Methods for genome editing and therapy by directed heterologous DNA insertion using retroviral integrase-Cas fusion proteins

Abstract

The present disclosure provides methods for editing proteins, nucleic acids, systems and genomic material, as well as methods of treatment.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 178,862, filed April 23, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] CRISPR-Cas has greatly improved our ability to rapidly modify mammalian genomes for basic research and clinical applications. CRISPR-Cas uses guide RNAs to guide Cas to specific DNA target sequences where it induces double-stranded DNA breaks and activates cellular repair pathways to introduce frameshift mutations or insert donor sequences via homology-directed repair (HDR). Despite these significant advances, targeted delivery of large DNA sequences for genome editing using CRISPR-Cas-mediated HDR remains inefficient, requires donor templates containing sizable flanking homologous regions, and induces the p53 DNA damage pathway (Byrne et al., 2015, NAR 43:e21; Happaniemi et al., 2018, Nat Med 24:927-30; Ihry et al., 2018, Nat Med 24:939-46). Taken together, these significantly limit the efficiency of CRISPR-Cas genome editing. Therefore, there is a need for improved integrated genome editing.

[0003] In contrast, the lentiviral enzyme integrase (IN) is necessary and sufficient to catalyze the insertion of the large lentiviral genome into host cell DNA through a process that does not require target sequence homology. IN-mediated insertion of lentiviral DNA occurs with little specificity of DNA target sequences, due in part to the C-terminal domain that binds nonspecifically to DNA (Lutzke & Plasterk 1998, J Virol 72:4841-48).

[0004] The current limitations of gene therapy technology prevent the treatment of most human single gene diseases. CRISPR-Cas gene editing has been attracting attention in recent years for the development of therapeutic approaches to repair harmful mutations in mammalian genomes. This remains a considerable challenge due to the large number of patient-specific mutations in the human genome that can cause diseases and disorders. CRISPR guide RNAs designed to target exon-intron boundaries enable exon skipping strategies to target these mutation groups, however, the effectiveness of these strategies remains untested and is not applicable to all patients.

[0005] Although transgenic expression of many genes can prevent and reverse disease outcomes in animal models, the large size of some genes significantly exceeds the size limitations of traditional gene editing approaches such as CRISPR-Cas, or traditional viral gene therapy approaches such as AAV (limit of about 4.9 kb), preventing their use in human gene therapy.Approaches using smaller engineered genes delivered by AAV are currently undergoing clinical trials, but it remains to be seen whether these strategies can bring about long-term recovery and whether they are applicable only to patients with specific mutations.

[0006] In contrast, lentiviral vectors can deliver large genes and allow permanent repair upon integration into the host genome. However, the current random nature of lentiviral integration can lead to off-target mutations and disease, which has hindered their use in clinical applications (Milone et al., 2018, Leukemia 23:1529-41). Lentiviral sequences are inserted into the host genome by the virally encoded enzyme integrase (IN), which utilizes a non-specific DNA binding domain required for genome integration (Andrake et al., 2015, Annu Rev Virol 2:241-64).

[0007] Thus, there is a need for improved editing of genomic material. The present invention meets this need. Summary of the Invention

[0008] In one aspect, the invention provides a method of treating Friedreich's ataxia in a subject, the method comprising administering to the subject: a) a fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein, and a nuclear localization signal (NLS), or a nucleic acid molecule having one or more nucleic acid sequences encoding a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein, and a nuclear localization signal (NLS); b) a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the subject's genome; and c) a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence, wherein the donor template sequence comprises a nucleic acid sequence encoding frataxin.

[0009] In one embodiment, the retrovirus is selected from the group consisting of human immunodeficiency virus (HIV), rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), moloney murine leukemia virus (MoLV), bovine leukemia virus (BLV), human T-lymphotropic virus (HTLV), avian sarcoma leukemia virus (ASLV), feline leukemia virus (FLV), xenotropic murine leukemia virus-related virus (XMLV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), prototype foamy virus (PFV), simian foamy virus (SFV), human foamy virus (HFV), walleye dermal sarcoma virus (WDSV), and bovine immunodeficiency virus (BIV). In one embodiment, the virus comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 9-48.

[0010] In one embodiment, the Cas protein is selected from the group consisting of Cas9, Cas14 and Cpf1. In one embodiment, the Cas protein comprises a sequence at least 70% identical to one of SEQ ID NOs: 1-8. In one embodiment, the Cas protein is catalytically deficient (dCas). In one embodiment, the Cas protein comprises a sequence at least 70% identical to one of SEQ ID NOs: 2, 4, 6 and 8.

[0011] In one embodiment, the NLS is a Ty1 NLS or a Ty2 NLS. In one embodiment, the NLS comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 53-54 and 361-973.

[0012] In one embodiment, the target region in the subject's genome is a safe harbor site. In one embodiment, the donor template sequence encodes a protein that is at least 70% identical to SEQ ID NO: 357. In one embodiment, the donor template sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 358. In one embodiment, the IN is HIV IN and the donor template sequence comprises the U3 sequence of SEQ ID NO: 258, the U5 sequence of SEQ ID NO: 259, or both.

[0013] In one aspect, the invention provides a fusion protein comprising a) a CRISPR-associated (Cas) 14 protein, and b) a nuclear localization signal (NLS). In one embodiment, the Cas14 protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 7-8.

[0014] In one embodiment, the NLS is a Ty1 NLS or a Ty2 NLS. In one embodiment, the NLS comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 53-54 and 361-973.

[0015] In one embodiment, the fusion protein comprises a sequence that is at least 70% identical to SEQ ID NO:145.

[0016] In one embodiment, the fusion protein further comprises a retroviral integrase (IN), or a fragment thereof. In one embodiment, the IN comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 9-48.

[0017] In one embodiment, the Cas14 protein is catalytically deficient (dCas). In one embodiment, the fusion protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 63-102 and 146.

[0018] In one aspect, the invention includes a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein.

[0019] In one aspect, the invention provides a method of editing genetic material, the method comprising administering to the genetic material a fusion protein as described herein, or a nucleic acid molecule encoding a fusion protein as described herein, a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region within the genetic material, and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.

[0020] In one embodiment, the guide nucleic acid comprises a tracrRNA and a CRISPR RNA (crRNA). In one embodiment, the tracrRNA comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 336-339.

[0021] In one aspect, the invention provides a system for editing genetic material, the system comprising: a) a nucleic acid sequence encoding a fusion protein comprising a CRISPR-associated (Cas) 14 protein and a nuclear localization signal (NLS); b) a nucleic acid sequence encoding a CRISPR-Cas system guide RNA; and c) a nucleic acid sequence encoding a donor template nucleic acid, the donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence, in one or more vectors.

[0022] In one embodiment, the Cas14 protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 7-8.

[0023] In one embodiment, the NLS is a Ty1 NLS or a Ty2 NLS. In one embodiment, the NLS comprises a sequence at least 70% identical to one of SEQ ID NOs: 53-54, and 361-973. In one embodiment, the Cas14 protein is catalytically deficient (dCas). In one embodiment, the fusion protein further comprises a retroviral integrase (IN) or a fragment thereof. In one embodiment, the IN comprises a sequence at least 70% identical to one of SEQ ID NOs: 9-48. In one embodiment, the Cas14 protein is catalytically deficient (dCas).

[0024] In one embodiment, the fusion protein comprises a sequence at least 70% identical to one of SEQ ID NOs: 63-102 and 146. In one embodiment, the fusion protein comprises a sequence at least 70% identical to SEQ ID NO: 145. In one embodiment, the guide RNA comprises a tracrRNA and a CRISPR RNA (crRNA). In one embodiment, the tracrRNA comprises a sequence at least 70% identical to one of SEQ ID NOs: 336-339. [Brief description of the drawings]

[0025] The following detailed description of the embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1A] 1 shows experimental results demonstrating enhanced nuclear localization of a retroviral integrase-dCas9 fusion protein for editing mammalian genomic DNA. A schematic diagram of the IN-dCas9 fusion protein is shown. [Figure 1B] 1 shows experimental results demonstrating enhanced nuclear localization of a retroviral integrase-dCas9 fusion protein for editing mammalian genomic DNA. 2 shows nuclear localization of the IN-dCas9 fusion protein. [Figure 1C]1 shows experimental results demonstrating improved nuclear localization of a retroviral integrase-dCas9 fusion protein for editing mammalian genomic DNA. 2 shows experimental results demonstrating the enzymatic activity of the INΔC-dCas9 fusion protein for integrating an IRES-mCherry template targeting the 3'UTRE of EF1-alpha in HEK293 cells. [Diagram 2] A schematic diagram of a nucleic acid editing technique demonstrating that fusion of viral integrase (IN) with CRISPR-dCas9 can integrate large DNA sequences in a target-specific manner. This approach allows the safe and durable delivery of large gene sequences that normally exceed the limitations of non-integrating AAV vectors. [Diagram 3] We present the experimental design and results of a GFP reporter cell line used to quantify and characterize the fidelity of individual integration events in mammalian cells. [Figure 4] Schematic diagram of CRISPR-Cas9-mediated homology-directed repair and retroviral integrase-mediated random DNA integration. [Diagram 5] Schematic diagram of integrase-Cas genome editing. [Figure 6] Schematic diagram of donor vector, generation of blunt-ended template, and generation of 3' processed template are shown. [Figure 7] The experimental design of co-transfection is shown, in which the INsrt template and the IN-dCas9 vector targeting the amilCP sequence were co-transfected into Cos7 cells. [Figure 8] Experimental design of paired guide RNA specific for the 3'UTR of human EF1-alpha locus to knock-in the IGR-mCherry-2A-puromycin-pA cassette into human HEK293 cell line and images of mCherry positive cells 48 hours post-transfection are shown. [Figure 9] 1 shows a schematic diagram illustrating directional editing. [Figure 10] FIG. 1 shows a schematic illustrating multiplex genome editing to create floxed alleles. [Figure 11] Figures 1A through 1C include experimental results demonstrating the efficiency of the Ty1 NLS-like sequence for nuclear localization of INΔC-Cas9 fusion proteins. A shows the detection of INΔC-dCas9 fusion proteins containing the C-terminal classical SV40 NLS, Ty1 NLS, or Ty2 NLS expressed in Cos-7 cells using an anti-FLAG antibody. B shows that Ty1 NLS-like sequences isolated from yeast proteins can either confer strong nuclear localization (MAK11) or no apparent localization activity (INO4 and STH1). C shows the sequences of Ty1, Ty2, and the Ty1 NLS-like sequence. Ty1 and Ty2 are highly conserved in both length and residue composition. Scale bar = 10 μm. [Figure 12] Figures 11A-11C, including A-C, show experimental results demonstrating that the Ty1 NLS enhances Cas9 DNA editing in mammalian cells. A shows a diagram of the px330 CRISPR-Cas9 expression plasmid encoding a hU6-driven single guide RNA (sgRNA) and a CAG-driven Cas9 protein containing an N-terminal 3xFLAG tag, an SV40 NLS, and a C-terminal NPM NLS. The Ty1 NLS was cloned in place of the NPM NLS in px330 (px330-Ty1). B shows that a frameshift-activated luciferase reporter was created in which an upstream 20 nt target sequence (ts) interrupts open reading from the downstream luciferase open reading frame. The frameshift induced by non-homologous end joining (NHEJ) reconstitutes the downstream reporter and allows luciferase to be expressed. C shows that co-expression of a frameshift-responsive luciferase reporter with px330 containing a single guide RNA specific for the target sequence resulted in approximately 20-fold activation of luciferase activity compared to non-targeting sgRNA. Co-expression of px330-Ty1 resulted in approximately 44% improvement compared to px330. [Figure 13]Included are A through E, which show genome targeting strategies for editing. Integration of DNA donor sequences can be targeted to various genome locations depending on the desired application. A shows that delivery of DNA donor sequences carrying gene cassettes can be targeted to intergenic "safe harbor" loci, preventing disruption of expression of adjacent or essential genes. B shows that delivery of DNA donor sequences carrying gene cassettes can be targeted to non-essential "safe harbor" loci, preventing disruption of expression of adjacent or essential genes. C shows that integration of DNA sequences encoding splice acceptor sequences (SA) can be delivered to intronic regions of genes (e.g., disease loci), allowing expression of the integrated sequence and preventing expression of downstream sequences. D shows that integration of DNA sequences encoding splice acceptor sequences (SA) can be delivered to intronic regions of genes (e.g., disease loci), allowing expression of the integrated sequence and preventing expression of downstream sequences. E indicates that incorporation of a DNA donor sequence containing an internal ribosome entry sequence (IRES) into the 3'UTR allows expression without disrupting expression from the endogenous locus. [Figure 14] A diagram of the lentivirus life cycle is shown. Lentiviruses, a subclass of retroviruses, are single-stranded RNA viruses that integrate a persistent double-stranded DNA (dsDNA) copy of their proviral genome into host cell DNA. After viral transduction, the lentiviral RNA genome is copied as blunt-ended dsDNA by virally encoded reverse transcriptase (RT) and inserted into the host genome by integrase I (IN). At its U3 and U5 ends, the lentiviral genome is flanked by short (approximately 20 base pairs) sequence motifs required for integration of the proviral genome by IN. IN-mediated insertion of retroviral DNA occurs with little DNA target sequence specificity and can integrate into active loci, which can disrupt normal gene function and cause disease in humans. [Figure 15A]Figure 1 shows genome editing in mammalian cells. Fusion of lentiviral integrase to dCas9 allows targeted non-homologous insertion of donor DNA sequences containing short viral ends. A diagram of a mammalian expression vector encoding a human U6-driven single guide RNA (sgRNA) and an integrase-dCas9 fusion protein is shown. [Figure 15B] Figure 1 shows genome editing in mammalian cells. Fusion of lentiviral integrase to dCas9 allows targeted non-homologous insertion of donor DNA sequences containing short viral ends. A diagram showing a dsDNA donor template containing an IGR IRES-mCherry-2A-puromycin (puro) cassette flanked by U3 / U5 viral motifs. [Figure 15C] Figure 1 shows genome editing in mammalian cells. The fusion of lentiviral integrase to dCas9 allows targeted non-homologous insertion of donor DNA sequences containing short viral ends. A schematic diagram of integrase-Cas9-mediated integration of this donor template into a CMV-eGFP reporter transgene stably expressed in COS-7 cells is shown. [Figure 15D] Figure 1 shows genome editing in mammalian cells. The fusion of lentiviral integrase to dCas9 allows targeted non-homologous insertion of donor DNA sequences containing short viral ends. Schematic diagram showing that integrase-Cas9-mediated integration of this donor template into the CMV-eGFP reporter transgene stably expressed in COS-7 results in the expression of mCherry and the simultaneous disruption of eGFP expression. [Figure 15E] Genome editing in mammalian cells. Fusion of lentiviral integrase to dCas9 allows targeted non-homologous insertion of donor DNA sequences containing short viral ends. Experimental results showing loss of eGFP expression and gain of mCherry expression in edited COS-7 cells are shown. [Figure 16A] FIG. 1 shows a conventional lentiviral gene delivery system. Diagram of the lentiviral genome encoding viral proteins between flanking long terminal repeats (LTRs). [Figure 16B] FIG. 1 shows a conventional lentiviral gene delivery system. FIG. 2 shows a schematic diagram showing that the lentiviral genome is utilized as a powerful gene delivery tool. Lentiviral particles can be used to package, deliver, and stably express donor transgene sequences. In the lentiviral vector gene expression system, the viral polyprotein is removed from the viral genome and expressed using a separate mammalian expression plasmid. The donor DNA sequence of interest can then be cloned between the adjacent LTR sequences in place of the viral polyprotein. Co-transfection of these vectors in mammalian packaging cells results in the formation of lentiviral particles that can deliver and incorporate the encoded donor sequence, but do not require coding information for integrase and other viral proteins required for subsequent viral propagation. Lentiviral particles are natural vectors for the delivery of both viral proteins (e.g., integrase and reverse transcriptase) as well as dsDNA donor sequences, and contain the necessary viral terminal sequences required for integrase-mediated insertion into mammalian cells. FIG. 3 shows the creation of lentiviral vectors. [Figure 16C]FIG. 1 shows a conventional lentiviral gene delivery system. FIG. 2 shows a schematic diagram showing that the lentiviral genome is utilized as a powerful gene delivery tool. Lentiviral particles can be used to package, deliver, and stably express donor transgene sequences. In the lentiviral vector gene expression system, the viral polyprotein is removed from the viral genome and expressed using a separate mammalian expression plasmid. The donor DNA sequence of interest can then be cloned between the adjacent LTR sequences in place of the viral polyprotein. Co-transfection of these vectors in mammalian packaging cells results in the formation of lentiviral particles that can deliver and incorporate the encoded donor sequence, however, without the coding information for integrase and other viral proteins required for subsequent viral propagation. Lentiviral particles are natural vectors for the delivery of both viral proteins (e.g., integrase and reverse transcriptase) as well as dsDNA donor sequences, and contain the necessary viral terminal sequences required for integrase-mediated insertion into mammalian cells. FIG. 3 shows the transduction of lentiviral particles that deliver and stably express donor transgene sequences. [Figure 17A] Targeted lentiviral integration is shown. Existing lentiviral delivery systems can be modified to introduce editing components for targeted lentiviral donor template integration for genome editing in mammalian cells. One approach is shown in which dCas9 is directly fused to integrase (or integrase lacking its C-terminal non-specific DNA binding domain) within the lentiviral packaging plasmid (e.g., psPax2) that encodes the gag-pol polyprotein. [Figure 17B]We show targeted lentiviral integration. Existing lentiviral delivery systems can be modified to introduce editing components for targeted integration of lentiviral donor templates for genome editing in mammalian cells. We show that modified gag-pol polyproteins are translated as polyproteins along with other viral components and loaded with guide RNAs and packaged into lentiviral particles. In this approach, the IN-dCas9 fusion protein retains sequences required for protease cleavage (PR), which allows it to be successfully cleaved from the gag-pol polyprotein during particle maturation. Transduction of mammalian cells delivers viral proteins including the IN-dCas9 fusion protein, sgRNA, and lentiviral donor sequences. [Figure 17C] Shows targeted lentivirus integration. Existing lentivirus delivery systems can be modified to introduce editing components for targeted integration of lentivirus donor templates for genome editing in mammalian cells. During lentivirus transduction, reverse transcriptase reverse-transcription of ssRNA genome generates dsDNA sequence containing correct viral terminal sequences (U3 and U5), which is integrated into mammalian genome by IN-dCas9 fusion protein. [Figure 18A] Shown is targeted lentivirus integration via fusion to viral protein.As one approach to achieve targeted lentivirus gene integration, show expression and packaging of IN-dCas9 as N- and C-terminal fusion with viral protein (e.g., viral protein R, VPR).A viral protease cleavage sequence is included between VPR and IN-dCas9 fusion protein, which allows IN-dCas9 to be released from VPR after maturation. [Figure 18B]This shows targeted lentivirus integration via fusion to viral proteins. Co-transfection of packaging cells with lentivirus components produces viral particles containing VPR-IN-dCas9 protein and sgRNA. The packaging plasmid (e.g., psPax2) required for viral particle formation contains a mutation in the integrase in the packaging plasmid that inhibits its catalytic activity, thereby inhibiting non-integrase-Cas9 mediated integration. [Figure 18C] Figure 1 shows targeted lentiviral integration via fusion to viral protein. Upon viral transduction, the IN-dCas9 protein is delivered as a protein and mediates the integration of the lentiviral donor sequence. The advantage of delivering the IN-dCas9 fusion and sgRNA as a riboprotein is that it is only expressed transiently in target cells. [Figure 19A] 1 shows targeted lentiviral integration via introduction into a transfer plasmid. Expression of IN-dCas9 fusion proteins and / or guide RNAs from within a viral transfer plasmid (or other viral vectors such as AAV) is one approach to achieve targeted lentiviral gene integration. [Figure 19B] This example shows targeted lentivirus integration via introduction into transfer plasmid.In this approach, the transfer plasmid containing IN-dCas9 fusion protein and sgRNA is co-transfected with the packaging and envelope plasmids required to generate lentivirus particles.When using lentivirus, the packaging plasmid contains catalytic mutations in integrase to inhibit non-specific integration. [Figure 19C]We show targeted lentiviral integration via introduction into a transfer plasmid. When transduced into mammalian cells, expression of the IN-dCas9 fusion protein and sgRNA produces a component that can target its own viral donor vector for targeted integration (self-integration). This method is used for targeted gene disruption or as a gene drive. [Figure 20A] 1 shows co-delivery of lentiviral donor sequences, with lentiviral particles encoding donor DNA sequences functioning as integrated donor templates. [Figure 20B] We show co-delivery of lentiviral donor sequences. In this approach, we show that self-integration of the virus-encoding sequences can be prevented by using integrase enzymes from different members of the retroviridae family and their corresponding transfer plasmids. We show the generation of HIV lentiviral particles encoding IN(FIV)-dCas9 fusion proteins. [Figure 20C] We show co-delivery of lentiviral donor sequences. In this approach, we show that self-integration of the viral coding sequence can be prevented by using integrase enzymes from different members of the retroviridae family and their corresponding transfer plasmids. We show the generation of FIV lentiviral particles containing FIV transfer plasmids. [Figure 20D] 1 shows co-delivery of lentiviral donor sequences. HIV lentiviral particles encoding an IN(FIV)-dCas9 fusion protein are used to incorporate an FIV donor template encoded within the FIV lentiviral particle. [Figure 21]Targeted lentiviral integration in mammalian primary cells. The data show lentiviral packaging, delivery and targeted integration of a lentiviral donor template encoding an IRES-tdTO cassette into the ROSA26mG / + locus of mouse embryonic fibroblasts. After 2 days, ubiquitous red fluorescent protein expression was detectable in MEFs transduced with lentivirus encoding an IRES-tdTO reporter, while GFP fluorescence was retained. Notably, 7 days after transduction, tdTO red fluorescent cells lacking green fluorescence in ROSA26mG / + primary cells were detectable in the culture medium. [Figure 22] Showing targeted lentiviral integration in stable mammalian cell lines. This data shows lentiviral packaging, delivery and targeted integration of a lentiviral donor template encoding an IRES-tdTO cassette into stably expressed CMV-eGFP in COS-7 cells. [Figure 23A] DNA binding domains for targeted integration in lentiviral particles are shown. Replacing the non-specific DNA binding domain of integrase with the programmable DNA binding domain of dCas9 allows targeted integration of dsDNA donor templates via delivery in lentiviral particles. Alternative DNA binding domains (such as TALENs) can be utilized for targeted integration as fusions to viral integrase. Using a similar lentiviral production approach, dCas9 in our previous packaging strategy is replaced with TALENs targeting specific sequences. TALENs are shown packaged and delivered as fusions to integrase in the context of the gag-pol polyprotein. [Figure 23B]DNA binding domains for targeted integration in lentiviral particles are shown. Replacing the non-specific DNA binding domain of integrase with the programmable DNA binding domain of dCas9 allows for targeted integration of dsDNA donor templates via delivery in lentiviral particles. Alternative DNA binding domains (such as TALENs) can be utilized for targeted integration as fusions to viral integrase. Using a similar lentiviral production approach, dCas9 in our previous packaging strategy is replaced with TALENs targeting specific sequences. TALENs packaged and delivered as fusions to integrase as fusions to viral proteins are shown. [Figure 23C] DNA binding domains for targeted integration in lentiviral particles are shown. Replacing the non-specific DNA binding domain of integrase with the programmable DNA binding domain of dCas9 allows targeted integration of dsDNA donor templates via delivery in lentiviral particles. Alternative DNA binding domains (such as TALENs) can be utilized for targeted integration as fusions to viral integrase. Using a similar lentiviral production approach, we replace dCas9 in our previous packaging strategy with TALENs that target specific sequences. TALENs packaged and delivered as fusions to integrase encoded within the transfer plasmid are shown. [Figure 24A] Figures 11A-11C, including A-C, show experimental results demonstrating that the Ty1 NLS enhances Cas9 DNA editing in mammalian cells. A shows a diagram of the px330 CRISPR-Cas9 expression plasmid encoding a hU6-driven single guide RNA (sgRNA) and a CAG-driven Cas9 protein containing an N-terminal 3xFLAG tag, an SV40 NLS, and a C-terminal NPM NLS. The Ty1 NLS was cloned in place of the NPM NLS in px330 (px330-Ty1). [Figure 24B](B) Results showing that a frameshift-activated luciferase reporter was created in which an upstream 20 nt target sequence (ts) interrupts open reading from the downstream luciferase open reading frame. The frameshift induced by non-homologous end joining (NHEJ) reconstitutes the downstream reporter and allows luciferase expression. [Figure 24C] C shows results demonstrating that co-expression of a frameshift-responsive luciferase reporter with px330 containing a single guide RNA specific for the target sequence resulted in approximately 20-fold activation of luciferase activity compared to non-targeting sgRNA. Co-expression of px330-Ty1 resulted in approximately 44% improvement compared to px330. [Diagram 25] FIG. 1 shows a schematic illustrating that TALENs can be utilized to induce retroviral integrase-mediated integration of a donor DNA template. [Figure 26] Schematic diagram of plasmid DNA integration assay. [Figure 27] Experimental data are presented demonstrating that TALEN pairs separated by 16 bp gave rise to approximately 6-fold more chloramphenicol-resistant colonies, while TALEN pairs separated by 28 bp were similar to non-targeted integrase. [Figure 28] The experimental design of co-transfection is shown, in which the INsrt template and the IN-dCas9 vector targeting the amilCP sequence were co-transfected into Cos7 cells. [Figure 29]Figures A through C show experimental results. A shows that expression of the amilCP chromoprotein in e. coli results in purple e. coli (white arrowhead). Integrase-Cas-mediated integration of donor sequences containing viral ends prevents expression of amilCP (orange arrowhead) (growth on kanamycin plates). B shows integration of Insrt IGR-CAT donor templates with either blunt (ScaI cut) or 3' processing mimic (FauI cut) ends into the pCRII-amilCP reporter in mammalian cells. Interestingly, deletion of the C-terminal nonspecific DNA binding domain as a fusion to dCas9 does not inhibit integrase-Cas-mediated integration. Using 3' processing mimicking ends shows an approximately two-fold increase in CAT-resistant clones. C shows evaluation of integrase mutations on integrase-Cas-mediated integration in plasmid DNA. Dimerization-blocking mutations (E85G and E85F) do not interfere with integrase-Cas-mediated integration using dual guide RNA targeted integration of the IGR-CAT donor template into amilCP. However, the IN E87G mutation cannot be restored by paired targeting sgRNA. Interestingly, tandem INΔC fusion to dCas9 (tdINΔC-dCas9) shows approximately 2-fold improved integration. [Diagram 30] FIG. 1 shows a therapeutic schematic for treating Friedreich's ataxia using Cas-IN fusion-mediated frataxin gene therapy. [Diagram 31] 1 shows the adaptation of CRISPR-Cas14 guide RNA sequences for expression from Pol III promoters in mammalian cells. [Diagram 32] Figure 1 shows the activity of different CRISPR-Cas14 guide RNA sequences to edit and activate frameshift-activated luciferase in mammalian cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure relates to fusion proteins, nucleic acids encoding the fusion proteins, systems and methods for editing genetic material. In one embodiment, the disclosure relates to nucleic acid molecules encoding retroviral integrase (IN)-CRISPR associated (Cas) fusion proteins and retroviral IN-Cas fusion proteins. In one embodiment, the IN-Cas fusion proteins further comprise a nuclear localization signal (NLS). In one embodiment, the disclosure relates to nucleic acid molecules encoding Cas-NLS fusion proteins and retroviral Cas-NLS fusion proteins. In some embodiments, the disclosure provides a method of treating Friedreich's ataxia, the method comprising administering a fusion protein or nucleic acid of the disclosure.

