425 kHz, about 450 kHz, about 475 kHz, about 500 kHz, about 550 kHz, about 600 kHz, about 650 kHz, about 700 kHz, about 750 kHz, about 800 kHz, about 850 kHz, about 900 kHz, about 950 kHz, about 1 MHz, about 2 MHz, about 3 MHz, about 4 MHz, about 5 MHz, about 6 MHz, about 7 MHz, about 8 MHz, about 9 MHz, about 10 MHz, about 11 MHz, about 12 MHz, about 13 MHz, about 14 MHz, about 15 MHz, etc., and ranges formed from any of the above values (e.g., a range from about 100 kHz to about 10 MHz, a range from about 50 kHz to about 1 MHz, a range from about 1 MHz to about 10 MHz, a range from about 50 kHz to about 500 kHz, a range from about 100 kHz to about 300 kHz, a range from about 150 kHz to about 300 kHz, etc.), and ranges formed by combining two integers between two of the above values (e.g., a range from about 122 kHz to about 313 kHz, a range from about 78 kHz to about 298 kHz, etc.).

[0037] In certain (non-limiting) embodiments, the alternating electric field can be applied at two or more different frequencies simultaneously or sequentially during treatment (i.e., the frequency can be changed during treatment). When two or more frequencies are present, each frequency is selected from any of the above values, or a range formed from any of the above values, or a range formed by combining two integers included between two of the above values.

[0038] According to the present invention, the alternating electric field can have any electric field strength as long as it can down-regulate at least one DNA damage repair pathway in at least a part of the cell(s) / subject. For example (but not limited to these), the alternating electric field can be at least about 0.1 V / cm, about 0.5 V / cm, about 1 V / cm, about 1.5 V / cm, about 2 V / cm, about 2.5 V / cm, about 3 V / cm, about 3.5 V / cm, about 4 V / cm, about 4.5 V / cm, about 5 V / cm, about 5.5 V / cm, about 6 V / cm, about 6.5 V / cm, about 7 V / cm, about 7.5 V / cm, about 8 V / cm, about 9 V / cm, about 9.5 V / cm, about 10 V / cm, about 10.5 V / cm, about 11 V / cm, about 11.5 V / cm, about 12 V / cm, about 12.5 V / cm, about 13 V / cm, about 13.5 V / cm, about 14 V / cm, about 14.5 V / cm, about 15 V / cm, about 15.5 V / cm, about 16 V / cm, about 16.5 V / cm, about 17 V / cm, about 17.5 V / cm, about 18 V / cm, about 18.5 V / cm, about 19 V / cm, about 19.5 V / cm, about 20 V / cm, etc., as well as ranges formed from any of the above values (e.g., a range from about 1 V / cm to about 20 V / cm, a range from about 1 V / cm to about 10 V / cm, a range from about 1 V / cm to about 4 V / cm, etc.), and ranges formed by combining two integers falling between two of the above reference values (e.g., a range from about 1.1 V / cm to about 18.6 V / cm, a range from about 1.2 V / cm to about 9.8 V / cm, a range from about 1.3 V / cm to about 4.7 V / cm, etc.).

[0039] An alternating electric field can be applied for any period sufficient to down-regulate at least one DNA damage repair pathway in at least a part of the cell(s) / subject. For example, but not limited to, the alternating electric field can be applied for at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, etc., also ranges formed from any of the above values (e.g., a range from about 24 hours to about 72 hours, etc.), and ranges formed by combining two integers falling between two of the above values (e.g., a range from about 14 hours to about 68 hours, etc.) can be applied.

[0040] The total time for which the alternating electric field is applied can be achieved continuously or intermittently. That is, when the alternating electric field is applied for a shorter period (e.g., less than about 12 hours or 24 hours), the alternating electric field can be applied continuously over that period. However, when the alternating electric field is applied for a longer period (e.g., a period of about 24 hours or more), the treatment period can include one or more interruptions during the application cycles that separate two or more application sections, whereby the application sections and interruptions are combined to form the total application period. If there are interruptions, the interruptions should typically account for about 50%, about 40%, about 30%, about 20% or less of the treatment time, such that the alternating electric field is applied for at least about 50%, about 60%, about 70%, about 80% or more of the treatment time. For example, but not limited to, the alternating electric field needs to be applied for at least about 19 hours out of 24 hours. In addition, the longer the time for which the alternating electric field is applied, the higher the efficacy.

[0041] Any CRISPR-Cas system known in the art or otherwise contemplated herein may be utilized in accordance with the present disclosure, so long as the system is capable of creating double-stranded DNA nicks in a target sequence in a subject. CRISPR-Cas systems are widely known and have been extensively reviewed (see, e.g., Adli, M. (2018) Nat Commun, 9:1911; Pickar-Oliver et al. (2019) Nature Reviews Molecular Cell Biology, 20:490-507; Xu et al. (2020) Comput Struct Biotechnol J., 18:2401-2415; Nidhi et al. (2021) Int J Mol Sci., 22(7):3327). In addition, CRISPR-Cas systems are commercially available from a variety of sources, including, for example, Merck / Millipore Sigma (Burlington, MA), Takara Bio USA (San Jose, CA), Thermo Fisher Scientific (Waltham, MA), Integrated DNA Technologies (Coralville, IA), GenScript Biotech (Piscataway, NJ), ACRO Biosystems (Newark, DE), Miras Bio LLC (Madison, WI), and the like.

[0042] The CRISPR-Cas system includes a CRISPR-related endonuclease or a gene encoding the same, which is known in the art or contemplated herein for use within the CRISPR-Cas system to recognize and cleave a target DNA complementary to the guide RNA. Non-limiting examples of Cas endonucleases that may be utilized in accordance with the present disclosure include Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, Cas9 nickase (nCas9), and the like, as well as variants and derivatives thereof, and any combination thereof.

[0043] Depending on the specific Cas endonuclease used, the sgRNA of the CRISPR-Cas system contains the sequence(s) specific to a particular Cas endonuclease that is recognized by that endonuclease and instructs the endonuclease to make a sequence-specific cleavage within the target DNA sequence. Selection and / or creation of an sgRNA containing a combination of these endonuclease-derived sequence(s) and the sequence(s) that hybridize to the target DNA sequence is within the knowledge of those skilled in the art and thus no further explanation is considered necessary.

