Systems and methods for treating conditions and diseases using alternating electric field and CRISPR-Cas system

Abstract

Systems and methods for inducing apoptosis and treating or reducing the onset of at least one condition, disease, disorder or infection in a subject are disclosed. The systems and methods rely on the application of an alternating electric field and the administration of a clustered regular spaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system. Kits for performing the methods disclosed herein are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS / INCORPORATION BY REFERENCE STATEMENT

[0002] not applicable.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] not applicable. Background Art

[0005] Tumor treatment fields (TTFields) are low intensity (e.g., 1 V / cm to 3 V / cm) alternating electric fields in the medium frequency range (100 kHz to 500 kHz) that target solid tumors by interfering with mitosis. This non-invasive treatment targets solid tumors and is described, for example, 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, and 10,441,776. TTFields are typically delivered by two pairs of transducer arrays that generate perpendicular fields within the tumor being treated; the transducer arrays that make up each of these pairs are positioned on opposite sides of the body part being treated. More specifically, for In a TTFields system, one pair of electrodes of the transducer array is located at the left and right sides (LR) of the tumor, and the other pair of electrodes of the transducer array is located at the anterior and posterior sides (AP) of the tumor. TTFields is approved for the treatment of glioblastoma multiforme (GBM) and can be used, for example, by system (Novocure Limited, St. Helier, Jersey), which The system includes a transducer array placed on the subject's head.

[0006] Used in Each transducer array in the device that delivers TTFields comprises a set of ceramic disc electrodes that are coupled to the subject's skin (e.g., but not limited to, the shaved head of a subject being treated for GBM) via a layer of conductive medical gel. The medical gel is intended to deform to match the contours of the body and provide good electrical contact between the array and the skin; thus, the gel interface bridges the skin and reduces interference. The device is intended to be worn continuously by the subject for 2 to 4 days, then removed, allowing the subject to perform hygiene care and re-shave the head (if necessary), followed by reapplication of a new set of arrays. In this way, the medical gel remains in essentially continuous contact with the subject's skin area for 2-4 days at a time, and the skin area is only uncovered and exposed to the environment for a brief period of time before more medical gel is applied to the skin.

[0007] TTFields have been shown to delay DNA damage repair mechanisms, particularly those responsible for repairing double-stranded DNA breaks; specifically, but not limited to, BRCA1 and Fanconi anemia proteins have been shown to have downregulated gene and protein expression following application of alternating electric fields to them (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).

[0008] Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-associated nuclease protein (called CRISPR-Cas9) is a gene editing system from prokaryotes and a key component of their adaptive immunity. This gene editing mechanism of the CRISPR-Cas9 system has been adapted and used in mammalian cells to silence or promote gene expression of precise sequences in DNA. The working mechanism of CRISPR-Cas9 is to recognize DNA elements with single guide RNAs for specific sequences of interest, and then the Cas9 enzyme cuts the double-stranded DNA gap (Zhang et al. (2021) Mol. Cancer, 20: 126; Afolabi et al. (2019) Immunology, 158 (2): 63-69). At this point, DNA repair mechanisms are used in the current use of the CRISPR-Cas9 system (Richardson et al. (2018) Nature Genetics, 50 (8): 1132-1139).

[0009] However, there is a need in the art for new and improved systems and methods to treat or reduce the occurrence of cancer and other conditions and diseases. The present invention is directed to such new and improved systems and methods. DETAILED DESCRIPTION

[0010] Before explaining at least one embodiment of the inventive concept in detail through exemplary language and results, it should be understood that the application of the inventive concept is not limited to the details of the construction and arrangement of the components set forth in the following description. These inventive concepts can have other embodiments or be practiced or implemented in various ways. Therefore, the language used herein is intended to give the broadest possible scope and meaning; and these embodiments are intended to be exemplary rather than exhaustive. In addition, it should be understood that the words and terms used herein are for descriptive purposes and should not be considered restrictive.

[0011] Unless otherwise defined herein, scientific and technical terms used in conjunction with the inventive concepts disclosed herein shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art, as described in various general and more specific references cited and discussed throughout this specification. Terms and laboratory procedures and techniques associated with analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry as described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0012] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the technical level of those skilled in the art to which the inventive concepts disclosed herein belong. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, as if each individual patent or publication was specifically and individually indicated to be incorporated herein by reference.

[0013] All compositions, assemblies, systems, kits and / or methods disclosed herein can be prepared and performed according to the present invention without undue experimentation. Although the compositions, assemblies, systems, kits and methods of the inventive concept have been described according to specific embodiments, it will be apparent to those skilled in the art that the steps or sequence of steps of the compositions and / or methods and methods described herein may be varied without departing from the concept, spirit and scope of the inventive concept. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the inventive concept as defined by the appended claims.

[0014] As used in accordance with the present invention, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0015] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Therefore, unless the context clearly indicates otherwise, the terms "a", "an", and "the" include plural referents. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or an increased number of compounds. The term "plurality" means "two or more".

[0016] The use of the term "at least one" should be understood to include one as well as any amount more than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, quantities of 100 / 1000 should not be considered limiting, as higher limits may also produce satisfactory results. Furthermore, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is used solely for the purpose of distinguishing two or more items and is not meant to imply, for example, any order or sequence or importance of one item relative to another or any order of addition.

[0017] The term "or" as used in the claims is intended to mean the inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0018] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. For example, the appearances of the phrase "in some embodiments" or "an example" in different places in the specification do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be interpreted as non-limiting with respect to the claims.

[0019] In this application, the term "about" is used to indicate that a value includes the inherent error variation of the components / devices / equipment, the method used to determine the value, or the variation that exists in the subject matter. For example, but not as a limitation, when the term "about" is used, the specified value can vary within 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 of the specified value, as such variations are suitable for performing the disclosed methods and as understood by those of ordinary skill in the art.

[0020] As used in this specification and claims, the words "comprising" (and any forms of including, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of including, such as "includes" and "include"), or "containing" (and any forms of including, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

[0021] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations containing repetitions of one or more terms or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of terms or terms in any combination unless otherwise apparent from the context.

[0022] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a large extent. For example, when related to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or 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, or that the two items are very close to each other, but not 100% adjacent to each other, 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.

[0023] The term "single guide RNA" or "sgRNA" refers to a DNA-targeting RNA that contains a guide sequence that targets a CRISPR-associated nuclease to a target genomic DNA and a scaffold sequence that interacts with the endonuclease (e.g., tracrRNA).

[0024] The terms "CRISPR-associated endonucleases", "Cas polypeptides" and "Cas nucleases" refer to a family of clustered regularly interspaced short palindromic repeat-associated polypeptides or nucleases that cleave DNA to produce blunt ends at double-strand breaks at sites specified by nucleotide guide sequences contained in crRNA transcripts. CRISPR-associated endonucleases require both crRNA and tracrRNA for site-specific DNA recognition and cleavage. The crRNA binds to the tracrRNA through a partially complementary region, guiding the endonuclease into a region of the target DNA that is homologous to the crRNA, called the "protospacer".