[0027] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0028] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

[0029] Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally performed according to conventional methods in the art and various general references provided throughout the specification (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0030] The nomenclature used herein and the laboratory procedures utilized in analytical chemistry and organic synthesis described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0031] As used in the context of the present invention (particularly in the context of the claims), the terms "a," "an," "the," and similar terms are to be construed to include both the singular and the plural, unless otherwise specified in the specification or clearly contradicted by context.

[0032] As used herein, "about" when referring to a measurable value, such as an amount, duration of time, and the like, is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, where such variations are appropriate for performing the disclosed methods.

[0033] "Antisense" refers specifically to the nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule that codes for a protein, or a sequence that is substantially homologous to the non-coding strand.As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule that codes for a protein.Antisense sequences do not necessarily have to be complementary to only the coding portion of the coding strand of a DNA molecule.Antisense sequences can be complementary to regulatory sequences designated on the coding strand of a DNA molecule that codes for a protein, which regulatory sequences control the expression of the coding sequence.

[0034] A "disease" is a condition in the health of an animal where the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health will continue to deteriorate.

[0035] In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be in the absence of the disorder. A disorder, if left untreated, does not necessarily result in a further deterioration of the animal's health state.

[0036] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such sign or symptom is experienced by a patient, or both, are reduced.

[0037] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene gives rise to the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0038] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof amenable to the methods described herein, whether in vitro or in vivo. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0039] The term "specifically binds" as used herein with respect to an antibody refers to an antibody that recognizes a particular antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific.

[0040] In some cases, the terms "specific binding" or "specifically binds" may be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a particular protein structure, rather than the entire protein. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0041] The "coding region" of a gene consists of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of the mRNA molecule produced by transcription of the gene.

[0042] The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that coincide with the anticodon region of a transfer RNA molecule or that code for a stop codon during translation of the mRNA molecule. Thus, a coding region can include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein transport signal sequence).

[0043] "Complementary" as used herein to refer to nucleic acids refers to the broad concept of sequence complementarity between regions of two nucleic acid strands, or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can base pair with a residue in a second nucleic acid strand that is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if the two regions are arranged in an antiparallel fashion and at least one nucleotide residue in the first region can base pair with a residue in the second region. In one embodiment, the first region comprises a first portion and the second region comprises a second portion such that when the first and second portions are arranged in an antiparallel fashion, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion can base pair with nucleotide residues of the second portion, hi one embodiment, all nucleotide residues of the first portion can base pair with nucleotide residues of the second portion.

[0044] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0045] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0046] As used herein, the term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, etc. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that perform other functions as well.

[0047] As used herein, the term "wild-type" is a term of the art understood by those of skill in the art and means the typical form of an organism, strain, gene or characteristic as found in nature, as distinguished from mutant or variant forms.

[0048] The term "homology" refers to the degree of complementarity. There may be partial or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions usually include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0049] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that naturally occurs in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0050] The term "isolated", when used in reference to a nucleic acid, such as "isolated oligonucleotide" or "isolated polynucleotide", refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is normally associated in its source. Thus, an isolated nucleic acid is present in a form or context that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state in which they occur in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome in close proximity to adjacent genes, and an RNA sequence (e.g., a particular mRNA sequence that codes for a particular protein) is found in a cell as a mixture with multiple other mRNAs that code for multiple proteins. However, an isolated nucleic acid includes, by way of example, such a nucleic acid in a cell that normally expresses that nucleic acid, where the nucleic acid is in a different chromosomal location than that of the natural cell or is otherwise adjacent to a different nucleic acid sequence than that found in nature. An isolated nucleic acid or oligonucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide will minimally contain a sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both a sense and an antisense strand (i.e., the oligonucleotide may be double-stranded).

[0051] The term "isolated," when used with respect to a polypeptide, as in "isolated protein" or "isolated polypeptide," refers to a polypeptide that is identified and separated from at least one contaminant with which it is normally associated in its source. Thus, an isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state they exist in nature.

[0052] "Nucleic acid" refers to any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester bonds or modified bonds, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone bonds, and combinations of such bonds. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil). The term "nucleic acid" generally refers to large polynucleotides.

[0053] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction.

[0054] The direction of 5' to 3' addition of nucleotides to the nascent RNA transcript is referred to as the transcription direction. The DNA strand that has the same sequence as the mRNA is referred to as the "coding strand," the sequence on the DNA strand that is 5' to a reference point on the DNA strand is referred to as the "upstream sequence," and the sequence on the DNA strand that is 3' to a reference point on the DNA strand is referred to as the "downstream sequence."

[0055] By "expression cassette" is meant a nucleic acid molecule containing a coding sequence operably linked to promoter / regulatory sequences necessary for the transcription and optionally translation of the coding sequence.

[0056] As used herein, the term "operably linked" refers to the linking of nucleic acid sequences in such a way that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linking of amino acid coding sequences in such a way that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibiting, etc.) protein or polypeptide is produced.

[0057] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that causes the gene product to be expressed in an inducible manner.

[0058] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on several factors. In general, the longer the sequence, the higher the temperature at which it specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0059] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific method. The complex can include two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence that can hybridize to a given sequence is referred to as the "complement" of the given sequence.

[0060] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be substantially produced only in the presence of an inducer that corresponds to the promoter.

[0061] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0062] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, any nucleic acid sequence obtained by any means available in the art, including, but not limited to, cloning of nucleic acid sequences from recombinant means, i.e., recombinant libraries or cell genomes, using conventional cloning techniques and PCR, etc., as well as by synthetic means.

[0063] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0064] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0065] The term "RNA" as used herein is defined as ribonucleic acid.

[0066] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally linked together. The amplified or assembled recombinant polynucleotide can be included in an appropriate vector, which can be used to transform a suitable host cell.

[0067] A recombinant polynucleotide may serve a non-coding function as well (eg, promoter, origin of replication, ribosome binding site, etc.).

[0068] The term "recombinant polypeptide," as used herein, is defined as a polypeptide produced by using recombinant DNA methods.

[0069] As used herein, "transcription activator-like effector nucleases (TALENs)" are artificial restriction enzymes created by fusing a TAL effector DNA binding domain to a DNA cleavage domain. These reactants allow efficient and programmable specific DNA cleavage and represent a powerful tool for editing genetic material in situ. Transcription activator-like effectors (TALEs) can be rapidly engineered to bind to almost any DNA sequence. The term TALENs as used herein is broad and includes monomeric TALENs that can cleave double-stranded DNA without the assistance of another TALEN. The term TALENs is also used to refer to one or both members of a TALEN pair that are engineered to function together to cleave DNA at the same site. TALENs that function together may be referred to as left TALENs and right TALENs, referring to the handedness of the DNA. See U.S. Serial No. 12 / 965,590, U.S. Serial No. 13 / 426,991 (U.S. Patent No. 8,450,471), U.S. Serial No. 13 / 427,040 (U.S. Patent No. 8,440,431), U.S. Serial No. 13 / 427,137 (U.S. Patent No. 8,440,432), and U.S. Serial No. 13 / 738,381, all of which are incorporated by reference in their entireties.

[0070] A "variant", as the term is used herein, is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in the sequence of a peptide variant are usually limited or conservative, such that the sequences of the reference peptide and variant are closely similar overall, and in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions, in any combination. A variant of a nucleic acid or peptide may be naturally occurring, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be made by mutagenesis techniques or by direct synthesis.

[0071] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0072] Ranges: Throughout this disclosure, various aspects of the invention can be expressed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an indefinite limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges in addition to the individual numerical values ​​within that range. For example, description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., in addition to the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0073] protein In one aspect, the present disclosure is based on the development of novel fusions of editing proteins that are effectively delivered to the nucleus. In one aspect, the proteins contain a nuclear localization signal (NLS). In one embodiment, the fusion protein comprises an editing protein and an integrase protein. In one embodiment, the fusion protein comprises a purification and / or detection tag.

[0074] In one aspect, the present disclosure is based in part on the development of a novel fusion of an editing protein that is effectively delivered to the nucleus. In one embodiment, the editing protein is a CRISPR-associated (Cas) protein. In one embodiment, the protein further comprises a nuclear localization signal. Thus, in one aspect, the present disclosure comprises a Cas protein and a nuclear localization signal (NLS).

[0075] In one aspect, the present disclosure is based in part on the development of novel fusions of editing proteins and retroviral integrase proteins that are effectively delivered to the nucleus. These fusion proteins combine the programmable DNA targeting ability of catalytically dead Cas with viral integrase. Thus, because the fusion proteins do not rely on cellular pathways for DNA insertion or require a cellular energy source such as ATP, the enzyme can function in many contexts, for example, from in vitro to prokaryotic, dividing eukaryotic or non-dividing eukaryotic cells. Furthermore, because integrases do not require homology regions for insertion, only small terminal motif sequences specific for each integrase family, the editing of these fusion proteins can utilize a single DNA donor template for multiple genome integration when guided by multiple guide RNAs. Thus, in one aspect, the present disclosure provides fusion proteins comprising a Cas protein, a nuclear localization signal (NLS), and a retroviral integrase (IN) or a fragment or variant thereof.

[0076] Edited Proteins In one embodiment, the fusion protein comprises an editing protein, in one embodiment, the editing protein includes, but is not limited to, a CRISPR-associated (Cas) protein.

[0077] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3. , Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRER SpCas9, VQR SpCas9, xCas9 3.7, their homologs, their orthologs, or modified forms thereof. In some embodiments, the Cas protein has DNA cleavage activity. In some embodiments, the Cas protein induces cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the Cas protein induces cleavage of one or both strands within about 1 base pair, within about 2 base pairs, within about 3 base pairs, within about 4 base pairs, within about 5 base pairs, within about 6 base pairs, within about 7 base pairs, within about 8 base pairs, within about 9 base pairs, within about 10 base pairs, within about 15 base pairs, within about 20 base pairs, within about 25 base pairs, within about 50 base pairs, within about 100 base pairs, within about 200 base pairs, within about 500 base pairs, or within more than about 5 base pairs of the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9 or Cas14. In one embodiment, the Cas protein is Cas9. In one embodiment, the Cas protein is Cas14. In one embodiment, the Cas protein is catalytically deficient (dCas).