[0044] In certain non-limiting embodiments, the methods of the disclosure can be applied to a variety of conditions where a mutation has occurred resulting in silencing or overactivation of a damaged gene, or conditions involving foreign genes / sequences. For example, but not limited to, at least one condition, disease, disorder, or infection is selected from the group consisting of cancer, autoimmune diseases, viral infections, bacterial infections, fungal infections, parasitic infections, metabolic syndrome, Huntington's disease, von Hippel-Lindau syndrome, etc., and any combination thereof.

[0045] Specific non-limiting examples of conditions, diseases, disorders, and infections that can be treated in accordance with the disclosure include the following: · Cancers associated with oncologically functional gain-of-function gene mutations (multiple possible) in cancer genes (e.g., Kontomanolis et al. (2020) Anticancer Research, 40(11):6009-6015; Frank et al. (1998) Journal of Clinical Oncology 16:7:2417-2425) and / or loss of function mutation(s) in a tumor suppressor gene (see, for example (but not by way of limitation) Chen et al. Chenetal. (2020) Sig Transduct Target Ther, 5:90); (however, not limited to these); · An autoimmune disease in which the sgRNA of the CRISPR-Cas system is directed to a specific sequence of a self-antigen, thereby causing the death of immune cells (i.e., immune cells that produce autoantibodies) that are inappropriately directed against "self" cells; · Infections in which the sgRNA of the CRISPR-Cas system targets one or more genes / sequences of infectious microorganisms (such as bacteria, parasites, fungi, viruses, etc.) (see, for example, Hu et al. (2014) BioMed Research International, Article ID 612823). In addition, the CRISPR-Cas9 system targets the LTR region at positions T5 and T6 of the HIV flow virus (Ebina et al. (2013) Sci Rep, 3:2510); · Metabolic syndromes such as hereditary tyrosinemia (however, not limited to these) (Pankowicz et al. (2016) Nat Commun, 7:12642); and have been used to target Huntington's disease. In metabolic syndromes, the sgRNA of the CRISPR-Cas system targets mutations in the FAH gene, TAT gene, and HPD gene that cause tyrosinemia type I, type II, and type III, respectively (however, not limited to these). · In Huntington's disease, the sgRNA of the CRISPR-Cas system targets CAG repeat expansions, because Huntington's disease is a disorder characterized by the loss of striatal neurons resulting from the expansion of CAG repeats in the huntingtin protein (see, e.g., Cattaneo et al., (2001) Trends in Neurosciences, 24(3):182-188, but not limited thereto).

[0046] In certain (non-limiting) embodiments, the target DNA sequence is led to a gene mutation, and this mutation is p53, retinoblastoma (Rb), phosphatase and tensin homolog (PTEN) deleted on chromosome 10, Ras-related domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis coli (APC), ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein (BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partner and localizer (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class O3a (FOXO3a), Ras (H-Ras, K-Ras, N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, von Hippel-Lindau (VHL), fumaroylacetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS variant transcription factor 1 (ETV1), β-catenin (CTNNB1), E2F1, T cell acute lymphoblastic leukemia 1 (TAL1), etc., and combinations thereof selected from the group consisting of.

[0047] In certain (non-limiting) embodiments, the target DNA sequence is a loss-of-function mutation of a tumor suppressor gene. Examples of tumor suppressor genes include p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras-associated domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis coli (APC), ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein (BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partner and localizer (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class O3a (FOXO3a), p38, von Hippel-Lindau (VHL), and any combination thereof, but are not limited thereto.

[0048] In certain (non-limiting) embodiments, the target DNA sequence is an oncogenic gain-of-function genetic mutation (i.e., an oncogene). Examples of oncogenes include Ras (H-Ras, K-Ras, and N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, fumarate acetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS variant transcription factor 1 (ETV1), β-catenin (CTNNB1), E2F1, T-cell acute lymphoblastic leukemia 1 (TAL1), etc., as well as any combination thereof, but are not limited thereto.

[0049] In certain (non-limiting) embodiments, the target DNA sequence is a gene of a microorganism selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), SARS-CoV-2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV-8), human T-lymphotropic virus type 1 (HTLV-1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Shigella flexneri, Streptococcus agalactiae, Mycoplasma, and any combination thereof.

[0050] The CRISPR-Cas system cargo can be administered to a cell(s) / subject via any form, any delivery vehicle(s), and any delivery mechanism(s) / pathway(s) known or envisioned herein for the delivery of the CRISPR-Cas system cargo, as long as its form and delivery vehicle(s) can deliver the CRISPR-Cas system to the target cell(s) and facilitate penetration into the nucleus to promote the creation of double-stranded DNA nicks at the target sequence of interest. The CRISPR-associated endonuclease or the gene encoding it can exist in the form of a protein, peptide, DNA molecule, RNA molecule (such as, but not limited to, an mRNA molecule), or any combination thereof, depending on the delivery vehicle used.

[0051] The sgRNA and the CRISPR-related endonuclease (or the gene encoding the same) may be delivered via a single delivery vehicle or may be divided into two different delivery vehicles (which may be of the same type or different types). In certain non-limiting embodiments, the CRISPR-Cas system can be delivered as a ribonucleoprotein (RNP) complex formed from the sgRNA and an endonuclease peptide or protein. In certain non-limiting embodiments, one or both components of the CRISPR-Cas system can be delivered via one or more DNA plasmids containing the sequence(s) of the sgRNA and / or the sequence(s) encoding the endonuclease. In other non-limiting embodiments, one or both components of the CRISPR-Cas system can be delivered via one or more DNA-based or RNA-based viral vectors (e.g., adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia, and / or herpes simplex virus vectors, etc.) or artificial viruses. In other non-limiting embodiments, one or both components of the CRISPR-Cas system can be delivered via liposomes and / or lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, or via poloxamers, polycations, virosomes, gold nanoparticles or other inorganic nanoparticles, etc. In yet other non-limiting embodiments, one or both components of the CRISPR-Cas system are delivered in naked form via direct physical delivery. In yet other non-limiting embodiments, the sgRNA is delivered via one of the above vehicles and the CRISPR-related endonuclease (or the nucleic acid sequence encoding the same) is delivered via another of the above vehicles.

[0052] In certain non-limiting embodiments, one or both components of the CRISPR-Cas system and / or the delivery vehicle can be modified to further assist, for example (but not limited to), delivering the CRISPR-Cas system to cancer cells, enhancing solubility, monitoring transport, etc. For example (but not limited to), the sgRNA, endonuclease (or the gene encoding it), and / or the delivery vehicle can be designed to include a nuclear localization sequence (NLS), a cell-penetrating peptide or peptide sequence, an affinity tag, a detectable tag (such as, but not limited to, a fluorescent tag), a cancer cell targeting ligand or receptor, etc., and any combination thereof.