[0025] The term "ribonucleoprotein complex" or "RNP complex" refers to a complex comprising an sgRNA and a CRISPR-associated nuclease polypeptide.

[0026] The term "pharmaceutically acceptable" refers to compounds and compositions that are suitable for administration to humans and / or animals without undue side effects (such as, but not limited to, toxicity, irritation, and / or allergic response) and at a reasonable benefit / risk ratio.

[0027] As used herein, the term "patient" or "subject" includes both human and veterinary subjects. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including but not limited to humans, domestic and farm animals, non-human primates, and any other animal with mammary tissue.

[0028] The term "treatment" refers to therapeutic treatment as well as prophylactic or preventive measures. Those in need of treatment include, but are not limited to, individuals who already have a particular condition / disease / infection and individuals at risk of acquiring a particular condition / disease / infection (e.g., individuals in need of prophylactic / preventive measures). The term "treatment" refers to the administration of an agent / element / method to a patient for therapeutic and / or prophylactic / preventive purposes.

[0029] As used herein, the term "therapeutic composition" or "pharmaceutical composition" refers to an agent that can be administered in vivo to produce a therapeutic and / or prophylactic / preventive effect.

[0030] Administration of a therapeutically effective amount or a prophylactically effective amount is to provide a therapeutic benefit in the treatment, prevention and / or management of a disease, disorder and / or infection. The specific amount that is therapeutically effective can be readily determined by an ordinary physician and can vary according to factors known in the art, such as, but not limited to, the type of disorder / disease / infection, the patient's medical history and age, the stage of the disorder / disease / infection, and the combined administration of other drugs.

[0031] The term "effective amount" refers to the amount of a biologically active molecule or its conjugate or derivative, or the amount of a treatment regimen (i.e., an alternating electric field), which, when used in the manner of the inventive concept, is sufficient to show a detectable therapeutic effect without excessive adverse side effects (such as, but not limited to, toxicity, irritation, and allergic reactions), and a reasonable benefit / risk ratio is comparable. The therapeutic effect may include, for example, but not limited to, preventing, inhibiting, or reducing the occurrence of at least one disorder, disease, and / or infection. The effective amount of the subject will depend on the type of subject, the size and health of the subject, the nature and severity of the disorder / disease / infection to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulation used, etc. Therefore, it is impossible to predetermine an exact effective amount. However, the effective amount for a given situation can be determined by a person of ordinary skill in the art using routine experiments based on the information provided herein.

[0032] As used herein, the term "concurrent therapy" is used interchangeably with the terms "combination therapy" and "adjuvant therapy" and will be understood to mean that a patient in need of treatment is treated with or administered another drug for the condition / disease / infection in combination with the treatment of the present invention. Such concurrent therapy may be sequential therapy, wherein the patient is first treated with one therapeutic regimen / drug composition and then treated with another therapeutic regimen / drug composition, or both therapeutic regimens / drug compositions are administered simultaneously.

[0033] Turning now to the inventive concept, systems, kits and methods for inducing apoptosis and / or treating or reducing the occurrence of at least one condition, disease, disorder or infection in a subject are provided. The system, kit and method combine the application of an alternating electric field with a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system. Alternating electric fields (such as but not limited to TTFields) delay DNA damage repair mechanisms, particularly mechanisms responsible for repairing double-stranded DNA breaks. CRISPR-Cas is a gene editing system that has been used in mammalian cells to silence or promote gene expression of precise sequences in DNA. The CRISPR-Cas9 system recognizes DNA elements by single guide RNA (sgRNA) for a specific sequence of interest, and then the Cas9 enzyme cuts in the double-stranded DNA gap. Currently, the CRISPR-Cas9 system uses a DNA repair mechanism. However, in the present invention, since the application of an alternating electric field inhibits the DNA damage repair mechanism, the DNA gap formed when exposed to the CRISPR-Cas system will not be repaired; therefore, cells exposed to a combination of an alternating electric field and a CRISPR-Cas system will undergo apoptosis.

[0034] This ability to efficiently induce apoptosis only in mutant cells (or other target cells of interest that uniquely possess a specific sequence to which the sgRNA can be directed) provides patients with a treatment option that has fewer toxicities and / or side effects compared to systemic therapies such as (but not limited to) chemotherapy.

[0035] Certain non-limiting embodiments of the present invention include methods of inducing apoptosis in at least one cell. In the method, an alternating electric field is applied to at least one cell for a period of time, and the application of the alternating electric field downregulates at least one DNA damage repair pathway in at least one cell. At least one cell is also exposed to a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system, which includes: (i) a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence in at least one cell; and (ii) a CRISPR-associated nuclease or a gene encoding it.

[0036] Certain non-limiting embodiments of the present invention include methods for treating or reducing the occurrence of at least one condition, disease, disorder, or infection in a subject. In the method, an alternating electric field is applied to at least a portion of the subject for a period of time, and the application of the alternating electric field downregulates at least one DNA damage repair pathway in at least a portion of the subject. A CRISPR-Cas system is also administered to the subject, and the CRISPR-Cas system includes: (i) a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one condition, disease, disorder, or infection; and (ii) a CRISPR-associated nuclease or a gene encoding the same.

[0037] In accordance with the present invention, any type of conductive or non-conductive electrodes and / or transducer arrays that can be used to generate an alternating electric field may be applied to the cells / subject to generate the alternating electric field, the electrodes and / or transducer arrays being known in the art or otherwise contemplated herein. According to the present invention, non-limiting examples of electrodes and transducer arrays that can be used to generate alternating electric fields include electrodes and transducer arrays as part of a TTFields system, such as, but not limited to, those described 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, 10,441,776, and 11,452,863; and U.S. Patent Application Nos. US2018 / 0001078, US2018 / 0160933, and US No. 2019 / 0117956, No. US2019 / 0307781 and No. US2019 / 0308016.

[0038] According to the present invention, the alternating electric field can be generated at any frequency that downregulates at least one DNA damage repair pathway in at least a portion of the cells / subjects. For example (but not limited to), the frequency of the alternating electric field can be about 50kHz, about 75kHz, about 100kHz, about 125kHz, about 150kHz, about 175kHz, about 200kHz, about 225kHz, about 250kHz, about 275kHz, about 300kHz, about 325kHz, about 350kHz, about 375kHz, about 400kHz, about 425kHz, about 650kHz, about 700kHz, about 750kHz, about 800kHz, about 850kHz, about 900kHz, about 950kHz, about 1MHz, about 2MHz, about 3MHz, about 4MHz, about 5MHz, about 6MHz, about 7MHz, 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 by any of the above values ​​(for example, 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 a range combining two integers falling between the above two values ​​(for example, a range from about 122 kHz to about 313 kHz, a range from about 78 kHz to about 298 kHz, etc.).

[0039] In certain specific (but non-limiting) embodiments, the alternating electric field may be applied at two or more different frequencies simultaneously or sequentially during treatment (i.e., the frequency may be changed during treatment). When there are two or more frequencies, each frequency is selected from any of the above values, or a range formed by any of the above values, or a range combining two integers falling between two of the above values.