[0078] In one embodiment, the Cas protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-8. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs: 1, 3, 5, or 7. The Cas protein comprises a sequence of one of SEQ ID NOs: 2, 4, 6, or 8. The Cas protein comprises a sequence of one of SEQ ID NOs: 1 or 2. The Cas protein comprises one of the sequences of SEQ ID NO:6 or SEQ ID NO:7.

[0079] Nuclear localization signal In some embodiments, the protein contains a nuclear localization signal (NLS). In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is derived from Ty1, yeast GAL4, SKI3, L29, or histone H2B protein, polyomavirus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or SV40 VP2 capsid protein, adenovirus E1a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleophosmin (NPM1), or simian virus 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or MAK11 or a MAK11-derived NLS.

[0080] In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 53. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 54. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 56. In one embodiment, the NLS comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-62 and 361-973. In one embodiment, the NLS comprises one of SEQ ID NOs: 49-62 and 361-973.

[0081] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX motif and a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises at least 20 amino acids. In one embodiment, the Ty1-like NLS comprises 20 to 40 amino acids. In one embodiment, the Ty1-like NLS comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 361-973. In one embodiment, the Ty1-like NLS protein comprises one of SEQ ID NOs: 361-973, wherein the sequence comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more insertions, deletions, or substitutions. In one embodiment, the Ty1-like NLS comprises one of SEQ ID NOs: 361-973.

[0082] In one embodiment, the NLS comprises a combination of two different NLSs. For example, in one embodiment, the NLS comprises an NLS from Ty1 and an NLS from SV40. In one embodiment, the NLS is Ty1 or an NLS from Ty1, Ty2 or an NLS from Ty2, or MAK11 or an NLS from MAK11. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 53. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 54. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 56.

[0083] In one embodiment, the NLS comprises two copies of the same NLS, for example, in one embodiment, the NLS comprises a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0084] In one embodiment, the NLS is a first sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to one of SEQ ID NOs: 49-62, and 361-973. and a second sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to one of SEQ ID NOs: 49-62 and 361-973. In one embodiment, the NLS comprises a first sequence of SEQ ID NOs: 49-62, and 361-973, and a second sequence of SEQ ID NOs: 49-62, and 361-973. In one embodiment, the first sequence and the second sequence are the same. In one embodiment, the first sequence and the second sequence are different.

[0085] In one embodiment, the NLS comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to one of SEQ ID NOs: 58-61. In one embodiment, the NLS comprises a sequence of SEQ ID NOs: 58-61.

[0086] Retroviral integrase In one embodiment, the retrovirus is a human immunodeficiency virus (HIV), a rous sarcoma virus (RSV), a mouse mammary tumor virus (MMTV), a moloney murine leukemia virus (MoLV), a bovine leukemia virus (BLV), a human T-lymphotropic virus (HTLV), an avian sarcoma leukemia virus (ASLV), a feline leukemia virus (FLV), a xenotropic murine leukemia virus-related virus (XMLV), a simian immunodeficiency virus (SIV), a feline immunodeficiency virus (FIV), an equine infectious anemia virus (EIAV), a prototype foamy virus (PFV), a simian foamy virus (SFV), a human foamy virus (HFV), a walleye cutaneous sarcoma virus (WDSV), or a bovine immunodeficiency virus (BIV).

[0087] In one embodiment, the integrase is a retrotransposon integrase. In one embodiment, the retrotransposon integrase is Ty1 or Ty2. In one embodiment, the integrase is a bacterial integrase. In one embodiment, the bacterial integrase is insF.

[0088] In one embodiment, the retroviral IN is HIV IN. In one embodiment, the HIV IN comprises one or more amino acid substitutions that improve catalytic activity, improve solubility, or increase interaction with one or more host cell cofactors. In one embodiment, the HIV IN comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine amino acid substitutions selected from the group consisting of E85G, E85F, D116N, F185K, C280S, T97A, Y134R, G140S, and Q148H. In one embodiment, the HIV IN comprises the amino acid substitutions F185K and C280S. In one embodiment, the HIV IN comprises the amino acid substitutions T97A and Y134R. In one embodiment, the HIV IN comprises the amino acid substitutions G140S and Q148H.

[0089] In one embodiment, the retroviral IN fragment comprises an IN N-terminal domain (NTD) and an IN catalytic core domain (CCD). In one embodiment, the retroviral IN fragment comprises an IN CCD and an IN C-terminal domain (CTD). In one embodiment, the retroviral IN fragment comprises an IN NTD. In one embodiment, the retroviral IN fragment comprises an IN CCD. In one embodiment, the retroviral IN fragment comprises an IN CTD. In one embodiment, the fragment of integrase retains at least one activity of full-length integrase. Functions and fragments of retroviral integrase are known in the art and can be found, for example, in Li, et al., 2011, Virology 411:194-205, and Maertens et al., 2010, Nature 468:326-29, which are incorporated herein by reference.

[0090] In one embodiment, the retroviral IN comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 9-48. In one embodiment, the retroviral IN comprises a sequence of one of SEQ ID NOs: 9-48.

[0091] Purification and / or Detection Tags In some embodiments, the protein comprises a purification and / or detection tag. In one embodiment, the tag is at the N-terminus of the protein. In one embodiment, the tag is a 3xFLAG tag. In one embodiment, the tag comprises an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:51. In one embodiment, the tag comprises the amino acid sequence of SEQ ID NO:51.

[0092] Fusion proteins In one aspect, the disclosure provides a fusion protein comprising a Cas protein as described herein and a nuclear localization signal (NLS). In one embodiment, the fusion protein comprises an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 147-149. In one embodiment, the protein comprises the sequence of one of SEQ ID NOs: 147-149. In one embodiment, the protein comprises the amino acid sequence of SEQ ID NO: 149.

[0093] In one aspect, the disclosure provides a fusion protein comprising a Cas protein as described herein, a nuclear localization signal (NLS), and a retroviral integrase (IN) or a fragment or variant thereof. In one embodiment, the fusion protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63-142. In one embodiment, the fusion protein comprises a sequence of one of SEQ ID NOs: 63-142. In one embodiment, the fusion protein comprises a generation and / or detection tag. In one embodiment, the fusion protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 143-146. In one embodiment, the fusion protein comprises a sequence of one of SEQ ID NOs: 143-146.

[0094] Proteins, peptides and fusion proteins The proteins of the present disclosure can be produced using chemical methods. For example, peptides can be synthesized by solid-phase techniques (Roberge JY et al (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be performed, for example, using an ABI 431 A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0095] The proteins of the present disclosure can be made using recombinant protein expression. The recombinant expression vector of the present disclosure comprises the nucleic acid of the present disclosure in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more control sequences selected based on the host cell used for expression, operably linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the control sequence in such a way that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in the host cell when the vector is introduced into the host cell).

[0096] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct intermittent expression of a nucleotide sequence in many types of host cells, and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector depends on such factors as the choice of the host cell to be transformed, the expression level of the desired protein, and the like. The expression vectors of the present disclosure are introduced into host cells, thereby producing the proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0097] The recombinant expression vectors of the present disclosure can be designed for the production of mutant proteins in prokaryotic or eukaryotic cells. For example, the proteins of the present disclosure can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector system can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0098] Expression of proteins in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add several amino acids to the protein encoded therein, e.g., at the amino or C-terminus of the recombinant protein. Such fusion vectors can typically serve three purposes, e.g., (i) to increase the expression of the recombinant protein, (ii) to increase the solubility of the recombinant protein, and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein so that the recombinant protein can be separated from the fusion moiety after purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, PreScission, TEV, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0099] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89) - not exactly, pET11a-d have an N-terminal T7 tag.

[0100] One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium with a reduced capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid to be inserted into the expression vector so that the individual codons for each amino acid are those preferentially used in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20:2111-2118). Such modifications of the nucleic acid sequence of the present disclosure can be performed with standard DNA synthesis techniques. Another strategy for resolving codon bias is by using BL21 Codon Plus strains (Invitrogen) or Rosetta strains (Novagen), which contain additional copies of rare E. coli tRNA genes.

[0101] In another embodiment, the expression vector encoding the protein of the present disclosure is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30:933-943), pJRY88 (Schultz et al., 1987. Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.) and picZ (InVitrogen Corp, San Diego, Calif.).

[0102] Alternatively, the polypeptides of the present disclosure can be produced in insect cells using baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0103] In yet another embodiment, the nucleic acid of the present disclosure is expressed in mammalian cells using a mammalian expression vector. Mammalian cell lines available in the art for expression of heterologous polypeptides include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human fetal kidney cells, human fetal retina cells, and many others. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195), pIRESpuro (Clontech), pUB6 (Invitrogen), pCEP4 (Invitrogen) pREP4 (Invitrogen), pcDNA3 (Invitrogen). When used in mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, Rous sarcoma virus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0104] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., expressing the nucleic acid using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific, Pinkert, et al., 1987. Genes Dev. 1:268-277), lymphocyte-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular the promoters of the T cell receptor (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulin promoters (Banerji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter, Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreatic-specific promoters (Edlund, et al., 1989. al., 1985. Science 230:912-916), as well as mammary gland-specific promoters (e.g., the whey promoter, U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166). Developmentally-regulated promoters, such as the murine hox promoters (Kessel and Gruss, 1990. Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546), are also included.

[0105] The present disclosure should be construed to include any form of protein that has substantial homology to the protein of the present disclosure. In one embodiment, a "substantially homologous" protein is about 50% homologous, about 70% homologous, about 80% homologous, about 90% homologous, about 91% homologous, about 92% homologous, about 93% homologous, about 94% homologous, about 95% homologous, about 96% homologous, about 97% homologous, about 98% homologous, or about 99% homologous to the amino acid sequence of the fusion protein of the present disclosure.

[0106] The protein may alternatively be made by recombinant means, or by truncation from a longer polypeptide. The composition of the protein may be confirmed by amino acid analysis or sequencing.

[0107] Variants of proteins according to the present disclosure may be (i) those in which one or more amino acid residues have been replaced with a conserved or non-conserved amino acid residue, where such replaced amino acid residues may or may not be encoded by the genetic code, (ii) those in which one or more modified amino acid residues are present, e.g., those residues modified by the addition of a substituent group, (iii) those in which the peptide is an alternative splice variant of the protein of the present disclosure, (iv) fragments of the peptide, and / or (v) those in which the protein is fused to another peptide, e.g., a leader or secretory sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally or chemically modified. Such variants are considered to be within the scope of one of skill in the art from the teachings herein.

[0108] As known in the art, "similarity" between two fusion proteins is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutes to the sequence of a second polypeptide. A variant is defined as comprising a peptide sequence that differs from the original sequence. In one embodiment, the variant differs from the original sequence by less than 40% of the residues per segment of interest, by less than 25% of the residues per segment of interest, by less than 10% of the residues per segment of interest, or by only a few residues per segment of interest, while being sufficiently homologous to the original sequence to maintain the functionality of the original sequence and / or the ability to stimulate differentiation of stem cells into the osteoblast lineage. The present disclosure provides variants that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or more similar to the original amino acid sequence. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % Similar or identical amino acid sequences. The degree of identity between two peptides is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences can be determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0109] The proteins of the present disclosure may be post-translationally modified. For example, post-translational modifications within the scope of the present disclosure include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require the introduction of additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation are examined by adding dog microsomal membranes or Xenopus egg extracts (US Pat. No. 6,103,489) to standard translation reactions.

[0110] Proteins of the disclosure can include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0111] A peptide or protein of the present disclosure can be phosphorylated using conventional methods, such as those described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0112] Cyclic derivatives of the fusion proteins of the present disclosure are also part of the present disclosure. Cyclization allows the protein to assume a more favorable conformation in association with other molecules. Cyclization can be performed using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between an amino group of one component and a carboxyl group of another component. Cyclization can also be performed using azobenzene-containing amino acids as described in Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The bond-forming component can be the side chain of an amino acid, a non-amino acid component, or a combination of the two. In one embodiment of the present disclosure, the cyclic peptide can include a beta turn at the right position. A beta turn can be introduced into the peptide of the present disclosure by adding the amino acid Pro-Gly to the right position.

[0113] It may be desirable to generate a more flexible cyclic peptide than the cyclic peptides containing the protein bond linkages described above. A more flexible peptide can be prepared by introducing cysteines at the left and right positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are positioned so as not to distort the beta sheets and turns. The peptide is more flexible as a result of the length of the disulfide bonds and the reduced number of hydrogen bonds in the beta sheet portion. The relative flexibility of the cyclic peptide can be measured by molecular dynamics simulations.

[0114] The present disclosure also provides chimeric proteins comprising Cas13 and a targeting domain fused or incorporated to a target protein and / or a targeting domain capable of directing the chimeric protein to a desired cellular component or cell type or tissue. Fusion proteins containing RNase (fusion proteins are then poor) Chimeric proteins may also contain additional amino acid sequences or domains. Chimeric proteins are recombinant in the sense that the various components are derived from different sources and therefore are not found together in nature (i.e., heterologous).

[0115] In one embodiment, the targeting domain can be a transmembrane domain, a membrane-binding domain, or a sequence that induces the protein to associate with, for example, vesicles or the nucleus. In one embodiment, the targeting domain can target the peptide to a specific cell type or tissue. For example, the targeting domain can be an antibody against a cell surface ligand or a cell surface antigen of the target tissue. The targeting domain can target the peptide of the present disclosure to a cellular component.

[0116] The peptides of the present disclosure can be synthesized by conventional techniques. For example, the peptide or chimeric protein can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods use either solid-phase or solution-phase synthesis methods (for example, for solid-phase synthesis techniques, see JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254, and for classical solution synthesis, see M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1). As an example, the peptides of the present disclosure can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry, which directly introduces phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0117] N- or C-terminal fusion proteins comprising the peptide or chimeric protein of the present disclosure conjugated with other molecules can be prepared by recombinantly fusing the N- or C-terminus of the peptide or chimeric protein with the sequence of a selected protein or selectable marker having a desired biological function. The resulting fusion protein contains the protein fused to the selected protein or marker protein described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA) and truncated myc.

[0118] The peptides of the present disclosure can be developed using biological expression systems. The use of these systems allows the creation of large libraries of random peptide sequences and screening of these libraries for peptide sequences that bind to specific proteins. Libraries can be created by cloning synthetic DNA encoding random peptide sequences into appropriate expression vectors (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries can also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0119] The peptides and chimeric proteins of the present disclosure can be converted into pharmaceutical salts by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0120] nucleic acid In one embodiment, a nucleic acid molecule is disclosed that encodes a fusion protein of the present disclosure. In one embodiment, the nucleic acid encodes a fusion protein that includes a nuclear localization signal (NLS). In one embodiment, the nucleic acid encodes a fusion protein that includes an editing protein and an integrase protein. In one embodiment, the nucleic acid encodes a fusion protein that includes a purification and / or detection tag.

[0121] The present disclosure also provides targeting nucleic acids comprising guide RNAs (gRNAs) for targeting proteins of the present disclosure to a target nucleic acid sequence.

[0122] Edited Proteins In one embodiment, the nucleic acid molecule comprises a sequence of a nucleic acid encoding an editing protein, including, but not limited to, CRISPR-associated (Cas) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA or RNA binding domains.

[0123] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3. , Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRER SpCas9, VQR SpCas9, xCas9 3.7, their homologs, their orthologs, or modified forms thereof. In some embodiments, the Cas protein has DNA cleavage activity. In some embodiments, the Cas protein induces cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the Cas protein induces cleavage of one or both strands within about 1 base pair, about 2 base pairs, about 3 base pairs, about 4 base pairs, about 5 base pairs, about 6 base pairs, about 7 base pairs, about 8 base pairs, about 9 base pairs, about 10 base pairs, about 15 base pairs, about 20 base pairs, about 25 base pairs, about 50 base pairs, about 100 base pairs, about 200 base pairs, about 500 base pairs, or more base pairs from the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9 or Cas14. In one embodiment, the Cas protein is Cas9. In one embodiment, the Cas protein is Cas14. In one embodiment, the Cas protein is catalytically deficient (dCas).

[0124] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-8. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1, 3, 5, or 7. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 2, 4, 6, or 8. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 1-2. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 7-8.

[0125] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 153-160. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 153, 155, 157, or 159. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises one of the nucleic acid sequences of SEQ ID NOs: 154, 156, 158 or 160. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises one of the nucleic acid sequences of SEQ ID NOs: 153-154. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises one of the nucleic acid sequences of SEQ ID NOs: 159-160.

[0126] Nuclear localization signal In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a nuclear localization signal (NLS). In one embodiment, the protein comprises an NLS. In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is Ty1,The NLS is derived from yeast GAL4, SKI3, L29, or histone H2B protein, polyomavirus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or SV40 VP2 capsid protein, adenovirus E1a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmin (NPM2), nucleophosmin (NPM1), or simian virus 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or an NLS from Ty1, Ty2 or an NLS from Ty2, or MAK11 or an NLS from MAK11.

[0127] In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 53. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 54. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 56. In one embodiment, the nucleic acid sequence encoding the NLS comprises an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-62, and 361-973. In one embodiment, the nucleic acid sequence encoding the NLS encodes an amino acid sequence of one of SEQ ID NOs: 49-62, and 361-973.

[0128] In one embodiment, the nucleic acid sequence encoding the NLS comprises an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 201-210. In one embodiment, the nucleic acid sequence encoding the NLS comprises one of SEQ ID NOs: 201-210.

[0129] In one embodiment, the NLS is a Ty1-like NLS. For example, in one embodiment, the Ty1-like NLS comprises a KKRX motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus. In one embodiment, the Ty1-like NLS comprises a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises a KKRX motif and a KKR motif. In one embodiment, the Ty1-like NLS comprises a KKRX motif at the N-terminus and a KKR motif at the C-terminus. In one embodiment, the Ty1-like NLS comprises at least 20 amino acids. In one embodiment, the Ty1-like NLS comprises 20 to 40 amino acids. In one embodiment, the nucleic acid sequence encoding the Ty1-like NLS encodes an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 361-973. In one embodiment, the nucleic acid sequence encoding the Ty1-like NLS encodes the amino acid sequence of one of SEQ ID NOs: 361-973, and the sequence comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more insertions, deletions, or substitutions. In one embodiment, the nucleic acid sequence encoding the Ty1-like NLS encodes the amino acid sequence of one of SEQ ID NOs: 361-973.

[0130] In some embodiments, the nucleic acid molecule comprises one or more nucleic acid sequences that encode a combination of two different NLSs.