[0053] The components of the CRISPR-Cas system can be delivered by any delivery mechanism / pathway known in the art or contemplated herein. Non-limiting examples of delivery mechanisms / pathways include injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, etc., and any combination thereof.

[0054] An alternating electric field can be applied to any part(s) of a subject that results in down - regulation of at least one DNA damage repair pathway in the desired part(s) of the cell(s) / subject. In certain (non - limiting) embodiments, the alternating electric field is targeted to a part of the subject that contains the target gene or genetic mutation. For example (but not limited to), if there is at least one solid tumor, the alternating electric field can be applied to a part of the subject that contains the solid tumor. Alternatively, if the treatment is targeted at an infection in the subject, the alternating electric field can be applied to at least a part of the surface or internal infection site of the subject (e.g., the cervix in the case of HPV infection, the thymus or the infection site in the case of HIV infection, the thymus or the infection site (such as HIV - induced pneumonia) in the case of AIDS - induced diseases, etc.). In yet another alternative, if the treatment is targeted at an autoimmune disease, the alternating electric field is applied to the draining lymph nodes of the inflammatory target, and if the treatment is targeted at a metabolic syndrome (such as, but not limited to, hereditary tyrosinemia), the alternating electric field is applied to the liver, and if the treatment is targeted at Huntington's disease, the alternating electric field can be applied to the brain.

[0055] The methods of the present disclosure can include one or more additional steps. For example, but not limited to, the method can include one or more steps to assist in identifying the subject to be treated, one or more additional administration steps (such as, but not limited to, administration of at least one substance for co - treatment), and / or one or more steps to analyze the effectiveness of the treatment.

[0056] In a non - limiting embodiment, the method can include examining a sample and the target within the sample. This step can be performed before and / or after one or both of the step of applying the alternating electric field and the step of administering the CRISPR - Cas system.

[0057] In certain (non-limiting) embodiments, the CRISPR-Cas system can be administered after the application of an alternating electric field has been initiated. In particular (but not limited to these), the CRISPR-Cas system can be administered during and / or after the application of the alternating electric field has elapsed.

[0058] For example (but not limited to these), the CRISPR-Cas system can be administered after a period of at least about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, etc., a range formed from any of the above values (e.g., a range from about 24 hours to about 96 hours, a range from about 24 hours to about 48 hours, etc.), and a range combining two integers falling between two of the above values (e.g., a range from about 14 hours to about 94 hours, etc.) after the application of the alternating electric field has been initiated. In certain (non-limiting) embodiments, at least one substance is orally administered at least about 24 hours after the application of the alternating electric field has been initiated.

[0059] In other non-limiting examples, the CRISPR-Cas system may be administered after the period during which the alternating electric field was applied has elapsed, in which case at least one substance is administered within about 3 hours, about 6 hours, about 9 hours, about 12 hours, about 15 hours, about 18 hours, about 21 hours, about 24 hours, about 27 hours, about 30 hours, about 33 hours, about 36 hours, about 39 hours, about 42 hours, about 45 hours, about 48 hours, about 51 hours, about 54 hours, about 57 hours, about 60 hours, about 63 hours, about 66 hours, about 69 hours, about 72 hours, about 75 hours, about 78 hours, about 81 hours, about 84 hours, about 87 hours, about 90 hours, about 93 hours, about 96 hours, etc. from the time the period has elapsed. In certain (non-limiting) embodiments, the CRISPR-Cas system is administered within about 96 hours after the period has elapsed.

[0060] In certain (non-limiting) embodiments, the method includes one or more additional steps. For example (but not limited to), the method may further include the step of stopping the application of the alternating electric field (such as, but not limited to) so that the cells / tissues can recover. In addition, any step can be repeated one or more times. Each step can be repeated the required number of times. When the step involving the application of the alternating electric field is repeated, the transducer array can be placed at a position slightly different from the original placement on the subject. By repositioning the array in this way, the therapeutic effect can be further enhanced, and the adverse effects on the skin caused by the alternating electric field can also be minimized. In addition, the step involving the administration of the CRISPR-Cas system can be repeated a variety of times and at a variety of intervals according to known and / or generally accepted regimens of the system.

[0061] Certain non-limiting embodiments of the present disclosure relate to a kit comprising any component of an alternating electric field generating system (e.g., one or more transducer arrays and / or one or more hydrogel compositions, etc., as disclosed in U.S. Pat. Nos. 7,016,725, 7,089,054, 7,333,852, 7,565,205, 8,244,345, 8,715,203, 8,764,675, 10,188,851, 10,441,776, and 11,452,863, and U.S. Patent Application Nos. 2018 / 0001078, 2018 / 0160933, 2019 / 0117956, 2019 / 0307781, and 2019 / 0308016, but not limited thereto) and one or more of any CRISPR-Cas system disclosed or contemplated herein. In particular (but not limited thereto), the kit may comprise (i) at least one pair of transducer arrays for generating an alternating electric field therebetween when applying the transducer array to at least one cell and / or disposing the transducer array on a subject, and (ii) a CRISPR-Cas system comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one symptom, disease, disorder, or infection, and a single guide RNA (sgRNA) comprising a CRISPR-associated endonuclease or a gene encoding the same. The symptom, disease, disorder, or infection, and the target DNA sequence can be any of those disclosed herein or otherwise contemplated.

[0062] In certain (non-limiting) embodiments, the kit may further include instructions for performing any of the methods disclosed or contemplated herein. For example (but not limited to), the kit may include instructions for applying one or more components of the alternating electric field generating system to the patient's skin, instructions for applying an alternating electric field to a particular part of the patient, instructions on when and how to administer the CRISPR-Cas system, and / or instructions on when to activate and turn off the alternating electric field in relation to the administration of the CRISPR-Cas system.

[0063] In addition to the components described in detail above, the kit may further contain other component(s) / reagent(s) for performing any of the specific methods described herein or otherwise contemplated. For example (but not limited to), the kit may further include (i) components (such as a razor, cleaning composition or wipe / towel, etc.) for preparing the skin before discarding the hydrogel composition and / or transducer array on the skin, (ii) components for removing the gel / transducer array(s), and / or (iii) components for cleaning the skin after removing the gel / transducer array(s). Alternatively (and / or in addition), the kit may further include at least one device and / or at least one reagent for examining a sample and recognizing whether a target DNA sequence is present in the sample, and / or at least one device and / or at least one reagent for analyzing the effectiveness of a treatment protocol.