[0040] According to the present invention, the alternating electric field may have any field strength as long as the alternating electric field is capable of down-regulating at least one DNA damage repair pathway in at least a portion of the cells / subject. For example, but not limited to, the strength of 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., and ranges formed by any of the above values ​​(for example, a range of about 1 V / cm to about 20 V / cm, a range of about 1 V / cm to about 10 V / cm, a range of about 1 V / cm to about 4 V / cm, etc.), and a range combining two integers falling between two of the above values ​​(for example, a range of about 1.1 V / cm to about 18.6 V / cm, a range of about 1.2 V / cm to about 9.8 V / cm, a range of about 1.3 V / cm to about 4.7 V / cm, etc.).

[0041] The alternating electric field can be applied for any period of time sufficient to downregulate at least one DNA damage repair pathway in at least a portion of the cells / 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 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, about 100 hours, about 101 hours, about 102 hours, about 103 hours, about 104 hours, about , 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., as well as ranges formed by any of the above values ​​(e.g., a range of about 24 hours to about 72 hours), and a range combining two integers falling between two of the above values ​​(e.g., a range of about 14 hours to about 68 hours, etc.).

[0042] The total period of time for applying the alternating electric field can be implemented in a continuous or intermittent manner. That is, when the time for applying the alternating electric field is short (for example, but not limited to, less than about 12 or 24 hours), the alternating electric field can be applied continuously during this period of time. However, when the alternating electric field is applied for a longer period of time (for example, but not limited to, a period of about 24 hours or longer), the treatment period can include one or more interruptions during the application period, which separates two or more application parts, so that the application parts and the interruption combination form the total application period. When there is an interruption, the interruption should generally account for about 50%, about 40%, about 30%, about 20% or less of the treatment time, so 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, in each 24-hour period, the alternating electric field should be applied for at least about 19 hours. In addition, the longer the time of applying the alternating electric field, the higher the efficacy.

[0043] According to the present invention, any CRISPR-Cas system known in the art or contemplated herein can be used, as long as the system is capable of generating double-stranded DNA gaps on the target sequence of interest. CRISPR-Cas systems are well known and have been extensively reviewed (e.g., see 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; see, for example, Merck / MilliporeSigma (Burlington, MA), Takara Bio USA, Inc. (San Jose, CA), ThermoFisher Scientific (Waltham, MA), Integrated DNA Technologies, Inc. (Coralville, IA), GenScript Biotech Corp (Piscataway, NJ), ACROBiosystems (Newark, DE), Mirus Bio LLC (Madison, WI), etc.

[0044] The CRISPR-Cas system will include any CRISPR-associated nuclease or gene encoding the enzyme, which is known in the art or contemplated herein for use in a CRISPR-Cas system to recognize and cleave a target DNA complementary to a guide RNA. Non-limiting examples of Cas endonucleases that can be used according to the present invention include Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, Cas9 nickase (nCas9), etc., and variants and derivatives thereof, and any combination thereof.

[0045] Depending on the particular Cas endonuclease used, the sgRNA of the CRISPR-Cas system will contain a specific sequence specific to the Cas endonuclease, which is recognized by the endonuclease and guides the endonuclease to perform sequence-specific cleavage in the target DNA sequence. The selection and / or generation of sgRNAs containing these endonuclease guide sequences and sequences that hybridize to the target DNA sequence are well within the knowledge of those of ordinary skill in the art, and therefore no further description thereof is deemed necessary.

[0046] In certain non-limiting embodiments, the methods of the present invention can be applied to various disorders in which mutations occur and result in silencing or overactivation of damaged genes, or disorders involving exogenous genes / sequences. For example (but not limited to), at least one disorder, disease, disorder or infection is selected from cancer, autoimmune disease, viral infection, bacterial infection, fungal infection, parasitic infection, metabolic syndrome, Huntington's disease, Hippel-Lindau syndrome, and combinations thereof.

[0047] Specific non-limiting examples of conditions, diseases, disorders and infections that may be treated according to the present invention include:

[0048] Cancers involving oncologically acquired functional gene mutations in oncogenes (e.g., but not limited to, see 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 functional mutations in tumor suppressor genes (e.g., but not limited to, see Chen et al. (2020) Sig Transduct Target Ther, 5:90);

[0049] Autoimmune diseases, in which the sgRNAs of the CRISPR-Cas system are directed against specific sequences of self-antigens, thereby killing immune cells that inappropriately target “self” cells (i.e., immune cells that produce autoantibodies);

[0050] Infection, where the sgRNA of the CRISPR-Cas system is directed against one or more genes / sequences from an infectious microorganism (e.g., but not limited to, bacteria, parasites, fungi, or viruses)—see, for example, Hu et al. (2014) BioMed Research International, Article ID 612823. In addition, the CRISPR-Cas9 system has been used to target the LTR region of the HIV provirus at positions T5 and T6 (Ebina et al. (2013) Sci Rep, 3:2510);

[0051] Metabolic syndrome, such as (but not limited to) hereditary tyrosinemia (Pankowicz et al. (2016) Nat Commun, 7: 12642), wherein the sgRNA of the CRISPR-Cas system targets (for example, but not limited to) mutations in the FAH, TAT, and HPD genes that cause type I, type II, and type III tyrosinemia, respectively;

[0052] Huntington's disease, wherein the sgRNA of the CRISPR-Cas system targets a CAG repeat expansion, because Huntington's disease is characterized by the loss of brain striatal neurons due to a CAG repeat expansion in the huntingtin protein (for example (but not limited to), see Cattaneo et al., (2001) Trends in Neurosciences, 24(3): 182-188).

[0053] In specific (but non-limiting) embodiments, the target DNA sequence is directed against a mutation in a gene selected from the group consisting of: p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), chaperone and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), 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), p38, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, VHL, fumaryl 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. and combinations thereof.

[0054] In certain specific (but non-limiting) embodiments, the target DNA sequence is a loss-of-function mutation in a tumor suppressor gene; examples of tumor suppressor genes include (but are not limited to) p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), partner and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), p38, VHL disease (VHL), etc. and any combination thereof.

[0055] In certain specific (but non-limiting) embodiments, the target DNA sequence is an oncological gain of function gene mutation (i.e., an oncogene); examples of oncogenes include (but are not limited to) Ras (H-Ras, K-Ras and N-Ras), mitogen-activated protein 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, fumaryl 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. and any combination thereof.

[0056] In certain specific (but non-limiting) embodiments, the target DNA sequence is a gene from a microorganism selected from human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), new coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis, Mycoplasma, etc. and any combination thereof.

[0057] CRISPR-Cas system cargo can be administered to cells / subjects in any form, by any delivery vector, and by any delivery mechanism / route known or otherwise contemplated herein for delivering CRISPR-Cas system cargo, as long as the form and delivery vector are capable of transporting the CRISPR-Cas system into the cell of interest and facilitating penetration into the nucleus, thereby facilitating the generation of double-stranded DNA nicks at the target sequence of interest. CRISPR-associated nucleases or genes encoding them can be present in the form of proteins, peptides, DNA molecules, RNA molecules (e.g., but not limited to mRNA molecules), or any combination thereof, depending on the delivery vector used.