[0131] For example, in one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences encoding a Ty1-derived NLS and a SV40-derived NLS. In one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences encoding two or more of Ty1 or a Ty1-derived NLS, Ty2 or a Ty2-derived NLS, or a MAK11 or a MAK11-derived NLS. In one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences encoding a Ty1 NLS comprising the amino acid sequence of SEQ ID NO:53. In one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences encoding a Ty2 NLS comprising the amino acid sequence of SEQ ID NO:54. In one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences encoding a MAK11 NLS comprising the amino acid sequence of SEQ ID NO:56.

[0132] In one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences encoding two copies of the same NLS. For example, in one embodiment, the nucleic acid molecule comprises one or more nucleic acid sequences, each encoding an NLS comprising a multimer of a first Ty1-derived NLS and a second Ty1-derived NLS.

[0133] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a first NLS sequence and a nucleic acid sequence encoding a second NLS sequence, in one embodiment, the first NLS sequence is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 47-56, 254-257, and 275-887. In one embodiment, the second NLS sequence is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-62 and 361-973. In one embodiment, the first NLS sequence and the second NLS sequence are the same. In one embodiment, the first NLS sequence and the second NLS sequence are different.

[0134] Retroviral integrase In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a retroviral integrase (IN). In one embodiment, the retroviral IN is human immunodeficiency virus (HIV), rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), moloney murine leukemia virus (MoLV), bovine leukemia virus (BLV), human T-lymphotropic virus (HTLV), avian sarcoma leukemia virus (ASLV), feline leukemia virus (FLV), xenotropic murine leukemia virus-related virus (XMLV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), prototype foamy virus (PFV), simian foamy virus (SFV), human foamy virus (HFV), walleye dermal sarcoma virus (WDSV), or bovine immunodeficiency virus (BIV). In one embodiment, the integrase is a retrotransposon integrase. In one embodiment, the retrotransposon integrase is Ty1 or Ty2. In one embodiment, the integrase is a bacterial integrase. In one embodiment, the bacterial integrase is insF.

[0135] In one embodiment, the retroviral IN is HIV IN. In one embodiment, the HIV IN comprises one or more amino acid substitutions that improve catalytic activity, improve solubility, or increase interaction with one or more host cell cofactors. In one embodiment, the HIV IN comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine amino acid substitutions selected from the group consisting of E85G, E85F, D116N, F185K, C280S, T97A, Y134R, G140S, and Q148H. In one embodiment, the HIV IN comprises the amino acid substitutions F185K and C280S. In one embodiment, the HIV IN comprises the amino acid substitutions T97A and Y134R. In one embodiment, the HIV IN comprises the amino acid substitutions G140S and Q148H.

[0136] In one embodiment, the retroviral IN fragment comprises an IN N-terminal domain (NTD) and an IN catalytic core domain (CCD). In one embodiment, the retroviral IN fragment comprises an IN CCD and an IN C-terminal domain (CTD). In one embodiment, the retroviral IN fragment comprises an IN NTD. In one embodiment, the retroviral IN fragment comprises an IN CCD. In one embodiment, the retroviral IN fragment comprises an IN CTD. In one embodiment, the fragment of integrase retains at least one activity of full-length integrase. Functions and fragments of retroviral integrase are known in the art and can be found, for example, in Li, et al., 2011, Virology 411:194-205, and Maertens et al., 2010, Nature 468:326-29, which are incorporated herein by reference.

[0137] In one embodiment, the nucleic acid sequence encoding the retroviral IN comprises a nucleic acid sequence encoding an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 9-48. In one embodiment, the nucleic acid sequence encoding the retroviral IN comprises a nucleic acid sequence encoding an amino acid sequence of one of SEQ ID NOs: 9-48.

[0138] In one embodiment, the nucleic acid sequence encoding the retroviral IN comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 161-200. In one embodiment, the nucleic acid sequence encoding the retroviral IN comprises a nucleic acid sequence of one of SEQ ID NOs: 161-200.

[0139] Purification and / or Detection Tags In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a purification and / or detection tag. In one embodiment, the tag is at the N-terminus of the protein. In one embodiment, the tag is a 3xFLAG tag.

[0140] In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag encodes an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51. In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag encodes the amino acid sequence of SEQ ID NO:51.

[0141] In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 203. In one embodiment, the nucleic acid sequence encoding the purification and / or detection tag comprises the sequence of SEQ ID NO:203.

[0142] Fusion proteins In one aspect, the disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising a Cas protein as described herein and a nuclear localization signal (NLS). In one embodiment, the nucleic acid molecule encoding the fusion protein encodes an amino acid sequence that is 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 147-149. In one embodiment, the nucleic acid molecule encoding the fusion protein encodes an amino acid sequence of one of SEQ ID NOs: 147-149. In one embodiment, the nucleic acid molecule encoding the fusion protein encodes the amino acid sequence of SEQ ID NO:149.

[0143] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 255-257. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises one of SEQ ID NO: 255-257, and the nucleic acid sequence encoding the fusion protein comprises the sequence of SEQ ID NO: 257.

[0144] In one aspect, the disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising a Cas protein described herein, a nuclear localization signal (NLS), and a retroviral IN or a fragment or variant thereof. In one embodiment, the nucleic acid sequence encoding the fusion protein encodes an amino acid sequence that is 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63-142. In one embodiment, the nucleic acid molecule encoding the fusion protein encodes an amino acid sequence of one of SEQ ID NOs: 63-142.

[0145] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211-250. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises the sequence of SEQ ID NO: 211-250.

[0146] In one embodiment, the fusion protein further comprises a purification and / or detection tag. In one embodiment, the nucleic acid sequence encoding the fusion protein encodes an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 143-146. In one embodiment, the nucleic acid sequence encoding the fusion protein encodes an amino acid sequence of one of SEQ ID NOs: 143-146.

[0147] In one embodiment, the nucleic acid sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 251-254. In one embodiment, the nucleic acid sequence comprises one of SEQ ID NOs: 251-254.

[0148] Guide Nucleic Acid In one aspect, the disclosure provides a guide nucleic acid for targeting Cas to a target nucleic acid. In one embodiment, the disclosure provides a tracrRNA and a CRISPR RNA (cRNA). In one embodiment, the tracrRNA and the cRNA are fused to form a single guide RNA (sgRNA). The crRNA or the crRNA portion of the gRNA double stranded comprises a DNA targeting segment of the gRNA. The tracrRNA or the tracrRNA portion of the gRNA comprises a segment that interacts with a Cas protein. In one embodiment, the crRNA or the crRNA portion of the gRNA comprises a tracr mate sequence that hybridizes with a portion of the tracrRNA and a spacer sequence that is substantially complementary to the target sequence such that it hybridizes to the target sequence.

[0149] As used herein, "target sequence" refers to a sequence that the guide RNA is designed to have complementarity with, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence may include any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be present in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used for recombination into a target locus that includes a target sequence is referred to as an "editing template" or an "editing polynucleotide" or an "editing sequence". In aspects of the invention, the foreign template polynucleotide may also be referred to as an editing template. In one aspect of the invention, the recombination is a homologous recombination.

[0150] "tracrRNA" sequence or similar terminology includes any polynucleotide sequence that has sufficient complementarity with the crRNA to hybridize. In some forms, the degree of complementarity between the tracrRNA sequence and the crRNA sequence along the shorter of the two lengths is about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more. In some embodiments, the tracr sequence is about 5 nucleotides or more, about 6 nucleotides or more, about 7 nucleotides or more, about 8 nucleotides or more, about 9 nucleotides or more, about 10 nucleotides or more, about 11 nucleotides or more, about 12 nucleotides or more, about 13 nucleotides or more, about 14 nucleotides or more, about 15 nucleotides or more, about 16 nucleotides or more, about 17 nucleotides or more, about 18 nucleotides or more, about 19 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 40 nucleotides or more, about 50 nucleotides or more, or longer. In some forms, the tracr sequence and the crRNA sequence are contained within a single transcript, such that hybridization between the two generates a transcript with a secondary structure, such as a hairpin. In some forms, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred forms, the transcript has two, three, four, or five hairpins. In some forms, the transcript has up to five hairpins, in which part of the sequence 5' of the final "N" and upstream of the loop corresponds to the tracr mate sequence, and part of the sequence 3' of the loop corresponds to the tracr sequence.

[0151] Generally, the degree of complementarity refers to optimal alignment of the sea and tracr sequences along the length of the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm and can further account for secondary structure, such as self-complementarity, either within the sea or tracr sequences. In some forms, when optimally aligned, the degree of complementarity between the tracr and sea sequences along the length of the shorter of the two is about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more.

[0152] Guide sequences can be selected to target any target sequence. In some embodiments, the target sequence is a sequence in the genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for S.pyogenes Cas9, a unique target sequence in a genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T, or C, and X can be any) occurs once in the genome. A unique target sequence in a genome can include a S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNXGG (N is A, G, T, or C, and X can be any) occurs once in the genome. For S. thermophilus CRISPR1 Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO:1), which occurs once in the genome, NNNNNNNNNNNNXXAGAAW (SEQ ID NO:2), where N is A, G, T or C, X is any, and W is A or T. A unique target sequence in a genome may include a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO:3), which occurs once in the genome, NNNNNNNNNNNXXAGAAW (SEQ ID NO:4), where N is A, G, T or C, X is any, and W is A or T. For S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T or C, and X can be anything) occurs once in the genome. A unique target sequence in a genome may include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNXGGXG (N is A, G, T or C, and X can be anything) occurs once in the genome.In each of these sequences, "M" can be A, G, T or C, and this need not be taken into consideration when identifying a sequence as unique.

[0153] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure in the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. One example of such an algorithm is mFold, described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry, University of Vienna, using a centroid structure prediction algorithm (see, for example, A. R. Gruber et al., 2008, Cell 106(1):23-24, and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

[0154] In general, the tracr mate sequence includes any sequence that has sufficient complementarity with the tracr sequence to promote one or more of: (1) excision of the guide sequence adjacent to the tracr mate sequence in cells containing the corresponding tracr sequence, and (2) formation of a CRISPR complex at the target sequence that includes the tracr mate sequence hybridized to the tracr sequence. In general, the degree of complementarity refers to optimal alignment of the tracr mate sequence with the tracr sequence along the length of the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm and can further account for secondary structures such as self-complementarity either within the tracr sequence or within the tracr mate sequence. In some embodiments, when optimally aligned, the degree of complementarity between the tracr sequence and the tracr mate sequence along the length of the shorter of the two is about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 97.5% or more, about 99% or more, or more. In some embodiments, the tracr sequence is about 5 nucleotides or more, about 6 nucleotides or more, about 7 nucleotides or more, about 8 nucleotides or more, about 9 nucleotides or more, about 10 nucleotides or more, about 11 nucleotides or more, about 12 nucleotides or more, about 13 nucleotides or more, about 14 nucleotides or more, about 15 nucleotides or more, about 16 nucleotides or more, about 17 nucleotides or more, about 18 nucleotides or more, about 19 nucleotides or more, about 20 nucleotides or more, about 25 nucleotides or more, about 30 nucleotides or more, about 40 nucleotides or more, about 50 nucleotides or more, or longer. In some embodiments, the tracr sequence and the tracr mate sequence are contained within a single transcript, such that hybridization between the two generates a transcript with a secondary structure, such as a hairpin. In one embodiment, the loop-forming sequence for use in a hairpin structure is 4 nucleotides long. In one embodiment, the loop-forming sequence for use in a hairpin structure has the sequence GAAA. However, longer or shorter loop sequences can be used, as well as alternative sequences.The sequence may include a nucleotide triplet (e.g., AAA) and another nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In one embodiment of the disclosure, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In some embodiments, the transcript has two, three, four, or five hairpins. In further embodiments of the disclosure, the transcript has up to five hairpins. In some embodiments, the single transcript further comprises a transcription termination sequence, which in some embodiments is a poly-T sequence, e.g., six T nucleotides.

[0155] In one embodiment, the Cas14 tracr sequence comprises a sequence that is at least 90% homologous to SEQ ID NO: 336. In one embodiment, the Cas14 tracr comprises a stretch of consecutive stretches of 5T that function as a stop sequence recognized by the Pol III promoter, and thus may prevent guide RNA expression in mammalian cells. Thus, in some embodiments, the Cas14 tracr sequence comprises a single mutation in the polyT sequence. For example, in one embodiment, the Cas14 tracr comprises a sequence that is at least 90% homologous to one of SEQ ID NOs: 337-339. In one embodiment, the Cas14 tracr comprises a sequence of one of SEQ ID NOs: 337-339.

[0156] In one embodiment, the Cas14 crRNA comprises a tracr mate sequence. In one embodiment, the tracr mate sequence comprises a sequence that is at least 90% homologous to one of SEQ ID NOs: 340-343. In one embodiment, the tracr mate sequence comprises a sequence that is at least 90% homologous to one of SEQ ID NOs: 340-343. In one embodiment, the Cas14 crRNA comprises a tracr mate sequence and a spacer sequence. In one embodiment, the Cas14 crRNA comprises a tracr mate sequence that is at least 90% homologous to one of SEQ ID NOs: 340-343, and the spacer sequence substantially hybridizes to the target. In one embodiment, the Cas14 crRNA comprises a tracr mate sequence of one of SEQ ID NOs: 340-343, and a spacer sequence, and the spacer sequence substantially hybridizes to the target.

[0157] In one embodiment, the Cas14 sgRNA comprises a tracr sequence that binds to the tracr mate sequence of the crRNA via a loop-forming sequence. In one embodiment, the sgRNA comprises a sequence that is at least 90% homologous to one of SEQ ID NOs: 344-349. In one embodiment, the sgRNA comprises a sequence that is at least 90% homologous to one of SEQ ID NOs: 344-349, and further comprises a spacer sequence. In one embodiment, the sgRNA comprises a sequence that is at least 90% homologous to one of SEQ ID NOs: 344-349, and further comprises a spacer sequence.

[0158] In one embodiment, the Cas14 sgRNA comprises a sequence that is at least 90% homologous to SEQ ID NOs: 350 to 355. In one embodiment, the Cas14 sgRNA comprises one of SEQ ID NOs: 350 to 355.

[0159] Ef1a2 promoter In one aspect, the nucleic acid molecule of the present disclosure comprises an Ef1a2 promoter that drives expression of a protein or gene described herein. In one embodiment, the Ef1a2 promoter can drive expression in heart, skeletal muscle, and neural tissues such as the brain and motor neurons. In one embodiment, the Ef1a2 promoter comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 333-335. In one embodiment, the Ef1a2 promoter comprises one of the sequences set forth in SEQ ID NOs: 333-335.

[0160] nucleic acid An isolated nucleic acid sequence of the disclosure can be obtained using any of a number of recombinant methods known in the art, for example, by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain the gene, or by direct isolation from cells and tissues containing the gene using standard techniques, etc. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0161] The isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to, DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including, for example, an isolated cDNA molecule, that encodes a protein of the present disclosure. In one embodiment, the composition comprises an isolated RNA molecule, or a functional fragment thereof, that encodes a protein of the present disclosure.

[0162] The nucleic acid molecules of the present disclosure can be modified to improve stability in serum or growth medium for cell culture. Modifications can be made to improve the stability, functionality and / or specificity of the nucleic acid molecules of the present disclosure and minimize immune stimulatory properties. For example, to improve stability, the 3' residues can be stabilized against degradation, e.g., they can be selected to consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0163] In one embodiment of the present disclosure, the nucleic acid molecule may comprise at least one modified nucleotide analogue. For example, the termini may be stabilized by introducing modified nucleotide analogues.

[0164] Non-limiting examples of nucleotide analogs include sugar-modified and / or backbone-modified ribonucleotides (i.e., modifications to the phosphate-sugar backbone). For example, the phosphodiester linkage of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate group linking adjacent ribonucleotides is replaced by a modified group, e.g., a phosphothioate group modified group. In exemplary sugar-modified ribonucleotides, the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0165] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O-alkylated and N-alkylated nucleotides, such as N6-methyladenosine, are preferred. It should be noted that the above modifications can be combined.

[0166] In some cases, the nucleic acid molecule comprises one or more of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modifications of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present disclosure may have improved resistance to nucleases. To increase nuclease resistance, the nucleic acid molecules may comprise, for example, 2'-modified ribose units and / or phosphorothioate bonds. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. To increase nuclease resistance, the nucleic acid molecules of the present disclosure may comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate bonds. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, such as 2-amino-A modifications, 2-thio (e.g., 2-thio-U) modifications, G-clamp modifications, can also increase binding affinity to targets.

[0167] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0168] In certain embodiments, the nucleic acid molecules of the present disclosure have one or more of the following properties.

[0169] Nucleic acid agents discussed herein include specifically unmodified RNA and DNA, as well as RNA and DNA modified, for example to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the components of nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those found in nature or naturally occurring in the human body. Rare or unusual but naturally occurring RNAs are referred to in the art as modified RNAs, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often referred to as modified RNAs, are usually the result of post-transcriptional modifications and fall within the scope of the term unmodified RNA as used herein. Modified RNAs as used herein refer to molecules in which one or more of the components of nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those found in nature or different from those found in the human body. Although they are referred to as "modified RNAs", it is to be understood that molecules that are not strictly speaking RNAs due to modifications are also included. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with non-ribophosphate constructs, e.g., uncharged mimics of the ribophosphate backbone, that allow the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with the ribophosphate backbone.

[0170] Modifications of the nucleic acids of this disclosure can be at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0171] The present disclosure also includes vectors into which an isolated nucleic acid of the present disclosure is inserted. The art is replete with suitable vectors that are useful in the present disclosure.

[0172] In brief summary, expression of natural or synthetic nucleic acid encoding the protein of the present disclosure is usually carried out by operably linking the nucleic acid encoding the protein of the present disclosure or a part thereof to a promoter and introducing the construct into an expression vector. The vector used is suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0173] The vector of the present disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present disclosure provides gene therapy vectors.

[0174] The isolated nucleic acids of the present disclosure can be cloned into numerous types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0175] Furthermore, vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. In general, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0176] Delivery Systems and Methods In one aspect, the present disclosure relates to the development of a novel lentiviral packaging and delivery system. Lentiviral particles deliver viral enzymes as proteins. In this way, the short-lived nature of lentiviral enzymes limits the possibility of off-target editing due to their long-term expression throughout the life of the cell. Introducing editing components, or traditional CRISPR-Cas editing components, as proteins into lentiviral particles is advantageous, considering that their required activity is only required for a short period of time. Thus, in one embodiment, the present disclosure provides a lentiviral delivery system and a method of using lentiviral delivery system to deliver the composition of the present disclosure, edit genetic material, and deliver nucleic acid.