[0064] The nature of the various additional component(s) / reagent(s) that may optionally be included in the kit depends on the particular form of treatment, and since the recognition thereof is within the skill of those in the art, no further explanation is considered necessary.

[0065] The components / reagents included in the kit may be in separate containers / compartments, or depending on the sterility, cross-reactivity, and stability of the components / reagents, various components / reagents may be combined in one or more containers / compartments.

[0066] The kit can be placed in any package that enables the components contained therein to function in accordance with the present disclosure. In certain non-limiting embodiments, the kit further includes a sealed package in which the components are placed. In certain (non-limiting) embodiments, the sealed package is substantially impermeable to air and / or substantially impermeable to light.

[0067] In addition, the kit may also include a series of written instructions that explain how to use one or more components of the kit. Such a kit can be used with any of the methods described herein or methods being considered separately.

[0068] In certain non-limiting embodiments, the shelf life of the kit is at least about 6 months, for example (but not limited to) at least about 9 months, or at least about 12 months. Non-limiting exemplary embodiments of the concept(s) of the present invention

[0069] Exemplary Embodiment 1. A method for inducing apoptosis in at least one cell, comprising: (1) applying an alternating current electric field to at least one cell for a certain period, wherein the application of the alternating current electric field down-regulates at least one DNA damage repair pathway in at least one cell; and (2) exposing to a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system, the system comprising a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence in at least one cell, and a CRISPR-associated endonuclease or a gene encoding the same.

[0070] Exemplary Embodiment 2. The method according to Exemplary Embodiment 1, wherein the CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

[0071] Exemplary Embodiment 3. The method according to Exemplary Embodiment 1 or 2, wherein the target DNA sequence is selected from the group consisting of a gain-of-function genetic mutation in an oncogene, a loss-of-function mutation in a tumor suppressor gene, a gene of an infectious microorganism, and a self-antigen sequence in an immune cell that produces autoantibodies.

[0072] Exemplary Embodiment 4. The method according to Exemplary Embodiment 3, wherein the target DNA sequence is induced by a gene mutation, and this displacement is p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras-related domain family (RASSF), ADP ribosylation factor (ARF), adenomatous polyposis coli (APC), ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein (BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partner and localizer (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class O3a (FOXO3a), Ras (H-Ras, K-Ras, N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, von Hippel-Lindau (VHL), fumarate acetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS variant transcription factor 1 (ETV1), β-catenin (CTNNB1), E2F1, T cell acute lymphoblastic leukemia 1 (TAL1), selected from the group consisting of.

[0073] Exemplary Embodiment 5. The method according to Exemplary Embodiment 3, wherein the target DNA sequence is a gene of a microorganism selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), SARS-CoV-2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV-8), human T-lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Shigella flexneri, group B streptococcus, and Mycoplasma.

[0074] Exemplary Embodiment 6. The method according to any one of Exemplary Embodiments 1 to 5, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed from an sgRNA and an endonuclease peptide or protein.

[0075] Exemplary Embodiment 7. The method according to any one of Exemplary Embodiments 1 to 6, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA plasmid.

[0076] Exemplary Embodiment 8. The method according to any one of Exemplary Embodiments 1 to 7, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA or RNA-based viral vector.

[0077] Exemplary Embodiment 9. The method according to Exemplary Embodiment 8, wherein the viral vector is selected from the group consisting of an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, a vaccinia, and / or a herpes simplex virus vector, or an artificial virus.

[0078] Exemplary Embodiment 10. The method according to any one of Exemplary Embodiments 1 to 9, wherein at least one component of the CRISPR-Cas system is delivered via at least one of liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

[0079] Exemplary Embodiment 11. The method according to any one of Exemplary Embodiments 1 to 10, wherein at least one component of the CRISPR-Cas system is administered in a naked form via direct physical delivery.

[0080] Exemplary Embodiment 12. The method according to any one of Exemplary Embodiments 1 to 11, wherein the sgRNA and the CRISPR-associated endonuclease or the gene encoding the same are administered by different delivery vehicles.

[0081] Exemplary Embodiment 13. The method according to any one of Exemplary Embodiments 1 to 12, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated endonuclease is modified to include a cell-penetrating peptide sequence.

[0082] Exemplary Embodiment 14. The method according to any one of Exemplary Embodiments 1 to 13, wherein at least one of the sgRNA and the CRISPR-associated endonuclease or the gene encoding the same is administered by a route selected from the group consisting of injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

[0083] Exemplary Embodiment 15. The method according to any one of Exemplary Embodiments 1 to 14, wherein the alternating electric field is applied at a frequency in the range of about 100 kHz to about 10 MHz.

[0084] Exemplary Embodiment 16. The method according to any one of Exemplary Embodiments 1 to 15, wherein the electric field strength of the alternating electric field is at least 1 V / cm.

[0085] Exemplary Embodiment 17. The method according to any one of Exemplary Embodiments 1 to 16, wherein at least one cell is part of a biological tissue, and the method further comprises the step of examining a sample of the biological tissue and identifying a target DNA sequence in the sample.

[0086] Exemplary Embodiment 18. A method for treating or alleviating the occurrence of at least one symptom, disease, disorder, or infection in a subject, comprising: (1) applying an alternating electric field to at least a part of the subject for a certain period, wherein at least one DNA damage repair pathway in at least a part of the subject is downregulated by the application of the alternating electric field; and (2) administering to the subject a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system, the system comprising a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one symptom, disease, disorder, or infection, and a CRISPR-associated endonuclease or a gene encoding the same.

[0087] Exemplary Embodiment 19. The method according to Exemplary Embodiment 18, wherein the CRISPR-associated endonuclease is selected from the group consisting of Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

[0088] Exemplary Embodiment 20. The method according to Exemplary Embodiment 18 or 19, wherein at least one symptom, disease, disorder, or infectious disease is selected from the group consisting of cancer, autoimmune disease, viral infection, bacterial infection, fungal infection, parasitic infection, metabolic syndrome, Huntington's disease, von Hippel-Lindau syndrome, and combinations thereof.