[0058] sgRNA and CRISPR-associated nuclease (or gene encoding it) can be delivered by a single delivery vector, or can be separated between two different delivery vectors (which can be the same or different types of vectors). In certain non-limiting embodiments, the CRISPR-Cas system can be delivered as a ribonucleoprotein (RNP) complex formed by sgRNA and nuclease peptide or protein. In certain non-limiting embodiments, one or two components of the CRISPR-Cas system can be delivered by one or more DNA plasmids containing sgRNA sequences and / or encoding nuclease sequences. In other non-limiting embodiments, one or two components of the CRISPR-Cas system can be delivered by one or more DNA or RNA-based viral vectors (such as but not limited to adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia virus and / or herpes simplex virus vectors, etc.) or artificial viruses. In other non-limiting embodiments, one or two components of the CRISPR-Cas system can be delivered by liposomes and / or lipid nanoparticles, immunoliposomes, lipid complexes, polycomplexes or poloxamer, polycations, virosomes, gold nanoparticles or other inorganic nanoparticles. In other non-limiting embodiments, one or both components of the CRISPR-Cas system are delivered in naked form by direct physical delivery. In further non-limiting embodiments, sgRNA is delivered by one of the above-mentioned vectors, while CRISPR-associated nuclease (or nucleic acid sequence encoding it) is delivered by another of the above-mentioned vectors.

[0059] In certain non-limiting embodiments, one or both components of the CRISPR-Cas system and / or the delivery vector may be modified, for example (but not limited to) to further aid in the transport of the CRISPR-Cas system into cancer cells, to enhance solubility, to monitor transport, etc. For example (but not limited to), the sgRNA, the endonuclease (or the gene encoding the same), and / or the delivery vector may be engineered 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.

[0060] The components of the CRISPR-Cas system can be delivered by any delivery mechanism / route known in the art or contemplated herein. Non-limiting examples of delivery mechanisms / routes include injection, infusion, topical administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, etc. and any combination thereof.

[0061] The alternating electric field can be applied to any part of the subject, which will result in downregulating at least one DNA damage repair pathway in the desired part of the cell / subject. In certain specific (but non-limiting) embodiments, the alternating electric field is targeted to a portion of the subject containing a gene or gene mutation to be targeted. For example (but not limited to), when there is at least one solid tumor, the alternating electric field can be applied to a portion of the subject containing the solid tumor. Alternatively, when the treatment is for an infection of the subject, the alternating electric field can be applied to at least a portion of the infected site on or in the subject (for example, but not limited to, the thymus or infected site of the human papillomavirus-infected cervix, HIV infection, and AIDS-induced conditions (for example, but not limited to, HIV-induced pneumonia)). In other alternatives, when the treatment is targeted at autoimmune diseases, the alternating electric field can be applied to the drainage lymph nodes of the inflammatory target; when the target of the treatment is metabolic syndrome such as (but not limited to) hereditary tyrosinemia, the alternating electric field can be applied to the liver; when the treatment is for Huntington's disease, the alternating electric field can be applied to the brain.

[0062] The method of the present invention may include one or more additional steps. For example, but not limited to, the method may include one or more steps that help identify the subject to be treated, one or more additional administration steps (for example, but not limited to, the administration of at least one substance for simultaneous therapy), and / or one or more steps for analyzing the effect of treatment.

[0063] In a non-limiting embodiment, the method further comprises a step of examining the sample and identifying the target DNA sequence in the sample. This step can be performed before and / or after one or both of the alternating electric field application and the CRISPR-Cas system application steps.

[0064] In certain specific (but non-limiting) embodiments, the CRISPR-Cas system can be applied after the application of the alternating electric field has begun. In particular (but not limited to), the CRISPR-Cas system can be applied during the application of the alternating electric field and / or after the application of the alternating electric field has passed.

[0065] For example, but not limited to, the CRISPR-Cas system can be administered after the application of the alternating electric field has begun for 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., as well as ranges formed by any of the above values ​​(e.g., a range of about 24 hours to about 96 hours, a range of 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 of about 14 hours to about 94 hours, etc.). In a specific (but non-limiting) embodiment, the at least one substance is orally administered at least about 24 hours after application of the alternating electric field has been initiated.

[0066] In other non-limiting embodiments, the CRISPR-Cas system can be administered after a period of time has elapsed since the alternating electric field was applied, wherein 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. of the period of time. In a specific (but non-limiting) embodiment, the CRISPR-Cas system is administered within about 96 hours of the period of time.

[0067] In certain specific (but 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 ceasing to apply the alternating electric field (for example, but not limited to) to allow cells / tissue to recover. In addition, any step may be repeated one or more times. Each step may be repeated as many times as desired. When the steps involving applying the alternating electric field are repeated, the transducer array may be placed at a slightly different location on the subject than its original location; repositioning the array in this manner may further improve the effectiveness of the treatment and may also minimize any adverse skin effects caused by the alternating electric field. In addition, the steps involving the administration of the CRISPR-Cas system may be repeated different times and at different intervals to follow any known and / or generally accepted system protocol.

[0068] Certain non-limiting embodiments of the present invention are directed to kits comprising any components of an alternating electric field generating system (e.g., but not limited to, one or more transducer arrays and / or one or more hydrogel compositions, 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, 8,764,676, 8,764,677, 8,764,678, 8,764,679, 8,764,671, 8,764,673, 8,764,674, 8,764,675, 8,764,676, 8,764,67 ... 10,188,851, 10,441,776 and 11,452,863; and U.S. Patent Application Nos. US2018 / 0001078, US2018 / 0160933, US2019 / 0117956, US2019 / 0307781 and US2019 / 0308016) and one or more of any CRISPR-Cas systems disclosed or otherwise contemplated herein. In particular (but not by way of limitation), the kit may include: (i) at least one pair of transducer arrays for generating an alternating electric field between the transducer arrays when applied to at least one cell and / or placed on a subject; and (ii) a CRISPR-Cas system comprising: a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one condition, disease, disorder or infection, and a CRISPR-associated nuclease or a gene encoding the same. The condition, disease, disorder or infection and the target DNA sequence may be any sequence disclosed or contemplated herein.

[0069] In a specific (but non-limiting) embodiment, the kit may also include instructions for practicing 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 the alternating electric field to a specific part of the patient, instructions on when and how to apply the CRISPR-Cas system, and / or instructions on when to activate and deactivate the alternating electric field associated with applying the CRISPR-Cas system.

[0070] In addition to the components described in detail above, the kit may also contain other components / reagents for practicing any specific method described or contemplated herein. For example (but not by way of limitation), the kit may additionally include: (i) components for preparing the skin prior to treatment of the hydrogel composition and / or the transducer array thereon (e.g., a razor, a cleaning composition, or a wipe / towel, etc.); (ii) components for removing the gel / sensor array; and / or (iii) components for cleaning the skin after removing the gel / transducer array. Alternatively (and / or in addition), the kit may additionally include at least one device and / or at least one reagent for examining a sample to identify the presence of a target DNA sequence in the sample, and / or at least one device and / or at least one reagent for analyzing the effectiveness of a treatment regimen.

