Crispr compositions and methods for treating african swine fever

Abstract

The disclosure relates to compositions comprising and methods of administration to a mammal of a nucleic acid vector encoding one or more single guide RNAs (sgRNAs), tracrRNAs and / or crRNAs used individually or in combination with Cas system components in order to treat African Swine Fever Virus (ASFV). The disclosure also relates to systems for assaying prophylactically and therapeutically effective amounts of nucleic acid that inhibit ASFV replication, reduce ASFV viral load, and lead to treatment-related reduction in disease burden that enables the subject to develop durable immunity to ASFV.

Description

CRISPR COMPOSITIONS AND METHODS FOR TREATING AFRICAN SWINEFEVERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 624,775, filed on January 24, 2024, and U.S. Provisional Patent Application No. 63 / 683,976, filed on August 16, 2024, both of which are incorporated by reference herein in their entireties.TECHNICAL FIELD

[0002] The present disclosure is directed, to compositions, nucleic acids, pharmaceutical compositions comprising the same, kits comprising the same, and methods for preventing or treating African Swine Fever Virus (ASFV) infection by administering a nucleic acid encoding one or more RNA guide sequences (gRNAs or sgRNAs), comprised of the CRISPR RNA (crRNA) and the trans-activating crispr RNAs (tracrRNAs) or functional fragments thereof, optionally, with a Cas protein or Cas variant protein or with a nucleic acid encoding a Cas protein or Cas variant protein. The present disclosure also relates to methods for preventing or treating African Swine Fever comprising cleaving or degrading of an ASFV double-stranded DNA (dsDNA), an ASFV single-stranded RNA (ssRNA), an ASFV dsDNA and an ASFV ssRNA, or one or more target loci involved in ASFV replication by administering an ASFV- targeting CRISPR vector to a subject. The present disclosure also relates to CRISPR compositions for use in a subject and methods for generating a durable immune response directed against ASFV infection.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory materialBACKGROUND

[0004] Therapeutic genome editing has great potential to benefit a range of diseases. A key challenge is the efficient and clinically suitable delivery of genome editing biomacromolecules. The CRISPR (clustered regularly interspersed short palindromic repeats) / Cas9 system is a transforming and powerful genome editing tool. CRISPR / Cas9 comprises a short guide RNA (sgRNA) and an RNA-guided nuclease (e.g., a Cas9 protein). Cas9-sgRNA complex recognizes the protospacer-adjacent motif (PAM) and a 20-nucleotide sequence in the genome by Watson-Crick base pairing. Site-specific double-stranded DNA breaks (DSB) generated by Cas9 are repaired by endogenous cellular mechanisms, including homology-directed repair (HDR) or nonhomologous end-joining (NHEJ). The therapeutic potential of CRISPR / Cas9 requires safe and efficient delivery.

[0005] An ideal genome editing delivery system would limit the duration of exposure to editing machinery in order to minimize potential side effects. Cas9-sgRNA ribonucleoprotein (RNP)-based delivery of CRISPR has been tested for cell culture or local delivery in mouse inner ear cells, but these methods are not amenable for systemic in vivo delivery to target major organs such as the liver. Viral vehicles including the adeno-associated virus (AAV) have been used as the delivery agents for long-term CRISPR expression. However, spCas9, as the most commonly used form of Cas9, is difficult to fit in typical AAV constructs with strong promoters. A smaller form of Cas9 has been shown to be capable of being packaged into a single AAV construct. However, concerns regarding potential off-target effects remain if Cas9 is stably expressed by AAV delivery. Moreover, the T cell responses to AAV capsid can limit repeat dosing in patients. The long-term presence of Cas9, a protein from bacteria, in human tissue also increases the risk of immunogenicity. These limitations can be substantively addressed using non-viral delivery system. Previously, lipid nanoparticles (LNP) encapsulated Cas9 mRNA in combination with an AAV carrying an sgRNA and a repair template inducing efficient genome editing in the mouse liver.

[0006] African Swine Fever Virus (ASFV) has been a virus causing significant damage to the domesticated and commercial pig populations in Asia and Africa. Recently, ASFV has been detected in pig populations within the Dominican Republic, potentially threatening commercial and domesticated pig populations within North and South America. Current prophylactics or treatments for the virus remain unknown.SUMMARY

[0007] In accordance with the purpose(s) of the disclosure, as embodied and broadly described herein, the disclosure, in some embodiments, relates to compounds and compositions useful in the treatment of ASFV such as, for example, sgRNA or tracrRNA molecules designed to bind an ASFV target sequence and a Cas protein that cuts the target sequence leaving the virus diminished in an ability of replication, assembly, or replication and assembly or incapable of replication, assembly, or replication and assembly. In other embodiments, a Cas protein is activated by crRNA guiding to a target mRNA sequence and initiating indiscriminate degradation of ssRNA, ssDNA, and dsDNA within target cells. The elimination of virus or increased inhibition of viral replication or virus production in host cells greatly improves outcomes for subject treated with such compositions. Furthermore, even alleviation of symptomsby administration of the disclosed compositions can significantly delay infection rates and decrease the spread of virus among a drift of pigs.

[0008] Provided herein are methods of disrupting viral genome to reduce viral replication and reduce disease burden, thereby enabling the healthier host to mount a productive and specific immune response against a pathogen. In an aspect described herein are methods for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, the methods comprising: a) administering to the subject an effective amount of an isolated nucleic acid, wherein the isolated nucleic acid comprises: i) a first nucleic acid sequence comprising at least about 90% complementarity to a portion of an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding a conserved ASFV open reading frame, and ii) a second nucleic acid sequence encoding a programmable Cas endonuclease protein or functional fragment thereof; b) expressing in a cell of the subject a (i) guide RNA (gRNA) comprising the first nucleic acid sequence from a first promoter in the isolated nucleic acid and (ii) the programmable Cas endonuclease protein or functional fragment thereof from a second promoter in the isolated nucleic acid, thereby activating the programmable Cas endonuclease protein or functional fragment thereof in the cell of the subject; c) using the activated programmable Cas endonuclease protein or functional fragment thereof to cleave or digest an ASFV nucleic acid at or near the portion of the endogenous ASFV nucleic acid sequence encoding the conserved ASFV open reading frame, wherein the cleaving or digesting reduces ASFV viral load in the subject; and d) generating a durable immune response in the subject to ASFV infection. In some embodiments, the using the activated programmable Cas endonuclease protein or functional fragment thereof in c) comprises mitigating one or more symptoms of ASFV infection following the reduction in ASFV viral load from the cleaving or digesting. In some embodiments, the generating the durable immune response comprises mitigating one or more symptoms of ASFV infection following an increase in ASFV-specific antibody response in the subject. In some embodiments, the programmable Cas endonuclease protein or functional fragment thereof is selected from a Cas9, a Casl2a2, a Cas 12a, a Cas 13d, or a CasX. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid at least about 90%, 95%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-6 or 108-111. In some embodiments, the programmable Cas endonuclease comprises a Casl2a. In some embodiments, the Casl2a is EnAsCasl2a. In some embodiments, the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 123-128. In some embodiments, the first nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, or six gRNA sequences each comprising a spacer sequence selected fromSEQ ID NOs: 123-128. In some embodiments, the first nucleic acid sequence further comprises coding sequence for six gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 123-128. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143. In some embodiments, the programmable Cas endonuclease comprises a Cas9. In some embodiments, the Cas9 is Sniper2L-Cas9. In some embodiments, the Cas9 comprises a nuclear localization signal (NLS), a nuclear export signal (NES), or a nucleocytoplasmic shuttling sequence (NCS) comprising an NLS and an NES. In some embodiments, the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 15-32 or 118-121. In some embodiments, the first nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, or seven gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 140. In some embodiments, the first nucleic acid sequence further comprises coding sequence for seven gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142. In some embodiments, the programmable Cas endonuclease comprises a Casl2a2. In some embodiments, the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 37-45. In some embodiments, the first nucleic acid sequence further comprises coding sequence nine gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 37-45. In some embodiments, the administering comprises systemic administration. In some embodiments, systemic administration comprises oral administration, IV administration, or IM administration. In some embodiments, wherein the systemic administration comprises oral administration, the oral administration comprises adding one or more components of a CRISPR system to a food source of the subject. In some embodiments, the gRNA and the programmable Cas endonuclease protein or functional fragment thereof are transiently expressed in cells of the subject following the administering. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a pig, a swine, a minipig, or a wild boar. In some embodiments, the cleaving or digesting ASFV nucleic acid reduces ASFV viral load in the subject by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% by 30 days after the administering. In some embodiments, the cleaving or digesting ASFV nucleic acid reduces ASFV viral load in the subject ASFV viral load is decreased by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 25 days after the administering. In some embodiments, the cleaving or digesting ASFV nucleic acid reduced ASFV viral load in the subject to an undetectable level by about 25-30 dpi following the administering. In some embodiments, the mitigating one or more symptoms of ASFV infection comprises increased survival of the subject following exposure of ASFV, reduction of high fever, improvement in skin lesions, improvement in breathing, improvement in appetite, improvements in eye discharge, nasal discharge, and bleeding, or improvement in lethargy, or any combination thereof. In some embodiments, survival of the subject following exposure to ASFV is improved. In some embodiments, the subject develops an extent of posttreatment immunity to ASFV to produce the durable immune response. In some embodiments, post-treatment immunity to ASFV lowers a likelihood of the subject subsequently developing ASF. In some embodiments, post-treatment immunity to ASFV prevents the subject from subsequently developing ASF. In some embodiments, the subject develops durable immunity to ASFV challenge, by allowing the subject to develop a robust humoral immune response by producing antibodies against ASFV. In some embodiments, the subject achieves a total recovery from ASF following the administering. In some embodiments, the subject survives at least 30 dpi following the administering. In some embodiments, at least about 50% or more subjects survive at least 30 dpi following the administering. In some embodiments, a surviving subject demonstrates an increased ASFV-specific antibody response. In some embodiments, the increased ASFV-specific antibody response is significant by at least about 10 dpi. In some embodiments, the increased ASFV-specific antibody response remains significant until at least about 30 dpi. In some embodiments, subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge. In some embodiments, subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge and a survival rate of greater than 90% when rechallenged with an otherwise lethal dose of ASFV. In some embodiments, subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge and a survival rate of 100% when re-challenged with an otherwise lethal dose of ASFV.

[0009] Provided herein are compositions for generating long-lasting immunity to African Swine Fever Virus (ASFV). In an aspect described herein, are compositions for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, the compositions comprising: (i) an isolated nucleic acid sequence encoding a Clustered regularly interspaced short palindromic repeat (CRISPRj-associated endonuclease or functional fragment thereof and (ii) a guide RNA (gRNA) complementary to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the composition is configured to reduce a viral load of ASFV to an extent that generates a durable immune response to an ASFV infection in the subject. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a programmable Cas endonuclease selected from a Cas9, a Casl2a2, a Cast 2a, a Cast 3d, or a CasX. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Cast 3d. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a CasX. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Casl2a. In some embodiments, the Casl2a is EnAsCasl2a. In some embodiments, the isolated nucleic acid sequence encoding the gRNA comprises a sequence selected from SEQ ID NOs: 123-128. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, or six gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 123-128. In some embodiments, the composition further comprises coding sequence for six gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 123-128. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Cas9. In some embodiments, the Cas9 is Sniper2L-Cas9. In some embodiments, the Cas9 comprises a nuclear localization signal (NLS), a nuclear export signal (NES), or a nucleocytoplasmic shuttling sequence (NCS) comprising an NLS and an NES. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, or seven gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for three gRNAs each comprising one of spacer sequences of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 140. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for seven gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100%identical to SEQ ID NO: 141 or SEQ ID NO: 142. In some embodiments, the CRISPR- associated endonuclease or functional fragment thereof comprises a Casl2a2. In some embodiments, the Casl2a2 comprises an NLS, an NES, or an NCS comprising an NLS and an NES. In some embodiments, the Casl2a2 is lacking an NLS. In some embodiments, the isolated nucleic acid sequence encoding the gRNA comprises a sequence selected from SEQ ID NOs: 37-45. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 37-45. In some embodiments, the gRNA and the programmable Cas endonuclease protein or functional fragment thereof are transiently expressed in cells of the subject following administering of the composition to a subject. In some embodiments, transient expression of the gRNA and the programmable Cas endonuclease protein or functional fragment thereof produces cleaving or digesting of ASFV nucleic acid in cells of the subject and reduces ASFV viral load in the subject by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 30 days after the administering. In some embodiments, transient expression of the gRNA and the programmable Cas endonuclease protein or functional fragment thereof improves survival of the subject following exposure to ASFV. In some embodiments, reduction in ASFV viral load allows the subject to develop an extent of post-treatment immunity to ASFV and achieve a durable protective immunity upon ASFV re-challenge.

[0010] Provided herein are guide RNAs (gRNAs) for treating ASFV. Provided herein are CRISPR vectors for treating ASFV. In some aspects, the CRISPR vectors reduce ASFV viral load. In some aspects, the CRISPR vectors generate long-lasting immunity to ASFV in a subject. In some aspects, the vectors encode one or more gRNAs for treating ASFV. In some aspects, the vectors encode a programmable Cas endonuclease, wherein the one or more gRNAs are designed to activate and target the programmable Cas endonuclease to viral nucleic acid in cells. In some aspects, the CRISPR vectors are isolated single vectors. In an aspect described herein, are vectors comprising a nucleic acid sequence encoding (i) a programmable Cas endonuclease and (ii) three guide RNAs (gRNAs), each gRNA comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome; wherein the programmable Cas endonuclease comprises a Cas9 or functional fragment thereof, and wherein the three gRNAs each comprise a sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the vector further comprises nucleic acid sequence encoding one, two, three, or four additional gRNAs, the additional gRNAs each comprising acomplementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 118-121. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142. In an aspect described herein are guide RNAs (gRNAs) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 121. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas endonuclease comprises a Cas9 or functional fragment thereof. In some embodiments, the vector further comprises nucleic acid sequence encoding one, two, three, four, five, or six additional gRNAs, the additional gRNAs each comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 15-17 and 118-120. In an aspect described herein are guide RNAs (gRNAs) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 123. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein are guide RNAs (gRNAs) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 124. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas endonuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein are guide RNAs (gRNAs) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 125. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas endonuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein are guide RNAs (gRNAs) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 126. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas endonuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein are guide RNAs (gRNAs)comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 127. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas endonuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein are guide RNAs (gRNAs) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 128. In some embodiments of vectors described herein, a vector comprises a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA, wherein the programmable Cas endonuclease comprises a Casl2a or functional fragment thereof. In some embodiments, the vector further comprises nucleic acid sequence encoding one or more, two or more, three or more, four or more, or five additional gRNAs, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 123-128. In some embodiments, the vector comprises each of nucleic acid sequences of SEQ ID NOs 123-128. . In some embodiments of vectors described herein, the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143. In some embodiments of vectors described herein, are vectors for use in a method for producing long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, wherein the method comprises administering to the subject an effective amount of the vector for transient co-expression of the programmable Cas endonuclease and one or more gRNAs targeting a conserved sequence in the Asfarviridae genome. In some embodiments, the use reduces ASFV viral load in the subject. In some embodiments, the use mitigates one or more symptoms of ASFV infection. In some embodiments, the one or more symptoms of ASFV infection comprise: increased survival of the subject following exposure of ASFV, reduction of high fever, improvement in skin lesions, improvement in breathing, improvement in appetite, improvements in eye discharge, nasal discharge, and bleeding, or improvement in lethargy, or any combination thereof. In some embodiments, the use produces a durable immune response in the subject to ASFV infection. In some embodiments, the durable immune response allows the subject to achieve durable protective immunity upon ASFV re-challenge. In an aspect described herein are vectors comprising a nucleic acid sequence encoding: a Cas9 protein of SEQ ID NO: 1; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121. In an aspect described herein are vectors comprising a nucleic acid sequence encoding: a Cas9 protein of SEQ ID NO: 108; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121. In some embodiments of vectors described herein, each of theseven gRNAs further comprises a Cas9 tracrRNA / direct repeat of SEQ ID NO: 14. In an aspect described herein are vectors comprising a nucleic acid sequence encoding: an EnAsCasl2a protein of SEQ ID NO: 110; and six guide RNAs (gRNAs), wherein each of the six gRNAs comprises a spacer sequence of one of SEQ ID NOs: 123-128. In an aspect described herein are vectors comprising a nucleic acid sequence encoding: a Cas9 protein of SEQ ID NO: 1; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121. In some embodiments of vectors described herein, each of the six gRNAs further comprises a AsCasl2a Direct Repeat of crRNA of SEQ ID NO: 122. In some embodiments of vectors described herein, the programmable Cas endonuclease and the gRNA or gRNAs are configured for expression in cells of a mammalian subject.

[0011] Provided herein are kits for prophylaxis and / or treatment of ASFV. In an aspect described herein are kits comprising a pharmaceutical composition comprising a CRISPR vector described herein, and instructions for use. In some embodiments, the instructions for use designate ASFV prophylaxis, ASFV treatment, or ASFV prophylaxis and ASFV treatment as indications in a subject in need of treatment. In some embodiments, the kit further comprises a drug delivery device. In some embodiments, the pharmaceutical composition is formulated with lipid nano-particles (LNPs).

[0012] Provided herein are methods of disrupting viral genome to limit viral replication and reduce disease burden, thereby enabling the healthier host to mount a productive and specific immune response against a pathogen. In an aspect described herein are methods of directly disabling viral replication and indirectly enabling an immune response against a pathogen in a subject, wherein the methods comprise (a) cleaving one or more target loci of the pathogen with a Cas endonuclease protein or a Cas variant protein; and (b) disrupting a function of the one or more target loci of the pathogen, thereby limiting infection and enabling the host to produce an immune response against the pathogen in the subject. In an aspect described herein are methods of disrupting a function of one or more target loci of a pathogen, wherein the methods comprise (a) contacting the one or more target loci of the pathogen with a Cas endonuclease protein or a Cas variant protein; and (b) disrupting the function of the one or more target loci of the pathogen, thereby enabling an immune response against the pathogen in the subject. In some instances, the Cas endonuclease protein or Cas variant protein is directed to one or more target loci by one or more guide RNA (gRNA) molecules. In an aspect described herein are methods of disrupting a function of one or more target loci of a pathogen, wherein the methods comprise (a) contacting the one or more target loci of the pathogen with a Cas endonuclease protein-gRNA complex or a Cas variant protein-gRNA complex; and (b) disrupting the function of the one or more target loci of the pathogen, thereby enabling an immune response against the pathogen inthe subject. In some instances, the Cas endonuclease protein-gRNA complex or the Cas variant protein-gRNA complex is termed a ribonucleoprotein (RNP).

[0013] Provided herein are methods for treating ASFV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first nucleic acid, or a pharmaceutically acceptable salt thereof; wherein the first nucleic acid comprises: i) a first nucleic acid sequence that comprises at least about 70% complementarity to an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding an ASFV polymerase, an ASFV helicase, an ASFV topoisomerase, or any combination thereof; and ii) a second nucleic acid sequence encoding a Cas endonuclease protein or functional fragment thereof. Also provided herein in some aspects are methods for treating African Swine Fever in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a ribonucleoprotein (RNP) complex, wherein the RNP complex comprises a Cas protein in complex with a nucleic acid, wherein the nucleic acid comprises at least about 70% complementarity to an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding an ASFV polymerase, an ASFV helicase, an ASFV topoisomerase, or any combination thereof. In some embodiments, the nucleic acid comprises any one or plurality of sequences from Table 4A. In some embodiments the nucleic acid sequence comprises any one or plurality of sequences from Table 4A. In some embodiments, the Cas endonuclease protein or functional fragment thereof comprises Cas9, Casl2a2, Casl2a, Casl3d, or CasX. In some embodiments, the Cas endonuclease protein or functional fragment thereof comprises Casl2a2. In some embodiments, Casl2a2 comprises one or more nuclear localization sequences (NLSs). In some embodiments, Casl2a2 is lacking a nuclear localization sequence (NLS). In some embodiments Casl2a2 is not substantially concentrated to a nucleus of a cell of the subject following the administering. In some embodiments, the Cas endonuclease protein or functional fragment thereof comprises Cas9. In some embodiments, Cas9 comprises an NLS. In some embodiments, the Cas endonuclease protein or functional fragment thereof comprises Casl2a. In some embodiments, the Cas endonuclease protein or functional fragment thereof comprises Cas 13d. In some embodiments, the Cas endonuclease protein or functional fragment thereof comprises CasX. In some embodiments, the first nucleic acid sequence comprises a sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to one or more sequences selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the first nucleic acid sequence comprises a sequence identical to one or more sequence selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the first nucleic acid sequence comprises a sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to one or more sequences selected from SEQ ID NOs: 15-35. In some embodiments,the first nucleic acid sequence comprises a sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to one or more sequences selected from SEQ ID NOs: 37-45. In some embodiments, the first nucleic acid sequence comprises a sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to one or more sequences selected from SEQ ID NOs: 47-64. In some embodiments, the first nucleic acid sequence comprises a sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to one or more sequences selected from SEQ ID NOs: 66-83. In some embodiments, the first nucleic acid sequence comprises a sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to one or more sequences selected from SEQ ID NOs: 15-17. In some embodiments, the first nucleic acid sequence comprises sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to two or more sequences selected from SEQ ID NOs: 15-17. In some embodiments, the first nucleic acid sequence comprises sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. In some embodiments, the first nucleic acid sequence comprises sequence identical to SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. In some embodiments, the first nucleic acid sequence comprises sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to two or more sequences selected from SEQ ID NOs: 37-45. In some embodiments, the first nucleic acid sequence comprises sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to three, four, five, six, seven, eight, or more sequences selected from SEQ ID NOs: 37-45. In some embodiments, the first nucleic acid sequence comprises sequence identical to three, four, five, six, seven, eight, or more sequences selected from SEQ ID NOs: 37-45. In some embodiments, the first nucleic acid sequence comprises sequence identical all sequences represented from SEQ ID NOs: 37-45. In some embodiments, ASFV viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%,45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than one week after the administering. In some embodiments, ASFV virus being treated belongs to the family Asfarviridae. In some embodiments, the first nucleic acid sequence codes for one or more guide RNAs (gRNAs). In some embodiments, the one or more gRNAs activate a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of Cas9, Casl2a, Casl2a2, Casl3b, and CasX. In some embodiments, the Cas protein functions as nuclease to cleave or digest one or more ASFV nucleic acids. In some embodiments, one or more ASFV nucleic acids comprise genomic DNA sequences identical to a portion of any one of an ASFV topoisomerase gene, an ASFV helicase gene, or an ASFV DNA polymerase gene. In some embodiments, one or more ASFVnucleic acids comprise single-stranded RNA sequences identical to any one of an ASFV topoisomerase mRNA, an ASFV helicase mRNA, or an ASFV DNA polymerase mRNA. In some embodiments, the one or more ASFV nucleic acids comprise genomic DNA sequences identical to a portion of an ASFV topoisomerase gene. In some embodiments, the one or more ASFV nucleic acids comprise genomic DNA sequences identical to a portion of an ASFV helicase gene. In some embodiments, the one or more ASFV nucleic acids comprise genomic DNA sequences identical to a portion of an ASFV DNA polymerase gene. In some embodiments, the one or more ASFV nucleic acids comprise single-stranded RNA sequences identical to a portion of an ASFV topoisomerase mRNA transcript. In some embodiments, the one or more ASFV nucleic acids comprise single-stranded RNA sequences identical to a portion of an ASFV helicase mRNA transcript. In some embodiments, the one or more ASFV nucleic acids comprise single-stranded RNA sequences identical to a portion of an ASFV DNA polymerase mRNA transcript. In some embodiments, the one or more gRNAs and the Cas protein comprise a gene-binding moiety, wherein said gene binding moiety is configured to bind at least one essential gene of a virus belonging to the family Asfarviridae.