[0089] Exemplary Embodiment 21. The method according to Exemplary Embodiment 20, having at least one of: (a) at least one symptom, disease, disorder, or infectious disease is cancer and the target DNA sequence is at least one oncological gain-of-function gene mutation; (b) at least one symptom, disease, disorder, or infectious disease is cancer and the target DNA sequence is at least one loss-of-function mutation in a tumor suppressor gene; (c) at least one symptom, disease, disorder, or infectious disease is a bacterial infection and the target DNA sequence is a bacterial sequence; (d) at least one symptom, disease, disorder, or infectious disease is a viral infection and the target DNA sequence is a viral sequence; (e) at least one symptom, disease, disorder, or infectious disease is a fungal infection and the target DNA sequence is a fungal sequence; (f) at least one symptom, disease, disorder, or infectious disease is a parasitic infection and the target DNA sequence is a parasitic sequence; (g) at least one symptom, disease, disorder, or infectious disease is an autoimmune disease and the target DNA sequence is a self-antigen sequence in immune cells that produce autoantibodies; (h) at least one symptom, disease, disorder, or infectious disease is Huntington's disease and the target DNA sequence is a CAG repeat expansion; and (i) at least one symptom, disease, disorder, or infectious disease is metabolic syndrome and the target DNA sequence is at least one mutation in a gene selected from the group consisting of FAH, TAT, and HPD.

[0090] Exemplary Embodiment 22. The method according to Exemplary Embodiment 21, wherein the target DNA sequence is a loss-of-function mutation of a tumor suppressor gene, and the tumor suppressor gene is selected from the group consisting of p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras-related domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis coli (APC), ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein (BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partner and localizer (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class O3a (FOXO3a), p38, and von Hippel-Lindau (VHL).

[0091] Exemplary Embodiment 23. The method according to Exemplary Embodiment 21, wherein the target DNA sequence is an oncogenic gain-of-function gene mutation, and the gene is selected from the group consisting of Ras (H-Ras, K-Ras, and N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, fumarate acetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS variant transcription factor 1 (ETV1). Selected from the group consisting of β-catenin (CTNNB1), E2F1, and T cell acute lymphoblastic leukemia 1 (TAL1).

[0092] Exemplary Embodiment 24. The method according to Exemplary Embodiment 21, wherein the target DNA sequence is selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), SARS-CoV-2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV-8), human T-lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Shigella flexneri, Group A streptococcus, and Mycoplasma, a sequence of a bacterium or virus selected from the group.

[0093] Exemplary Embodiment 25. The method according to any one of Exemplary Embodiments 18 to 24, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed from an sgRNA and an endonuclease peptide or protein.

[0094] Exemplary Embodiment 26. The method according to any one of Exemplary Embodiments 18 to 25, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA plasmid.

[0095] Exemplary Embodiment 27. The method according to any one of Exemplary Embodiments 18 to 26, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA or RNA-based viral vector.

[0096] Exemplary Embodiment 28. The method according to Exemplary Embodiment 27, wherein the viral vector is selected from the group consisting of an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, a vaccinia, and / or a herpes simplex virus vector, or an artificial virus.

[0097] Exemplary Embodiment 29. The method according to any one of Embodiments 18 to 28, wherein at least one component of the CRISPR-Cas system is delivered via at least one of liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

[0098] Exemplary Embodiment 30. The method according to any one of Embodiments 18 to 29, wherein at least one component of the CRISPR-Cas system is administered in a naked form via direct physical delivery.

[0099] Exemplary Embodiment 31. The method according to any one of Embodiments 18 to 30, wherein the sgRNA and the CRISPR-related endonuclease or the gene encoding the same are administered by different delivery methods.

[0100] Exemplary Embodiment 32. The method according to any one of Embodiments 18 to 31, wherein at least one of the sgRNA and the gene encoding the CRISPR-related endonuclease is modified to include a cell-penetrating peptide sequence.

[0101] Exemplary Embodiment 33. The method according to any one of Embodiments 18 to 32, wherein at least one of the sgRNA and the CRISPR-related endonuclease or the gene encoding the same is administered by a route selected from the group consisting of injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

[0102] Exemplary Embodiment 34. The method according to any one of Embodiments 18 to 33, wherein the CRISP-Cas system is administered about 24 hours to about 48 hours after the start of application of an alternating electric field.

[0103] Exemplary Embodiment 35. The method according to any one of Embodiments 18 to 34, wherein the time during which the alternating electric field is applied is in the range of about 24 hours to about 72 hours.

[0104] Exemplary Embodiment 36. The method according to any one of Embodiments 18 to 35, wherein the CRISPR-Cas system is administered after a certain period of time has elapsed.

[0105] Exemplary Embodiment 37. The method according to any one of Embodiments 18 to 36, wherein the alternating electric field is applied at a frequency in the range of about 100 kHz to about 10 MHz.

[0106] Exemplary Embodiment 38. The method according to any one of Embodiments 18 to 37, wherein the electric field strength of the alternating electric field is at least 1 V / cm.

[0107] Exemplary Embodiment 39. The method according to any one of Embodiments 18 to 38, wherein steps (1) and (2) are repeated one or more times.

[0108] Exemplary Embodiment 40. The method according to any one of Embodiments 18 to 39, further comprising the step of examining a sample from a subject and identifying a target DNA sequence in the sample.

[0109] Exemplary Embodiment 41. A kit comprising at least one pair of transducer arrays for generating an alternating electric field therebetween by applying a transducer array to at least one cell and / or placing the transducer array on a subject, and a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system, the system comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one symptom, disease, disorder, or infection, and a single guide RNA (sgRNA) comprising a CRISPR-associated endonuclease or a gene encoding the same.

[0110] Exemplary Embodiment 42. The kit according to Exemplary Embodiment 41, wherein the CRISPR-related endonuclease is selected from the group consisting of Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

[0111] Exemplary Embodiment 43. The kit according to Exemplary Embodiment 41 or 42, having at least one of: (a) at least one symptom, disease, disorder, or infection is cancer, and the target DNA sequence is at least one oncological gain-of-function genetic mutation; (b) at least one symptom, disease, disorder, or infection is cancer, and the target DNA sequence is at least one loss-of-function mutation in a tumor suppressor gene; (c) at least one symptom, disease, disorder, or infection is a bacterial infection, and the target DNA sequence is a bacterial sequence; (d) at least one symptom, disease, disorder, or infection is a viral infection, and the target DNA sequence is a viral sequence; (e) at least one symptom, disease, disorder, or infection is a fungal infection, and the target DNA sequence is a fungal sequence; (f) at least one symptom, disease, disorder, or infection is a parasitic infection, and the target DNA sequence is a parasitic sequence; (g) at least one symptom, disease, disorder, or infection is an autoimmune disease, and the target DNA sequence is a self-antigen sequence in immune cells that produce autoantibodies; (h) at least one symptom, disease, disorder, or infection is Huntington's disease, and the target DNA sequence is a CAG repeat expansion; and (i) at least one symptom, disease, disorder, or infection is metabolic syndrome, and the target DNA sequence is at least one mutation of a gene selected from the group consisting of FAH, TAT, and HPD.