[0177] For example, in one aspect, the delivery system comprises (1) a packaging plasmid, (2) a transfer plasmid, and (3) an envelope plasmid. In one embodiment, the packaging plasmid comprises a nucleic acid sequence encoding a modified gag-pol polyprotein. In one embodiment, the modified gag-pol polyprotein comprises an integrase fused to an editing protein. In one embodiment, the modified gag-pol polyprotein comprises an integrase fused to a Cas protein. In one embodiment, the modified gag-pol polyprotein comprises an integrase fused to a catalytically-dead Cas protein (dCas). In one embodiment, the packaging plasmid further comprises a sequence encoding an sgRNA sequence.

[0178] In one embodiment, the transfer plasmid comprises a donor sequence. The donor sequence can be any nucleic acid sequence to be delivered to the genome. In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Thus, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence, but does not comprise a U3 sequence. In one embodiment, the 5' LTR and the 3' LTR are specific for the integrase in the packaging plasmid.

[0179] In one embodiment, the donor template is a wild-type FXN gene, or a fragment thereof. In one embodiment, the donor template nucleic acid encodes a protein having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO:357. In one embodiment, the donor template nucleic acid encodes a protein of SEQ ID NO:357. In one embodiment, the donor template nucleic acid comprises a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO: 358. In one embodiment, the donor template nucleic acid comprises the sequence of SEQ ID NO:358.

[0180] In one embodiment, the transfer plasmid comprises a promoter driving expression of the donor sequence. In one embodiment, the promoter is the Ef1a2 promoter for driving expression of a protein or gene described herein. In one embodiment, the Ef1a2 promoter can drive expression in heart, skeletal muscle, and neural tissues such as the brain and motor neurons. In one embodiment, the Ef1a2 promoter comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 333-335. In one embodiment, the Ef1a2 promoter comprises one of the sequences set forth in SEQ ID NOs: 333-335.

[0181] In one embodiment, the packaging plasmid contains a sequence encoding HIV IN and the transfer plasmid contains the U3 sequence of SEQ ID NO:258, the U5 sequence of SEQ ID NO:259, or both.

[0182] In one embodiment, the transfer plasmid comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to SEQ ID NO: 359. In one embodiment, the transfer plasmid comprises the sequence of SEQ ID NO:359.

[0183] In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g protein envelope protein. In one embodiment, the envelope protein can be selected based on the desired cell type.

[0184] In one embodiment, the packaging plasmid, transfer plasmid and envelope plasmid are introduced into the cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a modified gag-pol protein to produce a modified gag-pol protein. In one embodiment, the cell transcribes a nucleic acid sequence encoding an sgRNA. In one embodiment, the sgRNA binds to an integrase-Cas fusion protein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes a donor sequence to provide a donor sequence RNA molecule. In one embodiment, the modified gag-pol protein bound to the sgRNA, the envelope polyprotein and the donor sequence RNA are packaged into a viral particle. In one embodiment, the viral particle is harvested from the cell culture medium. In one embodiment, the viral particle transduces a target cell, where the sgRNA binds to a target region of the cellular DNA, thereby targeting the IN-Cas9 fusion protein, and the integrase catalyzes the integration of the donor sequence into the cellular DNA.

[0185] In one aspect, the delivery system comprises (1) a packaging plasmid, (2) a transfer plasmid, (3) an envelope plasmid, and (4) a VPR-IN-dCas plasmid. In one embodiment, the packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the gag-pol polyprotein comprises a catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises a D116N integrase mutation.

[0186] In one embodiment, the transfer plasmid comprises a donor sequence. The donor sequence can be any nucleic acid sequence to be delivered to the genome. In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Thus, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence, but does not comprise a U3 sequence. In one embodiment, the 5' LTR and the 3' LTR are specific for the integrase in the VPR-IN-dCas packaging plasmid.

[0187] In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g protein (VSV-g) envelope protein. In one embodiment, the envelope protein may be selected based on the desired cell type.

[0188] In one embodiment, the VPR-IN-dCas plasmid comprises a nucleic acid sequence encoding a fusion protein comprising a VPR, an integrase, and an editing protein. In one embodiment, the VPR-IN-dCas plasmid comprises a nucleic acid sequence encoding a fusion protein comprising a VPR, an integrase, and a Cas protein. In one embodiment, the VPR-IN-dCas plasmid comprises a nucleic acid sequence encoding a fusion protein comprising a VPR, an integrase, and a dCas protein. In one embodiment, the fusion protein comprises a protease cleavage site between the VPR and the integrase. In one embodiment, the VPR-IN-dCas plasmid packaging plasmid further comprises a sequence encoding an sgRNA sequence.

[0189] In one embodiment, the packaging plasmid, transfer plasmid, envelope plasmid, and VPR-IN-dCas plasmid are introduced into the cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes a donor sequence to provide a donor sequence RNA molecule. In one embodiment, the cell transcribes and translates a fusion protein to produce a VPR-integrase-editing protein fusion protein. In one embodiment, the cell transcribes and translates a fusion protein to produce a VPR-integrase-dCas fusion protein. In one embodiment, the cell transcribes a nucleic acid sequence encoding an sgRNA. In one embodiment, the sgRNA binds to the VPR-integrase-dCas fusion protein.

[0190] In one embodiment, the gag-pol protein, the envelope polyprotein, the donor sequence RNA, and the VPR-integrase-dCas9 protein that binds to the sgRNA are packaged into a viral particle. In one embodiment, the viral particle is harvested from the cell culture medium. In one embodiment, the VPR is cleaved from the fusion protein in the viral particle via a protease site, resulting in an IN-dCas fusion protein. In one embodiment, the viral particle transduces a target cell, where the sgRNA binds to a target region of the cellular DNA, thereby targeting the IN-dCas fusion protein, and the integrase catalyzes the integration of the donor sequence into the cellular DNA.

[0191] In one aspect, the delivery system comprises (1) a transfer plasmid, (2) a packaging plasmid, and (3) an envelope plasmid. In one embodiment, the packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the gag-pol polyprotein comprises a catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises a D116N integrase mutation.

[0192] In one embodiment, the transfer plasmid comprises a nucleic acid encoding a sgRNA and a nucleic acid sequence encoding a fusion protein comprising an integrase and an editing protein. In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Thus, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence but does not comprise a U3 sequence. In one embodiment, the 5' LTR and the 3' LTR are specific for the integrase of the fusion protein. In one embodiment, the fusion protein comprises an integrase and a Cas protein. In one embodiment, the fusion protein comprises an integrase and a dCas protein. In one embodiment, the 5' LTR and the 3' LTR flank the sequence encoding the fusion protein and the sequence encoding the sgRNA.

[0193] In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g protein (VSV-g) envelope protein. In one embodiment, the envelope protein may be selected based on the desired cell type.

[0194] In one embodiment, the packaging plasmid, transfer plasmid and envelope plasmid are introduced into the cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes a nucleic acid sequence encoding an sgRNA. In one embodiment, the cell transcribes a nucleic acid sequence encoding a fusion protein.

[0195] In one embodiment, the gag-pol protein, the envelope polyprotein, the donor sequence RNA, and the VPR-integrase-dCas9 protein that binds to the sgRNA are packaged into a viral particle. In one embodiment, the viral particle is harvested from the cell culture medium. In one embodiment, the viral particle transduces a target cell, where the virus reverse translates and the cell expresses the fusion protein and the sgRNA. In one embodiment, the sgRNA binds to the Cas protein of the fusion protein and to another viral DNA transcript, where the integrase catalyzes self-integration. In one embodiment, the sgRNA binds to the Cas protein of the fusion protein and to the target region of the cellular DNA, thereby disrupting the target gene.

[0196] In one aspect, the delivery system comprises: (1) a transfer plasmid, (2) a first packaging plasmid, (3) a first envelope plasmid, (4) a second packaging plasmid, (5) a second envelope plasmid, and (6) a transfer plasmid. In one embodiment, the first packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the second packaging plasmid comprises a nucleic acid sequence encoding a gag-pol polyprotein. In one embodiment, the gag-pol polyprotein comprises a catalytically dead integrase. In one embodiment, the gag-pol polyprotein comprises a D116N or D64V integrase mutation.

[0197] In one embodiment, the first envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the second envelope plasmid comprises a nucleic acid sequence encoding an envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding an HIV envelope protein. In one embodiment, the envelope plasmid comprises a nucleic acid sequence encoding a vesicular stomatitis virus g protein (VSV-g) envelope protein. In one embodiment, the envelope protein may be selected based on the desired cell type.

[0198] In one embodiment, the transfer plasmid comprises a nucleic acid encoding an sgRNA and a nucleic acid sequence encoding a fusion protein comprising an integrase and an editing protein. In one embodiment, the fusion protein comprises an integrase and a Cas protein. In one embodiment, the fusion protein comprises an integrase and a dCas protein. In one embodiment, the integrase of the fusion protein is derived from a different species of lentivirus compared to the gag-pol polyprotein of the first and second packaging plasmids. For example, in one embodiment, the transfer plasmid comprises a nucleic acid encoding a fusion protein comprising an FIV integrase and Cas, and the first and second packaging plasmids comprise a nucleic acid sequence encoding an HIV gag-pol polyprotein. In one embodiment, the use of different lentivirus species prevents self-integration.

[0199] In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Thus, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence, but does not comprise a U3 sequence. In one embodiment, the 5' LTR and the 3' LTR are specific for the integrase of the gag-pol polyprotein. In one embodiment, the 5' LTR and the 3' LTR flank the sequence encoding the fusion protein and the sequence encoding the sgRNA.

[0200] In one embodiment, the transfer plasmid comprises a donor sequence. The donor sequence can be any nucleic acid sequence to be delivered to the genome. In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, the 3' LTR is a self-inactivating (SIN) LTR. Thus, in one embodiment, the 5' LTR comprises a U3 sequence, an R sequence and a U5 sequence, and the 3' LTR comprises an R sequence and a U5 sequence, but does not comprise a U3 sequence. In one embodiment, the 5' LTR and the 3' LTR are specific for the integrase in the Inscrtipter transfer plasmid.

[0201] In one embodiment, the first packaging plasmid, the transfer plasmid and the first envelope plasmid are introduced into the cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol protein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes a nucleic acid sequence encoding an sgRNA. In one embodiment, the cell transcribes a nucleic acid sequence encoding a fusion protein. In one embodiment, the gag-pol protein, the envelope polyprotein, the gRNA and the fusion protein RNA are packaged into a first viral particle. In one embodiment, the first viral particle is harvested from the cell culture medium.

[0202] In one embodiment, the second packaging plasmid, the transfer plasmid and the second envelope plasmid are introduced into the cell. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding a gag-pol polyprotein to produce a gag-pol polyprotein. In one embodiment, the cell transcribes and translates a nucleic acid sequence encoding an envelope protein to produce an envelope protein. In one embodiment, the cell transcribes a donor sequence to provide a donor sequence RNA molecule. In one embodiment, the gag-pol polyprotein, the envelope polyprotein and the donor sequence RNA are packaged into a second viral particle. In one embodiment, the second viral particle is harvested from the cell culture medium.

[0203] In one embodiment, the first packaging plasmid, the transfer plasmid, the first envelope plasmid, the second packaging plasmid, the transfer plasmid and the second envelope plasmid are introduced into the same cell. In one embodiment, the first packaging plasmid, the transfer plasmid and the first envelope plasmid are introduced into different cells than the second packaging plasmid, the transfer plasmid and the second envelope plasmid.

[0204] In one embodiment, the first and second viral particles transduce a target cell. In one embodiment, the virus reverse translates and the cell expresses a fusion protein and an sgRNA, where the sgRNA binds to the dCas of the fusion protein. In one embodiment, the virus reverse translates the donor sequence RNA into a donor DNA sequence that binds to the integrase of the fusion protein. In one embodiment, the sgRNA binds to a target region of the cellular DNA, thereby targeting the IN-dCas fusion protein, and the integrase catalyzes the integration of the donor DNA sequence into the cellular DNA.

[0205] Furthermore, many additional virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0206] For example, vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term stable integration of transgenes and their proliferation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of being less immunogenic.

[0207] In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). The term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8 and AAV-9. AAV vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors have several characteristics that make them ideal vectors for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter and delivery method.

[0208] AAV vectors may have a deletion of one or more of the AAV wild-type genes, preferably the rep and / or cap genes, in whole or in part, but may retain functional flanking ITR sequences. Despite high homology, different serotypes have tropism for different tissues. Although the receptor for AAV1 is not clear, AAV1 is known to transduce skeletal and smooth muscle more efficiently than AAV2. Since most studies have been performed with pseudotyped vectors in which vector DNA flanked by AAV2 ITRs is packaged into the capsid of another serotype, it is clear that the biological differences are related to the capsid rather than the genome. Recent evidence shows that DNA expression cassettes packaged into AAV1 capsids are at least 1 log10 more efficient in transducing cardiomyocytes than those packaged into AAV2 capsids. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9).

[0209] AAV fragments that are desirable for assembly into vectors include cap proteins, including vp1, vp2, vp3 and hypervariable regions, rep proteins, including rep78, rep68, rep52, and rep40, and sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, an artificial AAV serotype includes, but is not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be made by any suitable technique using selected AAV sequences (e.g., fragments of vp1 capsid protein) in combination with heterologous sequences that can be obtained from selected different AAV serotypes, non-adjacent parts of the same AAV serotype, non-AAV viral sources, or non-viral sources. An artificial AAV serotype can be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Thus, exemplary AAVs, or artificial AAVs, suitable for expression of one or more proteins include, among others, AAV2 / 8 (see U.S. Pat. No. 7,282,199), AAV2 / 5 (available from the National Institutes of Health), AAV2 / 9 (International Patent Publication No. WO 2005 / 033321), AAV2 / 6 (U.S. Pat. No. 6,156,303), and AAVrh8 (International Patent Publication No. WO 2003 / 042397).

[0210] Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are well known in the art, see, for example, U.S. Pat. Nos. 7,790,449, 7,282,199, WO 2003 / 042397; WO 2005 / 033321, WO 2006 / 110689, and U.S. Pat. No. 7,588,772 B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and construct(s) encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV viral particles are produced in response to a helper adenovirus or herpesvirus, necessitating the separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to recover AAV. The required helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also automatically provided by the system. In these new systems, helper functions can be provided by transient transfection of cells with constructs encoding the required helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level. In yet another system, the transgene flanked by ITRs and / or the rep / cap genes are introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems in general, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which are incorporated herein by reference in their entirety.These and other AAV production systems are also described in the following patents, the contents of each of which are incorporated by reference in their entirety: U.S. Pat. Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065. See generally, e.g., Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10:717-733; and the following references, each of which is incorporated by reference in its entirety. The methods used to construct any embodiment of the present invention are well known to those of skill in the art, including nucleic acid manipulation and genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for generating rAAV viral particles are well known, and the selection of an appropriate method is not a limitation of the present invention. See, e.g., K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0211] In one aspect, the delivery system comprises an AAV transfer plasmid. In one embodiment, the AAV transfer plasmid comprises a nucleic acid sequence encoding a donor sequence. The donor sequence can be any nucleic acid sequence to be delivered to the genome. In one embodiment, the AAV transfer plasmid comprises a 5' inverted repeat (ITR) sequence and a 3' ITR sequence. The ITR sequence can be from the same AAV source as the capsid or from a different AAV source. In one embodiment, the ITR sequence is from AAV2 or a deleted version thereof (AITR).

[0212] In one embodiment, the donor sequence is a wild-type FXN gene, or a fragment thereof. In one embodiment, the donor sequence encodes a protein having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO:357. In one embodiment, the donor sequence encodes a protein of SEQ ID NO:357. In one embodiment, the donor sequence encodes a protein having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO: 358. In one embodiment, the donor sequence encodes a protein of SEQ ID NO:358.

[0213] In one embodiment, the transfer plasmid comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to SEQ ID NO: 360. In one embodiment, the transfer plasmid comprises the sequence of SEQ ID NO:360.

[0214] In certain embodiments, the vector also includes conventional regulatory elements operably linked to the transgene in a manner that allows transcription, translation, and / or expression of the transgene in cells transfected with the plasmid vector or infected with the virus produced by the present disclosure. As used herein, "operably linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences acting in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and, if necessary, sequences that improve secretion of the encoded product. Numerous expression control sequences, including natural promoters, constitutive promoters, inducible promoters, and / or tissue-specific promoters, are known and available in the art.

[0215] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Usually, these are located in the region 30-110 bp upstream of the start site, but in recent years, some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be as long as 50 bp apart, after which activity begins to decrease. Depending on the promoter, individual elements appear to be able to function either cooperatively or independently to activate transcription.

[0216] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when such expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0217] Enhancer sequences found on vectors also regulate the expression of genes contained therein. Enhancers usually bind to protein factors to enhance the transcription of genes. Enhancers can be located upstream or downstream of the gene they regulate. Enhancers can also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vector of the present disclosure contains one or more enhancers to enhance the transcription of genes present in the vector.

[0218] To evaluate the expression of the fusion protein of the present disclosure, the expression vector introduced into the cell may also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selection marker can be carried on an individual piece of DNA and used in the co-transfection procedure. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0219] Reporter genes are used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that code for a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, the construct whose minimal 5' flanking region shows the highest expression level of the reporter gene is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0220] Methods for introducing and expressing genes into cells are known in the art.In relation to expression vectors, the vectors can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art.For example, the expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0221] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or foreign nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). An exemplary method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0222] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for gene insertion into mammalian, e.g., human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0223] Chemical means for introducing polynucleotides into host cells include macromolecule complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0224] When utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acid into host cells (in vitro, ex vivo or in vivo). In another embodiment, the nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, enclosed in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in a suspension in lipid, contained in or complexed with a micelle, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as a micelle, or in a "collapsed" structure. They may also simply be dispersed in the solution, or form aggregates that are not uniform in size or shape.Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids.For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes, and their derivatives.

[0225] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO, dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more easily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures, incorporating water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids can be in micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0226] Regardless of the method used to introduce foreign nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present disclosure.

[0227] system In one aspect, the present disclosure provides a system for editing genetic material, such as a nucleic acid molecule, a genome, or a gene. In one embodiment, the system comprises a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein, and a nuclear localization signal (NLS); a nucleic acid sequence encoding a CRISPR-Cas system guide RNA; and a nucleic acid sequence encoding a donor template nucleic acid, the donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence, in one or more vectors. In one embodiment, the CRISPR-Cas system guide RNA substantially hybridizes to the target DNA sequence of a gene.

[0228] In one embodiment, the system comprises a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein and a nuclear localization signal (NLS); a nucleic acid sequence encoding a first CRISPR-Cas system guide RNA; a nucleic acid sequence encoding a second CRISPR-Cas system guide RNA; and a nucleic acid sequence encoding a donor template nucleic acid, the donor template nucleic acid comprising a U3 sequence, a U5 sequence and a donor template sequence, in one or more vectors. In one embodiment, the first CRISPR-Cas system guide RNA substantially hybridizes to the first DNA sequence and the second CRISPR-Cas system guide RNA substantially hybridizes to the second DNA sequence. In one embodiment, the first DNA sequence and the second DNA sequence flank the target insertion region. In one embodiment, the system catalyzes the insertion of the donor template nucleic acid into the target insertion region.