[0014] In some embodiments, the ASFV topoisomerase comprises ASFV Topoisomerase II. In some embodiments, the ASFV helicase comprises an ASFV RNA helicase. In some embodiments, the ASFV DNA polymerase comprises an G1211R ASFV DNA polymerase or functional fragment thereof. In some embodiments, the Topoisomerase II is pi 192R or a fragment thereof. In some embodiments, the RNA helicase is QP509L, A859L, F105L, B92L, DI 133LK, or Q706L. In some embodiments, the subject is a mammal. In some embodiments, the subject is a pig, a swine, a minipig, or a wild boar. In some embodiments, the subject is a pig. In some embodiments, the pig species is Sus scrofa, Sus ahenobarbus, Sus barbatus, Sus cebrifons, Sus celebensis, Sus oliveri, Sus philippensis, or Sus verrucosus. In some embodiments,, the i) first nucleic acid sequence and the ii) second nucleic acid sequence are transcribed from a single vector. In some embodiments, the i) first nucleic acid sequence and the ii) second nucleic acid sequence are each transcribed from a separate vector. In some embodiments, the single vector or the separate vector comprise a plasmid. In some embodiments, ASFV topoisomerase, ASFV helicase, and ASFV DNA polymerase are each targeted by at least one nuclease, wherein at least one nuclease comprises at least three genebinding moieties. In some embodiments, ASFV topoisomerase, ASFV helicase, and ASFV DNA polymerase are each targeted by at least one nuclease, wherein at least one nuclease comprises at least six gene-binding moieties. In some embodiments, ASFV topoisomerase, ASFV helicase, and ASFV DNA polymerase are each targeted by at least one nuclease, wherein at least one nuclease comprises at least nine gene-binding moieties. In some embodiments, ASFVtopoisomerase, ASFV helicase, and ASFV DNA polymerase are each targeted by at least two nucleases using at least three gene-binding moieties. In some embodiments, wherein ASFV topoisomerase, ASFV helicase, and ASFV DNA polymerase are each targeted by at least two nucleases using at least six gene-binding moieties. In some embodiments, ASFV topoisomerase, ASFV helicase, and ASFV DNA polymerase are each targeted by at least two nucleases using at least nine gene-binding moieties. In some embodiments, at least nine gene-binding moieties comprise at least nine distinct gRNAs.

[0015] Also provided are methods of inhibiting ASFV infection in a subject exposed to ASFV disclosed herein, the method comprising administering to the subject a therapeutically effective amount of a first nucleic acid, or a pharmaceutically acceptable salt thereof; wherein the first nucleic acid comprises: i) a first nucleic acid sequence that comprises at least about 70% complementarity to an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding an ASFV polymerase, an ASFV helicase, an ASFV topoisomerase, or any combination thereof; and ii) a second nucleic acid sequence encoding a Cas endonuclease protein or functional fragment thereof. In some embodiments the nucleic acid sequence comprises any one or plurality of sequences from Table 4A. In some embodiments, a risk of the subject developing acute ASFV infection is reduced. In some embodiments, the reduction in risk of developing acute ASFV infection is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than one week after the administering. In some embodiments, administering comprises systemic administration. In some embodiments, administering comprises oral administration. In some embodiments, administering comprises IV administration. In some embodiments, administering comprises IM administration. In some embodiments, oral administration comprises adding one or more components of a CRISPR system to a food source of the subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a pig, a swine, a minipig, or a wild boar. In some embodiments, administering comprising a combination treatment of administering a CRISPR Cas9 encoding single vector and a CRISPR Casl2a2 encoding single vector. In some embodiments, survival of the subject following exposure to ASFV is improved. In some embodiments, the subject develops an extent of post-treatment enabled immunity to ASFV. In some embodiments, posttreatment enabled immunity to ASFV lowers a likelihood of the subject subsequently developing ASF. In some embodiments, post-treatment enabled immunity to ASFV prevents the subject from subsequently developing ASF. In some embodiments, the subject develops durable immunity to ASFV challenge. In some embodiments, the subject develops durable immunity toASFV challenge, for example, by allowing the subject to develop antibodies against ASFV. In some embodiments, the subject achieves a total recovery from ASF following the administering. In some embodiments, the subject survives at least 30 dpi following the administering. In some embodiments, at least about 50% or more subjects survive at least 30 dpi following the administering. In some embodiments, ASFV viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by 30 days after the administering. In some embodiments, ASFV viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by 25 days after the administering. In some embodiments, ASFV viral load in the subject is decreased to an undetectable level by about 25-30 dpi following the administering. In some embodiments, disease burden-related symptoms are reduced in the subject following the administering. In some embodiments, a surviving subject demonstrates an increased ASFV-specific antibody response. In some embodiments, the increased ASFV-specific antibody response is significant by at least about 10 dpi. In some embodiments, the increased ASFV-specific antibody response remains significant until at least about 30 dpi. In some embodiments, subjects that survive ASFV infection following the administering achieve a durable protective immunity upon ASFV re-challenge. In some embodiments, subjects that survive ASFV infection following the administering achieve a durable protective immunity upon ASFV re-challenge and a survival rate of greater than 90% when re-challenged with an otherwise lethal dose of ASFV. In some embodiments, subjects that survive ASFV infection following the administering achieve a durable protective immunity upon ASFV re-challenge and a survival rate of 100% when rechallenged with an otherwise lethal dose of ASFV.

[0016] Also provided are methods of inhibiting ASFV replication or assembly in a cell, the method comprising administering to the subject a therapeutically effective amount of a first nucleic acid, or a pharmaceutically acceptable salt thereof; wherein the first nucleic acid comprises: i) a first nucleic acid sequence that comprises at least about 70% complementarity to an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding an ASFV polymerase, an ASFV helicase, an ASFV topoisomerase, or any combination thereof; and ii) a second nucleic acid sequence encoding a Cas endonuclease protein or functional fragment thereof. In some embodiments the nucleic acid sequence comprises any one or plurality of sequences from Table 4A.

[0017] Also provided herein are single vectors comprising a nucleic acid sequence encoding at least one programmable nuclease and a gRNA, wherein at least one programmable nuclease and the gRNA are configured to bind at least one essential viral gene of a virus from the family Asfarviridae. In some embodiments, said virus belongs to the genus Asfivirus. In some embodiments, said virus is African swine fever virus (ASFV). In some embodiments, at least one essential viral gene of the virus comprises an ASFV topoisomerase, an ASFV helicase, an ASFV DNA polymerase, or any combination thereof. In some embodiments, at least one essential viral gene of the virus comprises an ASFV topoisomerase. In some embodiments, at least one essential viral gene of the virus comprises an ASFV helicase. In some embodiments, at least one essential viral gene of the virus comprises an ASFV DNA polymerase. In some embodiments, at least one programmable nuclease comprises a Cas protein. In some embodiments, the Cas protein is selected from Cas9, Casl2a, Casl2a2, Casl3b, or CasX. In some embodiments, the Cas protein is Cas9 or a functional fragment thereof. In some embodiments, the Cas protein is Casl2a or a functional fragment thereof. In some embodiments, the Cas protein is Casl2a2 or a functional fragment thereof. In some embodiments, the Cas protein is Cas 13b or a functional fragment thereof. In some embodiments, the Cas protein is CasX or a functional fragment thereof. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas protein is Casl2a. In some embodiments, the Cas protein is Casl2a2. In some embodiments, the Cas protein is Casl3b In some embodiments, the Cas protein is CasX. In some embodiments, the gRNA comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the gRNA comprises a sequence selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the gRNA comprises at least two sequences selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the gRNA comprises at least six sequences selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the gRNA comprises at least nine sequences selected from SEQ ID NOs: 15-35, 37-45, 47-64, and 66-83. In some embodiments, the gRNA comprises sequence of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. In some embodiments, three distinct gRNAs are formatted to be transcribed from the single vector, where the three distinct gRNAs comprise the sequence of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 respectively. In some embodiments, the gRNA comprises sequence of SEQ ID NOs: 37-45. In some embodiments, nine distinct gRNAs are formatted to be produced from the single vector, where the nine distinct gRNAs comprise the sequence of SEQ ID NOs: 37-45 respectively.

[0018] Also provided are kits comprising a composition comprising: : i) a first nucleic acid sequence that comprises at least about 70% complementarity to an endogenous African SwineFever Virus (ASFV) nucleic acid sequence encoding an ASFV polymerase, an ASFV helicase, an ASFV topoisomerase, or any combination thereof; and ii) a second nucleic acid sequence encoding a Cas endonuclease protein or functional fragment thereof , or pharmaceutically acceptable salts thereof; and one or more selected from: (a) instructions for treating ASFV; and (b) instructions for administering the composition. In some embodiments, the instructions for use designate ASFV prophylaxis, ASFV treatment, or ASFV prophylaxis and ASFV treatment as indications in a subject in need of treatment. In some embodiments, the kit comprises a drug delivery device. In some embodiments, the pharmaceutical composition is formulated with lipid nanoparticles (LNPs).BRIEF DESCRIPTIONS OF THE DRAWINGS

[0019] FIG. 1 shows a graph of Day 6 qPCR results measuring ASFV viral load in blood samples from animals in a prophylactic study to evaluate CRISPR Cas9 and CRISPR Casl2a2 prophylactic monotherapy delivered via IV administration to reduce ASFV viral load.

[0020] FIG. 2 shows graphs of Day 6 qPCR results measuring ASFV viral load in blood samples from animals in a prophylactic study to evaluate CRISPR Cas9 and CRISPR Casl2a2 prophylactic combination treatment delivered either by IV administration or by IM administration to reduce ASFV viral load.

[0021] FIG. 3 shows a graph of Day 6 qPCR results measuring ASFV viral load in blood samples from animals in a prophylactic study to evaluate CRISPR Cas9 and CRISPR Casl2a2 prophylactic combination treatment delivered by IV administration or IM administration to reduce ASFV viral load. IV and IM datasets are combined.

[0022] FIG. 4 shows left and right diagrams of a test subject to show six locations for IM administration of drug in prophylactic treatment or in therapeutic treatment.

[0023] FIG. 5 depicts two panels, a left and right panel showing prophetic experiments to be performed. The left panel depicts two sets of in vitro experiments in which a first set shows steps involved in a method of treating or exposing porcine cells to Cas protein / nucleic acid embodiments after exposure to ASFV. This schematic depicts an experiment to identify dosage for therapeutically effective amounts of treatment. The second set of experiments depicts steps involved in a method of treating or exposing porcine cells to Cas protein / nucleic acid embodiments before exposure to ASFV, which can identify dosage for prophylactically effective amounts of treatment. The right-hand side of experiments shows an identical series of steps except in actual animals (e.g., pigs), rather than porcine cells.

[0024] FIG. 6 depicts two sets of experiments used to identify treatment dosage in pigs as well as a timeline for running the experiments. As shown in FIG. 6 on the left, 15 Pigs are injected intramuscularly with Cas protein and nucleic acid embodiments at a dose 1 (DS1) or dose 2(DS2). As shown in FIG. 6 on the right, 15 Pigs are injected intramuscularly with Cas protein and nucleic acid embodiments at a high dose (HD) or low dose (LD). Blood sampling is performed after therapeutic compositions are administered to the pigs to monitor viral load as compared to control animals (C) which are untreated with therapeutic compositions.

[0025] FIG. 7 shows an image of an agarose gel measuring fluorescently labelled plasmid DNA encoding the targeted ASFV gene that was incubated with the Cas 9 Ribonucleoprotein (RNP) complex alone (Lane 1) or RNP with 1 or more synthetic guide RNAs (Lanes 2 and 3). Uncut supercoiled plasmid migrates through the gel as multiple smeared bands while the plasmid that was cleaved by CRISPR at a 1 or more sites migrates as single or multiple linear fragments resolved at defined molecular sizes.

[0026] FIG. 8 shows graphs of qPCR results measuring ASFV gene expression levels in HEK293T cell co-transfected with a plasmid encoding a targeted versus non-targeted ASFV genes and a plasmid encoding the SL 1.52 CRISPR system (Cas effector plus ASFV- multiplexed guides) demonstrating targeted gene knockdown in vitro. Data represents three independent biological repeats.

[0027] FIG. 9 shows a graph of cell viability as measured by Resazurin levels in HEK293T cells following co-transfection of ASFV-gene reporter and CRISPR-expressing plasmids. CRISPR-mediated cleavage of the ASFV target gene had minimal impact on cell health in vitro. Data represents three independent biological repeats.

[0028] FIG. 10A-FIG. 10B shows overview of in vivo trial with different treatments and a diagram of a test subjects. FIG. 10A shows conditions within the trial design. FIG. 10B shows six locations for IM administration of drug in prophylactic (Px) mode or in therapeutic (Tx) mode.

[0029] FIG. 11 depicts Kaplan-Meier depicting 4 / 7 pigs surviving longer than untreated controls. 7 pigs are injected intramuscularly with Cas protein and nucleic acid embodiments at two doses (DS2). Blood sampling is performed after therapeutic compositions are administered to the pigs to monitor viral load as compared to control animals (C) which are untreated with therapeutic compositions.

[0030] FIG. 12A and 12B show graphs indicating that CRISPR treatment led to a reduction in ASFV viral load in surviving pigs that were less symptomatic and capable of mounting a robust pathogen-specific immune response FIG. 12A ASFV-viral loads in SL_1.52-DD-treated surviving animals at indicated days post-infection (dpi) versus control untreated pigs (average). FIG. 12B shows a graph of increased pathogen-specific antibody response as a function of time in blood of SL_1.52-DD-treated surviving pigs post infection as measured by ASFV ELISA.

[0031] FIG. 13 shows survival outcome of all surviving CRISPR-treated pigs following rechallenge with a second otherwise lethal dose of ASFV.

[0032] FIG. 14 shows a vector map of a CRISPR-Cas9 expression plasmid with Cas9 expressed from a CMV promoter and three gRNAs each targeting a conserved ASFV nucleotide sequence and each gRNA expressed separately by a U6 promoter.

[0033] FIG. 15 shows a vector map of a CRISPR-Cas9 expression plasmid with Cas9 expressed from a CMV enhancer and CMV promoter and seven gRNAs each targeting a conserved ASFV nucleotide sequence and each gRNA expressed separately by a U6 promoter.

[0034] FIG. 16 shows a vector map of a CRISPR-Sniper2L-Cas9 expression plasmid with Sniper2L-Cas9 expressed from a CMV enhancer and CMV promoter and seven gRNAs each targeting a conserved ASFV nucleotide sequence and each gRNA expressed separately by a U6 promoter.

[0035] FIG. 17 shows a vector map of a CRISPR-EnAsCasl2a expression plasmid with EnAsCasl2a expressed from a CMV enhancer and CMV promoter and six gRNAs each targeting a conserved ASFV nucleotide sequence and each gRNA expressed separately by a U6 promoter.

[0036] FIG. 18 shows a map for a G1211R (ASFV DNA polymerase) reporter plasmid for use of testing targeted CRISPR cleavage within a conserved ASFV open reading frame in vitro.

[0037] FIG. 19 shows a map for a C717R (ASFV uncharacterized protein) reporter plasmid for use of testing targeted CRISPR cleavage within a conserved ASFV open reading frame in vitro.

[0038] FIG. 20 shows a map for a NP1450L (ASFV DNA-dependent RNA polymerase subunit) reporter plasmid for use of testing targeted CRISPR cleavage within a conserved ASFV open reading frame in vitro.

[0039] FIG. 21 shows a map for a Pl 192R (ASFV Type II Topoisomerase) reporter plasmid for use of testing targeted CRISPR cleavage within a conserved ASFV open reading frame in vitro.

[0040] FIG. 22A shows a graph of relative gene expression of reporter gene mRNA levels using four ASFV reporter constructs and assaying mRNA expression of four conserved target ASFV ORFs (G1211R, C717R, NP1450L, and Pl 192R) following co-expression in vitro of one of three tested Cas9 ASFV targeting CRISPR constructs.

[0041] FIG. 22B shows a graph of relative gene expression of reporter gene mRNA levels using four ASFV reporter constructs and assaying mRNA expression of four conserved target ASFV ORFs (G1211R, C717R, NP1450L, and Pl 192R) following co-expression in vitro of oneof three Cas9 ASFV targeting CRISPR constructs and a Cast 2a ASFV targeting CRISPR construct.DETAILED DESCRIPTIONOverview

[0042] The present invention can be understood more readily by reference to the following detailed description of the disclosure and the Examples included therein.

[0043] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such, they may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.COMPOSITIONS

[0044] The CRISPR / Cas or the CRISPR-Cas system (both terms are used interchangeably throughout this application) does not require the generation of customized proteins to target specific sequences but rather a single Cas protein (or CRISPR enzyme) can be programmed by a short RNA molecule to recognize a specific DNA target or a specific RNA target, in other words the Cas enzyme (such as a Type II Cas9 protein or Cas 12 family) can be recruited to a specific DNA target using a short RNA molecule complementary to at least a portion of such specific ASFV DNA target. One aspect of the disclosure is a guide sequence. A different Cas enzyme (such as a Cas 13 family protein), can be recruited to a specific RNA target using a short RNA molecule complementary to at least a portion of such specific pathogen viral genome and / or mRNA target. In another aspect, a Cas enzyme or Cas variant enzyme can be recruited to a specific DNA target using a short RNA molecule complementary to at least a portion of one or more target loci of ASFV. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering and treatment of ASFV infection using these genome engineering tools, which are aspects of the disclosure.

[0045] In some embodiments, the disclosure relates to a nucleic acid sequence and compositions comprising the same. In another aspect, the disclosure relates to a nucleic acid sequence disclosed herein and compositions comprising the same with or without a vector that comprises a CRISPR enzyme or functional fragment thereof. In some embodiments, the CRISPR enzyme or functional fragment thereof is a Cas endonuclease. In some embodiments, the CRISPR enzyme or functional fragment thereof is a Cas variant protein. In someembodiments, the nucleic acid sequence is a ribonucleic sequence or an sgRNA sequence that comprises from about 0% to about 99% modified nucleic acids in one, two or three domains which, in the 5' to 3' orientation, are: a DNA-binding domain, or an RNA-binding domain, a Cas protein-binding domain, and a transcription terminator domain.

[0046] In some embodiments, the disclosure relates to a compositions comprising a guide sequence comprising, consisting essentially of, or consisting of a sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one or combination of sequences disclosed herein, wherein the guide sequence comprises a fragment or variant of the sequences disclosed herein but possesses the same or substantially the same function as the full-length sequence disclosed herein. For example, in the case of a fragment or variant of a guide RNA disclosed herein that comprises modified nucleotides in the DNA-binding domain, in some embodiments, the variant or fragment would be functional insomuch as it would exceed or retain some or all of its capacity to bind DNA at that domain as compared to the full-length sequence.

[0047] In some embodiments, the DNA-binding domain is free of modifications in any one of its first 1, 2, 3, 4, 5 or more nucleotides on its 5' end. In some embodiments the transcription terminator domain is free of modifications on any of its last 1, 2, 3, 4, 5 or more nucleotides on its 3' end.

[0048] The disclosure relates to a nucleic acid sequence comprising at least one or a combination of domains from a 5' to 3' orientation: a DNA-binding domain, a Cas proteinbinding domain, and a transcription terminator domain, wherein the DNA-binding domain comprises from about 1% to about 99% modified nucleotides and / or the transcription terminator domain comprises from about 1% to about 99% modified nucleotides. The disclosure relates to a nucleic acid sequence comprising a series of contiguous domains from a 5' to 3' orientation: a DNA-binding domain, a Cas protein-binding domain, and a transcription terminator domain, wherein the DNA-binding domain comprises from about 1% to about 99% modified nucleotides and / or the transcription terminator domain comprises from about 1% to about 99% modified nucleotides; and wherein the Cas protein-binding domain comprises from about 1% to about 99% modified nucleotides comprising one or a combination of the nucleotides in Table 4A. The disclosure relates to a nucleic acid sequence consisting essentially of or consisting of a series of contiguous domains from a 5' to 3' orientation: a DNA-binding domain, a Cas protein-binding domain, and a transcription terminator domain, wherein the DNA-binding domain comprises from about 1% to about 99% modified nucleotides and / or the transcription terminator domain comprises from about 1% to about 99% modified nucleotides; and wherein the Cas protein-binding domain comprises from about 1% to about 99% modified nucleotides comprising one or a combination of the nucleotides in Table 4A.

[0049] Any of the disclosed nucleic acid sequences may comprise any one or combination or set of modifications disclosed herein. In some embodiments, the guide nucleic acid, crRNA and / or tracr nucleic acid sequence comprises RNA, DNA, or combinations of both RNA and DNA. In some embodiments, the RNA, DNA, or combinations of both RNA and DNA, or a part thereof comprise a modified nucleobase or a modified sugar. Modifications to nucleotides are known in the art but include any of the disclosed modifications disclosed in the present application. Oligonucleotides particularly suited for the practice of one or more embodiments of the present disclosure comprise 2'-sugar modified oligonucleotides wherein one or more of the 2'-deoxy ribofuranosyl moi eties of the nucleoside is modified with a halo, alkoxy, aminoalkoxy, alkyl, azido, or amino group. For example, the substitutions which may be independently selected from F, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, SMe, SO2Me, ONO2, NO2, NH3, NH2, NH-alkyl, OCH3=CH2 and OCCH. In each of these, alkyl is a straight or branched chain of Cl to C20, having unsaturation within the carbon chain. A preferred alkyl group is C1-C9 alkyl. A further preferred alkyl group is C5-C20 alkyl. A first group of substituents includes 2'- deoxy-2'-fluoro substituents. A further preferred group of substituents include Cl through C20 alkoxyl substituents. An additional group of substituents include cyano, fluoromethyl, thioalkoxyl, fluoroalkoxyl, alkylsulfinyl, alkylsulfonyl, allyloxy or alkeneoxy substituents.