[0112] Exemplary Embodiment 44. The kit according to Exemplary Embodiment 43, wherein the target DNA sequence is a loss-of-function mutation of a tumor suppressor gene, and the tumor suppressor gene is selected from the group consisting of p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras-related domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis coli (APC), ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein (BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partner and localizer (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class O3a (FOXO3a), p38, von Hippel-Lindau (VHL).

[0113] Exemplary Embodiment 45. The kit according to Exemplary Embodiment 43, wherein the target DNA sequence is a tumorigenic gain-of-function gene mutation, and the gene is selected from the group consisting of Ras (H-Ras, K-Ras, and N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, fumarate acetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS variant transcription factor 1 (ETV1), and selected from the group consisting of β-catenin (CTNNB1), E2F1, and T cell acute lymphoblastic leukemia 1 (TAL1).

[0114] Exemplary Embodiment 46. A kit according to Exemplary Embodiment 43, wherein the target DNA sequence is a sequence of a bacterium or virus selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV respectively), Epstein - Barr virus (EBV), human immunodeficiency virus (HIV), SARS - CoV - 2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV - 8), human T - lymphotropic virus - 1 (HTLV - 1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Shigella flexneri, Group A streptococcus, and Mycoplasma.

[0115] Exemplary Embodiment 47. A kit according to any one of Exemplary Embodiments 41 - 46, wherein the CRISPR - Cas system is in the form of a ribonucleoprotein (RNP) complex formed from an sgRNA and an endonuclease peptide or protein.

[0116] Exemplary Embodiment 48. A kit according to any one of Exemplary Embodiments 41 - 47, wherein at least one component of the CRISPR - Cas system is disposed within at least one DNA plasmid.

[0117] Exemplary Embodiment 49. A kit according to any one of Exemplary Embodiments 41 - 48, wherein at least one component of the CRISPR - Cas system is disposed within at least one DNA or RNA - based viral vector.

[0118] Exemplary Embodiment 50. A kit according to Exemplary Embodiment 49, wherein the viral vector is selected from the group consisting of an adenovirus, an adeno - associated virus, a lentivirus, a retrovirus, a vaccinia, and / or a herpes simplex virus vector, or an artificial virus.

[0119] Exemplary Embodiment 51. A kit according to any one of Exemplary Embodiments 41 to 50, wherein at least one component of the CRISPR-Cas system is disposed in at least one of liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

[0120] Exemplary Embodiment 52. A kit according to any one of Exemplary Embodiments 41 to 51, wherein at least one component of the CRISPR-Cas system is provided in a naked form for direct physical delivery.

[0121] Exemplary Embodiment 53. A kit according to any one of Exemplary Embodiments 41 to 52, wherein the sgRNA and the CRISPR-related endonuclease or the gene encoding the same are disposed in different delivery vehicles.

[0122] Exemplary Embodiment 54. A kit according to any one of Exemplary Embodiments 41 to 53, wherein at least one of the sgRNA and the gene encoding the CRISPR-related endonuclease is modified to include a cell-penetrating peptide sequence.

[0123] Exemplary Embodiment 55. A kit according to any one of Exemplary Embodiments 41 to 54, wherein at least one of the sgRNA and the CRISPR-related endonuclease or the gene encoding the same is formulated for administration by a route selected from the group consisting of injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

[0124] Exemplary Embodiment 56. A kit according to any one of Exemplary Embodiments 41 to 55, wherein the alternating electric field is configured to be applied at a frequency in the range of about 100 kHz to about 10 MHz.

[0125] Exemplary Embodiment 57. A kit according to any one of Exemplary Embodiments 41 to 56, wherein the alternating current electric field is configured to be applied at an electric field strength of at least 1 V / cm.

[0126] Exemplary Embodiment 58. A kit according to any one of Exemplary Embodiments 41 to 57, further comprising at least one device and / or at least one reagent for examining a sample to recognize a target DNA sequence in the sample.

[0127] Exemplary Embodiment 59. A clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system for use in any of the methods of Exemplary Embodiments 1 to 40, wherein the CRISPR-Cas system comprises a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to at least one intracellular target DNA sequence, and a CRISPR-associated endonuclease or a gene encoding the same. Exemplary Embodiment 60. Use of a clustered regularly arranged short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system according to any one of Exemplary Embodiments 1 to 40, wherein the CRISPR-Cas system comprises a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to at least one intracellular target DNA sequence, and a CRISPR-associated endonuclease or a gene encoding the same.

[0128] The present invention describes the concept(s) of the present invention in conjunction with the specific experiments, results, and words shown below, but it is obvious to those skilled in the art that many alternatives, modifications, and variations are apparent. Therefore, the present invention is intended to encompass all alternatives, modifications, and changes that are included within the spirit and broad scope of the present disclosure.

Claims

1. It's a kit, A pair of transducer arrays for generating an alternating electric field between them by applying a transducer array to at least one cell and / or placing the transducer array on a subject, The system comprises a clustered, regularly arranged short palindromic sequence repeat (CRISPR)-associated protein (CRISPR-Cas) system, the system is A single guide RNA (sgRNA) comprising a guide sequence that hybridizes to at least one target DNA sequence associated with a symptom, disease, disorder, or infection, and A kit containing a CRISPR-related endonuclease or the gene encoding it.

2. The kit according to claim 1, wherein the CRISPR-related endonuclease is selected from the group consisting of Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

3. (a) The at least one symptom, disease, disorder, or infection is cancer, and the target DNA sequence is at least one oncological gain-of-function gene mutation. (b) The at least one symptom, disease, disorder, or infection is cancer, and the target DNA sequence is at least one loss-of-function mutation in a tumor suppressor gene. (c) The at least one symptom, disease, disorder, or infection is a bacterial infection, and the target DNA sequence is a bacterial sequence. (d) The at least one symptom, disease, disorder, or infection is a viral infection, and the target DNA sequence is a viral sequence. (e) The at least one symptom, disease, disorder, or infection is a fungal infection, and the target DNA sequence is a fungal sequence. (f) The at least one symptom, disease, disorder, or infection is a parasitic infection, and the target DNA sequence is a parasitic sequence. (g) The at least one symptom, disease, disorder, or infection is an autoimmune disease, and the target DNA sequence is an autoantigen sequence within an immune cell that produces autoantibodies. (h) The at least one symptom, disease, disorder, or infection is Huntington's disease, the target DNA sequence is a CAG repeat extension, and (i) The at least one symptom, disease, disorder, or infection is a metabolic syndrome, and the target DNA sequence is at least one mutation in a gene selected from the group consisting of FAH, TAT, and HPD. The kit according to claim 1, satisfying at least one of the following conditions.