[0071] The nature of the various additional components / agents that may optionally be present in the kit will depend on the particular therapeutic modality, the identification of which is well within the skills of one of ordinary skill in the art; therefore, further description thereof is not deemed necessary.

[0072] Depending on the sterility, cross-reactivity and stability of the components / reagents, the components / reagents present in the kit may each be located in a separate container / compartment, or the various components / reagents may be combined in one or more containers / compartments.

[0073] The test kit can be placed in any package that allows the components present therein to function according to the present invention. In certain non-limiting embodiments, the test kit also includes a sealed package in which the components are placed. In certain specific (but non-limiting) embodiments, the sealed package is substantially airtight and / or substantially lighttight.

[0074] In addition, the kit may also include a set of written instructions explaining how to use one or more components of the kit. Kits of this nature can be used in any method described or contemplated herein.

[0075] In certain non-limiting embodiments, the kit has a shelf life of at least about 6 months, for example (but not limited to), at least about 9 months, or at least about 12 months.

[0076] Non-limiting illustrative embodiments of the inventive concept

[0077] Illustrative Example 1. A method of inducing apoptosis in at least one cell, the method comprising the steps of: (1) applying an alternating electric field to at least one cell for a period of time, wherein the application of the alternating electric field downregulates at least one DNA damage repair pathway in the at least one cell; and (2) exposing the at least one cell to a clustered regularly interspaced short palindromic repeats (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 the at least one cell; and a CRISPR-associated nuclease or a gene encoding the same.

[0078] Illustrative embodiment 2. The method according to claim 1, wherein the CRISPR-associated nuclease is selected from Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9 and Cas9 nickase (nCas9).

[0079] Illustrative Example 3. The method according to Illustrative Example 1 or 2, wherein the target DNA sequence is selected from 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 producing autoantibodies.

[0080] Illustrative Example 4. According to the method of illustrative embodiment 3, the target DNA sequence is directed to a mutation in a gene selected from the group consisting of p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), chaperone and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), 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), p38, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, VHL, fumaryl 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, and T-cell acute lymphoblastic leukemia 1 (TAL1).

[0081] Illustrative Example 5. The method according to Illustrative Example 3, wherein the target DNA sequence is a gene from a microorganism selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), novel coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis and Mycoplasma.

[0082] Illustrative Example 6. The method according to any one of Illustrative Examples 1 to 5, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed by the sgRNA and the endonuclease peptide or protein.

[0083] Illustrative Example 7. The method according to any one of Illustrative Examples 1 to 6, wherein at least one component of the CRISPR-Cas system is delivered by at least one DNA plasmid.

[0084] Illustrative Example 8. The method according to any one of Illustrative Examples 1 to 7, wherein at least one component of the CRISPR-Cas system is delivered by at least one DNA or RNA based viral vector.

[0085] Illustrative Example 9. The method according to Illustrative Example 8, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia virus and / or herpes simplex virus vectors or artificial viruses.

[0086] Illustrative embodiment 10. The method according to any one of illustrative embodiments 1 to 9, wherein at least one component of the CRISPR-Cas system is delivered by at least one of the following: liposomes, lipid nanoparticles, immunoliposomes, lipid complexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

[0087] Illustrative Example 11. The method according to any one of Illustrative Examples 1 to 10, wherein at least one component of the CRISPR-Cas system is administered in naked form by direct physical delivery.

[0088] Illustrative Example 12. The method according to any one of Illustrative Examples 1 to 11, wherein the sgRNA and the CRISPR-associated nuclease or the gene encoding the same are administered by different delivery vectors.

[0089] Illustrative Example 13. The method according to any one of Illustrative Examples 1 to 12, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated nuclease is modified to include a cell penetrating peptide sequence.

[0090] Illustrative embodiment 14. The method according to any one of illustrative embodiments 1 to 13, wherein at least one of the sgRNA and the CRISPR-associated nuclease or the gene encoding the same is administered by a route selected from the group consisting of injection, infusion, local administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

[0091] Illustrative Embodiment 15. The method according to any one of Illustrative 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.

[0092] Illustrative Embodiment 16. The method according to any one of Illustrative Embodiments 1 to 15, wherein the alternating electric field has a field strength of at least 1 V / cm.

[0093] Illustrative embodiment 17. The method according to any one of illustrative embodiments 1 to 16, wherein the at least one cell is part of a biological tissue, and wherein the method further comprises the step of examining a sample of the biological tissue and identifying the target DNA sequence in the sample.

[0094] Illustrative Example 18. A method of treating or reducing the occurrence of at least one condition, disease, disorder, or infection in a subject, the method comprising the steps of: (1) applying an alternating electric field to at least a portion of the subject for a period of time, wherein the application of the alternating electric field downregulates at least one DNA damage repair pathway in at least a portion of the subject; and (2) administering to the subject a clustered regularly interspaced short palindromic repeats (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 condition, disease, disorder, or infection; and a CRISPR-associated nuclease or a gene encoding the same.

[0095] Illustrative embodiment 19. The method according to illustrative embodiment 18, wherein the CRISPR-associated nuclease is selected from Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9 and Cas9 nickase (nCas9).

[0096] Illustrative embodiment 20. The method according to illustrative embodiment 18 or 19, wherein at least one condition, disease, disorder or infection is selected from cancer, autoimmune disease, viral infection, bacterial infection, fungal infection, parasitic infection, metabolic syndrome, Huntington's disease, Hippel-Lindau syndrome, and combinations thereof.

[0097] Illustrative Example 21. The method according to Illustrative Example 20, wherein at least one of the following: (a) at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one oncological gain of function gene mutation; (b) at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one loss of function mutation in a tumor suppressor gene; (c) at least one condition, disease, disorder or infection is a bacterial infection, and wherein the target DNA sequence is a bacterial sequence; (d) at least one condition, disease, disorder or infection is a viral infection, and wherein the target DNA sequence is a viral sequence; (e) at least one condition, disease, disorder or infection is a fungal infection. (f) at least one condition, disease, disorder or infection is a parasitic infection and wherein the target DNA sequence is a parasitic sequence; (g) at least one condition, disease, disorder or infection is an autoimmune disease and wherein the target DNA sequence is a self-antigen sequence in an immune cell that produces an autoantibody; (h) at least one condition, disease, disorder or infection is Huntington's disease and wherein the target DNA sequence is a CAG repeat expansion; and (i) at least one condition, disease, disorder or infection is metabolic syndrome and wherein the target DNA sequence is at least one mutation in a gene selected from FAH, TAT and HPD.

[0098] Illustrative embodiment 22. The method according to illustrative embodiment 21, wherein the target DNA sequence is a loss-of-function mutation in a tumor suppressor gene, and wherein the tumor suppressor gene is selected from p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), partner and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), p38 and VHL.

[0099] Illustrative Example 23. The method according to Illustrative Example 21, wherein the target DNA sequence is an oncological gain of function gene mutation, wherein the gene is selected from Ras (H-Ras, K-Ras and N-Ras), mitogen-activated protein 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, fumaryl 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 and T-cell acute lymphoblastic leukemia 1 (TAL1).