[0050] " Cas binding domain" refers to a nucleic acid element or domain within a nucleic acid sequence or polynucleotide sequence that, in a biophysically effective amount, will bind or have an affinity for one or a plurality of proteins (or functional fragments thereof) encoded by one or a plurality of CRISPR-associated genes. In some embodiments, in the presence of a the one or a plurality of proteins (or functional fragments thereof) and a target sequence, the one or plurality of proteins and the nucleic acid element forms a biologically active CRISPR complex and / or can be enzymatically active on a target sequence. The terms "CRISPR-associated genes" refer to any nucleic acid that encodes a regulatory or expressible gene that regulates a component or encodes a component of the CRISPR system. In some embodiments, the terms "Cas-binding domain" or "Cas protein-binding domain" refers to a nucleic acid element or domain within a nucleic acid sequence or polynucleotide sequence that, in a biophysically effective amount, will bind to or have an affinity for one or a plurality of proteins in Table 1 (or functional fragments or variants thereof that are at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the sequences disclosed in Table 1). In some embodiments, the Cas binding domain consists of no more than about 10, 11, 12, 13, 14, 15, 16, 17 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250 or more nucleotides in length and comprises at least one sequence that is capable of forming a hairpin or duplex that partially associates or binds to a biologically active CRISPR system at a concentration and within microenvironment suitable for CRISPR system formation. In some embodiments, the composition or pharmaceutical compositions comprises one or a combination of sgRNA, crRNA, and / or tracrRNA that consists of no more than about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more nucleotides in length and comprises at least one sequence that is capable of forming a hairpin or duplex that partially associates or binds to a biologically active protein (or functional fragment thereof) of an amino acid sequence disclosed in Table 1 at a concentration and within microenvironment suitable for CRISPR system formation and CRISPR enzymatic activity on a target sequence. In some embodiments, the Cas protein is derived from the Cas9 family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Casl2(a) family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Casl2a2 family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Cas 12g family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Cas 13b family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the CasX family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Cast 3a family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Casl3c family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Casl3x family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Cas7-11 family of Cas proteins or a functional fragment thereof. In some embodiments, the Cas protein is derived from the Cas 12a family of Cas proteins or a functional fragment thereof.

[0051] The terms "transcription terminator domain" refers to a nucleic acid element or domain within a nucleic acid sequence (or polynucleotide sequence) that, in a biophysically effective amount, prevents bacterial transcription when the CRISPR complex is in a bacterial species and / or creates a secondary structure that stabilizes the association of the nucleic acid sequence to one or a plurality of Cas proteins (or functional fragments thereof) encoded by one or a plurality of CRISPR-associated genes such that, in the presence of the one or a plurality of proteins (or functional fragments thereof), the one or plurality of Cas proteins and the nucleic acid element forms a biologically active CRISPR complex and / or can be enzymatically active on a target sequence in the presence of such a target sequence and a DNA-binding domain. In some embodiments, the transcription terminator domain comprises the nucleotide sequence TTTTTT.In some embodiments, the transcription terminator domain consists of no more than about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more nucleotides in length and comprises at least one sequence that is capable of forming a hairpin or duplex that partially drives association of the nucleic acid sequence (e.g., sgRNA, crRNA with tracrRNA, or other nucleic acid sequence) to a biologically active CRISPR complex at a concentration and microenvironment suitable for CRISPR complex formation.

[0052] The terms "DNA-binding domain" refer to an element or refers to a nucleic acid element or domain within a nucleic acid sequence or sgRNA that is complementary to a target sequence. In some embodiments, in a biophysically effective amount upstream from a Cas- binding domain, the DNA-binding domain will bind or have an affinity for one or a plurality of target nucleic acid sequences such that, in the presence of a biologically active CRISPR complex, one or plurality of Cas proteins can be enzymatically active on the target sequence. In some embodiments, the DNA binding domain comprises about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more nucleotides in length and comprises at least one sequence that is capable of forming Watson Crick base pairs with a target sequence as part of a biologically active CRISPR system at a concentration and microenvironment suitable for CRISPR system formation. In some embodiments, the DNA binding domain consists of no more than about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more nucleotides in length and comprises at least one sequence that is capable of forming Watson Crick base pairs with a target sequence as part of a biologically active CRISPR system at a concentration and microenvironment suitable for CRISPR system formation.

[0053] " CRISPR system" refers collectively to transcripts or synthetically produced transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as aprotospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, "target sequence" refers to a nucleic acid sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is a DNA polynucleotide and is referred to as a DNA target sequence. In some embodiments, a target sequence comprises at least three nucleic acid sequences that are recognized by a Cas-protein when the Cas protein is associated with a CRISPR complex or system which comprises at least one sgRNA or one tracrRNA / crRNA duplex at a concentration and within a microenvironment suitable for association of such a system. In some embodiments the target DNA comprises at least one or more proto-spacer adjacent motifs which sequences are known in the art and are dependent upon the Cas protein system being used in conjunction with the sgRNA or crRNA / tracrRNAs employed by this work. In some embodiments, the target DNA comprises NNG, where G is a guanine and N is any naturally occurring nucleic acid. In some embodiments the target DNA comprises any one or combination of NNG, NNA, GAA, NNAGAAW and NGGNG, where G is a guanine, A is adenine, and N is any naturally occurring nucleic acid or nucleotide.

[0054] In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, a mitochondrion or a chloroplast. A sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In aspects of the disclosure, an exogenous template polynucleotide may be referred to as an editing template. In an aspect of the disclosure, the recombination is homologous recombination. In some embodiments, a composition disclosed herein comprises a recombination template. A recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme (or equivalently a "Cas protein") as a part of a CRISPR complex. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.

[0055] In one aspect, the disclosure provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a synthetic guide sequence comprising at least one of the nucleic acid sequences disclosed herein, wherein the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell (such as a human or a porcine cell), wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and, optionally (2) a tracr mate sequence that is hybridized to a tracr sequence; and (b) a first regulatory element “operatively linked” to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; wherein expressible components (the enzyme-coding sequence and the tracr sequences) are located on the same or different vectors of the system. Within a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the system comprises: (a) a synthetic guide sequence comprising at least one of the nucleic acid sequences disclosed herein, wherein the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell (such as a human or a porcine cell), wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and, optionally (2) a tracr mate sequence that is hybridized to a tracr sequence; and (b) a first regulatory element operatively linked to an enzyme-coding sequence encoding said CRISPR enzyme; wherein said CRISPR enzyme does not comprise a nuclear localization sequence; wherein expressible components (the enzyme-coding sequence and the tracr sequences) are located on the same or different vectors of the system.

[0056] In some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of a tracr regulatory element. In some embodiments, component (a) further comprises one or more additional guide sequences operatively linked to the tracr regulatory element, wherein when expressed, each the additional guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the system comprises the tracr sequence under the control of itsown, second regulatory element, such as a polymerase III promoter. In some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one who is skilled in the art. For example, there are publicly and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython and SeqMan. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences (NLS) of sufficient strength to drive accumulation of said CRISPR complex in a detectable amount in the nucleus of a eukaryotic cell. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for CRISPR complex activity in eukaryotes, but that including such sequences with coupled to particular Cas enzymes enhances activity of the system, especially as to targeting nucleic acid molecules in the nucleus. In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is S. pneumoniae, S. pyogenes, or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. In some embodiments, the CRISPR enzyme is a Sniper-Cas9 enzyme. In some embodiments, the CRISPR enzyme is a Sniper2L-Cas9 enzyme. In some embodiments, the CRISPR enzyme is a Casl2(a) enzyme. In some embodiments, the CRISPR enzyme is a Casl2a enzyme. In some embodiments, the CRISPR enzyme is an enhanced AsCasl2a enzyme (EnAsCasl2a). In some embodiments, the CRISPR enzyme is a Casl2a2 enzyme. In some embodiments, the CRISPR enzyme is a Casl2g enzyme. In some embodiments, the CRISPR enzyme is a Casl3a enzyme. In some embodiments, the CRISPR enzyme is a Casl3b enzyme. In some embodiments, the CRISPR enzyme is a Casl3c enzyme. In some embodiments, the CRISPR enzyme is a Casl3d enzyme. In some embodiments, the CRISPR enzyme is a Casl3x enzyme. In some embodiments, the CRISPR enzyme is a Cas7-11 enzyme. In some embodiments, the CRISPR enzyme is a Cas 13b enzyme. In some embodiments, the CRISPR enzyme is a CasX enzyme. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments the CRISPR enzyme is chosen from one or combination of Table 1.

[0057] In some embodiments, the CRISPR complex does not comprise one or more nuclear localization sequences and said CRISPR complex accumulates in a detectable amount in thecytosol of a eukaryotic cell. Without wishing to be bound by theory, it is believed that excluding one of more NLS for CRISPR complex activity in eukaryotes for particular Cas enzymes (e.g., Casl2a2 and functional derivatives thereof) may enhance activity of the system, especially as to targeting nucleic acid molecules in the cytosol. Unlike some other Cas nucleases, once activated, Casl2a2 indiscriminately degrades targeted double stranded DNA, targeted single-stranded DNA and targeted single-stranded RNA. In some embodiments, Casl2a2 is ideally suited for targeting ASFV because: (i) it can be programmed to indiscriminately destroy DNA upon detection of ASFV RNA; (ii) is a multi -turnover enzyme instead of single turnover like Cas9 and Casl2a, and; (iii) because ASFV is a DNA virus the multi -turnover nature of Casl2a2 DNase activity would be essential to degrade the viral genomic material of ASFV.

[0058] In some embodiments, the CRISPR enzyme is a type II CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Casl2(a) enzyme. In some embodiments, the CRISPR enzyme is a Casl2a2 enzyme. In some embodiments, the CRISPR enzyme is a Casl3b enzyme. In some embodiments, the CRISPR enzyme is a CasX enzyme. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In some embodiments, the CRISPR enzyme lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments the CRISPR enzyme is chosen from one or a combination of Cas proteins listed in Table 1. In some embodiments, the CRISPR enzyme comprises one or more functional fragments of one or a combination of Cas proteins listed in Table 1. In some embodiments, the CRISPR enzyme comprises one of more Cas proteins comprising an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of one or more Cas proteins listed in Table 1. In some embodiments, the CRISPR enzyme comprises one of more Cas proteins comprising the amino acid sequence of one or more Cas proteins listed in Table 1. In some embodiments, the CRISPR enzyme comprises one of more Cas proteins comprising the amino acid sequence of CasX (Casl2e).

[0059] Table 1 - Exemplary Cas proteins

[0060] In another embodiment, the disclosure provides a cell or a vector comprising one of the sgRNAs of the disclosure or functional fragments thereof. The cell may be an animal cell or a plant cell. In some embodiments, the cell is a mammalian cell, such as a human cell. In some embodiments, the cell is a mammalian cell, such as a porcine cell.

[0061] The disclosure relates to nucleic acid molecules comprising nucleic acid sequences that encode a Cas protein or functional fragment thereof. In some embodiments, the disclosure relates to a vector comprising a nucleic acid sequence that encodes a Cas protein or functional fragment thereof operatively linked to a regulatory sequence. In some embodiments, the vector comprises a nucleic acid sequence that encodes a Cas protein or functional fragment thereof, operatively linked to a regulatory sequence.

[0062] In general, and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In some embodiments, the viral vector used to transport another nucleic acid to which it has been linked comprises an Adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrhlO. In some embodiments, the viral vector used to transport another nucleic acid to which it has been linked comprises a lentiviral vector. Another type of vector is a viral-like particle (VLP). In some embodiments, the vector used to transport another nucleic acid to which it has been linked comprises an exosome. In some embodiments, the vector used to transport another nucleic acid to which it has been linked comprises a red blood cell extracellular vesicle (RBCEV).

[0063] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0064] Another aspect of the disclosure relates to a composition comprising a nucleic acid disclosed herein and one or a plurality of recombinant expression vectors. Generally, thedisclosure relates to compositions comprising a synthetic guide sequence and one or a plurality of recombinant expression vectors. Recombinant expression vectors can comprise a nucleic acid of the disclosure in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.

[0065] The term "regulatory element" is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol II 1 promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and HI promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (rRSV) LTR promoter (optionally with the rRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41 :521-530 (1985)), the SV40 promoter, the dihydro folate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term "regulatory element" are enhancer elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-1 (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit 3-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, e.g., the level of expression desired. One or more nucleic acid sequences and one or more vectors can be introduced into host cells to thereby form complexes with other cellular or nonnatural compounds, produce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

[0066] Compositions of the disclosure in nucleotide sequences comprising percent sequence identity to any DNA-binding region disclosed herein. In some embodiments, nucleic acid sequences comprise from about 1 to about 100 modifications at recited position or across several nucleotides. Nucleic acid sequences or oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6- azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3 -deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. These include 5- substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxy ethyl sugar modifications.

[0067] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941, and 5,750,692, each of which is herein incorporated by reference in its entirety.

[0068] In some embodiments, nucleic acid molecules encoding the disclosed nucleic acid sequences, or salts thereof are unmodified.

[0069] In some embodiments, nucleic acid molecules encoding the disclosed nucleic acid sequences, or salts thereof, are substantially isolated. Partial separation can include, for example, a composition enriched in the compound of the disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art, but include, as a non-limiting example, mini-prep, maxi-prep or column separation of nucleic acid molecules from cells after lysing those cells comprising a plurality of nucleic acids.

[0070] In some embodiments, nucleic acid molecules encoding the disclosed nucleic acid sequences, or salts thereof, are substantially isolated. Partial separation can include, for example, a composition enriched in the compound of the disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art, but include, as a non-limiting example, mini-prep, maxi-prep or column separation of nucleic acid molecules from cells after lysing those cells comprising a plurality of nucleic acids. Table 4A denotes Guide RNA sequences (paired in various combinations of repeat / tracrRNA sequences with Guide RNA spacer sequences) for use in combination with the specified Cas protein to target ASFV dsDNA in specific ASFV genes. In some embodiments, Guide RNA sequences (paired in various combinations of repeat / tracrRNA sequences with Guide RNA spacer sequences) for use in combination with the specified Cas protein target ASFV ssRNA transcribed from specific ASFV genes comprise the nucleic acid for use in the CRISPR system. In some embodiments, Guide RNA sequences (paired in various combinations of repeat / tracrRNA sequences with Guide RNA spacer sequences) for use in combination with the specified Cas protein target ASFV dsDNA in specific ASFV genes and target ASFV ssRNA transcribed from specific ASFV genes for use in the CRISPR system.

[0071] In some instances, ASFV target genes and mRNA transcripts are selected based on early expression during ASFV infection and essential functions in ASFV propagation. Table 2 lists protein sequences encoded by ASFV target genes to be targeted by CRISPR systems described herein. In some embodiments, an ASFV DNA polymerase gene or mRNA is targetedby the CRISPR system. In some embodiments, an ASFV Topoisomerase II gene or mRNA is targeted by the CRISPR system. In some embodiments, an ASFV helicase gene or mRNA is targeted by the CRISPR system. In some embodiments, an ASFV C717R gene or mRNA is targeted by the CRISPR system. In some embodiments, an ASFV NP1450L gene or mRNA is targeted by the CRISPR system In some embodiments, genes that have been experimentally shown to be essential for ASFV infection / replication are selected for targeting by a CRISPR single vector system. In some embodiments, this is determined by gene knockdown experiments. These are most valuable for the CRISPR DNA targeting systems that will most likely function by inducing knockout mutations in its target gene(s). These CRISPR DNA targeting systems include Cas9 systems, Cast 2a systems, and Casl2a2 systems. Potential target genes for DNA targeting Cas systems may include A104R, EP402R, EP153R, B119L, I177L, A137R, CP204L. In some embodiments, targets are selected for a CRISPR RNA targeting system. Examples of such systems use Casl2a2 or Cas 13 family nucleases. A focus on genes that are transcribed early in the infection cycle is used to determine RNA targets. The goal with the RNA targeting system is to activate the system as early as possible, so that their trans cleavage activity can keep the virus from replicating its DNA. Potential target genes for an RNA targeting Cas system may include A151R, A240L, A280R, A498R, A505R, A506R, A528R, A542R, CP204L, DP238L, G1207R, I73R, I215L, I267L, J268L, L270L, U104L, V82L, Y118L, and XP124L. In other instances, ASFV target genes and mRNA transcripts are selected based on genomic conservation throughout isolates of ASFV. By targeting conserved regions in the ASFV genome, ASFV may be less capable of adapting by natural selection to escape the CRISPR targeting due to selective pressure to maintain these conserved genetic elements to enable viral propagation. Targeting conserved genomic regions conserved throughout isolates of ASFV also enables the CRISPR constructs to effectively target a broad range of ASFV isolates having variation between genomes but sharing targeted conserved genomic sequences. In some instances, conserved genomic regions within ASFV open reading frames (ORFs) are targeted. In some instances, conserved genomic regions with ASFV G1211R are targeted. In some instances, conserved genomic regions with ASFV C717R are targeted. In some instances, conserved genomic regions with ASFV NP1450L are targeted. In some instances, conserved genomic regions with ASFV G1211R, C717R, and NP1450L are targeted. Spacer sequences within gRNAs are selected for complementarity to conserved genomic regions with ASFV to effectively direct the programmable Cas endonuclease to a nucleotide sequence for targeted nucleotide cleavage or digestion. Table 2 lists exemplary ASFV target proteins and their amino acid sequences.

[0072] Table 2: Exemplary ASFV target proteins

[0073] In some embodiments, an ASFV target gene or mRNA transcript comprises a sequence listed in Table 3. ASFV target mRNA transcripts listed in Table 3 can be used to design crRNA sequences or sgRNA sequences used to target ASFV DNA or RNA for RNA-guided nuclease activity with an activated Cas nuclease. ASFV target mRNA transcripts listed in Table 3 encode ASFV target proteins in Table 2.

[0074] In some embodiments, a nucleic acid sequence encodes a Guide RNA comprising: i) a repeat / tracrRNA, paired with ii) a Spacer RNA sequence corresponding to an ASFV Target gene. In some embodiments, the nucleic acid sequence is transcribed from a vector. In some embodiments, the vector further comprises a nucleic acid sequence encoding a Cas protein or functional fragment thereof. In some embodiments, the vector does not comprise a nucleic acid sequence encoding a Cas protein or functional fragment thereof. In some embodiments, the vector is administered simultaneously or sequentially with a second vector comprising a nucleic acid sequence encoding a Cas protein or functional fragment thereof. In some embodiments, the vector further comprises the nucleic acid sequence encoding the Cas protein or functional fragment thereof transcribes the Guide RNA corresponding to the sequence of the repeat / tracrRNA, paired with the Spacer RNA sequence corresponding to the ASFV Target gene. In some embodiments, a Cas protein or functional fragment thereof encloses the guideRNA and binds the structure with specific interactions in a number of domains. In some embodiments, the Guide RNA recognizes a target DNA or RNA region of interest corresponding to an ASFV target gene or ASFV target RNA and directs a Cas protein or functional fragment thereof to the ASFV target gene or ASFV target RNA. In some embodiments, the direction of the Cas protein or functional fragment thereof to the ASFV target gene or ASFV target RNA enables the directed Cas protein or functional fragment thereof to target endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA. In some embodiments, the direction of the Cas protein or functional fragment thereof to the ASFV conversed target genomic region or ASFV target RNA enables the directed Cas protein or functional fragment thereof to target endonuclease activity to a specific sequence of interest within the conserved ASFV target gene or ASFV target RNA. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA inhibits ASFV viral replication. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA abrogates ASFV viral replication. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA diminishes ASFV viral replication. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA decreases an ASFV viral load. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA inhibits an exposure to ASFV in a subject following the administering from said exposure later developing into an acute ASFV infection. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA serves as an antiviral agent preventing or minimizing ASFV infection in the subject. In some embodiments, the targeted endonuclease activity to a specific sequence of interest within the ASFV target gene or ASFV target RNA serves as an antiviral agent, wherein the antiviral agent i) prevents ASFV infection, or ii) reduces an extent of ASFV infection in the subject.

[0075] In some embodiments, the Guide RNA sequence transcribed from the nucleic acid comprises a repeat / tracrRNA sequence paired with a Spacer RNA sequence. In some embodiments, the paired repeat / tracrRNA sequence and Spacer RNA sequence are listed in Table 4A. In Table 4A, nucleotide sequence in a CRISPR vector are listed. In some embodiments, gRNA or crRNA molecules are transcribed from the CRISPR vector. In some embodiments, the Guide RNA associates with the Cas protein or functional fragment thereof to elicit site-directed Cas-mediated endonuclease activity to a specific ASFV Target DNA withinone or a plurality of cells in a subject. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 14 paired with Spacer RNA sequence selected from SEQ ID NOs: 15-35 that targets an ASFV Helicase DNA sequence, ASFV DNA polymerase DNA sequence, or ASFV Topoisomerase II DNA sequence when combined with Cas9 protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 14 paired with Spacer RNA sequence selected from SEQ ID NOs: 15-17 that targets an ASFV DNA Polymerase DNA sequence when combined with Cas9 protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 14 paired with Spacer RNA sequence selected from SEQ ID NOs: 15-17 and 118-121 that targets an ASFV DNA Polymerase DNA sequence, ASFV C717R DNA sequence, or ASFV NP1450L DNA sequence when combined with Cas9 protein. In some embodiments, the Cas9 is SpCas9 (corresponding to protein sequence of SEQ ID NO: 1). In some embodiments, the Cas9 is Sniper-Cas9 (corresponding to protein sequence of SEQ ID NO: 109). In some embodiments, the Cas9 is Sniper2L-Cas9 (corresponding to protein sequence of SEQ ID NO: 108).

[0076] In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 122 paired with Spacer RNA sequence selected from SEQ ID NO: 123-128 that targets a conserved ASFV G1211R, C717R, or NP1450L sequence when combined with Casl2a protein or variant thereof. In some embodiments, the Casl2a variant is Enhanced AsCasl2a (corresponding to protein sequence of SEQ ID NO: 110).

[0077] In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 36 paired with Spacer RNA sequence selected from SEQ ID NO: 40-42 that targets an ASFV topoisomerase RNA sequence when combined with Casl2a(2) protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 36 paired with Spacer RNA sequence selected from SEQ ID NO: 37-39 that targets an ASFV Polymerase RNA sequence when combined with Casl2a(2) protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 36 paired with Spacer RNA sequence selected from SEQ ID NO: 43-45 that targets an ASFV DNA Polymerase RNA sequence when combined with Casl2a2 protein.

[0078] In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 46 paired with Spacer RNA sequence selected from SEQ ID NO: 47-64 that targets an ASFV topoisomerase, ASFV DNA polymerase, or ASFV helicase sequence when combined with Cast 3d protein.

[0079] In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 65 paired with Spacer RNA sequence selected from SEQ ID NO: 66-83 that targets anASFV topoisomerase, ASFV DNA Polymerase, or ASFV helicase sequence when combined with CasX protein.

[0080] In some embodiments, the Guide RNA sequence transcribed from the nucleic acid comprises a repeat / tracrRNA sequence paired with a Spacer RNA sequence. In some embodiments, the paired repeat / tracrRNA sequence and Spacer RNA sequence are listed in Table 4A. In some embodiments, the Guide RNA associates with the Cas protein or functional fragment thereof to elicit site-directed Cas-mediated endonuclease activity specific ASFV Target RNA molecules within one or a plurality of cells in a subject. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 14 paired with Spacer RNA sequence selected from SEQ ID NOs: 15-35 and 118-122 that targets an ASFV sequence when combined with Cas9 protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 122 paired with Spacer RNA sequence selected from SEQ ID NOs: 123-128 that targets an ASFV sequence when combined with Casl2a protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 36 paired with Spacer RNA sequence selected from SEQ ID NO: 37-45 that targets an ASFV sequence when combined with Casl2a(2) protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 46 paired with Spacer RNA sequence selected from SEQ ID NO: 47-64 that targets an ASFV Polymerase RNA sequence when combined with Casl3d protein. In some embodiments, the Guide RNA sequence comprises a repeat / tracrRNA of SEQ ID NO: 65 paired with Spacer RNA sequence selected from SEQ ID NO: 66-83 that targets an ASFV Helicase RNA sequence when combined with Casl2a(2) protein.