4. The target DNA sequence is a loss-of-function mutation in a tumor suppressor gene, and the tumor suppressor gene is p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras-related domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis of the colon (APC), ataxia telangiectasia mutation (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein ( The kit according to claim 3, selected from the group consisting of BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partners and locators (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class 03a (FOXO3a), p38, and von Hippel-Lindau (VHL).

5. The aforementioned target DNA sequence is an oncological gain-of-function gene mutation, and the gene is Ras (H-Ras, K-Ras, and N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun The kit according to claim 3, selected from the group consisting of NH2-terminal kinase (JNK), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, fumarylacetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS mutant transcription factor 1 (ETV1), β-catenin (CTNNNB1), E2F1, and T-cell acute lymphoblastic leukemia 1 (TAL1).

6. The kit according to claim 3, wherein the target DNA sequence is the sequence of a bacterium or virus selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), SARS-CoV-2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV-8), human T lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Salmonella typhi, Streptococcus bovine, and Mycoplasma.

7. The kit according to claim 1, wherein the CRISPR-Cas system is in the form of a ribonucleoprotein (RNP) complex formed from sgRNA and an endonuclease peptide or protein.

8. The kit according to claim 1, wherein at least one component of the CRISPR-Cas system is located within at least one DNA plasmid.

9. The kit according to claim 1, wherein at least one component of the CRISPR-Cas system is located within at least one DNA or RNA-based viral vector.

10. The kit according to claim 9, wherein the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia, and / or herpes simplex virus vector, or artificial virus.

11. The kit according to claim 1, wherein at least one component of the CRISPR-Cas system is arranged in at least one of liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

12. The kit according to claim 1, wherein at least one component of the CRISPR-Cas system is provided in a bare form for direct physical delivery.

13. The kit according to claim 1, wherein the sgRNA and the CRISPR-related endonuclease or the gene encoding it are arranged on different delivery hibicles.

14. The kit according to claim 1, wherein at least one of the genes encoding the sgRNA and CRISPR-related endonuclease is modified to include a cell-permeable peptide sequence.

15. The kit according to claim 1, wherein the sgRNA and at least one CRISPR-related endonuclease or gene encoding it are formulated for administration by a route selected from the group consisting of injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, bioristics, particle gun acceleration, and combinations thereof.

16. The kit according to claim 1, wherein the AC electric field is configured to be applied at a frequency in the range of approximately 100 kHz to approximately 10 MHz.

17. The kit according to claim 1, wherein the alternating electric field is configured to be applied with an electric field strength of at least 1 V / cm.

18. The kit according to claim 1, further comprising at least one device and / or at least one reagent for examining a sample and recognizing a target DNA sequence in the sample.

19. An agent for use in a method of inducing apoptosis in at least one cell, comprising a clustered and regularly arranged short palindromic sequence repeat (CRISPR)-related protein (CRISPR-Cas) system, The CRISPR-Cas system is A single guide RNA (sgRNA) containing a guide sequence that hybridizes to at least one target DNA sequence within a cell, and CRISPR-related endonuclease or the gene encoding it Includes, The aforementioned method, (1) A step of applying an alternating electric field to at least one cell for a certain period of time, wherein the application of the alternating electric field causes at least one DNA damage repair pathway in the at least one cell to be downregulated. (2) an agent comprising the step of exposing at least one of the cells to the CRISPR-Cas system.

20. The agent according to claim 19, wherein the CRISPR-related endonuclease is selected from the group consisting of Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

21. The agent according to claim 19, wherein the target DNA sequence is selected from the group consisting of oncological gain-of-function gene mutations in oncogenes, loss-of-function mutations in tumor suppressor genes, genes of infectious microorganisms, and autoantigen sequences in immune cells that produce autoantibodies.

22. The target DNA sequences are gene mutations, and these mutations include p53, retinoblastoma (Rb), deleted phosphatase and tensin homolog (PTEN) on chromosome 10, Ras-related domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis (APC), ataxia telangiectasia mutation (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein (BRCA1), breast cancer 2 protein (BRCA2) 2) BRCA2 partners and locators (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class 03a (FOXO3a), Ras (H-Ras, K-Ras, and N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH ２ - The agent according to claim 21, selected from the group consisting of terminal kinase (JNK), p38, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, von Hippel-Lindau (VHL), fumarylacetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS mutant transcription factor 1 (ETV1), β-catenin (CTNNNB1), E2F1, and T-cell acute lymphoblastic leukemia 1 (TAL1).

23. The agent according to claim 21, wherein the target DNA sequence is a gene derived from a microorganism selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), SARS-CoV-2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV-8), human T lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Salmonella typhi, Streptococcus bovine, and Mycoplasma.

24. The agent according to claim 19, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed from sgRNA and an endonuclease peptide or protein.

25. The agent according to claim 19, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA plasmid.

26. The agent according to claim 19, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA or RNA-based viral vector.

27. The agent according to claim 26, wherein the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia, and / or herpes simplex virus vector, or artificial virus.

28. The agent according to claim 19, wherein at least one component of the CRISPR-Cas system is delivered via at least one of liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

29. The agent according to claim 19, wherein at least one component of the CRISPR-Cas system is administered in a naked form via direct physical delivery.

30. The agent according to claim 19, wherein the sgRNA and CRISPR-related endonuclease or the gene encoding it are administered by different delivery hibicules.

31. The agent according to claim 19, wherein at least one of the genes encoding the sgRNA and CRISPR-related endonuclease is modified to include a cell-permeable peptide sequence.

32. The agent according to claim 19, wherein the sgRNA and at least one of the CRISPR-related endonuclease or the gene encoding it are administered by a route selected from the group consisting of injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, bioristics, particle gun acceleration, and combinations thereof.

33. The agent according to claim 19, wherein the alternating electric field is applied at a frequency in the range of approximately 100 kHz to approximately 10 MHz.

34. The agent according to claim 19, wherein the electric field strength of the alternating electric field is at least 1 V / cm.