[0229] In one embodiment, the system comprises a nucleic acid sequence encoding a first fusion protein, the first fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR associated (Cas) protein and a nuclear localization signal (NLS); a nucleic acid sequence encoding a first CRISPR-Cas system guide RNA; a nucleic acid sequence encoding a second fusion protein, the second fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR associated (Cas) protein and a nuclear localization signal (NLS); a nucleic acid sequence encoding a first CRISPR-Cas system guide RNA; a nucleic acid sequence encoding a second CRISPR-Cas system guide RNA; and a nucleic acid sequence encoding a donor template nucleic acid, the donor template nucleic acid comprising a U3 sequence, a U5 sequence and a donor template sequence, in one or more vectors.

[0230] In one embodiment, the first fusion protein and the second fusion protein are the same or different. For example, in one embodiment, the first fusion protein comprises an HIV IN or a fragment thereof, a dCas9 protein and an NLS, and the second fusion protein comprises a BIV IN or a fragment thereof, a Cpf1 Cas protein and an NLS.

[0231] In one embodiment, U3 is specific for the retroviral IN of the first fusion protein and U5 is specific for the retroviral IN of the second fusion protein. For example, in one embodiment, the first fusion protein comprises an HIV IN or a fragment thereof, a dCas9 protein and an NLS, and the second fusion protein comprises a BIV IN or a fragment thereof, a Cpf1 Cas protein and an NLS, and the U3 sequence is specific for HIV IN and the U5 sequence is specific for BIV IN.

[0232] In one embodiment, the first CRISPR-Cas system guide RNA substantially hybridizes to a first DNA sequence and the second CRISPR-Cas system guide RNA substantially hybridizes to a second DNA sequence. In one embodiment, the first DNA sequence and the second DNA sequence flank a target insertion region. In one embodiment, the system catalyzes the insertion of a donor template nucleic acid into the target insertion region.

[0233] In one embodiment, the system includes a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein, and a nuclear localization signal (NLS); a CRISPR-Cas system guide RNA; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.

[0234] In one embodiment, the nucleic acid sequence encoding the fusion protein, the nucleic acid sequence encoding the CRISPR-Cas system guide RNA, and the nucleic acid sequence encoding the donor template nucleic acid are present on the same or different vectors.

[0235] In one embodiment, the nucleic acid sequence further comprises a promoter. In one embodiment, the promoter is the Ef1a2 promoter for driving expression of a protein or gene described herein. In one embodiment, the Ef1a2 promoter can drive expression in heart, skeletal muscle, and neural tissues such as the brain and motor neurons. In one embodiment, the Ef1a2 promoter comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 333-335. In one embodiment, the Ef1a2 promoter comprises one of the sequences set forth in SEQ ID NOs: 333-335.

[0236] In one embodiment, the nucleic acid sequence encoding the fusion protein encodes a fusion protein comprising a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63-142. In one embodiment, the nucleic acid sequence encoding the fusion protein encodes a fusion protein comprising a sequence of one of SEQ ID NOs: 63-142.

[0237] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 211-250. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence of one of SEQ ID NOs: 211-250.

[0238] In one embodiment, the U3 and U5 sequences are specific to retroviral IN. For example, in one embodiment, the retroviral IN is HIV IN and the U3 sequence is a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:258. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:259.

[0239] In one embodiment, the retroviral IN is RSV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:260. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:261.

[0240] In one embodiment, the retroviral IN is HFV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:262. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:263.

[0241] In one embodiment, the retroviral IN is EIAV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:264. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:265.

[0242] In one embodiment, the retroviral IN is MoLV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:266. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:267.

[0243] In one embodiment, the retroviral IN is an MMTV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:268. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:269.

[0244] In one embodiment, the retroviral IN is WDSV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:270. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:271.

[0245] In one embodiment, the retroviral IN is BLV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:272. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:273.

[0246] In one embodiment, the retroviral IN is SIV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:274. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:275.

[0247] In one embodiment, the retroviral IN is FIV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:276. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:277.

[0248] In one embodiment, the retroviral IN is a BIV IN and the U3 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:278. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:279.

[0249] In one embodiment, IN is TY1 and the U3 sequence is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:280. and U5 sequences include sequences that are at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:281.

[0250] In one embodiment, IN is InsF IN, the U3 sequence is an IS3 IRL sequence, and the U5 sequence is an IS3 IRR sequence. In one embodiment, IN is InsF IN and the U3 sequence is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:282. and the U5 sequence comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:283.

[0251] Systems and vectors can be designed for the expression of CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vector systems can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0252] Vectors can be introduced into and propagated in prokaryotes. In some embodiments, prokaryotes are used to amplify copies of vectors introduced into eukaryotic cells or as intermediate vectors in the production of vectors introduced into eukaryotic cells (e.g., amplifying plasmids as part of a viral vector packaging system). In some embodiments, prokaryotes are used to amplify copies of vectors and express one or more nucleic acids to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add several amino acids to a protein encoded therein, for example, to the amino terminus of the recombinant protein. Such fusion vectors can serve one or more purposes, for example, (i) to increase expression of the recombinant protein, (ii) to increase the solubility of the recombinant protein, and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In most cases, a proteolytic cleavage site is introduced into the fusion expression vector at the junction between the fusion moiety and the recombinant protein so that the recombinant protein can be separated from the fusion moiety after purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include factor Xa, thrombin, and enterokinase. Examples of fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0253] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0254] In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6:229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30:933-943), pJRY88 (Schultz et al., 1987. Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0255] In some embodiments, the vector uses a baculovirus expression vector to drive protein expression in insect cells. Baculovirus vectors available for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

[0256] In some embodiments, the vector can drive expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195). When used in mammalian cells, the control functions of the expression vector are usually provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0257] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., expressing the nucleic acid using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific, Pinkert, et al., 1987. Genes Dev. 1:268-277), lymphocyte-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular the promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulin promoters (Baneiji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter, Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreatic specific promoters (Edlund, et al., 1985. Science 230:912-916), and mammary gland specific promoters (e.g., whey promoter, U.S. Pat. No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters, such as murine hox promoters (Kessel and Gruss, 1990. Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546), are also included.

[0258] In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system to drive the expression of one or more elements of the CRISPR system. In general, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDR (Spacer Interspersed Direct Repeat), constitutes a family of DNA loci that are usually specific to certain bacterial species. CRISPR loci include different classes of interspersed short sequence repeats (SSRs) recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433

[1987] and Nakata et al., J. Bacteriol., 171:3553-3556

[1989] ) and related genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (see Groenen et al., Mol. Microbiol., 10:1057-1065

[1993] ; Hoe et al., Emerg. Infect. Dis., 5:254-263

[1999] ; Masepohl et al., Biochim. Biophys. Acta 1307:26-30

[1996] ; and Mojica et al., Mol. Microbiol., 17:85-93

[1995] ). CRISPR loci are usually referred to as short regularly spaced repeats (SRSRs) because they differ from other SSRs by the structure of the repeats (Janssen et al., OMICS J. Integ. Biol., 6:23-33

[2002] , and Mojica et al., Mol. Microbiol., 36:244-246

[2000] ). In general, the repeats are short elements that occur in clusters, regularly spaced apart by unique intervening sequences of substantially constant length (Mojica et al.,

[2000] , supra).Although the repeat sequences are highly conserved among strains, the number of interspersed repeats and the sequence of the spacer region usually vary from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401

[2000] ). CRISPR loci have been identified in over 40 prokaryotes (e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] and Mojica et al., 2003). al.,

[2005] ), which include, for example, Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Examples of suitable bacterium include, but are not limited to, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0259] Methods for editing and delivering nucleic acids In one embodiment, the present disclosure provides a method for editing genetic material, such as nucleic acid molecules, genomes or genes.For example, in one embodiment, the editing is integration.In one embodiment, the editing is CIRSPR-mediated editing.

[0260] In one embodiment, the method comprises administering to the genetic material a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genetic material; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the method comprises administering to the genetic material a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genetic material; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the method is an in vitro method or an in vivo method.

[0261] In one embodiment, the present disclosure provides a method for delivering a nucleic acid sequence to a genetic material.In one embodiment, the method comprises administering to a gene a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.In one embodiment, the method comprises administering to a genetic material a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genetic material; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.In one embodiment, the method is an in vitro method or an in vivo method.

[0262] In one embodiment, the method comprises administering to a cell a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region within the gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the method comprises administering to a cell a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region within the gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.

[0263] In one embodiment, the method of editing genetic material is a method of editing a gene. In one embodiment, the gene is located in the genome of the cell. In one embodiment, the method of editing genetic material is a method of editing a nucleic acid.

[0264] In one embodiment, the present disclosure provides a method for inserting a donor template sequence into a target sequence. In one embodiment, the method inserts a donor template sequence into a target sequence in a cell. In one embodiment, the method comprises administering to a cell a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a region in the target sequence; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the method comprises administering to a cell a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence that is complementary to a region in the target sequence; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.

[0265] Targeted delivery of large DNA sequences for genome editing using CRISPR-Cas9-mediated HDR remains inefficient.However, the present disclosure provides a method for inserting large donor template sequences into target sequences in cells.In one embodiment, the method inserts donor template sequences at least 1 kb or more, at least 2 kb or more, at least 3 kb or more, at least 4 kb or more, at least 5 kb or more, at least 6 kb or more, at least 7 kb or more, at least 8 kb or more, at least 9 kb or more, at least 10 kb or more, at least 11 kb or more, at least 12 kb or more, at least 13 kb or more, at least 14 kb or more, at least 15 kb or more, at least 16 kb or more, at least 17 kb or more, or at least 18 kb or more.In one embodiment, the method comprises administering to cells a fusion protein or a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a region in the target sequence; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.

[0266] In one embodiment, the target sequence is located within a gene. In one embodiment, the donor template sequence disrupts a gene sequence, thereby inhibiting or reducing the expression of the gene. In one embodiment, the target sequence has a mutation, and the donor template sequence inserts a repaired sequence into the target sequence, thereby repairing the gene mutation. In one embodiment, the donor template sequence is a gene sequence, and the donor template sequence is inserted into the target sequence in a cell to allow the expression of the gene.

[0267] In one embodiment, the donor template sequence is inserted into a safe harbor site. Thus, in one embodiment, the guide nucleic acid comprises a nucleotide sequence complementary to a safe harbor region within a gene. A safe harbor region allows expression of a therapeutic gene without affecting the expression of adjacent genes. A safe harbor region can include an intergenic region away from adjacent genes, such as H11, or within a "non-essential" gene, such as CCR5, hROSA26, or AAVS1. Exemplary safe harbor regions and guide nucleic acid sequences complementary to these sequences can be found, for example, in Pellenz et al., New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion, 2019, Hum Gene Ther 30(7):814-28, which is incorporated herein by reference.

[0268] In one embodiment, the donor template sequence is inserted into the 3' untranslated region (UTR), allowing expression of the donor template sequence to be controlled by the promoter of another gene.

[0269] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a CRISPR-associated (Cas) protein and a nucleic acid sequence encoding a nuclear localization signal (NLS).

[0270] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a retroviral integrase (IN) or a fragment thereof, a nucleic acid sequence encoding a CRISPR-associated (Cas) protein, and a nucleic acid sequence encoding a nuclear localization signal (NLS).

[0271] In one embodiment, the retrovirus is a human immunodeficiency virus (HIV), a rous sarcoma virus (RSV), a mouse mammary tumor virus (MMTV), a moloney murine leukemia virus (MoLV), a bovine leukemia virus (BLV), a human T-lymphotropic virus (HTLV), an avian sarcoma leukemia virus (ASLV), a feline leukemia virus (FLV), a xenotropic murine leukemia virus-related virus (XMLV), a simian immunodeficiency virus (SIV), a feline immunodeficiency virus (FIV), an equine infectious anemia virus (EIAV), a prototype foamy virus (PFV), a simian foamy virus (SFV), a human foamy virus (HFV), a walleye cutaneous sarcoma virus (WDSV), or a bovine immunodeficiency virus (BIV).

[0272] In one embodiment, the retroviral IN is HIV IN. In one embodiment, the HIV IN comprises one or more amino acid substitutions that improve catalytic activity, improve solubility, or increase interaction with one or more host cell cofactors. In one embodiment, the HIV IN comprises one or more amino acid substitutions selected from the group consisting of E85G, E85F, D116N, F185K, C280S, T97A, Y134R, G140S, and Q148H. In one embodiment, the HIV IN comprises the amino acid substitutions F185K and C280S. In one embodiment, the HIV IN comprises the amino acid substitutions T97A and Y134R. In one embodiment, the HIV IN comprises the amino acid substitutions G140S and Q148H.

[0273] In one embodiment, the retroviral IN fragment comprises an IN N-terminal domain (NTD) and an IN catalytic core domain (CCD). In one embodiment, the retroviral IN fragment comprises an IN CCD and an IN C-terminal domain (CTD). In one embodiment, the retroviral IN fragment comprises an IN NTD. In one embodiment, the retroviral IN fragment comprises an IN CCD. In one embodiment, the retroviral IN fragment comprises an IN CTD.

[0274] In one embodiment, the nucleic acid sequence encoding the retroviral IN encodes an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 9-48. In one embodiment, the nucleic acid sequence encoding the retroviral IN encodes an amino acid sequence of one of SEQ ID NOs: 9-48.

[0275] In one embodiment, the nucleic acid sequence encoding the retroviral IN comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 161-200. In one embodiment, the nucleic acid sequence encoding the retroviral IN comprises a nucleic acid sequence of one of SEQ ID NOs: 161-200.

[0276] In one embodiment, the Cas protein is Cas9, Cas13, Cas14 or Cpf1. In one embodiment, the Cas protein is catalytically deficient (dCas).

[0277] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a sequence encoding an amino acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 1-8. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a sequence encoding one of SEQ ID NOs: 1-8.

[0278] In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 153-160. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 153-160.

[0279] In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the NLS is derived from yeast GAL4, SKI3, L29, or histone H2B protein, polyomavirus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or SV40 VP2 capsid protein, adenovirus E1a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, or simian virus 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or an NLS derived from Ty1, Ty2 or an NLS derived from Ty2, or MAK11 or an NLS derived from MAK11. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO:53. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO:54. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO:56.

[0280] In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid encoding at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 49-62, and 361-973. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding one of SEQ ID NOs: 49-62, and 361-973.

[0281] In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 201-210. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence of one of SEQ ID NOs: 201-210.

[0282] In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 63-146. In one embodiment, the nucleic acid molecule encoding the fusion protein comprises a sequence of one of SEQ ID NOs: 63-146.

[0283] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 211-254. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence of one of SEQ ID NOs: 211-254.

[0284] In one embodiment, the U3 and U5 sequences are specific for retroviral IN.

[0285] In some embodiments, the gene is any target gene of interest. For example, in one embodiment, the gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method includes introducing a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the IN-Cas fusion protein binds to a target polynucleotide and causes cleavage of the target polynucleotide in the gene. In one embodiment, the IN-Cas fusion protein is complexed with a guide nucleic acid that hybridizes to a target sequence in the target polynucleotide. In one embodiment, the IN-Cas fusion protein is complexed with a nucleic acid sequence encoding the donor template nucleic acid. In one embodiment, the IN-Cas fusion protein is complexed with a nucleic acid sequence encoding the guide nucleic acid. In one embodiment, the IN-Cas fusion protein is complexed with a nucleic acid sequence encoding the guide nucleic acid and a nucleic acid sequence encoding the donor template nucleic acid. In one embodiment, the IN-Cas fusion protein is complexed with a guide nucleic acid and a donor template nucleic acid that hybridizes to a target sequence within a target polynucleotide. In one embodiment, the IN-Cas fusion protein is complexed with a donor template nucleic acid. In one embodiment, the IN-Cas fusion protein is complexed with a guide nucleic acid. In one embodiment, the IN-Cas fusion protein is complexed with a guide nucleic acid and a donor template nucleic acid.

[0286] In some embodiments, IN-Cas catalyzes the integration of a donor template into a gene. In one embodiment, the integration introduces one or more mutations into the gene. In some embodiments, the mutations result in one or more amino acid changes in the protein expressed from the gene containing the target sequence.

[0287] In one embodiment, IN-mediated integration of DNA sequences can occur in either direction of the target DNA sequence. In one embodiment, various combinations of Cas and IN retrovirus class proteins are used to facilitate directional editing. For example, in one embodiment, the fusion of IN from the retrovirus class binds to a first catalytically dead Cas, which allows binding to a specific target sequence using a Cas-specific guide RNA. In one embodiment, the donor sequence includes both HIV LTR and BIV LTR sequences. Thus, in one embodiment, this sequence is integrated into the target DNA in a single direction.

[0288] In one embodiment, LoxP-flanked (Floxed) sequences are introduced around the gene of interest. The inclusion of floxed sequences allows for CRE-mediated recombination and conditional mutagenesis. Current methods for generating Floxed alleles using CRISPR-Cas9 are inefficient. The most widely used approach is to use two guide RNAs to induce DNA cleavage at adjacent target sequences and homologous recombination repair to insert a ssDNA template containing LoxP sequences. However, when using double sgRNAs to induce cleavage, the most favorable response is the deletion of the intervening sequence, resulting in global gene deletion. Thus, in one embodiment, the use of integrase-Cas-mediated gene insertion increases the efficiency of tandem insertion of DNA sequences. In one embodiment, since IN-mediated integration can occur in either direction, integration of a sequence containing an inverted LoxP sequence allows for recombination of the sequence flanked by LoxP.

[0289] Methods of Treatment and Use In one aspect. The disclosure provides methods of treating Friedreich's ataxia, reducing symptoms of Friedreich's ataxia, and / or reducing the risk of developing Friedreich's ataxia. Friedreich's ataxia is an autosomal recessive genetic disease that causes difficulty walking, loss of sensation in the arms and legs, and dysphonia that worsens over time. Symptoms typically begin between the ages of 5 and 20. Many develop hypertrophic cardiomyopathy and require mobility aids such as canes, walkers, or wheelchairs in their teens. As the disease progresses, they lose their vision and hearing. Other complications include scoliosis and diabetes. The condition is caused by a mutation in the FXN gene on chromosome 9, which makes a protein called frataxin. In 98% of cases, the mutant FXN gene has a 90-1,300 GAA triplet repeat expansion in intron 1 of both alleles, and in the remaining 2%, the mutant FXN gene has a point mutation within the FXN gene. GAA expansion causes epigenetic changes and the formation of heterochromatin near the repeat. The length of the short GAA repeat correlates with age of onset and disease severity. The formation of heterochromatin leads to reduced transcription of the gene and lower levels of frataxin. Individuals affected by Friedreich's ataxia may have 5-35% less frataxin compared to healthy individuals. Heterozygous carriers of the mutant FXN gene have 50% lower frataxin levels, but this reduction is not sufficient to cause symptoms.