[0081] Alternative gRNA sequences may be selected based on intrinsic parameters of the corresponding Cas enzyme itself such as, PAM / PFS sequence preference and orientation, spacer length preference, specific scaffold or direct repeat sequences and orientation of scaffold / repeat and spacer components of the gRNA. Ideally, the spacer sequences would contain limited secondary structure and limited hard to transcribe segments, such as T-rich sequences that may prematurely terminate transcription of the gRNA. The PAM (protospacer adjacent motif) sequence for Cas9 enzyme, (e.g.,SpCas9), is 5'-NGG-3', wherein "N" can be any base. The PAM sequence for Casl2a is TTTV where "V" can be A, C, or G; meaning that Casl2a typically recognizes a thymine-rich sequence adjacent to the target site on the DNA strand.

[0082] There are also several existing software known in the art and available for free or for a fee, such as CRISPick, CHOPCHOP, GENEIOUS Prime, and Benchling CRISPR gRNA design tool, which can be used to generate a library of possible gRNA sequences for a specified target region based on known parameters.

[0083] In some embodiments, the Guide RNA sequence transcribed from the nucleic acid comprises a repeat / tracrRNA sequence paired with a Spacer RNA sequence. In some embodiments, the paired repeat / tracrRNA sequence and Spacer RNA sequence are listed in Table 4A. In some embodiments, the specific i) repeat / tracrRNA sequence, paired with a specific ii) Spacer RNA sequence, to be used in CRISPR complex with a specific iii) Cas protein is listed in Table 4A as a Pairing. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is at least nine of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is at least eight of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is at least seven of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is at least six of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is at least five of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may beused in a method described herein, is at least four of the following selected from 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, thePairing defining a composition described herein, which additionally may be used in a method described herein, is at least three of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is at least two of the following selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, and 75. In some embodiments, the Pairing defining a composition described herein, which additionally may be used in a method described herein, is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75.

[0084] The disclosure relates to a composition comprising a cell with any one or combination of nucleic acid sequences disclosed herein. In some embodiments, the cell is a plant, insect or mammalian cell. In some embodiments, the cell is a eukaryotic cell or a prokaryotic cell, the cell may be isolated from the body of a mammal, a component of a culture system, or part of an organism, such as a pig or minipig. In some embodiments, the system and methods described herein include at least two components: (1) the RNAs or DNA / RNA hybrid (guide nucleic acid, a crRNA, tracrRNA, and / or a single cr / tracrRNA hybrid) targeted to a particular ASFV sequence in a cell; and (2) a Cas protein disclosed herein. In some embodiments, the Cas protein may be in a complex with the tracrRNA / crRNA / sgRNA, also called a ribonucleoprotein (RNP) complex, before being introduced or administered into the target virus, cell or organism. In some cases, a system also can include a nucleic acid containing a donor sequence targeted to a sequence in the cell. The donor sequence and the guide sequence may be on one or a plurality of nucleic acid molecules. The Cas protein disclosed herein can create targeted DNA double-strand breaks at the desired viral nucleotide sequence (or loci), and the host cell can repair the doublestrand break using the provide donor DNA sequence, thereby incorporating the modification stably into the viral DNA. In some embodiments, the viral DNA is cleaved and repaired after removing DNA between two or more cleavage events, such that the viral DNA is ligated with asignificant deletion of genomic nucleic acid sequence. The resultant deletion can cause mutated or silenced viral genes and therefore disrupt viral expression and / or assembly.

[0085] Exemplary ASFV helicase protein sequence, ASFV topoisomerase protein sequence, ASFV DNA polymerase sequence, are listed in Table 2. Exemplary ASFV C717R uncharacterized protein sequence and ASFV NP1450L DNA-directed RNA polymerase subunit protein sequence are listed in Table 2. In some embodiments, the guide sequence comprises a DNA or RNA target region complementary for an ASFV protein listed in Table 2. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid encoding from about 3 to about 30 amino acids from ASFV helicase. In some embodiments, the ASFV helicase comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the guide sequence comprises a DNA target region complementary for an ASFV genomic DNA sequence listed in Table 3. In some embodiments, compositions described herein comprise a guide sequence comprising a DNA target region complementary to a nucleic acid encoding from about 3 to about 30 amino acids from ASFV topoisomerase. In some embodiments, the ASFV topoisomerase comprises the amino acid sequence of SEQ ID NO: 10. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid encoding from about 3 to about 30 amino acids from ASFV DNA polymerase. In some embodiments, the ASFV DNA polymerase comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 112. In some embodiments, compositions described herein comprise a guide sequence comprising a DNA target region complementary to a nucleic acid encoding from about 3 to about 30 amino acids from ASFV C717R. In some embodiments, the ASFV C717R comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, compositions described herein comprise a guide sequence comprising a DNA target region complementary to a nucleic acid encoding from about 3 to about 30 amino acids from ASFV NP1450L. In some embodiments, the ASFV topoisomerase comprises the amino acid sequence of SEQ ID NO: 114.

[0086] In some embodiments, the targeted ASFV sequence is in a conserved ASFV ORF. In embodiments, the conversed ASFV ORF that is targeted encodes an ASFV DNA polymerase (for instance, G1211R). In embodiments, the conversed ASFV ORF that is targeted encodes an ASFV Helicase. In embodiments, the conversed ASFV ORF that is targeted encodes an ASFV Topoisomerase type II. In embodiments, the conversed ASFV ORF that is targeted encodes C717R uncharacterized protein. In embodiments, the conversed ASFV ORF that is targeted encodes a DNA-directed RNA polymerase subunit (e.g., NP1450L). The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 11. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 115. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 11. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 12. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 13. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 116. The disclosure relates to a composition comprising a guide sequence comprising a DNA target region complementary to a nucleic acid comprising at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to from about 9 to about 90 nucleotide segment of the sequence of SEQ ID NO: 117. Table 3 lists exemplary ASFV target mRNA transcripts derived from conversed ASFV ORFs.

[0087] Table 3: Exemplary ASFV target mRNA transcripts

[0088] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Cleavage of ASFV DNA renders it inoperable and susceptible to degradation within a host cell. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wildtype tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operatively linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional (bind the Cas proteinor functional fragment thereof). In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that the presence and / or expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operatively linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. With at least some of the modifications contemplated by this disclosure, in some embodiments, the guide sequence or RNA or DNA sequences that form a CRISPR complex are at least partially synthetic. The CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. In some embodiments, the disclosure relates to a composition comprising a chemically synthesized guide sequence. In some embodiments, the chemically synthesized guide sequence is used in conjunction with a vector comprising a coding sequence that encodes a CRISPR enzyme, such as a type II Cas9 protein. In some embodiments, the chemically synthesized guide sequence is used in conjunction with one or more vectors, wherein each vector comprises a coding sequence that encodes a CRISPR enzyme, such as a type II Cas9 protein. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a repeat / tracrRNA, CRISPR enzyme and one or more additional (second, third, fourth, etc.) guide sequences, tracr mate sequence (optionally operatively linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and / or tracr sequence are each a component of different nucleic acid sequences. For instance, in the case of a tracr and tracr mate sequences and in some embodiments, the disclosure relates to a composition comprising at least a first and second nucleic acid sequence, wherein the first nucleic acid sequence comprises a tracr sequence and the second nucleic acid sequence comprises a tracr mate sequence, wherein the first nucleic acid sequence is at least partially complementary to the second nucleic acid sequence such that the first and second nucleic acid form a duplex and wherein the first nucleic acid and the secondnucleic acid either individually or collectively comprise a DNA-targeting domain, a Cas protein binding domain, and a transcription terminator domain. In some embodiments, the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are operatively linked to and expressed from the same promoter. In some embodiments, the disclosure relates to compositions comprising any one or combination of the disclosed domains on one guide sequence or two separate tracrRNA / crRNA sequences with or without any of the disclosed modifications. Any methods disclosed herein also relate to the use of tracrRNA / crRNA sequence interchangeably with the use of a guide sequence, such that a composition may comprise a single synthetic guide sequence and / or a synthetic tracrRNA / crRNA with any one or combination of modified domains disclosed herein. One or a plurality of vectors may also be components in any system or composition provided herein. In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operatively linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression, the guide sequence directs sequence- specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple, different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple, different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide- sequence-containing vectors may be provided, and optionally delivered to a cell. The disclosure relates to any composition comprising any of the aforementioned elements and one or more artificially synthesized guide sgRNA described herein. Another aspect of the disclosure relates to a CRISPR system comprising a modified CRISPR enzyme (or "Cas protein") or a nucleotide sequence encoding one or more Cas proteins. Any protein capable of enzymatic activity in cooperation with a guide sequence is a Cas protein. In some embodiments, the disclosure relates to a system comprising a vector comprising a regulatory element operatively linked to anenzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein from the Cas family of enzymes.

[0089] In some embodiments, the disclosure relates to a system, composition, or pharmaceutical composition comprising any one or plurality of Cas proteins either individually or in combination with one or a plurality of guide sequences. Compositions of one or a plurality of Cas proteins may be administered to a subject with any of the disclosed guide sequences sequentially or contemporaneously. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Casl2a, type V CRISPR-Cas systems, variants and fragments thereof, or modified versions thereof having at least 70% homology to the sequences of Table 1, wherein are incorporated by reference in their entireties. These enzymes are known; for example, amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. Amino acid sequence of S. pyogenes Cas9 protein may be found in the Genbank database under accession number AZQ25086. After an organism is infected with ASFV, the organism can be administered or transfected with a sequence encoding a Cas protein disclosed herein or a functional fragment thereof, a crRNA, a trRNA, a crRNA and a tracrRNA, a cr / tracrRNA hybrid, and / or a synthetic guide nucleic acid (and, in some cases, a donor sequence), any suitable method can be used to determine whether targeted mutagenesis has occurred at the target site. In some embodiments, a phenotypic change can indicate that a donor sequence has been integrated into the target site. PCR-based methods also can be used to ascertain whether a genomic target site contains targeted mutations or donor sequence, and / or whether precise recombination has occurred at the 5' and 3' ends of the donor. In some embodiments, a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about (or more than about) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the disclosure, the CRISPR enzyme comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest aminoacid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, but other types of NLS are known. Non- limiting examples of NLS include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence. In some embodiments, the CRISPR enzyme or Cas protein (used interchangeably) is free of a nuclear localization sequence. In some embodiments, any domain comprises hybrid RNA / DNA sequences of either unmodified or modified nucleotides. In some embodiments, the DNA- targeting domain comprises no less than about 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, or 20 nucleotides, wherein no more than about 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides is a modified or unmodified deoxyribonucleic acid. In some embodiments, the DNA-targeting domain comprises no less than about 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, or 20 nucleotides, wherein no more than about 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides from the 5' end of the guide sequence is a modified or unmodified deoxyribonucleic acid. In some embodiments, the Cas-binding domain comprises no less than about 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, or 20 nucleotides, wherein no more than about 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides is a modified or unmodified deoxyribonucleic acid. In some embodiments, the transcription terminator domain comprises no less than about 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, or 20 nucleotides, wherein no more than about 50, 45, 40, 35, 30, 25, 20, 15, 14, 13 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides is a modified or unmodified deoxyribonucleic acid. In some embodiments, the transcription terminator domain is free of modified or unmodified deoxyribonucleic acid. In some embodiments, the Cas-binding domain is free of modified or unmodified deoxyribonucleic acid. Table 1 lists Accession Numbers of Cas proteins (or those related with Cas-like function) and Nucleic Acids encoding the same. Any mutants or variants that comprise at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity to the encoded nucleic acids or acids set forth in the Accession Numbers below are also incorporated by reference in their entireties: AZQ25086, ATB19154.1, KIM12007.1, WP_117939725.1, 6NY3_Y , WP-124327589.1, 8I3Q_A , 5W1H_A , 6AAY A , WP_005959231.1, 7VTN_A , ERL19323.1, NC_O14644.1; NC_002967. 9, NC_007929.1; NC_000913. 3 NC_004547. 2, NC_009380.1; NC_011661.1; NC_010175.1; NC_010175.1; NC_010175.1; NC_003413.1; NC_000917.1; NC_002939. ,5 NC_018227.2; NC_004829.2, NC_021921.1; NC_014160.1; NC_011766.1; NC_007681.1; NC_021592. l; NC_021592. l; NC_021169. l; NC_020517.1; NC_018656.1 ; NC_018015.1 ; NC_018015.1 ; NC_017946. l; NC_017576. l; NC_017576.1; NC_015865.1; NC_015865.1; NC_015680.1; NC_015680.1; NC_015474.1; NC_015435.1;NC_013790.1; NC_013790.1; NC_012883.1; NC_012470.1; NC_016051.1; NC_010610.1; NC_009515.1; NC_008942.1 ; NC_007181.1; NC_007181.1; NC_006624.1 ; NC_006448.1 ; NC_002935.2; NC_002935.2; NC_002950.2, NC_002950. 2, NC_002663.1; NC_002663.1; NC_004557.1; NC_004557.1; NC_019943.1; NC_019943.1; NC_019943.1; NC_017459.1; NC_017459.1; NC_015518.1; NC_015460.1; NC_015416.1; NC_014933.1; NC_013961.1; NC_013202.1; NC_013158.1; NC_009464.1; NC_008508.1; NC_007426.1; NC_000917.1; NC_003901.1; NC_003901.1; NC_003106. .2, NC_009434.1; NC_005085.1; NC_005085.1; NC_020247.1; NC_020247.1; NC_020246.1; NC_020246.1; NC_018224.1; NC_015943.1; NC_011138.3; NC_009778.1; NC_006834.1; NC_014228.1; NC_010002.1; NC_013892.1; NC_010296.1; NC_009615.1; NC_012632.1; NC_012632.1; NC_012588.1; NC_012588.1; NC_007643.1 ; NC_002939.5; NC_011296.1; NC_011296.1; NC_018609.1; NC_021355.1; NC_021355.1; NC_020800.1 ; NC_019942.1 ; NC_019792.1 ; NC_015958.1; NC_015678.1 ; NC_015636.1; NC_015562.1; NC_014222.1; NC_014222.1; NC_014002.1; NC_013887.1; NC_013156.1; NC_011832.1; NC_009953.1; NC_009635.1; NC_009634.1; NC_008618.1; NC_007955.1; NC_007955.1; NC_007955.1; NC_007955.1; NC_007955.1; NC_007796.1; NC_002754.1; NC_002754.1; NC_011835.1; NC_013198.1; NC_000962.3; NC_002163.1; NC_017034.1; NC_009089.1; NC_008698.1; NC_020419.1; NC_020419.1; NC_020419.1; NC_015847.1; NC_014374.1; NC_013520.1; NC_010482.1; NC_009776.1; NC_009776.1; NC_009033.1; NC_000916.1; NC_018015.1; NC_015518.1; NC_014537.1; NC_009440.1 ; NC_007644.1; NC_007644.1; NC_022246.1; NC_019943.1; NC_016023.1; NC_016023.1; NC_015416.1; NC_013722.1 ; NC_013722.1 ; NC_009464.1 ; NC_007643.1 ; NC_007643.1 ; NC_007643.1; NC_003106.2; NC_004342.2; NC_018658.1; NC_017276.1; NC_017275.1; NC_016112.1; NC_016112.1; NC_003552.1; NC_003197.1; NC_003198.1; NC_012726.1; NC_012623.1; NC_015964.1; NC_023069.1; NC_023044.1; NC_022777.1; NC_022777.1; NC_022777.1; NC_013769.1; NC_013769.1; NC_011832.1; NC_011296.1; NC_009712.1;NC_009634.1; NC_009439.1; NC_009135.1; NC_008599.1; NC_007796.1; NC_007796.1; NC_007796.1; NC_007355.1; NC_021082.1; NC_018001.1; NC_009785 1; NC_022084.1; NC_018092.1 ; NC_014804.1 ; NC_014147.1; NC_009053.1 ; NC_000961.1; NC_ 000961.1; NC_021058.1; NC_018876.1; NC_018876.1; NC_018081.1; NC_011567.1; NC_016901.1; NC_ 014500.1; NC_013715.1; NC_019977.1; NC_019042.1; NC_017274.1; NC_015954.1; NC_015676.1; NC_ 015320.1; NC_014122.1; NC_014122.1; NC_013407.1; NC_014961.1; NC_013926.1; NC_013926.1; NC_ 021353.1; NC_008818.1; NC_021058.1; NC_015151.1; NC_013849.1; NC_009051.1; NC_018876.1; NC_ 018876.1; NC_014507.1; NC_015574.1; NC_014500.1; NC_012622.1; NC_012589.1; NC_009515.1; NC_ 017275.1; NC_000913.3; NC_017527.1; NC_018227.2; NC_007355.1; NC_014106.1; NC_010610.1; NC_ 008054.1;NC_007164.1; NC_015760.1; NC_009953.1; NC_010572.1; NC_009613.3; NC_014334.1;NC_ 008526.1; NC_026150.1; NC_015776.1; NC_007116.6; NC_012779 2; NC_003901.1;NC_020892.1 ; NC_ 011832.1; NC_003143.1 ; NC_003143.1 ; NC_008800.1 ; NC_011308.1;NC_008942.1; NC_007297.1; NC_ 005877.1; NC_005877.1; NC_002689 2; NC_006085.1;NC_004116 1; NC_010397.1; NC_009917.1; NC_ 012490.1; NC_006067.1; NW_004197518.1;NC_022777.1; NC_019042.1; NC_004547.2; NC_002695.1; NT_ 078267. 5; NC_002656.1;NC_022774.1; NC_01109 1.1; NC_005881.2; NC_011183.1; NC_ 015937.1; NC_008584.1;NC_024122.1 ; NC_022768.1 ; NC_022772.1 ; NC_013085.1; NC_010154.1; NC_ 010152.1;NC_010155.1; NC_009804.1; NC_009803.1; NC_005342.2; NC_004333 2; NC_023735.1;NC_ 023694.1; NC_027364.1; NC_019526.1; NC_023607.1; NC_021353.1; NC_021592.1;NC_012039.1; NC_ 008942.1; NC_002936.3; NC_005877.1; NC_021169.1; NC_021058.1;NC_020517.1; NC_020388.1; NC_ 020388.1; NC_018656.1; NC_015435.1; NC_014804.1;NC_013790.1; NC_013790.1; NC_009440.1; NC_ 009051.1; NC_007929.1; NC_007929.1;NC_005042.1; NC_003454.1; NC_003238 2; NC_021313.1; NC_ 019943.1; NC_019943.1;NC_017459.1; NC_017384.1; NC_015288.1; NC_015287.1; NC_015284.1; NC_ 015281.1;NC_015280.1; NC_014334.1; NC_014297.1; NC_013967.1; NC_013202.1; NC_011129.1;NC_ 007426.1; NC_007426.1; NC_003901.1; NC_003901.1; NC_004342 2; NC_014622 2;NC_023731.1; NC_ 023729.1; NC_023716.1; NC_017275.1; NC_015574.1; NC_015216.1;NC_015216.1; NC_013922.1; NC_ 013922.1; NC_013743.1; NC_012966.1; NC_012966.1;NC_011913.1; NC_010397.1; NC_010296.1; NC_ 009380.1; NC_006396.1; NC_006347.1;NC_002944 2; NC_003552.1; NC_004663.1; NW_006890135.1; NW_005819424.1;NW_005395962.1; NC_022273.1; NC_019466.1; NC_018739.2; NC_016132.1; NC_012593.1;NC_026744.1; NC_026585.1; NC_026584.1; NC_022067.1; NC_017274.1; NC_017274.1;NC_017274.1; NC_016563.1; NC_015562.1; NC_013769.1; NC_013769.1; NC_010175.1;NC_002754.1; NC_002754.1; NC_009089.1; NC_014374.1; NC_009776.1; NC_005877.1;NC_005877.1; NC_005877.1; NC_002689.2; NC_002689.2; NC_002689.2; NC_000918.1;NC_022093.1 ; NC_022093.1 ; NC_022093.1 ; NC_018092.1 ; NC_015931.1; NC_015931.1;NC_015931.1; NC_015865.1; NC_010482.1 ; NC_010482.1 ; NC_000916.1; NC_000961.1;NC_000961.1; NC_000853.1; NC_000853.1; NC_021313.1; NC_020388.1; NC_018876.1;NC_015151.1; NC_013849.1; NC_009440.1 ; NC_007426.1 ; NC_007181.1; NC_007181.1;NC_007181.1; NC_003106.2; NC_027207.1; NC_027206.1; NC_020247.1; NC_020247.1;NC_020247.1; NC_020246.1; NC_020246.1; NC_020246.1; NC_006347.1; NC_005140.1;NC_013486.1; NC_013486.1; NC_012726.1; NC_012632.1; NC_012589.1; NC_012588.1;NC_006038.1; and NC_012012.3.

[0090] Nucleotide sequences for components of the CRISPR systems described herein are listed in Table 4A.

[0091] Table 4A: Nucleotide sequences for gRNA and CRISPR vector components

[0092] Table 4B lists exemplary nucleotide sequences for ASFV targeting CRISPR plasmid vectors utilizing components listed in Table 4 A.

[0093] Table 4B: Nucleotide sequences for ASFV targeting CRISPR single vectors

[0094] In some instances, mutations or other potential changes in plasmid sequences described herein may be made to improve activity, either as improved prophylactic activity, improved therapeutic activity, or improved prophylactic and therapeutic activity as an ASF treatment. The suCasl2a2 enzyme possesses an RNA-triggered collateral / trans-nuclease activity — it indiscriminately cleaves dsDNA, ssDNA and RNA after binding its intended target sequence. This activity could potentially be reduced or abolished by mutations in suCasl2a2 nucleotide sequence that leads to a change in the transcribed RNA and ultimately the translated Casl2a2 protein. This collateral activity of Casl2a2 has been shown in bacterial cells and is what leads to ‘abortive infection,’ which is a process where a bacterial cell infected by a virus suffers damage and is potentially killed leading to less viral replication because the host cell itself is dead. This collateral activity has not been shown definitively in mammalian cells yet but there may be some indication that it may be present. If it is present, it may cause toxicity due to its off-target effects on the host cell DNA and / or RNA. In some embodiments, these mutations may be beneficial because they can lead to a reduction in off-target effects of Casl2a2. In some embodiments, the Cas gene nucleotide sequence can be mutated such that the expressed enzyme possesses increased target-binding specificity and / or nuclease activity. In some embodiments, these mutations may increase the effectiveness of a Cas-based therapy. Increased nuclease activity may allow the Cas enzyme to cleave nucleic acids more quickly, in an effort to better keep up with replication of the virus, while increased target-binding specificity will decrease eventswhere the Cas nuclease activity is incorrectly activated by a mismatched target. The Cas gene nucleotide sequence can be mutated such that the expressed enzyme possesses a modified ability to detach from the targeted sequence. The Casl2a2 enzyme leads to multiple turnover events — it binds its target, which opens the nuclease active site thereby permitting indiscriminate cleavage of ssDNA, dsDNA, and ssRNA. Cas9, on the other hand, leads to single turnover events — it binds its target, cleaves, and stays attached unless it gets kicked off its binding site due to other cellular elements / processes. In some embodiments, the modification of this property by mutations may lead to improved targeting. An increase in turnover by Cas9 may result in Cas9 being more efficient as it may be able to better target several copies of the target sequence in the same cell without having to rely on being passively being kicked off its target. A decrease in the turnover of Casl2a2 may potentially lead to reduced off-target effects of Casl2a2 because it may reduce the rate of trans-nuclease activity.