[0100] Illustrative Example 24. The method according to Illustrative Example 21, wherein the target DNA sequence is a sequence from a bacterium or virus selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), novel coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis and Mycoplasma.

[0101] Illustrative embodiment 25. The method according to any one of illustrative embodiments 18 to 24, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed by the sgRNA and the endonuclease peptide or protein.

[0102] Illustrative embodiment 26. The method according to any one of illustrative embodiments 18 to 25, wherein at least one component of the CRISPR-Cas system is delivered by at least one DNA plasmid.

[0103] Illustrative embodiment 27. The method according to any one of illustrative embodiments 18 to 26, wherein at least one component of the CRISPR-Cas system is delivered by at least one DNA or RNA based viral vector.

[0104] Illustrative Example 28. The method according to Illustrative Example 27, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia virus and / or herpes simplex virus vectors or artificial viruses.

[0105] Illustrative embodiment 29. The method according to any one of illustrative embodiments 18 to 28, wherein at least one component of the CRISPR-Cas system is delivered by at least one of the following: liposomes, lipid nanoparticles, immunoliposomes, lipid complexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

[0106] Illustrative embodiment 30. The method according to any one of illustrative embodiments 18 to 29, wherein at least one component of the CRISPR-Cas system is administered in naked form by direct physical delivery.

[0107] Illustrative Example 31. The method according to any one of illustrative examples 18 to 30, wherein the sgRNA and the CRISPR-associated nuclease or the gene encoding the same are administered by different delivery methods.

[0108] Illustrative embodiment 32. The method according to any one of illustrative embodiments 18 to 31, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated nuclease is modified to include a cell penetrating peptide sequence.

[0109] Illustrative embodiment 33. The method according to any one of illustrative embodiments 18 to 32, wherein at least one of the sgRNA and the CRISPR-associated nuclease or the gene encoding the same is administered by a route selected from the group consisting of injection, infusion, local administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

[0110] Illustrative embodiment 34. The method according to any one of illustrative embodiments 18 to 33, wherein the CRISP-Cas system is administered about 24 hours to about 48 hours after the start of application of the alternating electric field.

[0111] Illustrative Embodiment 35. The method according to any one of Illustrative Embodiments 18 to 34, wherein the alternating electric field is applied for a period of time in the range of about 24 hours to about 72 hours.

[0112] Illustrative embodiment 36. The method according to any one of illustrative embodiments 18 to 35, wherein the CRISP-Cas system is administered after a period of time has passed.

[0113] Illustrative Embodiment 37. The method according to any one of Illustrative 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.

[0114] Illustrative Embodiment 38. The method according to any one of Illustrative Embodiments 18 to 37, wherein the alternating electric field has a field strength of at least 1 V / cm.

[0115] Illustrative Example 39. The method according to any one of Illustrative Examples 18 to 38, wherein steps (1) and (2) are repeated one or more times.

[0116] Illustrative embodiment 40. The method according to any one of illustrative embodiments 18 to 39, wherein the method further comprises the step of examining a sample from the subject and identifying the target DNA sequence in the sample.

[0117] Illustrative Example 41. A kit comprising: at least one pair of transducer arrays for generating an alternating electric field therebetween when the transducer arrays are applied to at least one cell and / or when the transducer arrays are placed on a subject; and a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system comprising: a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one condition, disease, disorder, or infection; and a CRISPR-associated nuclease or a gene encoding the same.

[0118] Illustrative embodiment 42. The kit according to illustrative embodiment 41, wherein the CRISPR-associated nuclease is selected from Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9 and Cas9 nickase (nCas9).

[0119] Illustrative Example 43. The kit of Illustrative Example 41 or 42, wherein at least one of the following: (a) at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one oncological gain of function gene mutation; (b) at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one loss of function mutation in a tumor suppressor gene; (c) at least one condition, disease, disorder or infection is a bacterial infection, and wherein the target DNA sequence is a bacterial sequence; (d) at least one condition, disease, disorder or infection is a viral infection, and wherein the target DNA sequence is a viral sequence; (e) at least one condition, disease, disorder or infection is a viral infection. (f) at least one condition, disease, disorder or infection is a parasitic infection and wherein the target DNA sequence is a parasitic sequence; (g) at least one condition, disease, disorder or infection is an autoimmune disease and wherein the target DNA sequence is an autoantigen sequence in an immune cell that produces an autoantibody; (h) at least one condition, disease, disorder or infection is Huntington's disease and wherein the target DNA sequence is a CAG repeat expansion; and (i) at least one condition, disease, disorder or infection is metabolic syndrome and wherein the target DNA sequence is at least one mutation in a gene selected from FAH, TAT and HPD.

[0120] Illustrative Example 44. A kit according to illustrative embodiment 43, wherein the target DNA sequence is a loss-of-function mutation in a tumor suppressor gene, and wherein the tumor suppressor gene is selected from p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), partner and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), p38 and VHL.

[0121] Illustrative Example 45. A kit according to illustrative example 43, wherein the target DNA sequence is an oncological gain of function gene mutation, wherein the gene is selected from Ras (H-Ras, K-Ras and N-Ras), mitogen-activated protein 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, fumaryl 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 and T-cell acute lymphoblastic leukemia 1 (TAL1).

[0122] Illustrative embodiment 46. The kit according to illustrative embodiment 43, wherein the target DNA sequence is a sequence from a bacterium or virus, and the bacterium or virus is selected from human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), new coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis and Mycoplasma.

[0123] Illustrative embodiment 47. The kit according to any one of illustrative embodiments 41 to 46, wherein the CRISPR-Cas system is in the form of a ribonucleoprotein (RNP) complex formed by sgRNA and endonuclease peptide or protein.

[0124] Illustrative embodiment 48. The kit according to any one of illustrative embodiments 41 to 47, wherein at least one component of the CRISPR-Cas system is located in at least one DNA plasmid.

[0125] Illustrative embodiment 49. The kit according to any one of illustrative embodiments 41 to 48, wherein at least one component of the CRISPR-Cas system is located in at least one DNA or RNA based viral vector.

[0126] Illustrative embodiment 50. The kit according to illustrative embodiment 49, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia virus and / or herpes simplex virus vectors or artificial viruses.

[0127] Illustrative embodiment 51. The kit according to any one of illustrative embodiments 41 to 50, wherein at least one component of the CRISPR-Cas system is located in at least one of the following: liposomes, lipid nanoparticles, immunoliposomes, lipoplexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

[0128] Illustrative embodiment 52. The kit according to any one of illustrative embodiments 41 to 51, wherein at least one component of the CRISPR-Cas system is provided in naked form for direct physical delivery.

[0129] Illustrative Example 53. The kit according to any one of illustrative examples 41 to 52, wherein the sgRNA and the CRISPR-associated nuclease or the gene encoding the same are located in different delivery vectors.

[0130] Illustrative embodiment 54. The kit according to any one of illustrative embodiments 41 to 53, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated nuclease is modified to include a cell penetrating peptide sequence.

[0131] Illustrative embodiment 55. The kit according to any one of illustrative embodiments 41 to 54, wherein at least one of the sgRNA and the CRISPR-associated nuclease or the gene encoding the same is formulated for administration by a route selected from the group consisting of injection, infusion, local administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

[0132] Illustrative Embodiment 56. The kit according to any one of Illustrative 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.