[0290] In one embodiment, the method comprises administering a fusion protein of the present disclosure or a nucleic acid molecule encoding the fusion protein of the present disclosure; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genetic material, and a donor template nucleic acid comprising a U3 sequence, a U5 sequence. In one embodiment, the method further comprises administering a donor template sequence. In one embodiment, the targeting nucleotide is a safe harbor site.

[0291] Safe harbor regions allow expression of a therapeutic gene without affecting the expression of adjacent genes. Safe harbor regions can include intergenic regions away from adjacent genes, such as H11, or within "non-essential" genes, such as CCR5, hROSA26, or AAVS1. Exemplary safe harbor regions and guide nucleic acid sequences complementary to these sequences can be found, for example, in Pellenz et al., New Human Chromosomal Sites with "Safe Harbor" Potential for Targeted Transgene Insertion, 2019, Hum Gene Ther 30(7):814-28, which is incorporated herein by reference.

[0292] In one embodiment, the target region is within the FXN gene and the donor template is a wild-type FXN gene or a fragment thereof, whereby through the activity of the fusion protein, the wild-type FXN gene or a fragment thereof is integrated into the FXN gene, correcting the mutation(s) within the FXN gene, which in turn restores FXN expression to normal levels and ameliorates the symptoms of Friedreich's ataxia.

[0293] In one embodiment, the donor template comprises a wild-type FXN gene or a fragment thereof. In one embodiment, the donor template comprises a nucleic acid sequence encoding a wild-type frataxin or a fragment thereof. In one embodiment, the donor template nucleic acid encodes a protein having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO:357. In one embodiment, the donor template nucleic acid encodes a protein of SEQ ID NO:357. In one embodiment, the donor template nucleic acid comprises a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO: 358. In one embodiment, the donor template nucleic acid comprises the sequence of SEQ ID NO:358.

[0294] The present disclosure provides a method for treating, reducing symptoms, and / or reducing the risk of developing a disease or disorder and / or genetic modification to produce a desired phenotypic outcome. For example, in one embodiment, the method of the present disclosure treats, reduces symptoms, and / or reduces the risk of developing a disease or disorder in a mammal. In one embodiment, the method of the present disclosure treats, reduces symptoms, and / or reduces the risk of developing a disease or disorder in a plant. In one embodiment, the method of the present disclosure treats, reduces symptoms, and / or reduces the risk of developing a disease or disorder in a yeast organism.

[0295] In one embodiment, the disease or disorder is caused by one or more mutations in a genomic locus.Therefore, in one embodiment, the disease or disorder can be treated, reduced, or reduced risk through the introduction of a nucleic acid sequence corresponding to the wild-type sequence of the region having one or more mutations, and / or the introduction of an element that prevents or reduces the expression of the genomic sequence having one or more mutations.Therefore, in one embodiment, the method includes the manipulation of the target sequence within the coding element, non-coding element, or regulatory element of the genomic locus within the target sequence.

[0296] For example, in one embodiment, the disease is a monogenic disease. In one embodiment, the disease includes, but is not limited to, Duchenne muscular dystrophy (mutations occurring in dystrophin), Limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (mutations occurring in dysferlin), Cystic fibrosis (mutations occurring in CFTR), Wilson's disease (mutations occurring in ATP7B), and Stargardt's macular degeneration (mutations occurring in ABCA4).

[0297] The present disclosure also provides a method for regulating the expression of a gene or genetic material. For example, in one embodiment, the method of the present disclosure provides for the delivery of genetic material to confer a phenotype to a cell or organism. For example, in one embodiment, the method provides resistance to a pathogen. In one embodiment, the method provides for the regulation of a metabolic pathway. In one embodiment, the method provides for the production and use of a substance in an organism. For example, in one embodiment, the method produces substances such as biological substances, pharmaceutical substances, and biofuels in organisms such as eukaryotes, yeast, bacteria, or plants.

[0298] In one embodiment, the method comprises administering a fusion protein or a nucleic acid molecule encoding the fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in a gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence. In one embodiment, the method further comprises administering a donor template sequence.

[0299] In one embodiment, the target sequence is located within a gene. In one embodiment, the donor template sequence disrupts a gene sequence, thereby inhibiting or reducing the expression of the gene. In one embodiment, the target sequence has a mutation, and the donor template sequence inserts a repaired sequence into the target sequence, thereby repairing the gene mutation. In one embodiment, the donor template sequence is a gene sequence, and the donor template sequence is inserted into the target sequence in a cell to allow the expression of the gene.

[0300] In some embodiments, the gene is any target gene of interest. For example, in one embodiment, the gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method includes introducing a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the IN-Cas9 fusion protein binds to a target polynucleotide and causes cleavage of the target polynucleotide in the gene. In one embodiment, the IN-Cas9 fusion protein is complexed with a guide nucleic acid that hybridizes to a target sequence in the target polynucleotide. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding the donor template nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding the guide nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding the guide nucleic acid and a nucleic acid sequence encoding the donor template nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding the guide nucleic acid and a nucleic acid sequence encoding the donor template nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a guide nucleic acid and a donor template nucleic acid that hybridizes to a target sequence within a target polynucleotide. In one embodiment, the IN-Cas9 fusion protein is complexed with a donor template nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a guide nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a guide nucleic acid and a donor template nucleic acid.

[0301] In some embodiments, IN-Cas9 catalyzes the integration of the donor template into a gene. In one embodiment, the integration introduces one or more mutations into the gene. In some embodiments, the mutations result in one or more amino acid changes in the protein expressed from the gene containing the target sequence.

[0302] array Table 1 provides a summary of the sequences. [Table 1-1]

Table 1-2

Table 1-3

Table 1-4

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[0303] The present disclosure will be described in more detail by referring to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.Therefore, the present disclosure should not be interpreted as being limited to the following examples in any way, but rather as encompassing any and all variations that become evident as a result of the teachings provided herein.

[0304] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present disclosure and practice the claimed methods. The following examples therefore specifically illustrate certain embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0305] Example 1: Improved nuclear localization of retroviral integrase-dCas9 fusion proteins for editing mammalian genomic DNA Efficient CRISPR-Cas9 editing of mammalian genomic DNA requires nuclear localization of Cas9, a large bacterial RNA-guided endonuclease that normally functions in prokaryotic cells that lack a nuclear membrane. It has been shown that efficient nuclear localization of Cas9 in mammalian cells requires the addition of at least two mammalian nuclear localization signals, one located at the N-terminus and one at the C-terminus (Cong et al., 2013, Science 339:819-23).

[0306] To promote nuclear localization of retroviral integrase-dCas9 fusion proteins for editing, we included an N-terminal SV40 NLS in the integrase in addition to the C-terminal SV40 NLS on dCas9 (Figure 1A). Unexpectedly, when expressed in mammalian cells, only a small fraction of the IN-dCas9 fusion protein localized to the nucleus, as detected using a FLAG antibody that recognizes the C-terminal 3xFLAG epitope on dCas9 (Figure 1B). Interestingly, while the full-length IN-dCas9 fusion protein gave rise to cytoplasmic aggregates, deletion of the C-terminal domain of integrase (INΔC) resulted in increased solubility and nuclear localization (Figure 1B).

[0307] Fusion of retroviral integrase to dCas9 appears to dramatically reduce its ability to localize to the nucleus. To further improve the nuclear localization of the integrase-dCas9 fusion protein, several different mammalian nuclear localization sequences were tested for their ability to induce nuclear transport of IN-dCas9 (Figure 1B). Multimerization of three copies of the SV40 NLS (3xSV40) had no apparent effect on the degree of nuclear localization of IN-dCas9 or INΔC-dCas9. However, addition of a bipartite NLS derived from nucleoplasmin (NPM) increased the nuclear localization of the INΔC-dCas9 fusion protein but not the full-length IN fusion protein. The combination of 3xSV40 and the NPM NLS appeared to be similar to NPM alone.

[0308] Interestingly, yeast LTR retrotransposons (e.g., Ty1) are the evolutionary ancestors of retroviruses and replicate their genomes through reverse transcription of an RNA intermediate in the cytoplasm (Curcio et al., 2015, Microbiol Spectr 3:MDNA3-0053-2014). LTR retrotransposons contain the integrase enzyme required for insertion of the retrotransposon genome. In contrast to higher eukaryotes that undergo open mitosis during cell division, yeast undergoes closed mitosis, so the nuclear membrane remains intact. Thus, in the case of Ty1 neogenesis, active nuclear transport is required for nuclear transport of the integrase / retrotransposon genome complex. Thus, in contrast to mammalian integrase enzymes, Ty1 integrase contains a large C-terminal bipartite NLS required for retrotransposition (Moore et al., 1998, Mol Cell Biol 18:1105-14). Interestingly, the results presented herein demonstrate that fusion of the Ty1 NLS to the C-terminus of both IN-dCas9 fusion proteins resulted in strong nuclear localization in mammalian cells (Figure 1B).

[0309] Increased nuclear localization of the INΔC-dCas9 fusion protein significantly improved editing in dividing mammalian cells in culture. Addition of the Ty1 NLS enhanced the activity of the INΔC-dCas9 fusion protein to integrate an IRES-mCherry template targeting the 3'UTRE of EF1-alpha in HEK293 cells (Figure 1C). Utilizing the strong Ty1 NLS may further enable editing in non-dividing cells that constantly maintain the nuclear membrane (e.g., in vivo therapeutic applications).

[0310] Example 2: An integrated gene editing approach for the correction of muscular dystrophy As shown elsewhere herein, fusion of lentiviral integrase to CRISPR-Cas9 allows sequence-specific integration of large DNA sequences into genomic DNA. This approach can be utilized to deliver therapeutically beneficial genes to non-pathogenic genomic locations (safe harbors) to permanently repair genetic diseases in humans (Figure 2). This technology allows sequence-specific integration of large DNA donor sequences containing short viral terminal motifs.

[0311] The main advantage of the gene therapy approach of the present disclosure is the ability to deliver donor DNA sequence to target genome location.In addition, this approach does not require homology arms and relies on guide RNA targeting, greatly simplifying genome editing.Therefore, once a specific reporter donor sequence is created, it can be directed to any location (or multiple locations) for a wide variety of applications.

[0312] Fusion of lentiviral integrase to dCas9 is sufficient to insert donor DNA sequences containing short viral ends into target sequences using CRISPR guide RNA in mammalian cells (Figure 3). To monitor integrase-Cas mediated integration in mammalian cells, we generated a donor vector containing an IGR IRES sequence followed by an mCherry-2a-puromycin gene and an SV40 polyadenylation sequence (Figure 3). We then designed an sgRNA to target a stable human CMV-eGFP stable cell line in COS-7 cells. The stable transgene of hCMV-eGFP provided a heterologous target sequence that could be used to determine editing at a strongly expressed, but nonessentially expressed, locus. The donor mCherry-2a-puro template was purified and co-transfected with sgRNA and IN-dCas9 into GFP stable cells and cultured for 48 hours. After 48 hours, mCherry positive cells were found in the culture, replacing the GFP positive signal (Figure 3).

[0313] Efficiency and fidelity of integrase-Cas-mediated integration of human dystrophin into mammalian genomes. Integrase-Cas mediated gene delivery induces sequence-specific integration of large DNA sequences into mammalian genomic DNA. Integrase-Cas is used to deliver the human dystrophin gene under the control of the human α-skeletal actin (HSA) promoter to a safe harbor location using CRISPR guide RNA specific for human AAVS1 and mouse ROSA26 genomic DNA in cultured cells. Correct targeting of dystrophin is assessed using PCR-based genotyping.

[0314] Integrase-Cas-mediated dystrophin gene therapy restores muscle function in a mouse model of Duchenne muscular dystrophy. The efficacy of Inscritpr-mediated delivery of human dystrophin is determined in the MDX mouse line, the most commonly used mouse model for muscular dystrophy. After systemic delivery, the levels of dystrophin expression are quantified and measured in limb skeletal muscle, heart and diaphragm using anti-dystrophin antibodies over a time course of 2, 4 and 6 months. Alleviation of DMD disease pathology is assessed by quantifying serum creatine kinase (CK) levels (a marker of skeletal muscle damage and a diagnostic marker for DMD patients), grip strength, and histological analysis of limb skeletal muscle, heart and diaphragm.

[0315] Histological analysis of gene expression. At 8 weeks of age, the left hind quadriceps, heart, and diaphragm are harvested, weighed, fixed in 4% formaldehyde in PBS, and processed using routine methods for paraffin histology. The percentage of myofibers expressing HSA-dystrophin / GFP fusion protein was used to assess the DMD. Mdx / y Immunofluorescence assays are performed using anti-GFP antibodies in both WT and WT mice. Right hindlimb muscles are flash frozen in liquid nitrogen for subsequent PCR-based genotyping, gene expression by RT-PCR, and protein expression analysis by Western blot.

[0316] Integrase-Cas-mediated delivery attenuates disease pathology in a mouse model of Duchenne muscular dystrophy. Use hematoxylin and eosin (H&E), von Kossa and Masson's trichrome staining of transverse tissue sections to identify muscle fibers containing central nuclei, calcification and endomysial fibrosis, respectively. Use quantitative comparison and statistical analysis to compare the proportion of muscle fibers with central nuclei or areas of stained calcification or fibrosis in quadriceps limb muscles. Compare at least three different planar sections for each muscle from three different mice of each genotype. Dmd mice treated with integrase-Cas exhibited a less severe phenotype. mdx / y There is a decreased proportion of myofibers with central nuclei and a decreased total area of ​​fibrosis and calcification.

[0317] Serum creatine kinase (CK) measurement. Serum CK is a correlative marker of skeletal muscle damage and a diagnostic marker for DMD patients. CK measurements are performed at 2, 4, 6 and 8 weeks in the aforementioned animal cohorts using a non-lethal procedure. Briefly, blood is collected directly from the periorbital vascular plexus into microhematocrit tubes and allowed to clot for 30 minutes at room temperature before centrifugation at 1,700 x g for 10 minutes. mdx / y Treated mice, which display a less severe phenotype than KO, have significantly reduced serum CK levels.

[0318] Example 3: Genome editing - directed non-homologous DNA integration The data presented here demonstrate an optimized integrase-Cas to enable efficient editing of mammalian genomes.

[0319] Optimized Editing To optimize IN-mediated integration, we will determine whether amino acid mutations that enhance integrase catalytic activity, solubility, or interaction with host cell cofactors improve editing, and we will evaluate the efficiency and fidelity of IN proteins isolated from seven unique classes of retroviruses.

[0320] To quantify and characterize IN-dCas9-mediated integration in mammalian cells, we use a plasmid-based reporter system that utilizes a blue chromoprotein from the coral Acropora millepora (amilCP), which produces deep blue colonies when expressed in Escherichia coli. Disruption of the amilCP open reading frame abolishes expression of the blue protein, which can be used as a direct readout of targeting fidelity. In addition, we generated a donor template that encodes a chloramphenicol antibiotic resistance gene flanked by U3 and U5 retroviral terminal sequences from HIV. Integration of this donor template confers resistance to chloramphenicol, which can be used to monitor integrase-Cas-mediated DNA integration. In this reporter assay, an expression plasmid containing the IN-dCas9 fusion protein, an sgRNA targeting amilCP, and the donor template is co-transfected into mammalian COS-7 cells with the bacterial amilCP reporter. After 48 hours, total plasmid DNA is recovered using column purification and transformed into E. coli. IN-dCas9 is sufficient to integrate a template DNA encoding chloramphenicol into the amilCP reporter plasmid, thereby disrupting expression of amilCP and conferring resistance to chloramphenicol. This rapid assay, which allows quantification and clonal sequence analysis of individual integration events, is used to optimize editing.

[0321] Enhanced integrase activity: Although most mutations in IN abolish its activity, research over the past few decades has identified several mutations that improve IN integration by increasing IN catalytic activity (D116N), dimerization (E85F), solubility (F185K / C280S), and interaction with host cell proteins (K71R). IN-dCas9 fusion proteins containing activating IN mutations are used to determine whether the fusion protein enhances activity using a plasmid-based reporter assay.

[0322] Modulation of integrase activity by host cell proteins: Although IN is the only protein necessary and sufficient to integrate proviral DNA in vitro, interactions with host cell proteins can significantly alter IN-mediated DNA integration. Notably, LEDGF / p75, VBP1 and SNF5 are well-characterized HIV IN-interacting proteins that can promote IN-mediated integration. These factors are expressed using plasmid reporter assays to determine whether they promote integration of the donor template.

[0323] Compare and contrast integrases from various retrovirus classes: Although all IN enzymes from retrovirus classes contain a conserved core catalytic D,D(35)E residue, they differ significantly in genome size, complexity, U3 and U5 terminal sequences, and DNA binding efficiency. To determine the editing efficiency of various retrovirus IN, model examples from each retrovirus class, including alpha (RSV), beta (MMTV), gamma (MoLV), delta (BLV), epsilon (WDSV), and spumavirus (HFV), were cloned as fusions to dCas9. Donor plasmids containing the respective U3 and U5 terminal motifs are generated. Protein expression is verified by Western blot and nuclear localization is verified using immunocytochemistry using a FLAG antibody to detect the 3xFLAG epitope located at the C-terminus of dCas9.

[0324] Mammalian genomic DNA editing efficiency The efficiency and fidelity of mammalian genomic DNA editing is determined using a stable CMV-driven GFP reporter cell line to generate donor templates containing RFP and puromycin selection cassettes. Integration events are quantified and clonally characterized to determine the efficiency and fidelity of this method as a novel genome editing technology.

[0325] Creation of a cell-based reporter assay: A donor template containing an IRES-RFP-2A-puromycin cassette and a guide RNA targeting the GFP coding sequence is used to quantify integration events at this locus. Insertion of the donor cassette into the CMV-GFP locus results in RFP expression replacing GFP expression and resistance to the antibiotic puromycin. FACS sorting is used to measure the percentage of cells that are RFP+ / GFP- (targeted integration) after transfection and 48 hours of culture to quantify the efficiency and fidelity of Inscripr editing. Puromycin is used to select for clonal integration events. This is characterized by using PCR primers to amplify the sequence between the GFP locus and the donor cassette.

[0326] Editing at multiple endogenous loci: Integrase-Cas is used to knock-in an RFP-2A puromycin cassette using sgRNAs specific for the CMV-GFP locus and the 3'UTR of the human EF1-alpha locus in a HEK293 human cell line. Targeting the 3'UTR allows expression of an IRES-dependent vector without disrupting normal gene expression. After clonal selection with puromycin, PCR genotyping is used to measure the percentage of clones that have integrated the donor template at both loci.