[0095] In some embodiments, the Cas gene nucleotide sequences may be modified to make them shorter. Shorter gene sequences would equate to shorter plasmid sequences, which can improve their encapsulation in nanoparticles and other delivery methods like viral vectors. Shorter sequences may also lead to more plasmids per nanoparticle or smaller nanoparticles, both of which can potentially increase dose delivered in vivo, the former by increased number of plasmids delivered per transfection event, and the latter via increased transfection efficiency. Reduced Cas gene sequence size can be achieved by eliminating sequences corresponding to domains of the Cas enzyme that are determined to be non-essential or dispensable to the specific binding and nuclease activity of the enzyme.

[0096] In some embodiments, a promoter sequence controlling the expression of the Cas enzyme, or targeting RNA sequences (or both), or other regulatory sequences, may be replaced with a different promoter sequence. Conditional or cell / tissue-specific promoters may be used instead of the current constitutive mammalian promoters. A conditional promoter may be such that it allows for the expression of the Cas gene, or targeting RNAs, or both, only under certain conditions that could potentially be controlled by humans. For example, the promoter may be such that it is activated by a small molecule that can be delivered to the pigs via feed. In such a scenario, all pigs could be given a dose of the Cas-based therapeutic / prophylactic treatment, but the treatment does not take effect until the pigs are fed a special feed that contains the small molecule. This may allow farmers to blanket treat animals without side-effects in healthy animals. A cell or tissue-specific promoter may be such that the Cas-based therapeutic may be delivered systemically (IV, IM, Nasal, Feed, other methods) and makes it into a wide variety of cell types, but the Cas enzyme, or targeting RNAs, or both, are only expressed in specific cell types or tissue types. This may be beneficial because the ASFV virus infects certain cell typesmore than others and if we are able to limit the effects of the therapeutic in those tissues or cells, we may also be able to limit side-effects and off-target effects. Translation of Cas enzymes may also be modulated by insertion of RNA aptamer motifs within the 5’ untranslated region of the mRNA sequence that regulate translation initiation based on the presence or absence of external factors, such as specific metabolites or specific viral RNA. In some embodiments, specific multiplexed gRNAs that target conserved sequences in the ASF genome are used in methods of treatment described herein as an effective universal cure for ASFV. In some instances, the treatment is effective against Genotype II ASFV virus. In some instances, guide RNAs specific for conserved ASF genomic sequences are utilized to target one or more ASFV nucleic acid sequences. In some instances, ASFV RNA is targeted. In some instances, ASFV DNA is targeted. In some instances, the compositions described herein are an effective therapeutic for ASF. In some instances, the compositions described herein are used in methods of treating ASFV and form an effective therapeutic strategy for ASF.

[0097] In an aspect, compositions described herein comprise: (i) a first nucleic acid encoding a Cas endonuclease protein or a Cas variant protein configured to disrupt a function of one or more target loci of a pathogen in a subject and (ii) a second nucleic acid encoding one or more guide RNA (gRNA) molecules, wherein the disrupting the function of the target locus results in an increase in immune response of the subject. In some embodiments, the immune response is an innate immune response. In some embodiments, the composition disrupts the function of the one or more target loci by contacting the one or more target loci (e.g., genes or mRNA involved in pathogen replication) with one or more RNA-guide Cas proteins or one or more RNA-guided Cas variant proteins. In some embodiments, disrupting the function of the target loci involves targeted cleavage from a CRISPR RNP complex. In some embodiments, disrupting the function of the target loci involves indiscriminate nuclease activity from an activated Cas endonuclease (e.g., Casl2a2) or an activated Cas variant protein. In some embodiments, the increase in the immune response comprises an increase in a number of antibodies specific to the pathogen. In some embodiments, the Cas endonuclease protein or the Cas variant protein comprises a Cas9 enzyme. In some embodiments, the Cas endonuclease protein or the Cas variant protein comprises a Casl2 enzyme. In some embodiments, the Casl2 enzyme is a Casl2a enzyme. In some embodiments, the Casl2 enzyme is a Casl2g enzyme. In some embodiments, the Cas endonuclease protein or the Cas variant protein comprises a Casl3a, Casl3b, Casl3c, Casl3d, Casl3x, or any combination thereof. In some embodiments, the Cas endonuclease protein or the Cas variant protein comprises Cas7-11. In some embodiments, each of the one or more gRNA molecules comprise at least about 90% sequence complementarity to at least one of the one or more target loci. In some embodiments, the one or more target loci are selected based on beingessential for replication of the specific pathogen (e.g., genes or mRNA essential for replication of ASFV). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a pig, a swine, a minipig, or a wild boar. In some embodiments, the pig species is Sus scrofa, Sus ahenobarbus, Sus barbatus, Sus cebrifons, Sus celebensis, Sus oliveri, Sus philippensis, or Sus verrucosus.

[0098] In an aspect, CRISPR compositions described herein are designed for generating long- lasting immunity to African Swine Fever Virus (ASFV) in a subject. In an aspect, the compositions for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject comprise: (i) an isolated nucleic acid sequence encoding a Clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease or functional fragment thereof and (ii) a guide RNA (gRNA) complementary to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the composition is configured to reduce a viral load of ASFV to an extent that generates a durable immune response to an ASFV infection in the subject. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a programmable Cas endonuclease selected from a Cas9, a Casl2a2, a Cast 2a, a Cas 13d, or a CasX. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Casl2a. In some embodiments, the Casl2a selected from use in designing the composition is EnAsCasl2a. In some embodiments, the isolated nucleic acid sequence encoding the gRNA comprises a sequence selected from SEQ ID NOs: 123-128. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, or six gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 123-128. In some embodiments, the isolated nucleic acid sequence six gRNA sequences each comprising a spacer sequence of one of SEQ ID NOs: 123-128. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence identical to SEQ ID NO: 143. In some embodiments, the isolated nucleic acid is encoded with a plasmid consists essentially of a nucleotide sequence of SEQ ID NO: 143. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Casl3d. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a CasX. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Cas9. In some embodiments, the Cas9 is Sniper-Cas9. In some embodiments, the Cas9 is Sniper2L-Cas9. In some embodiments, the Cas9 comprises a nuclear localization signal (NLS), a nuclear export signal (NES), or a nucleocytoplasmic shuttling sequence (NCS) comprising an NLS and anNES. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, or seven gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for three gRNAs each comprising one of spacer sequences of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 140. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence identical to SEQ ID NO: 140. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of the nucleotide sequence of SEQ ID NO: 140. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for seven gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence identical to SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of a nucleotide sequence of SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 142. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence identical to SEQ ID NO: 142. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of a nucleotide sequence of SEQ ID NO: 142. In some embodiments, the CRISPR-associated endonuclease or functional fragment thereof comprises a Casl2a2. In some embodiments, the Casl2a2 comprises an NLS, an NES, or an NCS comprising an NLS and an NES. In some embodiments, the Casl2a2 is lacking an NLS. In some embodiments, the isolated nucleic acid sequence encoding the gRNA comprises a sequence selected from SEQ ID NOs: 37-45. In some embodiments, the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 37-45. In some embodiments of compositions described herein, the gRNA and the programmable Cas endonuclease protein or functional fragment thereof are transiently expressed in cells of the subject following administering of the composition to a subject. In some embodiments, transient expression of the gRNA and the programmable Cas endonuclease protein or functional fragment thereof produces cleaving or digesting of ASFV nucleic acid in cells of the subject and reducesASFV viral load in the subject by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 30 days after the administering. In some embodiments, transient expression of the gRNA and the programmable Cas endonuclease protein or functional fragment thereof improves survival of the subject following exposure to ASFV. In some embodiments, reduction in ASFV viral load allows the subject to develop an extent of posttreatment immunity to ASFV and achieve a durable protective immunity upon ASFV rechallenge.

[0099] In an aspect described herein are gRNAs comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome. Designed gRNAs can be used for activating and targeting programmable Cas endonuclease to a conversed target nucleic acid sequence in an Asfarviridae genome. In some embodiments of compositions described herein, a gRNA is designed to be produced from a vector. In some aspects described here are vectors comprising a nucleic acid sequence encoding (i) a programmable Cas endonuclease and (ii) three guide RNAs (gRNAs), each gRNA comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome; wherein the programmable Cas endonuclease comprises a Cas9 or functional fragment thereof, and wherein the three gRNAs each comprise a sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the vector further comprises nucleic acid sequence encoding one, two, three, or four additional gRNAs, the additional gRNAs each comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 118-121. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 141. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 142. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 121. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) a programmable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 121, wherein the programmable Cas endonuclease comprises a Cas9 or functional fragment thereof. In some embodiments, the vector further comprises nucleic acid sequence encoding one, two, three, four, five, or six additionalgRNAs, the additional gRNAs each comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 15-17 and 118-120. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 123. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) a programmable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 123, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 124. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) a programmable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 124, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 125. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) a programmable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 125, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 126. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) a programmable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 126, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 127. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) a programmable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 127, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In an aspect described herein is a guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 128. In some embodiments of vectors described herein, the vector comprises a nucleic acid sequence encoding i) aprogrammable Cas endonuclease, and ii) a gRNA comprising a spacer sequence of SEQ ID NO: 128, wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof. In some embodiments, the vector further comprises nucleic acid sequence encoding one or more, two or more, three or more, four or more, or five additional gRNAs, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 123-128. In some embodiments, the vector further comprises nucleic acid sequence comprising each of nucleic acid sequences of SEQ ID NOs 123-128. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, identical to SEQ ID NO: 143. In some embodiments, the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence identical to SEQ ID NO: 143. In some embodiments, the nucleic acid sequence is contained in a plasmid consisting essentially of the nucleotide sequence of SEQ ID NO: 143. In some aspects, vectors described herein are for use in a method for producing long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, wherein the method comprises administering to the subject an effective amount of the vector for transient coexpression of the programmable Cas endonuclease and one or more gRNAs targeting a conserved sequence in the Asfarviridae genome. In some embodiments, the use reduces ASFV viral load in the subject. In some embodiments, the use mitigates one or more symptoms of ASFV infection. In some embodiments, the one or more symptoms of ASFV infection comprise: increased survival of the subject following exposure of ASFV, reduction of high fever, improvement in skin lesions, improvement in breathing, improvement in appetite, improvements in eye discharge, nasal discharge, and bleeding, or improvement in lethargy, or any combination thereof. In some embodiments, the use reduces high fever (about 105°F or higher) in the subject. In some embodiments, the use reduces an extent of malaise in the subject. In some embodiments, the use improves appetite in the subject. In some embodiments, the use decreases an extent or appearance of red, blotchy skin in the subject. In some embodiments, the use decreases an extent of skin lesions in the subject. In some embodiments, the use decreases hemorrhagic disease or hemorrhagic symptoms in the subject. In some embodiments, the use decreases diarrhea in the subject. In some embodiments, the use decreases vomiting in the subject. In some embodiments, the use improves respiratory distress in the subject. In some embodiments, the use prevents sudden death in the subject. In some embodiments, the use prevents sudden death in the subject prior to the onset of one or more clinical signs of ASFV infection. In some embodiments, the use improves ataxia in the subject.

[0100] In some embodiments, the use produces a durable immune response in the subject to ASFV infection. In some embodiments, the durable immune response allows the subject to achieve durable protective immunity upon ASFV re-challenge. In some embodiments, the vector comprises a nucleic acid sequence encoding a Cas9 protein of SEQ ID NO: 1; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121. In some embodiments, the vector comprises a nucleic acid sequence encoding: a Cas9 protein of SEQ ID NO: 108; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121. In some embodiments, each of the seven gRNAs further comprises a Cas9 tracrRNA / direct repeat of SEQ ID NO: 14. In some embodiments, the vector comprises a nucleic acid sequence encoding an EnAsCasl2a protein of SEQ ID NO: 110; and six guide RNAs (gRNAs), wherein each of the six gRNAs comprises a spacer sequence of one of SEQ ID NOs: 123-128. In some embodiments, the vector comprises a Cas9 protein of SEQ ID NO: 1; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121. In some embodiments, each of the six gRNAs further comprises a AsCasl2a Direct Repeat of crRNA of SEQ ID NO: 122. In some embodiments of vectors described herein, the programmable Cas endonuclease and the gRNA or gRNAs are configured for expression in cells of a mammalian subject. In some embodiments of vectors described herein, the gRNA or gRNAs are configured as an sgRNAs.METHODS

[0101] In some embodiments, compounds and compositions described herein are useful in treating an ASFV infection or symptoms associated with ASFV infection in a cell, and symptoms associated with ASFV infection in a subject. Thus, provided herein are methods for treating an ASFV infection, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition described herein or a pharmaceutically acceptable salt thereof, or a composition comprising a disclosed compound or pharmaceutically acceptable salt thereof. In some embodiments, the methods comprise a step of administering a composition comprising disclosed nucleic acid sequences that comprise a DNA targeting domain specific for an ASFV protein, such as an enzyme. Disorders treatable by the present compounds and compositions comprise fever, malaise, decrease in appetite, weakness, red blotchy skin, skin lesions, diarrhea, vomiting, coughing, difficulty breathing, spontaneous abortion, and sudden death. In some embodiments, the disclosure relates to any of the above disclosed methods disclosed herein, wherein the administrating step comprises administering a pharmaceutical composition comprising: (i) a pharmaceutically effective amount of any of the disclosed compounds; and (ii) a pharmaceutically acceptable carrier. Thus, in variousembodiments, disclosed are methods for treating ASFV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the Cas protein and / or guide or tracer RNA disclosed herein and a therapeutically effective amount of a compound selected from carprofen, mefenamic acid, phenacetin, valdecoxib, fenoldopam mesylate, Feverphenazine hydrochloride, bupivacaine HC1, phenazopyridine HC1, alverine citrate, nitenpyram, 4-aminobutyric acid (GABA), PF-3845, esmolol HC1, cimetidine, conivaptan HC1, (+)-MK-801 maleate, MK-0752, R04929097, rosmarinic acid, theophylline, aripiprazole, flopropione, latrepirdine 2HC1, ADX-47273, MPEP, nefopam HC1, phenazopyridine HC1, epinephrine, (+)-matrine, phenothiazine, naproxen sodium, AMG-517, isoliquiritigenin, nilvadipine, prednisone, and simvastatin, or a pharmaceutically acceptable salt thereof. In a further embodiment, the compound is selected from aripiprazole, dexmedetomidine, matrine, and MPEP, or a pharmaceutically acceptable salt thereof. In further embodiments, the compound is FDA approved. In further embodiments, the administering is accomplished by oral adminstration, parenteral administration, sublingual administration, transdermal administration, rectal administration, transmucosal administration, topical administration, inhalation, buccal administration, intrapleural administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intranasal administration, intrathecal administration, and intraarticular administration, or combinations thereof. In various embodiments, the method further comprises administering an effective amount of an agent associated with the treatment of an ASFV infection in addition to the one or plurality of nucleic acid sequences and Cas proteins disclosed herein. Thus, in various embodiments, the method further comprises administering an agent known for the treatment of an ASFV infection. In some embodiments, the compound and the agent are administered simultaneously. In some embodiments, the compound and the agent are administered sequentially. In some embodiments, the compound and the agent are co-packaged. In some embodiments, the compound and the agent are co-formulated. The disclosure also relates to a method of altering expression of at least one ASFV gene or protein in a cell comprising introducing into a cell an engineered, non-naturally occurring CRISPR associated (Cas) (CRISPR-Cas) system comprising: (a) a vector comprising a nucleotide sequence encoding any CRISPR enzyme disclosed herein, any mutated CRISPR enzyme having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 9%, 97%, 98%, or 99% sequence identity to any CRISPR enzyme disclosed herein, or functional fragment thereof; and (b) a nucleic acid sequence disclosed herein, wherein components (a) and (b) are located on same or different vectors of the system; wherein the cell contains and / or expresses an ASFV DNA molecule having a target sequence and encoding the gene product; and wherein the guide RNAtargets and hybridizes with a DNA target sequence, the CRISPR enzyme or functional fragment thereof cleaves the DNA molecule, whereby expression of the at least one ASFV gene or protein is altered. In some embodiments, the ASFV gene is silenced by enzymatic cleavage of the ASFV genomic or endogenous DNA by the CRISPR-Cas system present in the cell after administration or exposure. The disclosure also relates to a method of altering expression of at least one viral gene product in a cell comprising introducing into a cell an engineered, non-naturally occurring CRISPR associated (Cas) (CRISPR-Cas) system comprising: (a) a vector comprising a nucleotide sequence encoding a Type I, Type-II, or Type III Cas9 protein or functional fragment thereof; and (b) a nucleic acid sequence disclosed herein, wherein components (a) and (b) are located on same or different vectors of the system; wherein the cell contains and expresses a DNA molecule having an ASFV target sequence and encoding the gene product; and wherein the guide RNA targets and hybridizes with a DNA target sequence and the Cas protein or functional fragment thereof cleaves the DNA molecule, whereby expression of the at least one gene product is altered. In some embodiments, the Cas protein is a Cas9, a Casl2, a Casl2a, a Casl3, a CasX, a Casl2a2, a Casl2g, a Casl3a, a Casl3b, a Casl3c, a Casl3d, a Casl3x, or a Cas7-1 Iprotein, or a functional fragment or variant thereof. In some embodiments, Casl2a2 is a multi -turnover enzyme that degrades double stranded DNA, single-stranded DNA and singlestranded RNA. In some embodiments, Casl2a2 enzyme from Sulfuricurvum sp. is activated when its CRISPR RNA guide base pairs with an RNA target. In some embodiments, Casl2a2 indiscriminately degrades double stranded DNA, single-stranded DNA and single-stranded RNA once activated. In some embodiments, Casl2a2 indiscriminately degrades targeted double stranded DNA, targeted single-stranded DNA and targeted single-stranded RNA once activated. In some embodiments, like Casl2a2, Cas 13 is a multi -turnover enzyme. In some embodiments, Cas 13 only targets RNA instead of DNA. In some embodiments, Cas9 or Cas 12 function as single turnover endonucleases. In some embodiments, the composition or pharmaceutical composition comprises a nucleic acid molecule encoding a nucleic acid sequence encoding one or a plurality of Cas proteins chosen from: a Cas9, a Cas 12, a Cas 12a, a Cas 13, a CasX, a Casl2a2, a Casl2g, a Casl3a, a Casl3b, a Casl3c, a Casl3d, a Casl3x, or a Cas7-1 Iprotein, or a functional fragment or variant thereof. In some embodiments, methods of the disclosure comprise administering or exposing a cell or animal to the compositions or pharmaceutical compositions disclosed herein comprising: (i) a combination of Cas proteins chosen from: a Cas9, a Casl2, a Casl2a, a Casl3, a CasX, a Casl2a2, a Casl2g, a Casl3a, a Casl3b, a Casl3c, a Casl3d, a Casl3x, or a Cas7-1 Iprotein, or a functional fragment or variant thereof; or (ii) a plurality of nucleic acid molecule comprising a nucleic acid sequence encoding one or a combination of Cas proteins chosen from: a Cas9, a Cas 12, a Cas 12a, a Cas 13, a CasX, aCasl2a2, a Casl2g, a Casl3a, a Casl3b, a Casl3c, a Casl3d, a Casl3x, or a Cas7-1 Iprotein , or a functional fragment or variant thereof. The disclosure also relates to methods of treating or preventing growth and / or proliferation of ASFV in a subject diagnosed with or suspected of having ASFV infection, the methods comprising administering to a subject diagnosed with ASFV or suspected of having ASFV one or more pharmaceutical compositions disclosed herein. The disclosure also relates to methods for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject. In some aspects, the methods described herein reduce ASFV viral load in a treated subject by cleaving or digested targeted ASFV nucleic acids with a CRISPR system described herein. In some aspects, the methods described herein generate a durable humoral immune response in a treated subject to ASFV infection. In some aspects, the methods described herein mitigate one or more symptoms of ASFV infection in a subject following a treatment. In some aspects, the methods described herein mitigate one or more symptoms of ASFV infection in a subject following a treatment, enabling the subject mount an effective and durable immune response. In some aspects, the methods described herein mitigate one or more symptoms of ASFV infection in a subject following a treatment thereby extending a period of time in which the subject survives ASFV infection, enabling the subject mount an effective and durable immune response.