[0133] Illustrative Embodiment 57. The kit according to any one of Illustrative Embodiments 41 to 56, wherein the alternating electric field is configured to be applied with a field strength of at least 1 V / cm.

[0134] Illustrative embodiment 58. The kit according to any one of illustrative embodiments 41 to 57, further comprising at least one device and / or at least one reagent for examining a sample to identify a target DNA sequence in the sample.

[0135] Illustrative Example 59. A clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (CRISPR-Cas) system for use in the methods of any one of Illustrative Examples 1 to 40, the CRISPR-Cas 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 nuclease or a gene encoding the same.

[0136] Illustrative Example 60. Use of a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system in the method of any one of Illustrative Examples 1 to 40, the CRISPR-Cas 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 nuclease or a gene encoding the same.

[0137] Although the attached disclosure describes the concept of the present invention in conjunction with the specific experiments, results and language set forth below, it is apparent that many substitutions, modifications and variations are apparent to those skilled in the art. Therefore, it is intended to include all such substitutions, modifications and variations that fall within the spirit and broad scope of the present invention.

Claims

1. A kit comprising: at least one pair of transducer arrays for generating an alternating electric field therebetween when the transducer arrays are applied to at least one cell and / or when the transducer arrays are placed on a subject; as well as A clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system comprising: a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one condition, disease, disorder, or infection; as well as CRISPR-associated endonuclease or a gene encoding the same.

2. The method of claim 1, wherein the CRISPR-associated nuclease is selected from Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

3. The kit according to claim 1 or 2, wherein at least one of the following: (a) the at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one oncological gain of function gene mutation; (b) the at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one loss-of-function mutation in a tumor suppressor gene; (c) the at least one condition, disease, disorder or infection is a bacterial infection, and wherein the target DNA sequence is a bacterial sequence; (d) the at least one condition, disease, disorder or infection is a viral infection, and wherein the target DNA sequence is a viral sequence; (e) the at least one condition, disease, disorder or infection is a fungal infection, and wherein the target DNA sequence is a fungal sequence; (f) the at least one condition, disease, disorder or infection is a parasitic infection, and wherein the target DNA sequence is a parasitic sequence; (g) the at least one condition, disease, disorder or infection is an autoimmune disease, and wherein the target DNA sequence is an autoantigen sequence in an autoantibody-producing immune cell; (h) the at least one condition, disease, disorder or infection is Huntington's disease, and wherein the target DNA sequence is a CAG repeat expansion; and (i) the at least one condition, disease, disorder or infection is metabolic syndrome, and wherein the target DNA sequence is at least one mutation in a gene selected from the group consisting of FAH, TAT and HPD.

4. The kit according to claim 3, wherein the target DNA sequence is a loss-of-function mutation in a tumor suppressor gene, and wherein the tumor suppressor gene is selected from p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), partner and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), p38 and VHL.

5. The kit of claim 3, wherein the target DNA sequence is an oncological gain of function gene mutation, wherein the gene is selected from Ras (H-Ras, K-Ras and N-Ras), mitogen-activated protein 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, fumaryl 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 and T-cell acute lymphoblastic leukemia 1 (TAL1).

6. A kit according to claim 3, wherein the target DNA sequence is a sequence from a bacterium or a virus, and the bacterium or virus is selected from human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), new coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis and Mycoplasma.

7. The kit according to any one of claims 1 to 6, wherein the CRISPR-Cas system is in the form of a ribonucleoprotein (RNP) complex formed by sgRNA and an endonuclease peptide or protein.

8. The kit according to any one of claims 1 to 7, wherein at least one component of the CRISPR-Cas system is located in at least one DNA plasmid.

9. The kit according to any one of claims 1 to 8, wherein at least one component of the CRISPR-Cas system is located in at least one DNA or RNA based viral vector.

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

11. The kit of any one of claims 1 to 10, wherein at least one component of the CRISPR-Cas system is located in at least one of the following: liposomes, lipid nanoparticles, immunoliposomes, lipid complexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

12. The kit according to any one of claims 1 to 11, wherein at least one component of the CRISPR-Cas system is provided in naked form for direct physical delivery.

13. The kit according to any one of claims 1 to 12, wherein the sgRNA and CRISPR-associated nuclease or the gene encoding the same are located in different delivery vectors.

14. The kit according to any one of claims 1 to 13, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated nuclease is modified to include a cell penetrating peptide sequence.

15. The kit of any one of claims 1 to 14, wherein at least one of the sgRNA and CRISPR-associated nuclease or the gene encoding the same is formulated for administration by a route selected from the group consisting of injection, infusion, local administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

16. The kit according to any one of claims 1 to 15, wherein the alternating electric field is configured to be applied at a frequency in the range of about 100 kHz to about 10 MHz.

17. The kit according to any one of claims 1 to 16, wherein the alternating electric field is configured to be applied with a field strength of at least 1 V / cm.

18. The kit according to any one of claims 1 to 17, further comprising at least one device and / or at least one reagent for examining a sample to identify the target DNA sequence in the sample.

19. A clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system for use in the method of any one of claims 21 to 60, the CRISPR-Cas system comprising: a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence in at least one cell; as well as CRISPR-associated endonuclease or a gene encoding the same.

20. Use of a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system in a method according to any one of claims 21 to 60, the CRISPR-Cas system comprising: a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence in at least one cell; as well as CRISPR-associated endonuclease or a gene encoding the same.

21. A method of inducing apoptosis in at least one cell, the method comprising the steps of: (1) applying an alternating electric field to the at least one cell for a period of time, wherein the application of the alternating electric field downregulates at least one DNA damage repair pathway in the at least one cell; as well as (2) exposing the at least one cell to a clustered regularly interspaced short palindromic repeats (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; as well as CRISPR-associated endonuclease or a gene encoding the same.

22. The method of claim 21, wherein the CRISPR-associated nuclease is selected from Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

23. The method of claim 21 or 22, wherein the target DNA sequence is selected from 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.

24. The method of claim 23, wherein the target DNA sequence is directed against a mutation in a gene selected from the group consisting of: p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), partner and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), 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), p38, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), c-Myc, VHL, fumaryl 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, and T-cell acute lymphoblastic leukemia 1 (TAL1).

25. The method of claim 23, wherein the target DNA sequence is a gene from a microorganism selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), novel coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis and Mycoplasma.

26. The method of any one of claims 21 to 25, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed by an sgRNA and an endonuclease peptide or protein.

27. The method of any one of claims 21 to 26, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA plasmid.

28. The method of any one of claims 21 to 27, wherein at least one component of the CRISPR-Cas system is delivered by at least one DNA or RNA based viral vector.

29. The method of claim 28, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia virus and / or herpes simplex virus vectors or artificial viruses.

30. The method of any one of claims 21 to 29, wherein at least one component of the CRISPR-Cas system is delivered by at least one of liposomes, lipid nanoparticles, immunoliposomes, lipid complexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

31. The method of any one of claims 21 to 30, wherein at least one component of the CRISPR-Cas system is administered in naked form by direct physical delivery.