35. The agent according to claim 19, further comprising the step of examining a sample of the biological tissue and identifying a target DNA sequence in the sample, wherein the at least one cell is part of a biological tissue.

36. An agent for use in a manner to treat or alleviate the occurrence of at least one symptom, disease, disorder, or infection in a subject, comprising a clustered and regularly arranged short palindromic sequence repeat (CRISPR)-related protein (CRISPR-Cas) system, The CRISPR-Cas system is A single guide RNA (sgRNA) comprising a guide sequence that hybridizes to at least one target DNA sequence associated with a symptom, disease, disorder, or infection, and CRISPR-related endonuclease or the gene encoding it Includes, The aforementioned method, (1) A step of applying an alternating electric field to at least a portion of the subject for a certain period of time, wherein the application of the alternating electric field causes at least one DNA damage repair pathway in at least a portion of the subject to be downregulated; (2) an agent comprising the step of administering the CRISPR-Cas system to the subject.

37. The agent according to claim 36, wherein the CRISPR-related endonuclease is selected from the group consisting of Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

38. The agent according to claim 36, wherein the at least one symptom, disease, disorder, or infection is selected from the group consisting of cancer, autoimmune disease, viral infection, bacterial infection, fungal infection, parasitic infection, metabolic syndrome, Huntington's disease, von Hippel-Lindau syndrome, and combinations thereof.

39. (a) The at least one symptom, disease, disorder, or infection is cancer, and the target DNA sequence is at least one oncological gain-of-function gene mutation. (b) The at least one symptom, disease, disorder, or infection is cancer, and the target DNA sequence is at least one loss-of-function mutation in a tumor suppressor gene. (c) The at least one symptom, disease, disorder, or infection is a bacterial infection, and the target DNA sequence is a bacterial sequence. (d) The at least one symptom, disease, disorder, or infection is a viral infection, and the target DNA sequence is a viral sequence. (e) The at least one symptom, disease, disorder, or infection is a fungal infection, and the target DNA sequence is a fungal sequence. (f) The at least one symptom, disease, disorder, or infection is a parasitic infection, and the target DNA sequence is a parasitic sequence. (g) The at least one symptom, disease, disorder, or infection is an autoimmune disease, and the target DNA sequence is an autoantigen sequence within an immune cell that produces autoantibodies. (h) The at least one symptom, disease, disorder, or infection is Huntington's disease, the target DNA sequence is a CAG repeat extension, and (i) The at least one symptom, disease, disorder, or infection is a metabolic syndrome, and the target DNA sequence is at least one mutation in a gene selected from the group consisting of FAH, TAT, and HPD. The agent according to claim 38, which satisfies at least one of the following conditions.

40. The target DNA sequence is a loss-of-function mutation in a tumor suppressor gene, and the tumor suppressor gene is p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras-related domain family (RASSF), ADP-ribosylation factor (ARF), adenomatous polyposis of the colon (APC), ataxia telangiectasia mutation (ATM), checkpoint kinase 2 (CHK2), breast cancer 1 protein ( The agent according to claim 39, selected from the group consisting of BRCA1), breast cancer 2 protein (BRCA2), BRCA2 partners and locators (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box class 03a (FOXO3a), p38, and von Hippel-Lindau (VHL).

41. The aforementioned target DNA sequence is an oncological gain-of-function gene mutation, and the gene is Ras (H-Ras, K-Ras, and N-Ras), mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase (ERK), c-Jun NH ２ The agent according to claim 39, selected from the group consisting of terminal kinase (JNK), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, fumarylacetoacetate hydrolase (FAH), TAT, 4-hydroxyphenylpyruvate dioxygenase (HPD), fms-related tyrosine kinase 3 (FLT3), c-KIT, BRAF, phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), telomerase reverse transcriptase (TERT), ETS mutant transcription factor 1 (ETV1), β-catenin (CTNNNB1), E2F1, and T-cell acute lymphoblastic leukemia 1 (TAL1).

42. The agent according to claim 39, wherein the target DNA sequence is the sequence of a bacterium or virus selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV and HCV, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), SARS-CoV-2 virus, herpes simplex virus (HSV), human herpesvirus 8 (HHV-8), human T lymphotropic virus-1 (HTLV-1), Merkel cell polyomavirus (MCPyV), cytomegalovirus (CMV), Helicobacter pylori, Chlamydia trachomatis, Salmonella typhi, Salmonella paratyphi A, Salmonella typhi, Streptococcus bovine, and Mycoplasma.

43. The agent according to claim 36, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed from sgRNA and an endonuclease peptide or protein.

44. The agent according to claim 36, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA plasmid.

45. The agent according to claim 36, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA or RNA-based viral vector.

46. The agent according to claim 45, wherein the viral vector is selected from the group consisting of adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia, and / or herpes simplex virus vector, or artificial virus.

47. The agent according to claim 36, wherein at least one component of the CRISPR-Cas system is delivered via at least one of liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

48. The agent according to claim 36, wherein at least one component of the CRISPR-Cas system is administered in a naked form via direct physical delivery.

49. The agent according to claim 36, wherein the sgRNA and the CRISPR-related endonuclease or the gene encoding it are administered by different delivery methods.

50. The agent according to claim 36, wherein at least one of the genes encoding the sgRNA and CRISPR-related endonuclease is modified to include a cell-permeable peptide sequence.

51. The agent according to claim 36, wherein the sgRNA and at least one of the CRISPR-related endonuclease or the gene encoding it are administered by a route selected from the group consisting of injection, infusion, topical application, transplantation, electroporation, lipofection, microinjection, microfluidics, bioristics, particle gun acceleration, and combinations thereof.

52. The agent according to claim 36, wherein the CRISPR-Cas system is administered approximately 24 to 48 hours after the start of application of an alternating electric field.

53. The agent according to claim 36, wherein the time for which the alternating electric field is applied is in the range of approximately 24 hours to approximately 72 hours.

54. The CRISPR-Cas system is administered after a certain period of time has elapsed, according to claim 36.

55. The agent according to claim 36, wherein the alternating electric field is applied at a frequency in the range of approximately 100 kHz to approximately 10 MHz.

56. The agent according to claim 36, wherein the electric field strength of the alternating electric field is at least 1 V / cm.

57. The agent according to claim 36, wherein steps (1) and (2) are repeated once or more times.

58. The agent according to claim 36, further comprising the step of examining a sample from the subject and identifying a target DNA sequence in the sample.