[0102] In some embodiments, the method for treating infection with a pathogen in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a first nucleic acid decreases a pathogen count in the subject. In some embodiments, the method for treating ASFV infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a first nucleic acid that decreases ASFV viral load in the subject. In some embodiments, the viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than three weeks after the administering. In some embodiments, the viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%,65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than two weeks after the administering. In some embodiments, the viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%,25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%,60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than one week after the administering. In some embodiments, the viral load is decreased byat least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than five days after the administering. In some embodiments, the viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%,55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99%, less than four days after the administering. In some embodiments, the viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%,50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%, less than three days after the administering. In some embodiments, the viral load is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than two days after the administering. In some embodiments, the viral load in the subject is decreased by at least about 10% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 15% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 20% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 25% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 30% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 40% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 50% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 55% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 60% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 62% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 64% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 65% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 66% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 67% less than oneI l lweek after the administering. In some embodiments, the viral load in the subject is decreased by at least about 68% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 69% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 70% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 75% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 80% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 85% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 90% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 95% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 97% less than one week after the administering. In some embodiments, the viral load in the subject is decreased by at least about 99% less than one week after the administering. In some embodiments, the first nucleic acid comprises: i) a first nucleic acid sequence that comprises at least about 70% complementary to an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding an ASFV helicase, an ASFV isomerase, or an ASFV topoisomerase; and ii) a second nucleic acid sequence encoding a Cas endonuclease protein or functional fragment thereof. In some embodiments, wherein the second nucleic acid sequence encodes a multi -turnover Cas endonuclease, the viral load in the subject is decreased by at least about 67% less than one week after the administering. In some embodiments, wherein the second nucleic acid sequence encodes a multi -turnover Casl2a2 endonuclease, the viral load in the subject is decreased by at least about 67% less than one week after the administering. In some embodiments, wherein the second nucleic acid sequence encodes a Cas9 endonuclease or functional variant thereof or functional fragment thereof, the viral load in the subject is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than one week after the administering. In some embodiments, wherein the second nucleic acid sequence encodes a Cas9 endonuclease or functional variant thereof or functional fragment thereof, the viral load in the subject is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by about 10 days after the administering. In some embodiments, wherein the second nucleic acid sequenceencodes a Casl2a endonuclease or functional variant thereof or functional fragment thereof, the viral load in the subject is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than one week after the administering. In some embodiments, wherein the second nucleic acid sequence encodes a Cast 2a endonuclease or functional variant thereof or functional fragment thereof, the viral load in the subject is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by about 10 days after the administering. In some embodiments, ASFV viral load is decreased to undetectable levels by about 25 days after the administering. In some embodiments, ASFV viral load is decreased to undetectable levels by about 30 days after the administering. In some embodiments, wherein the second nucleic acid sequence encodes a multi -turnover Cast 3b endonuclease, the viral load in the subject is decreased by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 57%, 59%, 60%, 62%, 65%, 67%, 70%, 72%, 75%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, less than one week after the administering. In some embodiments, ASFV viral replication is muted following the administering. ASFV viral replication can be measured from as viral load in a blood sample from the subject by qPCR detection and quantitation of ASFV nucleic acid levels. In some embodiments, the ASFV p72 is detected and quantitated by qPCR from a blood sample from the subject to determine ASFV viral load at a selected time point. In some embodiments, ASFV replication is muted by 10 dpi, 11 dpi, 12 dpi, 13 dpi, 14 dpi, 15 dpi, 16 dpi, 17 dpi, 18 dpi, 19 dpi, 20 dpi, 21 dpi, 22 dpi, 23 dpi, 24 dpi, 25 dpi, 26 dpi, 27 dpi, 28 dpi, 29 dpi, 30 dpi, or more following the administering. In some embodiments, surviving subjects have ASFV replication muted to undetectable levels by 20 dpi, 21 dpi, 22 dpi, 23 dpi, 24 dpi, 25 dpi, 26 dpi, 27 dpi, 28 dpi, 29 dpi, 30 dpi, or more following the administering. In some embodiments, surviving subjects have ASFV replication muted to undetectable levels by 25 dpi. In some embodiments, surviving subjects have ASFV replication muted to undetectable levels by 30 dpi. In some instances, subjects that were treated are observed to be less symptomatic of ASF compared to untreated control subjects. In some embodiments, ASFV viral is decreased to a sufficient extent to increase survival in the subject. In some embodiments, ASFV viral is decreased to a sufficient extent to increase survival in the subject and allow for the subject to achieve an increased amount of circulating ASFV-specific antibodies. In some embodiments, theincreased amount of circulating ASFV-specific antibodies is present in treated animals by at least about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more than 14 days following the administering. In some embodiments, the increased amount of circulating ASFV-specific antibodies is present in treated animals by at least about 10 days following the administering. In some embodiments, surviving animals exhibit robust production of ASFV-specific antibodies and presence in the serum of ASFV-specific antibodies by at least about 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30 days, or more than 30 days following the administering. In some embodiments, surviving animals exhibit robust production of ASFV-specific antibodies and presence in the serum of ASFV-specific antibodies at 30 days or more following the administering. In some embodiments, ASFV viral load is not reduced to an undetectable level by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 days following the administering. In some embodiments, reduction of ASFV viral load following the administering is reduced to an extent that increases survival in the subject and ASFV viral load is maintained for a period of time at a non-lethal level, wherein the non-lethal level is sufficient for the humoral immune system of the subject to produce an adaptive immune response directed to one or more ASFV antigens. In some embodiments, the adaptive immune response comprises a robust production of ASFV-specific antibodies. In some embodiments, the robust production of ASFV-specific antibodies in the subject contributes to further reduction of ASFV viral load in the subject. In some embodiments, the robust production of ASFV-specific antibodies in the subject contributes to further reduction of ASFV viral load in the subject to an undetectable level. In some embodiments, the adaptive immune response comprises a durable immune response in the subject which contributes to further reduction of ASFV viral load in the subject. In some embodiments, the adaptive immune response comprises a durable immune response in the subject which contributes to the subject developing a durable immunity to ASFV re-challenge. In some embodiments, the durable immune response comprises mitigating one or more symptoms of ASFV infection following an increase in ASFV-specific antibody response in the subject. In some embodiments, the durable immune response generates a post-treatment immunity to ASFV in the subject. In some embodiments, the post-treatment immunity to ASFV lowers a likelihood of the subject subsequently developing ASF. In some embodiments, the post-treatment immunity to ASFV prevents the subject from subsequently developing ASF. In some embodiments, the subject develops durable immunity to ASFV challenge, by allowing the subject to develop a robust humoral immune response by producing antibodies against ASFV. In some embodiments, the subject achieves a total recovery from ASF following the administering. In some embodiments, the subject survives at least 30 dpi following the administering. In some embodiments, subjectsthat survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge. In some embodiments, the subject has a reduction in cellular inflammation, autophagy, or apoptosis, or any combination thereof following the administering. In some embodiments, the subject has an improved immune response to ASFV following the administering, wherein the improved immune response comprises an increase in interferon production, an increase in antigen presentation, or an increase in cellular immunity, or any combination thereof. In some embodiments, the administering prevent ASFV virions from escaping an immune response from the subject. In some embodiments, the administering improves the function of monocytes in the subject. In some embodiments, the administering increases a total number of CD4+T cells in the peripheral blood of the subject. In some embodiments, the administering is completed according to a dosage schedule. In some embodiments, the dosage schedule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 administrations of the therapeutically effective amount of the first nucleic acid.

[0103] In an aspect described here are methods for treating African Swine Fever in a subject, the methods comprising: a) administering to the subject an effective amount of an isolated nucleic acid, wherein the isolated nucleic acid comprises: i) a first nucleic acid sequence comprising at least about 90% complementarity to a portion of an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding a conserved ASFV open reading frame, and ii) a second nucleic acid sequence encoding a programmable Cas endonuclease protein or functional fragment thereof; b) expressing in a cell of the subject a (i) guide RNA (gRNA) comprising the first nucleic acid sequence from a first promoter in the isolated nucleic acid and (ii) the programmable Cas endonuclease protein or functional fragment thereof from a second promoter in the isolated nucleic acid, thereby activating the programmable Cas endonuclease protein or functional fragment thereof in the cell of the subject; and c) using the activated programmable Cas endonuclease protein or functional fragment thereof to cleave or digest an ASFV nucleic acid at or near the portion of the endogenous ASFV nucleic acid sequence encoding the conserved ASFV open reading frame, wherein the cleaving or digesting reduces ASFV viral load in the subject. In some embodiments, a durable immune response in the subject to ASFV infection is generated following the administering.

[0104] In some embodiments, the method for treating infection with a pathogen in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a first nucleic acid, wherein the administering increases pathogen-specific antibody response in surviving subjects. In some embodiments, the method for treating ASFV infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a first nucleic acid, wherein the administering increases pathogen-specific antibody response insurviving subjects. In some embodiments, the method for treating African Swine Fever in a subject comprises administering to the subject a therapeutically effective amount of a first nucleic acid, wherein the administering increases pathogen-specific antibody response in surviving subjects. Pathogen-specific antibody response can be measured by ASFV ELISA to calculate sample / positive ratio as a percentage. In some embodiments, the increased pathogenspecific antibody response in surviving subjects is detected by a certain number of day post infection dpi). In some embodiments, the increased pathogen-specific antibody response in surviving subjects is detected at 2 dpi, 3 dpi, 4 dpi, 5 dpi, 6 dpi, 7 dpi, 8 dpi, 9 dpi, 10 dpi, 11 dpi, 12 dpi, 13 dpi, 14 dpi, 15 dpi, 16 dpi, 17 dpi, 18 dpi, 19 dpi, 20 dpi, 21 dpi, 22 dpi, 23 dpi, 24 dpi, 25 dpi, 26 dpi, 27 dpi, 28 dpi, 29 dpi, 30 dpi, or more. In some embodiments, a significantly increased pathogen-specific antibody response in surviving subjects is detected by 10 dpi. In some embodiments, a significantly increased pathogen-specific antibody response in surviving subjects is detected by 30 dpi. In some embodiments, a significantly increased pathogen-specific antibody response in surviving subjects produces a durable immune response. In some instances, the administering prolongs survival following infection with ASFV. In some instances, the administering prolongs survival following infection with what would otherwise be a lethal dose of ASFV. In some instances, the administering prolongs survival following exposure to ASFV. In some embodiments, a percentage of treated animals following the administering survive ASFV infection. In some embodiments, the percentage of treated animals following the administering surviving ASFV infection is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%. In some embodiments, the percentage of treated animals following the administering surviving ASFV infection is at least about 57%. In some instances, after an intramuscular injection of a lethal dose of ASFV, swine receiving anti -ASFV CRISPR based treatment have reduced viral loads, and 57% of infected swine result in complete clearance of the virus. In some instances, after an intramuscular injection of a lethal dose of ASFV, swine receiving anti -ASFV CRISPR based treatment have reduced viral loads, and 57% of infected swine result in complete clearance of the virus, and full recovery from ASF. In some embodiments, treated animals following the administering surviving ASFV infection achieve recovery. In some embodiments, recovered animals achieve muted ASFV replication. In some embodiments, recovered animals achieve muted ASFV replication to undetectable levels in a blood sample. In some embodiments, muted ASFV replication and increase length of survival enables the adaptive immune system of the subject to mount an effective adaptive immune response. In some instances, this effective adaptive immune response is a durable immune response. In some instances, following the administering, the subject acquires immunity to subsequent re-infectionfrom ASFV. In some instances, following the administering, the subject is immune to ASFV reinfection. In some instances, following the administering, the subject can mount an effective adaptive immune response to ASFV re-challenge. In some instances, following the administering, the subject is able to mount an effective immune response to ASFV re-challenge to an extent that ASFV replication is significantly reduced in the subject compared to challenge of an untreated subject with an equivalent dose of ASFV. In some instances, previously treated subjects have recovered from ASFV infection following the administering. In some embodiments, previously treated subjects have increased survival following re-challenge with an otherwise lethal dose of ASFV. In some embodiments, previously treated subjects have increased survival following re-challenge with a dose of ASFV. In some instances, previously treated subjects achieve a percentage of survival upon ASFV re-challenge. In some instances, greater than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of previously treated subjects survive ASFV re-challenge. In some instances, 100% of previously treated subjects survive ASFV re-challenge. In some instances, ASFV re-challenge leads to significantly reduced ASFV replication within the previously treated subject. In some instances, the previously treated subject is able to mount an effective immune response to ASFV rechallenge. In some instances, the previously treated subject is able to mount an effective immune response involving pathogen-specific antibodies produced by the subject to ASFV rechallenge. In some instances, the previously treated subject achieves a durable immune response to ASFV. In some instances, the previously treated subject achieves a durable immune response to ASFV which prevents re-infection from ASFV upon subsequent exposure to ASFV particles. In some instances, the previously treated subject shows no clinical symptoms upon ASFV rechallenge. In some instances, the previously treated subject shows no clinical symptoms upon ASFV rechallenge by intramuscular injection of an otherwise lethal dose of ASFV. In some instances, the previously treated subject is able to survive ASFV rechallenge by intramuscular injection of an otherwise lethal dose of ASFV. In some instances, surviving animals following the administering after exposure to ASFV develop a robust immunity from the initial exposure to ASF virus, and when rechallenged with a second intramuscular lethal dose of ASF, are fully protected, showing no clinical symptoms.

[0105] According to one aspect, the disclosure relates to a method of altering a eukaryotic cell comprising: transfecting the eukaryotic cell with a nucleic acid disclosed herein complementary to genomic DNA of the ASFV viral genome, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site-specific manner, wherein the cell expresses or comprises the RNA and the enzyme, the RNA binds to complementary genomic viral DNA and the enzyme cleaves the viralDNA in a site-specific manner. According to one aspect, the enzyme is Cas9 or modified Cas9 or a homolog of Cas9. According to one aspect, the enzyme is Casl2a or modified Casl2a or a homolog of Casl2a. According to one aspect, the enzyme is Casl2a2 or modified Casl2a2 or a homolog of Casl2a2. According to one aspect, the enzyme is Casl3b or modified Casl3b or a homolog of Cast 3b. According to one aspect, the enzyme is CasX or modified CasX or a homolog of CasX. According to one aspect, the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a porcine cell. According to one aspect, the nucleic acid disclosed herein comprises from about 10 to about 250 nucleotides. According to one aspect, the nucleic acid disclosed herein comprises from about 20 to about 100 nucleotides.

[0106] According to one aspect, a method of altering a human or porcine cell is provided including transfecting the human or porcine cell with a nucleic acid encoding RNA complementary to ASFV viral DNA in the eukaryotic cell, transfecting the human or porcine cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site-specific manner, wherein the human or porcine cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site-specific manner. According to one aspect, the enzyme is Cas9 or modified Cas9 or a homolog of Cas9. Modified Cas9 proteins or homologs of Cas9 are for instance disclosed in US Pat. No. 9,074,199, which is incorporated herein by reference. According to one aspect, the enzyme is Casl2a or modified Casl2a or a homolog of Casl2a. According to one aspect, the enzyme is Casl2a2 or modified Casl2a2 or a homolog of Casl2a2. According to one aspect, the enzyme is Casl3b or modified Casl3b or a homolog of Casl3b. According to one aspect, the enzyme is CasX or modified CasX or a homolog of CasX.According to one aspect, the RNA includes between about 10 to about 250 nucleotides.According to one aspect, the RNA includes between about 20 to about 100 nucleotides. The step of transfecting a nucleic acid encoding an RNA may be added to any method disclosed herein so that there is sequential or concurrent transfection of not only synthetic guide or tracer sequences such as those disclosed herein but also one or a plurality of vectors comprising a nucleic acid sequence encoding a Cas protein or variant or functional fragment thereof. The disclosure relates, among other things, to the rationale design of sgRNA, tracr / crRNA duplexes, and, generally, guide sequences that activate and / or catalyze the reaction of a CRISPR enzyme with a target nucleic acid sequence. The disclosure relates to the discovery that guide sequences (whether in the form of sgRNA, tracr / crRNA duplexes, or tracr / crRNA single strands) can be heavily modified to enhance on-target enzymatic efficiency as long as certain nucleotides that bind to the CRISPR enzyme, variant or functional fragments thereof are conserved at certainpositions and / or, in some cases, conserved in respect to certain substituents on each nucleotide that are capable of binding a Cas protein, variant or functional fragments thereof in the presence of such a the Cas protein, variant or functional fragments thereof. Certain positions of the guide sequence can be more heavily modified based upon their functional association to other components of the CRISPR complex. For instance, in some embodiments, the composition or pharmaceutical composition disclosed herein comprises one or a plurality of nucleic acid sequences on one or plurality of nucleic acid molecules wherein the nucleic acid sequences comprise contiguous domains in the 5' to 3' orientation.: a DNA-targeting domain, a Cas-binding domain, and a transcription terminator domain. Pharmaceutical compositions also provided herein are pharmaceutical compositions comprising a guide or tracrRNA as disclosed herein, or pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier. Thus, in various embodiments, disclosed are pharmaceutical compositions comprising a therapeutically effective amount of at least one disclosed nucleic acid sequence (e.g., SEQ ID NOs: 14-101 or SEQ ID NOs: 118-143, and a pharmaceutically acceptable carrier. In a further embodiment, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed compound. In a still further embodiment, a pharmaceutical composition can be provided comprising a prophylactically effective amount of at least one disclosed compound, wherein the compound comprises a nucleic acid sequence. In yet a further embodiment, the disclosure relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a disclosed compound, wherein the compound is present in an effective amount. In an even further embodiment, the pharmaceutical compositions are useful in inhibiting neurotoxicity in a subject. In a still further embodiment, the pharmaceutical compositions are useful in treating ASFV infection. In a still further embodiment, the pharmaceutical compositions are useful in preventing ASFV infection. In a still further embodiment, the pharmaceutical compositions are useful in preventing an acute ASFV infection following exposure to an active ASFV infectious agent. In some embodiments, the one or plurality of nucleic acid sequences comprise a nucleic acid sequence selected from SEQ ID NOs: 15-35, 37-45, 47-64, 66-83, 118-121, and 123-128, or a variant that comprises at least about 70%, 80%, 87%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence listed above. The present invention also provides vectors comprising any of the nucleic acids described above encoding guide RNAs described herein. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a retrovirus. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments the vector is an AAV vector. In some embodiments, the first nucleic acid sequence is selected from a sequence comprising at least about 75% sequence identity to any one of SEQ ID NOs: 15-35, 37-45, 47-64, 66-83, 118-121,and 123-128. In some embodiments, the first nucleic acid sequence is selected from a sequence comprising at least about 90% sequence identity to any one of SEQ ID NOs: 15-35, 37-45, 47- 64, 66-83, 118-121, and 123-128. In some embodiments, the first nucleic acid sequence is selected from a sequence comprising at least about 95% sequence identity to any one of SEQ ID NOs: 15-35, 37-45, 47-64, 66-83, 118-121, and 123-128. In some embodiments, the first nucleic acid sequence is selected from a sequence comprising any one of SEQ ID NOs: 15-35, 37-45, 47-64, 66-83, 118-121, and 123-128.

[0107] In some aspects described herein are methods of prophylaxis for ASF. In some embodiments, a composition described herein in administered to a subject prior to exposure to ASF. In some embodiments, repeat doses of the prophylactic treatment are administered to the subject. In some embodiments, repeat doses of a treatment are administered as a therapy following the development of one or more symptoms related to ASF. In some embodiments, the method of prophylaxis for ASF comprises administering a CRISPR Casl2a single vector described herein to the subject as a monotherapy. In some embodiments, the method of prophylaxis for ASF comprises administering a CRISPR Cas9 single vector described herein to the subject as a monotherapy. In some embodiments, the method of treatment for ASF comprises administering a CRISPR Casl2a2 single vector described herein to the subject as a monotherapy. In some embodiments, the method of prophylaxis for ASF comprises administering a CRISPR Cas9 single vector described and a CRISPR Casl2a2 single vector described herein to the subject as a combination therapy. In some embodiments, the CRISPR single vector DNA plasmid is formulated with LNPs for administering to the subject. In some embodiments, the formulated CRISPR single vector drug is administered by IV administration. In some embodiments, the formulated CRISPR single vector drug is administered by IM administration. In some embodiments, the formulated CRISPR single vector drug is administered by oral administration.

[0108] In some aspects described herein are methods of therapeutic treatment for ASF. In some embodiments, a composition described herein in administered to a subject following an exposure to ASFV. In some embodiments, repeat doses of the therapeutic treatment are administered to the subject. In some embodiments, repeat doses of a therapeutic treatment are administered following the development of one or more symptoms related to ASF. In some embodiments, the method of treatment for ASF comprises administering a CRISPR Cas9 single vector described herein to the subject as a monotherapy. In some embodiments, the method of treatment for ASF comprises administering as CRISPR Casl2a2 single vector described herein to the subject as a monotherapy. In some embodiments, the method of treatment for ASFV comprises administering a CRISPR Cas9 single vector described and a CRISPR Casl2a2 singlevector described herein to the subject as a combination therapy. In some embodiments, the CRISPR single vector DNA plasmid is formulated with LNPs for administering to the subject. In some embodiments, the formulated CRISPR single vector drug is administered by IV administration. In some embodiments, the formulated CRISPR single vector drug is administered by IM administration. In some embodiments, the formulated CRISPR single vector drug is administered by oral administration.

[0109] Described herein are methods for treating African Swine Fever Virus (ASFV) in a subject. In one aspect, the methods for treating ASFV generated a long-lasting immunity to ASFV injection in the treated subject. In an aspect described herein, are methods for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, the methods comprising: a) administering to the subject an effective amount of an isolated nucleic acid, wherein the isolated nucleic acid comprises: i) a first nucleic acid sequence comprising at least about 90% complementarity to a portion of an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding a conserved ASFV open reading frame, and ii) a second nucleic acid sequence encoding a programmable Cas endonuclease protein or functional fragment thereof; b) expressing in a cell of the subject a (i) guide RNA (gRNA) comprising the first nucleic acid sequence from a first promoter in the isolated nucleic acid and (ii) the programmable Cas endonuclease protein or functional fragment thereof from a second promoter in the isolated nucleic acid, thereby activating the programmable Cas endonuclease protein or functional fragment thereof in the cell of the subject; using the activated programmable Cas endonuclease protein or functional fragment thereof to cleave or digest an ASFV nucleic acid at or near the portion of the endogenous ASFV nucleic acid sequence encoding the conserved ASFV open reading frame, wherein the cleaving or digesting reduces ASFV viral load in the subject; and d) generating a durable immune response in the subject to ASFV infection. In some embodiments, the using the activated programmable Cas endonuclease protein or functional fragment thereof in c) comprises mitigating one or more symptoms of ASFV infection following the reduction in ASFV viral load from the cleaving or digesting. In some embodiments, the generating the durable immune response comprises mitigating one or more symptoms of ASFV infection following an increase in ASFV-specific antibody response in the subject. In some embodiments, the programmable Cas endonuclease protein or functional fragment thereof is selected from a Cas9, a Casl2a2, a Cas 12a, a Cas 13d, or a CasX. In some embodiments, the programmable Cas endonuclease protein or functional fragment thereof is a Casl3d. In some embodiments, the programmable Cas endonuclease protein or functional fragment thereof is a CasX. In some embodiments, the programmable Cas endonuclease protein or functional fragment thereof is a Cas9. In some embodiments, the programmable Cas endonuclease proteinor functional fragment thereof is a Casl2a. In some embodiments, the programmable Cas endonuclease protein or functional fragment thereof is a Casl2a2. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid at least about 90%, 95%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-6 or 108-111. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 1. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 3. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 4. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 5. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 6. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 102. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 108. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 109. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 110. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid sequence at least about 90% identical to SEQ ID NO: 111. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 102. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 108. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 109. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 110. In some embodiments, the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising the amino acid sequence of SEQ ID NO: 111. In some embodiments, the programmable Cas endonuclease comprises a Casl2a. In some embodiments, the Casl2a is EnAsCasl2a. In some embodiments, the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 123-128. In some embodiments, the first nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, or six gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 123-128. In some embodiments, the first nucleic acid sequence further comprises coding sequence for six gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 123-128. In some embodiments, the first nucleic acid sequence comprises each of SEQ ID NOs: 123-128, wherein the sequences of each of SEQ ID NOs: 123-128 are configured for use as spacer sequences in a gRNA directed to a conserved ASFV genomic target sequence. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence at least about 90% identical to SEQ ID NO: 143. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprising a nucleotide sequence identical to SEQ ID NO: 143. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of the nucleotide sequence of SEQ ID NO: 143. In some embodiments, the programmable Cas endonuclease comprises a Cas9. In some embodiments, the Cas9 is Sniper2L-Cas9. In some embodiments, the Cas9 comprises a nuclear localization signal (NLS), a nuclear export signal (NES), or a nucleocytoplasmic shuttling sequence (NCS) comprising anNLS and an NES. In some embodiments, the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 15-32 or 118-121. In some embodiments, the first nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, or seven gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 140. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90% identical to SEQ ID NO: 140. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence identical to SEQ ID NO: 140. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of a nucleotide sequence of SEQ ID NO: 140. In some embodiments, the first nucleic acid sequence further comprises coding sequence for seven gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 15-17 and 118-121. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 142. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90% identical to SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90% identical to SEQ ID NO: 142. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence identical to SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence identical to SEQ ID NO: 142. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of a nucleotide sequence of SEQ ID NO: 141. In some embodiments, the isolated nucleic acid is encoded with a plasmid consisting essentially of a nucleotide sequence of SEQ ID NO: 142. In some embodiments, the programmable Cas endonuclease comprises a Casl2a2. In some embodiments, the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 37-45. In some embodiments, the first nucleic acid sequence further comprises coding sequence nine gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 37-45. In some embodiments, the administering comprises systemic administration. In some embodiments, systemic administration comprises oral administration,IV administration, or IM administration. In some embodiments, wherein the systemic administration comprises oral administration, the oral administration comprises adding one or more components of a CRISPR system to a food source of the subject. In some embodiments, the gRNA and the programmable Cas endonuclease protein or functional fragment thereof are transiently expressed in cells of the subject following the administering. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a pig, a swine, a minipig, or a wild boar. In some embodiments, the cleaving or digesting ASFV nucleic acid reduces ASFV viral load in the subject by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 30 days after the administering. In some embodiments, the cleaving or digesting ASFV nucleic acid reduces ASFV viral load in the subject ASFV viral load is decreased by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 25 days after the administering. In some embodiments, the cleaving or digesting ASFV nucleic acid reduced ASFV viral load in the subject to an undetectable level by about 25-30 dpi following the administering. In some embodiments, the mitigating one or more symptoms of ASFV infection comprises increased survival of the subject following exposure of ASFV, reduction of high fever, improvement in skin lesions, improvement in breathing, improvement in appetite, improvements in eye discharge, nasal discharge, and bleeding, or improvement in lethargy, or any combination thereof. In some embodiments, survival of the subject following exposure to ASFV is improved. In some embodiments, the subject develops an extent of posttreatment immunity to ASFV to produce the durable immune response. In some embodiments, post-treatment immunity to ASFV lowers a likelihood of the subject subsequently developing ASF. In some embodiments, post-treatment immunity to ASFV prevents the subject from subsequently developing ASF. In some embodiments, the subject develops durable immunity to ASFV challenge, by allowing the subject to develop a robust humoral immune response by producing antibodies against ASFV. In some embodiments, the subject achieves a total recovery from ASF following the administering. In some embodiments, the subject survives at least 30 dpi following the administering. In some embodiments, at least about 50% or more subjects survive at least 30 dpi following the administering. In some embodiments, a surviving subject demonstrates an increased ASFV-specific antibody response. In some embodiments, the increased ASFV-specific antibody response is significant by at least about 10 dpi. In some embodiments, the increased ASFV-specific antibody response remains significant until at leastabout 30 dpi. In some embodiments, subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge. In some embodiments, subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge and a survival rate of greater than 90% when rechallenged with an otherwise lethal dose of ASFV. In some embodiments, subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge and a survival rate of 100% when re-challenged with an otherwise lethal dose of ASFV.