32. The method of any one of claims 21 to 31, wherein the sgRNA and CRISPR-associated nuclease or gene encoding the same are administered by different delivery vectors.

33. The method of any one of claims 21 to 32, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated nuclease is modified to include a cell penetrating peptide sequence.

34. The method of any one of claims 21 to 33, wherein at least one of the sgRNA and the CRISPR-associated nuclease or the gene encoding the same is administered by a route selected from the group consisting of injection, infusion, local administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

35. The method of any one of claims 21 to 34, wherein the alternating electric field is applied at a frequency in the range of about 100 kHz to about 10 MHz.

36. The method of any one of claims 21 to 35, wherein the alternating electric field has a field strength of at least 1 V / cm.

37. The method according to any one of claims 21 to 36, wherein the at least one cell is part of a biological tissue, and wherein the method further comprises the step of examining a sample of the biological tissue and identifying the target DNA sequence in the sample.

38. A method of treating or reducing the incidence of at least one condition, disease, disorder or infection in a subject, the method comprising the steps of: (1) applying an alternating electric field to at least a portion of the subject for a period of time, wherein the application of the alternating electric field downregulates at least one DNA damage repair pathway in at least a portion of the subject; as well as (2) administering to the subject a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system, comprising: a single guide RNA (sgRNA) comprising a guide sequence that hybridizes to a target DNA sequence associated with at least one condition, disease, disorder, or infection; as well as CRISPR-associated endonuclease or a gene encoding the same.

39. The method of claim 38, wherein the CRISPR-associated nuclease is selected from Cas3, Cas9, Cas12, Cas12a, Cas12b, Cas12e, Cas12f, Cas13a, Cas13b, mini-Cas9, and Cas9 nickase (nCas9).

40. The method of claim 38 or 39, wherein the at least one condition, disease, disorder or infection is selected from cancer, autoimmune disease, viral infection, bacterial infection, fungal infection, parasitic infection, metabolic syndrome, Huntington's disease, Hippel-Lindau syndrome, and combinations thereof.

41. The method of claim 40, wherein at least one of the following: (a) the at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one oncological gain of function gene mutation; (b) the at least one condition, disease, disorder or infection is cancer, and wherein the target DNA sequence is at least one loss-of-function mutation in a tumor suppressor gene; (c) the at least one condition, disease, disorder or infection is a bacterial infection, and wherein the target DNA sequence is a bacterial sequence; (d) the at least one condition, disease, disorder or infection is a viral infection, and wherein the target DNA sequence is a viral sequence; (e) the at least one condition, disease, disorder or infection is a fungal infection, and wherein the target DNA sequence is a fungal sequence; (f) the at least one condition, disease, disorder or infection is a parasitic infection, and wherein the target DNA sequence is a parasitic sequence; (g) the at least one condition, disease, disorder or infection is an autoimmune disease, and wherein the target DNA sequence is an autoantigen sequence in an autoantibody-producing immune cell; (h) the at least one condition, disease, disorder or infection is Huntington's disease, and wherein the target DNA sequence is a CAG repeat expansion; and (i) the at least one condition, disease, disorder or infection is metabolic syndrome, and wherein the target DNA sequence is at least one mutation in a gene selected from the group consisting of FAH, TAT and HPD.

42. The method of claim 41, wherein the target DNA sequence is a loss-of-function mutation in a tumor suppressor gene, and wherein the tumor suppressor gene is selected from the group consisting of p53, retinoblastoma (Rb), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Ras association 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), partner and localizer of BRCA2 (PALB2), tuberous sclerosis complex 1 (TSC1), tuberous sclerosis complex 2 (TSC2), neurofibromatosis type 1 (NF1), liver kinase B1 (LKB1), E2F1, KLF5, forkhead box 03a class (FOXO3a), p38, and VHL.

43. The method of claim 41, wherein the target DNA sequence is an oncological gain of function gene mutation, wherein the gene is selected from Ras (H-Ras, K-Ras and N-Ras), mitogen-activated protein 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, fumaryl 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 and T-cell acute lymphoblastic leukemia 1 (TAL1).

44. The method of claim 41, wherein the target DNA sequence is a sequence from a bacterium or virus selected from the group consisting of human papillomavirus (HPV), hepatitis B virus and hepatitis C virus (HBV virus and HCV virus, respectively), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), novel coronavirus, herpes simplex virus (HSV), human herpes virus 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 typhimurium, Streptococcus bovis, and Mycoplasma.

45. The method of any one of claims 38 to 44, wherein the CRISPR-Cas system is administered in the form of a ribonucleoprotein (RNP) complex formed by an sgRNA and an endonuclease peptide or protein.

46. ​​The method of any one of claims 38 to 45, wherein at least one component of the CRISPR-Cas system is delivered via at least one DNA plasmid.

47. The method of any one of claims 38 to 46, wherein at least one component of the CRISPR-Cas system is delivered by at least one DNA or RNA based viral vector.

48. The method of claim 47, wherein the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, retrovirus, vaccinia virus and / or herpes simplex virus vectors or artificial viruses.

49. The method of any one of claims 38 to 48, wherein at least one component of the CRISPR-Cas system is delivered by at least one of liposomes, lipid nanoparticles, immunoliposomes, lipid complexes, polyplexes, poloxamers, polycations, virosomes, gold nanoparticles, other inorganic nanoparticles, and combinations thereof.

50. The method of any one of claims 38 to 49, wherein at least one component of the CRISPR-Cas system is administered in naked form by direct physical delivery.

51. The method of any one of claims 38 to 50, wherein the sgRNA and CRISPR-associated nuclease or gene encoding the same are administered by different delivery methods.

52. The method of any one of claims 38 to 51, wherein at least one of the sgRNA and the gene encoding the CRISPR-associated nuclease is modified to include a cell penetrating peptide sequence.

53. The method of any one of claims 38 to 52, wherein at least one of the sgRNA and CRISPR-associated nuclease or a gene encoding the same is administered by a route selected from the group consisting of injection, infusion, local administration, implantation, electroporation, lipofection, microinjection, microfluidics, biolistics, particle gun acceleration, and combinations thereof.

54. The method of any one of claims 38 to 53, wherein the CRISP-Cas system is administered from about 24 hours to about 48 hours after initiation of application of the alternating electric field.

55. The method of any one of claims 38 to 54, wherein the period of time for which the alternating electric field is applied is in the range of about 24 hours to about 72 hours.

56. The method of any one of claims 38 to 55, wherein the CRISP-Cas system is administered after the period of time has elapsed.

57. The method of any one of claims 38 to 56, wherein the alternating electric field is applied at a frequency in the range of about 100 kHz to about 10 MHz.

58. The method of any one of claims 38 to 57, wherein the alternating electric field has a field strength of at least 1 V / cm.

59. The method according to any one of claims 38 to 58, wherein steps (1) and (2) are repeated one or more times.

60. The method of any one of claims 38 to 59, wherein the method further comprises the step of examining a sample from the subject and identifying the target DNA sequence in the sample.