[0110] In further embodiments, the pharmaceutical composition is administered to a mammal. In still further embodiments, the mammal is a human. In some embodiments, the mammal is a pig, a swine, or a minipig. In some embodiments, the mammal is a pig. In further embodiments, the pharmaceutical composition is administered following identification of the mammal in need of treatment of a disorder associated signs of neurological dysfunction. In still further embodiments, the mammal has been diagnosed with a need for treatment of a disorder associated with ASFV prior to the administering step. In further embodiments, the pharmaceutical composition is administered following identification of the mammal in need of treatment of an ASFV infection. In still further embodiments, the mammal has been diagnosed with a need for treatment of an ASFV infection prior to the administering step. In various embodiments, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. The choice of carrier will be determined in part by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting. Formulations suitable for oral administration can comprise (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granule; (c) powders; (d) suspensions in an appropriate liquid;and (e) suitable emulsions. Liquid formulations may include diluents, such as water, cyclodextrin, dimethyl sulfoxide and alcohols, for example, ethanol, benzyl alcohol, propylene glycol, glycerin, and the polyethylene alcohols including polyethylene glycol, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard-or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, the addition to the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art. The compounds of the present disclosure alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethylene glycol) 400, glycerol ketals, such as 2,2-dimethyl-l, 3-dioxolane-4- methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropyl methylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. Oils which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isosteric acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamines, and polyoxymethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl P-amino propionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof. The parenteral formulations typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. Pharmaceutically acceptable excipients are also well-known to those who are skilled in the art. The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present disclosure. The following methods and excipients are merely exemplary and are in no way limiting. The pharmaceutically acceptable excipients preferably do not interfere with the action of the active ingredients and do not cause adverse side-effects. Suitable carriers and excipients include solvents such as water, alcohol, and propylene glycol, solid absorbants and diluents, surface active agents, suspending agent, tableting binders, lubricants, flavors, and coloring agents. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, Eds., 238-250(1982) and ASHP Handbook on Injectable Drugs, Toissel, 4thed., 622-630 (1986). Formulations suitable for topical administration include lozenges comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art. Additionally, formulations suitable for rectal administration may be presented as suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. One skilled in the art will appreciate that suitable methods of exogenously administering a compound of the present disclosure to an animal are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. As regards these applications, the present method includes the administration to an animal, particularly a mammal, and more particularly a human, of a therapeutically effective amount of the compound effective in the treatment (e.g., prophylactic or therapeutic) of an ASFV infection. The method also includes the administration of a therapeutically effect amount of the compound for the treatment of patient having a predisposition for being afflicted with an ASFV infection. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the animal over a reasonable timeframe. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition of the animal, the body weight of the animal, as well as the severity and stage of the disorder. The total amount of the compound of the present disclosure administered in a typical treatment is preferably from about 1 mg / kg to about 100 mg / kg of body weight for mice, and from about 10 mg / kg to about 50 mg / kg of body weight, and from about 20 mg / kg to about 40 mg / kg of body weight for humans per daily dose. This total amount is typically, but not necessarily, administered as a series of smaller doses over a period of about one time per day to about three times per day for about 24 months, and over a period of twice per day for about 12 months. The size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations. In certain embodiments, a composition described herein is formulated foradministration to a patient in need of such composition. Compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.

[0111] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound described herein in the composition will also depend upon the particular compound in the composition. A compound described herein can be administered alone or can be co-administered with an additional therapeutic agent. Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). Additional therapeutic agents include, but are not limited to, other active agents known to be useful in treating an ASFV infection as further described herein. In some embodiments, the compounds described herein can be delivered in a vesicle, in particular a liposome (see, Langer, Science, 1990, 249, 1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). Suitable compositions include, but are not limited to, oral non-absorbed compositions. Suitable compositions also include, but are not limited to saline, water, cyclodextrin solutions, and buffered solutions of pH 3-9. The compounds described herein, or pharmaceutically acceptable salts thereof, can be formulated with numerous excipients including, but not limited to, purified water, propylene glycol, PEG 400, glycerin, DMA, ethanol, benzyl alcohol, citric acid / sodium citrate (pH3), citric acid / sodium citrate (pH5), tris(hydroxymethyl)amino methane HC1 (pH7.0), 0.9% saline, 1.2% saline, acetate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, carbonate, chloride, citrate, decanoate, edetate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolate, hexanoate, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl sulfate, mucate, napsylate, nitrate, octanoate, oleate, pamoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, succinate, sulfate, tartrate, teoclate, tosylate, and any combination thereof. In someembodiments, an excipient is chosen from propylene glycol, purified water, and glycerin. In some embodiments, the formulation can be lyophilized to a solid and reconstituted with, for example, water prior to use. When administered to a mammal (e.g., to an animal such as a pig for veterinary use or to a human for clinical use) the compounds can be administered in isolated form. When administered to a pig or a human, the compounds can be sterile. Water is a suitable carrier when the compound of Formula I is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions described herein can take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing a liquid, powder, sustained-release formulation, suppository, aerosol, spray, or any other form suitable for use. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, A.R. Gennaro (Editor) Mack Publishing Co. In some embodiments, the compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to humans. In some embodiments, the compounds are formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to domesticated animals such as pigs. Typically, compounds are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration may optionally include a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The pharmaceutical compositions can be in unit dosage form. In such form, the composition can be divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. In some embodiments, acomposition of the present disclosure is in the form of a liquid wherein the active agent is present in solution, in suspension, as an emulsion, or as a solution / suspension. In some embodiments, the liquid composition is in the form of a gel. In other embodiments, the liquid composition is aqueous. In other embodiments, the composition is in the form of an ointment. In some embodiments, the composition is in the form of a solid article. For example, in some embodiments, the ophthalmic composition is a solid article that can be inserted in a suitable location in the eye, such as between the eye and eyelid or in the conjunctival sac, where it releases the active agent as described, for example, U.S. Pat. No. 3,863,633; U.S. Pat. No. 3,867,519; U.S. Pat. No. 3,868,445; U.S. Pat. No. 3,960,150; U.S. Pat. No. 3,963,025; U.S. Pat. No. 4,186,184; U.S. Pat. No. 4,303,637; U.S. Pat. No. 5,443,505; and U.S. Pat. No. 5,869,079. Release from such an article is usually to the cornea, either via the lacrimal gland that bathes the surface of the cornea, or directly to the cornea itself, with which the solid article is generally in intimate contact. Solid articles suitable for implantation in the eye in such fashion are generally composed primarily of polymers and can be bioerodible or non-bioerodible. Bioerodible polymers that can be used in the preparation of ocular implants carrying one or more of the compounds described herein in accordance with the present disclosure include, but are not limited to, aliphatic polyesters such as polymers and copolymers of poly(glycolide), poly(lactide), poly(epsilon-caprolactone), poly-(hydroxybutyrate) and poly(hydroxyvalerate), polyamino acids, polyorthoesters, polyanhydrides, aliphatic polycarbonates and polyether lactones. Suitable non-bioerodible polymers include silicone elastomers. The compositions described herein can contain preservatives. Suitable preservatives include, but are not limited to, mercury-containing substances such as phenylmercuric salts (e.g., phenylmercuric acetate, borate and nitrate) and thimerosal; stabilized chlorine dioxide; quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride; imidazolidinyl urea; parabens such as methylparaben, ethylparaben, propylparaben and butylparaben, and salts thereof; phenoxyethanol; chlorophenoxyethanol; phenoxypropanol; chlorobutanol; chlorocresol; phenylethyl alcohol; disodium EDTA; and sorbic acid and salts thereof. Compositions of the disclosure include particles comprising the nucleic acid sequences and / or molecule disclosed herein. As used herein, a "particle" refers to any entity having a diameter of less than 100 microns (pm). Typically, particles have a longest dimension (e.g., diameter) of 1000 nm or less. In some embodiments, particles have a diameter of 300 nm or less. In some embodiments, nanoparticles have a diameter of about 200 nm or less. In some embodiments, nanoparticles have a diameter of about 100 nm or less. In general, particles are greater in size than the renal excretion limit but are small enough to avoid accumulation in the liver. In some embodiments, a population of particles may be relativelyuniform in terms of size, shape, and / or composition. In general, inventive particles are biodegradable and / or biocompatible. Inventive particles can be solid or hollow and can comprise one or more layers. In some embodiments, particles are spheres, spheroids, flat, plate-shaped, cubes, cuboids, ovals, ellipses, cylinders, cones, or pyramids. In some embodiments, particles can be a matrix of polymers. In some embodiments, the matrix is cross-linked. In some embodiments, formation of the matrix involves a cross-linking step. In some embodiments, the matrix is not substantially cross-linked. In some embodiments, formation of the matrix does not involve a cross-linking step. In some embodiments, particles can be a non-polymeric particle (e.g., a metal particle, quantum dot, ceramic, inorganic material, bone, etc.). Components of the pharmaceutical compositions disclosed herein may comprise particles or may be microparticles, nanoparticles, liposomes, and / or micelles comprising one or more disclosed nucleic acid sequences. As used herein, the term "nanoparticle" refers to any particle having a diameter of less than 1000 nm. Examples of nanoparticles are disclosed in Nature Biotechnology 31, 638- 646, which is herein incorporated by reference in its entirety. It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of use.Kits

[0112] In some aspects, disclosed are kits comprising a nucleic acid described herein, or a pharmaceutically acceptable salt thereof, and one or more selected from: (a) instructions for treating an ASFV infection; and (b) instructions for administering the nucleic acid in connection with treating ASFV. In some embodiments, a pharmaceutical composition comprising a nucleic acid described herein can be packaged in a container. In some embodiments, the instructions can direct administration of the pharmaceutical composition. In some embodiments, the kit further comprises a drug delivery device for administering the pharmaceutical composition to a subject. In some embodiments, the drug delivery device is a syringe or a catheter. In some embodiments, the nucleic acid is formulated with LNPs. In further embodiments, the kit comprises the agent known for the treatment of an ASFV infection. Examples of agents known for the treatment of an ASFV infection include, but are not limited to, CRISPR enzyme and a guide RNA comprising a DNA-binding region complementary to one or more ASFV genes. In further embodiments, the compound and at least one agent are co-formulated. In further embodiments, the compound and at least one agent are co-packaged. The kits can also comprise compounds and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and / or product and another component for delivery to a patient. It is understood that the disclosed kits can be prepared from the disclosedcompounds, products, and pharmaceutical compositions. It is also understood that the disclosed kits can be employed in connection with the disclosed methods of use. Methods of making a kit can include placing a pharmaceutical composition comprising a nucleic acid described herein, salt thereof, formulation, or composition described herein in a container for packaging. A method can further comprise an inclusion of instructions for use.

[0113] The foregoing description illustrates and describes the disclosure. Additionally, the disclosure shows and describes only the preferred embodiments but, as mentioned above, it is to be understood that it is capable to use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the disclosure concepts as expressed herein, commensurate with the above teachings and / or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to explain best modes known by applicant and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the disclosure to the form disclosed herein. Also, it is intended to the appended claims be construed to include alternative embodiments. All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls.Definitions

[0114] Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0115] As used herein, the terms “a” or “an” means that “at least one” or “one or more” unless the context clearly indicates otherwise. The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, z.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in various embodiments, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet anotherembodiment, to both A and B (optionally including other elements); etc. The term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of’.

[0116] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0117] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0118] As used herein, the term “kit” refers to a set of components provided in the context of a system for delivering materials or diagnosing a subject with having been contaminated with a disclosed toxin or exposed to a disclosed toxin. Such delivery systems may include, for example, systems that allow for storage, transport, or delivery of various therapeutic reagents (e.g., oligonucleotides, enzymes, extracellular matrix components etc. in appropriate containers) and / or supporting materials (e.g., buffers, media, cells, written instructions for performing the assay etc.) from one location to another. For example, in some embodiments, kits include one or more enclosures (e.g., boxes) containing relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a kit comprising a therapeutically effective amount of the nucleic acid disclosed herein and wherein the kit comprises two or more separate containers that each contain a sub-portion of total kit components. Containers may be delivered to an intended recipient together or separately. For example, a first container may contain a petri dish or polystyrene plate for use in a cell culture assay, while a second container may contain an excipient or liposome. As another example, the kit may comprise a first container comprising a solid support such as a chip or slide with one or a plurality of ligands with affinities to one or a plurality of biomarkers disclosed herein (such as p72 of ASFV or other capsid protein correlatedto viral load) and a second container comprising any one or plurality of reagents necessary for the detection and / or quantification of the amount of biomarkers in a sample.

[0119] The term “fragmented kit” is intended to encompass kits containing Analyte Specific Reagents (ASR’s) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contain a sub-portion of total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all components in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits. In some embodiments, the kit comprises nucleic acids disclosed herein and, optionally, a container comprising a Cas protein or a nucleic acid sequence encoding one or a plurality of Cas proteins.

[0120] As used herein, the phrase "integer from about X to about Y" means any integer that includes the endpoints. That is, where a range is disclosed, each integer in the range including the endpoints is disclosed. For example, the phrase "integer from X to Y" discloses 1, 2, 3, 4, or 5 as well as the range 1 to 5.

[0121] As used herein, the term "animal" includes, but is not limited to, humans, invertebrates, and non-human vertebrates such as wild animals, rodents, such as rats, ferrets, and domesticated animals, and farm animals, such as dogs, cats, horses, pigs, cows, sheep, and goats. In some embodiments, the animal is a mammal. In some embodiments, the animal is a human. In some embodiments, the animal is a pig, a swine, or a minipig.

[0122] As used herein, the term "mammal" means any animal in the class Mammalia such as rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human. In some embodiments, the mammal refers to any non-human mammal. The present disclosure relates to any of the methods or compositions of matter disclosed herein wherein the sample is taken from a mammal or non- human mammal. The present disclosure relates to any of the methods or compositions of matter disclosed herein wherein the sample is taken from a human, pig or minipig.

[0123] The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. "Identical" or "identity" as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs inboth sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. As used herein, “specific for” or “specifically binds to” means that the binding affinity of a substrate to a specified target nucleic acid sequence, such as a nucleic acid that encodes an ASFV enzyme or structural protein, isstatistically higher than the binding affinity of the same substrate to a generally comparable, but non-target amino acid sequence. The substrate's Kd to each nucleotide sequence can be compared to assess the binding specificity of the substrate to a particular target nucleotide sequence.

[0124] Human or non-human variants of the enzymes above are contemplated by the methods, systems, and devices disclosed herein. Variants of these enzymes include sequences that comprise at least 70% of the sequence to the porcine or human sequences disclosed herein. As used herein, the term "variants" is intended to mean substantially similar sequences. For nucleic acid molecules, a variant comprises a nucleic acid molecule having deletions (e.g., truncations) at the 5' and / or 3' end; deletion and / or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitution of one or more nucleotides at one or more sites in the native polynucleotide.

[0125] As used herein, a "native" nucleic acid molecule or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For nucleic acid molecules, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the disclosure. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the disclosure. Generally, variants of a particular nucleic acid molecule or amino acid sequence of the disclosure will have at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide or guide RNA or target DNA as determined by sequence alignment programs and parameters as described elsewhere herein. Variants of a particular nucleic acid molecule of the disclosure (e.g., the reference amino acid sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant nucleic acid molecule and the polypeptide encoded by the reference nucleic acid molecule comprising an expressible nucleic acid sequence. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of nucleic acid molecule of the disclosure is evaluated by comparison of the percent sequence identity shared by the two polypeptides that they encode, the percent sequence identity between the two encoded polypeptides is at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the term "variant" protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and / or C-terminal end of the native protein; deletion and / or addition of one or more amino acids at one or more internal sitesin the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins, variant guide RNAs or nucleic acid sequences encompassed by the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a protein of the disclosure will have at least about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the disclosure may differ from that protein by as few as about 1 to aboutl5 amino acid residues, as few as about 1 to about 15, such as from about 6 to about 10, as few as about 20, 15, 10, 9, 8, 7, 6, 5, as few as about 4, 3, 2, or even about 1 amino acid residue. The nucleic acid sequences or molecules as well as the proteins or polypeptides of the disclosure may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the proteins can be prepared by mutations in the nucleic acid sequence that encode the amino acid sequence recombinantly.

[0126] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0127] The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0128] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X, and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0129] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0130] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0131] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician or veterinarian, and found to have acondition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. In some embodiments of the disclosed methods, the subject has been diagnosed with a need for treatment of a disorder caused by ASFV such as, for example, a fever, prior to the administering step. As used herein, the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. It is contemplated that the identification can, in some embodiments, be performed by a person different from the person making the diagnosis. It is also contemplated, in further embodiments, that the administration of a diagnosis can be performed by one who previously or subsequently performed the administration of a pharmaceutical composition disclosed herein.

[0132] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intra-aural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various embodiments, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various embodiments, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0133] As used herein, the terms “contacting” or “exposing” mean bringing together of two elements in an in vitro system or an in vivo system. For example, “contacting” a compound disclosed herein with an individual or subject or cell includes the administration of the compound to an individual or subject, such as a pig, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing the compounds or pharmaceutical compositions disclosed herein.

[0134] The nucleic acid sequences according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug forming moi eties. For instance, the hydroxymethyl position may form mono-, di- or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med. Chem. 1988, 31, 318; Aligas-Martin et al., PCT WO 2000 / 041531, p. 30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.

[0135] "Therapeutically effective amount" refers to an amount of a compound, material, or composition, as described herein effective to achieve a particular biological result such as, but not limited to, biological results disclosed, described, or exemplified herein. Such results may include, but are not limited to, the effective reduction of symptoms associated with any of the disease states mentioned herein, as determined by any means suitable in the art. The therapeutically effective amount of the composition may be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, the type and / or severity of the particular condition being treated, or the need to modulate the activity of the molecular pathway induced by association of the guide RNA or tracer RNA to the viral DNA target. The therapeutically appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art. A therapeutically effective dose of the saxiphilins described herein may provide partial or complete biological activity as compared to the biological activity induced by the wild-type or naturally occurring polypeptides upon which the saxiphilins are derived. A therapeutically effective dose of the proteins or amino acids described herein may provide a sustained biochemical or biological affect and / or an increased resistance to infection of ASFV when administered to a subject as compared with the normal affect observed in the absence of the administration. In some embodiments, the term “effective amount” or “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired result (e.g., that will elicit a biological or medical response of a subject e.g., a dosage from about 0.01 to about 100 mg / kg body weight / day) or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain suchamounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various embodiments, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition. The term "fragment" refers to any analog of a naturally occurring polypeptide disclosed herein that comprises at least 4 amino acids identical to the naturally occurring polypeptide upon which the analog is based.

[0136] The term "functional fragment" refers to any fragment of Cas protein disclosed herein that comprises at least about 75% sequence identity to any of those amino acid sequence chosen from Table Y, and shares the function of the naturally occurring polypeptide upon which the saxiphilin is based. In some embodiments, the functional nature of the fragment is to bind or associate a disclosed DNA target sequence, and, in some embodiments, cut the DNA target sequence if within a cell, in vitro or in a subject such as a pig.

[0137] The compounds described herein may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the compounds described herein refer to nontoxic “pharmaceutically acceptable salts.” Pharmaceutically acceptable salt forms include pharmaceutically acceptable acidic / anionic or basic / cationic salts. Suitable pharmaceutically acceptable acid addition salts of the compounds described herein include (e.g., salts of inorganic acids such as hydrochloric acid, hydrobromic, phosphoric, nitric, and sulfuric acids and of organic acids such as acetic acid, benzenesulfonic, benzoic, methanesulfonic, and p- toluenesulfonic acids). Examples of pharmaceutically acceptable base addition salts include e.g., sodium, potassium, calcium, ammonium, organic amino, or magnesium salt. The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. The pharmaceutical composition can comprise anypharmaceutically acceptable carrier or ingredient, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity- increasing agents, water-absorbing agents, water- miscible cosolvents, water softeners, or wetting agents. As used herein, the phrase “pharmaceutically acceptable” means those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of humans or other animals. In some embodiments, “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0138] “Disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In some embodiments, the disease is a disease related to (e.g., characterized by) modulation of ASFV viral load. In some embodiments, the disease is an ASFV infection. “Disease”, “disorder”, and “condition” are used interchangeably herein. In some embodiments, the condition is an ASFV infection. As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, (e.g., a therapeutic treatment). In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible subject prior to the onset of one or more symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a particular organism such as ASFV, or other susceptibility factors), as a prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to delay their recurrence.

[0139] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. The term “preventing” refers to preventing a disease, disorder, or condition from occurring in a human or an animal that may be predisposed to the disease, disorder and / or condition, but has not yet been diagnosed as having it; and / or inhibiting the disease, disorder, or condition, / .< ., arresting its development. In some embodiments the disclosure relates to a method of preventing ASFV viral replication or viral assembly or viral particle release an infected cell by administering to a subject a therapeutically effective amount of a Cas protein and a disclosed nucleic acid sequence (such as a guide or tracrRNA) comprising a sequence that associates or hybridizes a DNA target sequence of an ASFV genomic DNA or endogenous sequence or an ASFV mRNA transcript sequence. Nucleic acids that hybridize with a DNA target sequence or an RNA target sequence are exemplified in Table 4A. In some embodiments, the viral endogenous or genomic sequence is in the cytosol of the infected cell. In some embodiments, the endogenous or genomic sequence is in the nucleus of an infected cell.

[0140] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present disclosure. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. The term "salt" refers to acidic salts formed with inorganic and / or organic acids, as well as basic salts formed with inorganic and / or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally be pharmaceutically acceptable although salts of non- pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Salts include those formed from hydrochloric, hydrobromic, sulfuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulfonic and benzenesulfonic acids. In some embodiments, salts of the compositions comprising a saxiphilin or functional fragment thereof may be formed by reacting the free base, or a salt, enantiomer or racemate thereof, with one or more equivalents of the appropriate acid. In some embodiments, pharmaceutical acceptable salts of the present invention refer to analogs having at least one basic group or at least one basic radical. In some embodiments, pharmaceutical acceptable salts of the present invention comprise a free amino group, a free guanidino group, a pyrazinyl radical, or a pyridyl radical that forms acid addition salts. In some embodiments, the pharmaceutical acceptable salts of the present invention refer to analogs that are acid additionsalts of the subject compounds with (for example) inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono- or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such as nicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such as methane-, ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonic acids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid. When several basic groups are present mono- or poly-acid addition salts may be formed. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, for example, water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents, which may be removed in vacuo or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin. In some embodiments, the salts may be those that are physiologically tolerated by a patient. Salts according to the present invention may be found in their anhydrous or hydrated crystalline form (i.e., complexed or crystallized with one or more molecules of water).

[0141] The terms “subject” and “patient” and “subject in need thereof’ may be used interchangeably, and means a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, minipigs, horses, sheep, goats and the like), laboratory animals (e.g., rats, mice, guinea pigs and the like), and wild animal populations (e.g., wild boar Sos scrofa, or any one of a recognized subspecies of Sos scrofa)). Non-limiting examples include humans, other mammals, bovines, cats, rats, mice, dogs, monkeys, goats, sheep, cows, horses, pigs, and other non-mammalian animals. In some embodiments, the subject is a pig, a swine, or a minipig in need of treatment for ASFV or neurotoxicity.

[0142] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., a protein associated disease, a symptom associated with ASFV infection) means that the disease (e.g., the ASFV) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. For example, a symptom of a gut motility disease or condition may be a symptom that results (entirely or partially) from modulation of viral load (e.g., induction of colonic motility). As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease. For example, ASFVinfection may be treated with an agent (e.g., compound as described herein) effective for modulating inflammation or fever within a subject, such as a pig.

[0143] “ Control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects.

[0144] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g., chemical compounds including biomolecules, or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be an amino acid sequence disclosed herein and a nucleic acid sequence disclosed herein in complex with viral genomic DNA. In some embodiments contacting includes allows the CRISPR complex described herein to interact with a viral DNA and neutralize or inhibit its biological effect or effects. In some embodiments, the biological effect is the reduction of viral load in a subject and / or cell.

[0145] As defined herein, the term “inhibition,” “inhibit,” “inhibiting,” and the like in reference to a protein-inhibitor (e.g., antagonist) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein relative to the activity or function of a viral protein or viral genome in the absence of the inhibitor. In some embodiments inhibition refers to reduction of a disease or symptoms of disease caused by the presence or infection of the virus. In some embodiments, inhibition refers to a reduction in the activity of a signal transduction pathway or signaling pathway caused by the presence of a virus, such as ASFV. Thus, inhibition includes, at least in part, partially or totally blocking infection, decreasing, preventing, or delaying replication or viral assembly, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity of the amount of a viral protein in a cell as compared to the same metric in a cell not treated with the same inhibitor.

[0146] As defined herein, the term “activation,” “activate,” “activating,” and the like in reference to a protein-activator (e.g., agonist) interaction means positively affecting (e.g., increasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the activator. In some embodiments, activation refers to an increase in the activity of a signal transduction pathway or signaling pathway. Thus, activation may include, atleast in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease. Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up- regulating signal transduction or enzymatic activity or the amount of a protein that may modulate the level of another protein or increase / decrease cell survival.

[0147] The term “modulator” refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule. In some embodiments, the modulator is a modulator of neurotoxicity. In some embodiments, the modulator is a modulator of viral toxicity or viral load in an infected cell and is a compound that reduces the severity of one or more symptoms of a disease associated with viral load, such as ASFV. In some embodiments, a modulator is a compound that reduces the severity of one or more symptoms of ASFV infection caused by exposure to or contamination by ASFV.

[0148] The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0149] As used herein, the term “administering” means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Oral administration may include adding a composition described herein to a food source of the subject to be injected during feeding. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies (e.g., viral therapies including, for example, cholinesterase inhibitors, modafinil, or non-steroidal anti-inflammatory drugs. The composition of the disclosure can be administered alone or can be co-administered to the subject. Coadministration is meant to include simultaneous or sequential administration of the compound individually or in combination (more than one compound or agent). Thus, the preparations can also be combined,when desired, with other active substances (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral preparations include tablets, pills, powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions. The compositions of the present disclosure may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present disclosure can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In some embodiments, the formulations of the compositions of the present disclosure can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, (e.g., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis). By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present disclosure into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46: 1576- 1587, 1989). The compositions of the present disclosure can also be delivered as nanoparticles. Pharmaceutical compositions provided by the present disclosure include compositions wherein the active ingredient (e.g., compounds described herein, including embodiments or examples of saxiphilins and functional fragments thereof) is contained in a therapeutically effective amount, / .< ., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods to treat a disease, such compositions will contain an amount of active ingredient effective to achieve the desired result, e.g., modulating the activity of a target molecule, and / orreducing, eliminating, or slowing the progression of disease symptoms. Determination of a therapeutically effective amount of a compound of the disclosure is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein. The dosage and frequency (single or multiple doses) administered to a mammal can vary depending upon a variety of factors, for example, whether the mammal suffers from another disease, and its route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated (e.g., symptoms of ASFV infection), kind of concurrent treatment, complications from the disease being treated or other health- related problems. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds of Applicants' disclosure. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the ability of those skilled in the art. For any compound described herein, the therapeutically effective amount can be initially determined from in vitro cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art. As is well known in the art, therapeutically effective amounts for use in animal subjects can also be determined from designed test protocols in animal models. For example, a dose for pigs can be formulated to achieve a concentration that has been found to be effective in designed test protocols in animal models. The dosage in pigs can be adjusted by monitoring compo...

Claims

CLAIMSWhat is claimed is:

1. A method for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, the method comprising: a) administering to the subject an effective amount of an isolated nucleic acid, wherein the isolated nucleic acid comprises: i. a first nucleic acid sequence comprising at least about 90% complementarity to a portion of an endogenous African Swine Fever Virus (ASFV) nucleic acid sequence encoding a conserved ASFV open reading frame, and ii. a second nucleic acid sequence encoding a programmable Cas endonuclease protein or functional fragment thereof; b) expressing in a cell of the subject a (i) guide RNA (gRNA) comprising the first nucleic acid sequence from a first promoter in the isolated nucleic acid and (ii) the programmable Cas endonuclease protein or functional fragment thereof from a second promoter in the isolated nucleic acid, thereby activating the programmable Cas endonuclease protein or functional fragment thereof in the cell of the subject; c) using the activated programmable Cas endonuclease protein or functional fragment thereof to cleave or digest an ASFV nucleic acid at or near the portion of the endogenous ASFV nucleic acid sequence encoding the conserved ASFV open reading frame, wherein the cleaving or digesting reduces ASFV viral load in the subject; and d) generating a durable immune response in the subject to ASFV infection.

2. The method of claim 1, wherein the using the activated programmable Cas endonuclease protein or functional fragment thereof in c) comprises mitigating one or more symptoms of ASFV infection following the reduction in ASFV viral load from the cleaving or digesting.

3. The method of claim 1, wherein the generating the durable immune response comprises mitigating one or more symptoms of ASFV infection following an increase in ASFV-specific antibody response in the subject.

4. The method of claim 1, wherein the programmable Cas endonuclease protein or functional fragment thereof is selected from a Cas9, a Casl2a2, a Casl2a, a Casl3d, or a CasX.

5. The method of claim 4, wherein the second nucleic acid sequence encodes a programmable Cas endonuclease protein or functional fragment thereof comprising an amino acid at least about 90%, 95%, 98%, 99%, or 100% identical to a sequence selected from SEQ ID NOs: 1-6 or 108-111.

6. The method of claim 4, wherein the programmable Cas endonuclease comprises a Casl2a.

7. The method of claim 6, wherein the Casl2a is EnAsCasl2a.

8. The method of claim 6 or 7, wherein the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 123-128.

9. The method of claim 6-7, wherein the first nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, or six gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 123-128.

10. The method of claim 6-7, wherein the first nucleic acid sequence further comprises coding sequence for six gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 123-128.

11. The method of any one of claims 6-10 , wherein the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143.

12. The method of claim 4, wherein the programmable Cas endonuclease comprises a Cas9.

13. The method of claim 12, wherein the Cas9 is Sniper2L-Cas9.

14. The method of claim 12 or 13, wherein the Cas9 comprises a nuclear localization signal (NLS), a nuclear export signal (NES), or a nucleocytoplasmic shuttling sequence (NCS) comprising an NLS and an NES.

15. The method of any one of claims 12-14, wherein the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 15-32 or 118-121.

16. The method of any one of claims 12-15, wherein the first nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, or seven gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 15-17 and 118-121.

17. The method of any one of claims 12-16, wherein the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 140.

18. The method of any one of claims 12-17, wherein the first nucleic acid sequence further comprises coding sequence for seven gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 15-17 and 118-121.

19. The method of claim 18, wherein the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142.

20. The method of claim 4, wherein the programmable Cas endonuclease comprises a Casl2a2.

21. The method of claim 20, wherein the gRNA comprising the first nucleic acid sequence comprises a sequence selected from SEQ ID NOs: 37-45.

22. The method of claim 20 or 21, wherein the first nucleic acid sequence further comprises coding sequence nine gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 37-45.

23. The method of any one of claims 1-22, wherein the administering comprises systemic administration.

24. The method of claim 23, wherein systemic administration comprises oral administration, IV administration, or IM administration.

25. The method of claim 24, comprising oral administration, wherein oral administration comprises adding one or more components of a CRISPR system to a food source of the subject.

26. The method of any one of claims 1-25, wherein the gRNA and the programmable Cas endonuclease protein or functional fragment thereof are transiently expressed in cells of the subject following the administering.

27. The method of any one of claims 1-26, wherein the subject is a mammal.

28. The method of claim 27, wherein the mammal is a pig, a swine, a minipig, or a wild boar.

29. The method of any one of claims 1-28, wherein the cleaving or digesting ASFV nucleic acid reduces ASFV viral load in the subject by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 30 days after the administering.

30. The method of any one of claims 1-29, wherein the cleaving or digesting ASFV nucleic acid reduces ASFV viral load in the subject ASFV viral load is decreased by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 25 days after the administering.

31. The method of any one of claims 1-30, wherein the cleaving or digesting ASF V nucleic acid reduced ASFV viral load in the subject to an undetectable level by about 25-30 dpi following the administering.

32. The method of any one of claims 1-31, wherein the mitigating one or more symptoms of ASFV infection comprises increased survival of the subject following exposure of ASFV, reduction of high fever, improvement in skin lesions, improvement in breathing, improvement in appetite, improvements in eye discharge, nasal discharge, and bleeding, or improvement in lethargy, or any combination thereof.

33. The method of claim 32, wherein survival of the subject following exposure to ASFV is improved.

34. The method of any one of claims 1-33, wherein the subject develops an extent of posttreatment immunity to ASFV to produce the durable immune response.

35. The method of claim 34, wherein post-treatment immunity to ASFV lowers a likelihood of the subject subsequently developing ASF.

36. The method of claim 34 or 35, wherein post-treatment immunity to ASFV prevents the subject from subsequently developing ASF.

37. The method of any one of claims 1-36, wherein the subject develops durable immunity to ASFV challenge, by allowing the subject to develop a robust humoral immune response by producing antibodies against ASFV.

38. The method of any one of claims 1-37, wherein the subject achieves a total recovery from ASF following the administering.

39. The method of any one of claims 1-38, wherein the subject survives at least 30 dpi following the administering.

40. The method of any one of claims 1-39, wherein at least about 50% or more subjects survive at least 30 dpi following the administering.

41. The method of any one of claims 1-40, wherein a surviving subject demonstrates an increased ASFV-specific antibody response.

42. The method of claim 41, wherein the increased ASFV-specific antibody response is significant by at least about 10 dpi.

43. The method of claim 41 or 42, wherein the increased ASFV-specific antibody response remains significant until at least about 30 dpi.

44. The method of any one of claims 1-43, wherein subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge.

45. The method of any one of claims 1-44, wherein subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge and a survival rate of greater than 90% when re-challenged with an otherwise lethal dose of ASFV.

46. The method of any one of claims 1-45, wherein subjects that survive ASFV infection following the administering achieve durable protective immunity upon ASFV re-challenge and a survival rate of 100% when re-challenged with an otherwise lethal dose of ASFV.

47. A composition for generating long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, the composition comprising: (i) an isolated nucleic acid sequence encoding a Clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonucleaseor functional fragment thereof and (ii) a guide RNA (gRNA) complementary to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the composition is configured to reduce a viral load of ASFV to an extent that generates a durable immune response to an ASFV infection in the subject.

48. The composition of claim 47, wherein the CRISPR-associated endonuclease or functional fragment thereof comprises a programmable Cas endonuclease selected from a Cas9, a Casl2a2, a Cas 12a, a Cas 13d, or a CasX.

49. The composition of claim 48, wherein the CRISPR-associated endonuclease or functional fragment thereof comprises a Casl2a.

50. The composition of claim 49, wherein the Casl2a is EnAsCasl2a.

51. The composition of claim 48 or 49, wherein the isolated nucleic acid sequence encoding the gRNA comprises a sequence selected from SEQ ID NOs: 123-128.

52. The composition of any one of claims 49-51, wherein the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, or six gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 123-128.

53. The composition of claim 51 or 52, further comprising coding sequence for six gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 123-128.

54. The composition of any one of claims 49-53, wherein the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143.

55. The composition of claim 48, wherein the CRISPR-associated endonuclease or functional fragment thereof comprises a Cas9.

56. The composition of claim 55, wherein the Cas9 is Sniper2L-Cas9.

57. The composition of claim 55 or 56, wherein the Cas9 comprises a nuclear localization signal (NLS), a nuclear export signal (NES), or a nucleocytoplasmic shuttling sequence (NCS) comprising an NLS and an NES.

58. The composition of any one of claims 55-57, wherein the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, or seven gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 15-17 and 118-121.

59. The composition of any one of claims 55-58, wherein the isolated nucleic acid sequence further comprises coding sequence for three gRNAs each comprising one of spacer sequences of SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

60. The composition of any one of claims 55-59, wherein the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 140.

61. The composition of any one of claims 55-59, wherein the isolated nucleic acid sequence further comprises coding sequence for seven gRNA sequences each comprising one of spacer sequences selected from SEQ ID NOs: 15-17 and 118-121.

62. The composition of any one of claims 55-59 or 61, wherein the isolated nucleic acid is encoded with a plasmid comprises a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142.

63. The composition of claim 48, wherein the CRISPR-associated endonuclease or functional fragment thereof comprises a Casl2a2.

64. The composition of claim 63, wherein the Casl2a2 comprises an NLS, an NES, or an NCS comprising an NLS and an NES.

65. The composition of claim 63, wherein the Casl2a2 is lacking an NLS.

66. The composition of any one of claims 63-65, wherein the isolated nucleic acid sequence encoding the gRNA comprises a sequence selected from SEQ ID NOs: 37-45.

67. The composition of any one of claims 63-65, wherein the isolated nucleic acid sequence further comprises coding sequence for two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine gRNA sequences each comprising a spacer sequence selected from SEQ ID NOs: 37-45.

68. The composition of any one of claims 47-67, wherein the gRNA and the programmable Cas endonuclease protein or functional fragment thereof are transiently expressed in cells of the subject following administering of the composition to a subject.

69. The composition of claim 68, wherein transient expression of the gRNA and the programmable Cas endonuclease protein or functional fragment thereof produces cleaving or digesting of ASFV nucleic acid in cells of the subject and reduces ASFV viral load in the subject by at least about 5%, 6%, 7%, 85, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 20%, 22%, 24%, 25%, 27%, 29%, 30%, 32%, 35%, 37%, 40%, 42%, 45%, 47%, 50%, 52%, 55%, 60%, 62%, 65%, 67%, 70%, 72%, 55%, 77%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by 30 days after the administering.

70. The composition of claim 68 or 69, wherein transient expression of the gRNA and the programmable Cas endonuclease protein or functional fragment thereof improves survival of the subject following exposure to ASFV.

71. The composition of claim 69 or 70, wherein reduction in ASF V viral load allows the subject to develop an extent of post-treatment immunity to ASFV and achieve a durable protective immunity upon ASFV re-challenge.

72. A vector comprising a nucleic acid sequence encoding (i) a programmable Cas endonuclease and (ii) three guide RNAs (gRNAs), each gRNA comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome; wherein the programmable Cas endonuclease comprises a Cas9 or functional fragment thereof, and wherein the three gRNAs each comprise a sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

73. The vector of claim 72, further comprising nucleic acid sequence encoding one, two, three, or four additional gRNAs, the additional gRNAs each comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 118-121.

74. The vector of claim 73, wherein the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 141 or SEQ ID NO: 142.

75. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 121.

76. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 75, wherein the programmable Cas endonuclease comprises a Cas9 or functional fragment thereof.

77. The vector of claim 76, further comprising nucleic acid sequence encoding one, two, three, four, five, or six additional gRNAs, the additional gRNAs each comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 15-17 and 118- 120.

78. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 123.

79. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 78; wherein the programmable Cas nuclease comprises a Casl2a or functional fragment thereof.

80. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 124.

81. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 80, wherein the programmable Cas endonuclease comprises a Cas 12a or functional fragment thereof.

82. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 125.

83. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 82, wherein the programmable Cas endonuclease comprises a Cas 12a or functional fragment thereof.

84. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 126.

85. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 84, wherein the programmable Cas endonuclease comprises a Cas 12a or functional fragment thereof.

86. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 127.

87. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 86, wherein the programmable Cas endonuclease comprises a Cas 12a or functional fragment thereof.

88. A guide RNA (gRNA) comprising a complementary sequence to a conversed target nucleic acid sequence in an Asfarviridae genome, wherein the complementary sequence comprises SEQ ID NO: 128.

89. A vector comprising a nucleic acid sequence encoding a programmable Cas endonuclease and the gRNA of claim 88, wherein the programmable Cas endonuclease comprises a Cas 12a or functional fragment thereof.

90. The vector of any one of claims 73-78, further comprising nucleic acid sequence encoding one or more, two or more, three or more, four or more, or five additional gRNAs, wherein the additional gRNAs each comprise a sequence selected from SEQ ID NOs: 123-128.

91. The vector of claim 90, comprising each of nucleic acid sequences of SEQ ID NOs 123-128.

92. The vector or claim 91, wherein the nucleic acid sequence is contained in a plasmid comprising a nucleotide sequence at least about 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 143.

93. The vector of any one of claims 72-74, 76-77, 79, 81, 83, 85, 87, and 89-92, for use in a method for producing long-lasting immunity to African Swine Fever Virus (ASFV) in a subject, wherein the method comprises administering to the subject an effective amount of the vector for transient co-expression of the programmable Cas endonuclease and one or more gRNAs targeting a conserved sequence in the Asfarviridae genome.

94. The vector of claim 93, wherein the use reduces ASFV viral load in the subject.

95. The vector of claim 93 or 94, wherein the use mitigates one or more symptoms of ASFV infection.

96. The vector of claim 95, wherein the one or more symptoms of ASFV infection comprise: increased survival of the subject following exposure of ASFV, reduction of high fever, improvement in skin lesions, improvement in breathing, improvement in appetite, improvements in eye discharge, nasal discharge, and bleeding, or improvement in lethargy, or any combination thereof.

97. The vector of any one of claims 93-96, wherein the use produces a durable immune response in the subject to ASFV infection.

98. The vector of claim 97, wherein the durable immune response allows the subject to achieve durable protective immunity upon ASFV re-challenge.

99. A vector comprising a nucleic acid sequence encoding: a Cas9 protein of SEQ ID NO: 1; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121.

100. A vector comprising a nucleic acid sequence encoding: a Cas9 protein of SEQ ID NO: 108; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121.

101. The vector of claim 99 or 100, wherein each of the seven gRNAs further comprises a Cas9 tracrRNA / direct repeat of SEQ ID NO: 14.

102. A vector comprising a nucleic acid sequence encoding: an EnAsCasl2a protein of SEQ ID NO: 110; and six guide RNAs (gRNAs), wherein each of the six gRNAs comprises a spacer sequence of one of SEQ ID NOs: 123-128.

103. A vector comprising a nucleic acid sequence encoding:a Cas9 protein of SEQ ID NO: 1; and seven guide RNAs (gRNAs), wherein each of the seven gRNAs comprises a spacer sequence of one of SEQ ID NOs: 15-17 and 118-121.

104. The vector of claim 102 or 103, wherein each of the six gRNAs further comprises a AsCasl2a Direct Repeat of crRNA of SEQ ID NO: 122.

105. The vector of any one of claims 72-74, 76-77, 79, 81, 83, 85, 87, and 89-104, wherein the programmable Cas endonuclease and the gRNA or gRNAs are configured for expression in cells of a mammalian subject.

106. A kit comprising a pharmaceutical composition comprising the vector of any one of claims 72-74, 76-77, 79, 81, 83, 85, 87, and 89-105, and instructions for use.

107. The kit of claim 106, wherein the instructions for use designate ASFV prophylaxis, ASFV treatment, or ASFV prophylaxis and ASFV treatment as indications in a subject in need of treatment.

108. The kit of claim 106 or 107, comprising a drug delivery device.

109. The kit of any one of claim 106-109, wherein the pharmaceutical composition is formulated with lipid nano-particles (LNPs).

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