[0327] Example 4: Generation and characterization of IN-dCas9 Generation of functional IN-dCas9 fusion proteins. To generate a functional IN-dCas9 fusion protein for use in mammalian cells, full-length retroviral IN was separated from HIV-1 (amino acids 1148-1435 of the gag-pol polyprotein) by a flexible 15 amino acid linker [(GGGGS)3)] and cloned into the N-terminus of human codon-optimized dCas9 (Figure 6). An SV40 nuclear localization signal (NLS) was included at the N-terminus of IN, which, together with the C-terminal SV40 NLS on dCas9, resulted in nuclear localization of the IN-dCas9 fusion protein. To generate an IN-dCas9 fusion lacking the C-terminal nonspecific DNA binding domain, an additional construct was generated containing only the N-terminus of IN and the catalytic core domain (aa1148-1369) as an N-terminal fusion to dCas9 (Figure 6).

[0328] Construction of reporters to monitor plasmid DNA editing. To quantify and characterize IN-dCas9-mediated integration in mammalian cells, we designed a plasmid-based reporter assay that utilizes a blue pigment protein from the coral Acropora millepora (amilCP), which produces deep blue colonies when expressed in Escherichia coli (Figure 6). Disruption of the amilCP open reading frame abolishes expression of the blue protein, which can be used as a direct readout of targeting fidelity and as the target DNA for integrase-Cas-mediated integration. To promote efficient dimerization of the N-terminal dCas9 fusion protein with the target DNA, the single guide RNA (sgRNA) target sequence was designed in a "PAM-out" orientation, separated by a 16-bp spacer sequence (Figure 4).

[0329] Generation of viral terminal donor sequences for integrase-Cas-mediated integration. To construct a targeting vector that can be used to generate donor sequences for integrase-Cas mediated integration, 30 base pairs containing the U3 and U5 HIV ends were subcloned into pCRII (Figure 6). A multiple cloning site containing nine unique restriction enzymes was included between U3 and U5 to facilitate subcloning of the donor sequence. Because U3 and U5 share the same three nucleotides at their ends (ACT and AGT, respectively), additional half-site sequences were included to generate ScaI restriction sites at both ends that can be used to generate blunt-ended donor sequences from the plasmid backbone (Figure 6). In addition, flanking type IIS restriction enzyme sites were included for FauI, which cleaves and leaves two 5' nucleotide overhangs, mimicking the preprocessed viral 3' end containing an exposed CA dinucleotide (Figure 6). To aid in gel purification and separation of the FauI digested template from the plasmid backbone, multisite-directed mutagenesis was used to remove the six FauI sites present in the pCR II plasmid backbone.

[0330] Protocol: Preparation of INsrt donor templates for transfection 1) Set up restriction digestion of INsrt plasmid DNA 2) Restriction digestion reaction 3) Gel purify the donor template from the backbone DNA 4) Elute donor DNA for transfection

[0331] Integrase-Cas-mediated integration of donor sequences into plasmid DNA in mammalian cells. To allow for positive selection of cooperative IN-dCas9-mediated integration, we designed an INsrt donor vector carrying a chloramphenicol resistance gene (CAT) that is not present in the reporter expression plasmid (Figure 7). The IGR IRES from Plautia stali enteric virus (PSIV) was included in front of the CAT gene. This IGR IRES can initiate translation in both prokaryotic and eukaryotic cells to support translation at multiple integration sites. Templates containing the chloramphenicol resistance gene and viral ends were digested using either ScaI (blunt ends) or FauI (processed ends) and gel purified from the plasmid backbone DNA. The INsrt template, co-transfection of the IN-dCas9 vector targeting the amilCP sequence, was co-transfected into Cos7 cells (Figure 7). After 48 hours, total plasmid DNA was recovered using column purification and transformed into E. coli. Chloramphenicol-resistant clones were observed with both full-length IN and INDC-dCas9 fusion proteins. Plasmid sequencing revealed that the IG3-CAT plasmid sequence was integrated into the amilCP reporter. Interestingly, the use of a FauI-digested donor sequence, which mimics the 3' pre-processing of viral DNA ends, resulted in twice as many chloramphenicol-resistant clones compared to ScaI-digested blunt-end templates. Integrase-Cas-mediated integration contained hallmarks of HIV IN lentiviral integration, including five-base pair repeats of host DNA flanking the integration site. Interestingly, integration sites did not occur between the two sgRNA target sites, but on either side of the amilCP target sequence.

[0332] Integration of Insrt IGR-CAT donor templates with either blunt (ScaI cut) or 3' processing mimic (FauI cut) ends into pCRII-amilCP reporter in mammalian cells. Interestingly, deletion of the C-terminal non-specific DNA binding domain as a fusion to dCas9 does not inhibit integrase-Cas-mediated integration. Using 3' processing mimic ends shows an approximately 2-fold increase in CAT-resistant clones (Figure 29B). Dimerization-inhibiting mutations (E85G and E85F) do not interfere with integrase-Cas-mediated integration using dual guide RNA targeted integration of IGR-CAT donor templates into amilCP. However, the IN E87G mutation cannot be restored by paired targeting sgRNA. Interestingly, tandem INΔC fusions to dCas9 (tdINΔC-dCas9) show approximately 2-fold improved integration (Figure 29C).

[0333] Protocol: Integrase-Cas mediated integration of donor sequences into plasmid DNA in mammalian cells. 1) The multicistronic sgRNA and IN-dCas9 plasmids, bacterial amilCP reporter plasmid and INsrt donor template are co-transfected into mammalian (e.g., Cos7) cells. a. Set up the transfection reaction immediately before plating the cells. b. Harvest, plate and transfect the cells. 2) Recover plasmid DNA from the transfected cells. 3) Transform the recovered plasmid DNA into chemically competent E. coli.

[0334] Generation of CMV-GFP stable mammalian cell lines for integrase-Cas-mediated integration into genomic DNA. We generated a stable GFP reporter cell line that can be used to quantify and characterize the fidelity of individual integration events in mammalian cells (Figure 3). A plasmid encoding GFP under the control of the human CMV promoter (pcDNA3.1-GFP) was linearized and transfected into Cos7 cells, and stable clones were selected using G418 and serial dilutions. This artificial locus allows for strong gene expression that can be targeted for disruption without compromising the viability of normal cells, as might otherwise occur when targeting essential host genes.

[0335] Integrase-Cas mediated integration of donor sequences into mammalian genomic DNA. To quantify integration events at the CMV-GFP locus, a donor template was constructed containing an IGR-mCherry-2A-puromycin-pA cassette and a paired guide RNA targeting the GFP coding sequence (Figure 3). Integration of the donor cassette into the CMV-GFP locus drives expression of mCherry, disrupts expression of GFP, and confers resistance to the antibiotic puromycin. After transfection and 48 h of culture, mCherry-positive cells were observed, some of which still contained weak but detectable levels of GFP expression (Figure 3).

[0336] Integrase-Cas-mediated integration of donor sequences at endogenous loci. A targeting strategy was designed and a guide RNA specific to the 3'UTR of the human EF1-alpha locus was selected to knock-in the IGR-mCherry-2A-puromycin-pA cassette into human HEK293 cell lines (Figure 8). The 3'UTR was targeted to express the IGR-mCherry cassette without disrupting the open reading frame of EF1-alpha expression. After transfection and 48 hours of culture, mCherry-positive cells were observed in the culture medium (Figure 8).

[0337] Protocol: Integrase-Cas-mediated integration of donor sequences into mammalian genomic DNA 1) Using Fugene6 or Lipofectamine2000, co-transfect a plasmid encoding sgRNA, a plasmid encoding IN-dCas9, and a plasmid encoding an INsrt donor template into mammalian cells (COS7, HEK293, etc.) in a 1:1:1 ratio. a. Harvest, plate and transfect cells. 2) Antibiotic selection of the integrated sequence a. Wash the cells with 10 ml of medium containing antibiotic selection and plate in this medium. b. The cells are cultured and then cloned.

[0338] Directional editing. IN-mediated integration of DNA sequences can occur in either direction of the target DNA sequence. The use of various combinations of Cas and IN retrovirus class proteins provides the ability to promote directional editing. For example, fusion of IN from BIV (bovine immunodeficiency virus or other HIV-related viruses) fused to catalytically dead LbCpf1 (LbCpf1) allows binding to specific target sequences using Cpf1-specific guide RNAs. The use of donor sequences containing both HIV and BIV terminal sequences fixes unidirectional binding to the target DNA (Figure 9).

[0339] Multiplex genome editing for the generation of floxed alleles. Introduction of LoxP-flanked (Floxed) sequences around a gene of interest allows for CRE-mediated recombination and conditional mutagenesis. Current methods of generating Floxed alleles using CRISPR-Cas9 are inefficient. The most widely used approach is to use two guide RNAs to induce DNA cleavage at adjacent target sequences and homologous recombination repair to insert a ssDNA template containing LoxP sequences. However, when using double sgRNAs to induce cleavage, the most favorable response is the deletion of the intervening sequence, resulting in global gene deletion. When IN-mediated strand transfer with host DNA does not allow efficient deletion of the intervening sequence, the use of integrase-Cas-mediated gene insertion provides a more efficient alternative approach for tandem insertion of DNA sequences. Since IN-mediated integration can occur in either direction, integration of sequences containing inverted LoxP sequences allows recombination of sequences flanked by LoxP (Figure 10).

[0340] Example 5: Identification and activity of Ty1 NLS-like sequences The integrase enzyme from the yeast Ty1 retrotransposon contains a non-classical bipartite nuclear localization signal consisting of tandem KKR motifs separated by a relatively large linker sequence. Previous studies in yeast have demonstrated that these basic motifs are required for nuclear localization and Ty1 transposition (Kenna et al., 1998, Mol Cell Biol 18, 1115-1124; Moore et al., 1998, Mol Cell Biol 18, 1105-1114). Ty1 transposition is entirely dependent on the presence of the Ty1 NLS, and interestingly, a classical NLS is insufficient to reproduce the Ty1 NLS activity required for transposition. Interestingly, another yeast protein shares this tandem KKR motif, and considering that many of these proteins are localized to the nucleus, they may function as NLSs (Kenna et al., 1998, Mol Cell Biol 18, 1115-1124).

[0341] As shown in Example 1, the yeast Ty1 NLS confers strong nuclear localization of Cas proteins and Cas fusion proteins in mammalian cells. To determine whether this activity is an inherent property of the Ty1 NLS, we tested whether closely related NLSs derived from Ty2 integrase and other yeast Ty1 NLS-like motifs are sufficient to localize the integrase-dCas9 fusion protein (INΔC-Cas9) to the nucleus in mammalian cells. Interestingly, the Ty2 NLS, which is highly conserved in the Ty1 NLS, was as efficient as the Ty1 NLS in nuclear localization (Figure 11). Fusions of three different Ty1 NLS-like sequences identified in yeast that are distinct from the Ty1 / Ty2 NLS sequence (Kenna et al., 1998) either showed strong NLS activity (MAK11) or no obvious NLS activity (INO4 and STH1). The MAK11 sequence is derived from a yeast nuclear protein and is located at the C-terminus of the protein. Further screening suggested that this sequence indeed functions as an NLS. N20-40 A large number of potential Ty1 NLS-like sequences were identified across a wide variety of species by all proteins in the SWISS-PROT Protein Sequence Databank using KKR (SEQ ID NOs: 275-887). These data indicate that other Ty1 NLS-like sequences may have strong NLS activity and may be useful for localization of proteins, including Cas and Cas fusion proteins, in dividing and non-dividing eukaryotic cells.

[0342] Example 6: Improved CRISPR-Cas9 DNA editing using Ty1 NLS CRISPR-Cas DNA cleavage system is derived from bacteria, and Cas proteins are large and lack endogenous mammalian nuclear localization signals (NLS), which prevents their efficient nuclear localization in mammalian cells. Previously, it has been shown that efficient nuclear localization and DNA editing by CRISPR-Cas9 in mammalian cells requires the addition of two classical nuclear localization signals (N-terminal SV40 and C-terminal nucleoplasmin (NPM) bipartite NLS) (Cong et al., 2013, Science 339, 819-823). Due to the strong properties of the non-classical yeast retrotransposon Ty1 NLS to localize Cas fusion proteins in mammalian cells (Example 1), we tested whether Ty1 NLS could also function to improve the editing efficiency of conventional CRISPR-Cas9 in mammalian cells.

[0343] To determine whether Ty1 improves CRISPR-Cas9 editing, we modified an existing CRISPR-Cas9 expression plasmid (px330) by replacing the C-terminal NPM NLS with the non-classical Ty1 NLS (px330-Ty1) (Figure 12A). We then created a frameshift-responsive luciferase reporter that encodes an out-of-frame luciferase coding sequence downstream of the target sequence (ts) (Figure 12B). In this reporter assay, breaks near the target sequence and incomplete repair by the cellular non-homologous end joining (NHEJ) pathway can induce nucleotide insertions or deletions that can reconstitute the luciferase coding sequence and result in luciferase expression.

[0344] Co-expression of a luciferase reporter with a vector encoding Cas9 containing the NPM NLS and a single guide RNA specific for a 20-nucleotide target sequence resulted in an approximately 20-fold increase in luciferase activity over background compared to non-targeting guide RNA (Figure 12C). Notably, expression of Cas9 containing the Ty1 NLS significantly enhanced reporter activity (approximately 44%) in COS-7 cells compared to Cas9 containing the NPM NLS (Figure 12C).

[0345] Example 7: Genome targeting strategies for editing Targeted integration of DNA donor sequences using integrase-DNA binding fusion proteins can be targeted to various locations within the genome, depending on the desired outcome. For example, therapeutic DNA donor sequences consisting of gene expression cassettes (e.g., promoters, gene sequences, and transcription terminators) can be targeted to "safe harbor" locations that allow the therapeutic gene to be expressed without affecting the expression of adjacent genes (see Pellenz et al., 2019, Hum Gene Ther 30, 814-828 for a review and list of safe harbor sites in the human genome). These can include intergenic regions away from adjacent genes, such as H11, or within "non-essential" genes, such as CCR5, hROSA26, or AAVS1 (Figures 13A and 13B).

[0346] To restore the expression of disease-causing gene mutations, targeted integration of therapeutic gene sequences into endogenous disease loci may be advantageous because the locus is already defective and the spatial and temporal expression of the locus is under endogenous regulatory control. In one repeat, a DNA donor sequence encoding a therapeutic gene containing a splice acceptor can be integrated into the first intron of the endogenous locus, whereby splicing 1) allows expression of the introduced gene sequence and 2) prevents downstream expression of the mutant sequence (by termination from the integrated poly(A) sequence or LTR sequence) (Figure 13C). If it targets a downstream intron, a smaller DNA donor sequence can be delivered or expressed (Figure 13D).

[0347] Targeted insertion of a DNA donor sequence containing an IRES sequence into the 3' untranslated region (3'UTR) of a gene can be beneficial in that this approach allows expression with the same spatial and temporal expression as the target locus and is less likely to disrupt the target locus (Figure 13E).

[0348] Example 8: Targeted lentiviral integration into mammalian genomes using CRISPR-CAS The data presented here demonstrate three different approaches for the delivery and targeted integration of lentiviral donor sequences into the mammalian genome.

[0349] Lentivirus life cycle Lentiviruses are single-stranded RNA viruses that integrate persistent double-stranded DNA (dsDNA) copies of their proviral genome into host cell DNA (Figure 14). Lentiviral genomes are flanked by long terminal repeat (LTR) sequences that control transcription of viral genes and contain short (approximately 20 base pairs) sequence motifs at the U3 and U5 ends that are necessary for proviral genome integration. Following viral infection, the lentiviral RNA genome is copied as blunt-ended dsDNA by the virus-encoded reverse transcriptase (RT) and inserted into the host genome by integrase (IN). IN consists of three functional domains essential for IN activity, including a C-terminal domain (CTD) that binds DNA nonspecifically. IN-mediated insertion of retroviral DNA occurs with little DNA target sequence specificity and can integrate into active loci, which may disrupt normal gene function and cause disease in humans. This limits the utility of lentiviral vectors for gene therapy, despite the advantage of a large sequence payload capacity.

[0350] Genome editing CRISPR-Cas9 allows programmable DNA targeting by utilizing a short single guide RNA to recognize and bind to DNA. Catalytically inactive Cas9 (dCas9) retains the ability to target DNA and has recently been repurposed as a programmable DNA binding platform for a wide variety of applications for genome interrogation and regulation. As shown in Example 1, fusion of lentiviral integrase to dCas9 is sufficient to insert a donor DNA sequence containing short viral ends into a target sequence using CRISPR guide RNA in mammalian cells (Figure 15). To monitor integrase-Cas mediated integration in mammalian cells, a donor vector containing an IGR IRES sequence followed by an mCherry-2a-puromycin gene and an SV40 polyadenylation sequence was generated (Figure 15B). sgRNAs were designed to target a stable human CMV-eGFP stable cell line in COS-7 cells (Figures 15C and 15D). The stable transgenesis of hCMV-eGFP provided a heterologous target sequence that can be used to determine editing at strongly expressed, but nonessentially expressed, loci. The donor mCherry-2a-puro template was purified and co-transfected with sgRNA and IN-dCas9 into GFP-stable cells and cultured for 48 hours. After 48 hours, mCherry-positive cells were found in the culture medium, replacing the GFP-positive signal (Figure 15E). Introducing the editing components (integrase-CRISPR-Cas9 fusion) into lentiviral particles allows targeted and easily programmable integration of the lentiviral genome into host DNA, thus removing a major limitation of lentiviral gene therapy (i.e., non-specific lentiviral integration). This approach is useful for both basic research and therapeutic applications.

[0351] Lentiviral gene delivery system Lentiviral vectors have been adapted as powerful gene delivery tools for research applications (Figure 16). Lentiviral structural and enzymatic proteins are transcribed and translated as a large polyprotein (gag-pol and envelope) (Figure 16A). Once introduced into budding viral particles, the polyproteins are processed into individual proteins by viral proteases. In lentiviral vector gene expression systems, these polyproteins are removed from the viral genome and expressed using separate mammalian expression plasmids (Figure 16B). Donor DNA sequences of interest can then be cloned in place of the viral polyproteins, between the flanking LTR sequences. Co-transfection of these vectors in mammalian cells results in the formation of lentiviral particles that can deliver and integrate the encoded donor sequences, but do not require coding information for integrase and other viral proteins necessary for subsequent viral propagation (Figure 16B). Lentiviral particles are natural vectors for delivering both viral proteins (e.g., integrase and reverse transcriptase) as well as dsDNA donor sequences, and contain the necessary viral terminal sequences required for integrase-mediated insertion into mammalian cells (Figure 16C).

[0352] Packaging of the integrase-dCas9 fusion protein into lentiviral particles. Existing lentiviral delivery systems can be modified to introduce editing components for targeted integration of lentiviral donor templates for genome editing in mammalian cells (Figures 17-20). Here, we describe three different approaches for delivery and targeted integration of lentiviral donor sequences into mammalian genomes.

[0353] The first approach is to directly introduce dCas9 as a fusion to integrase (or integrase lacking its C-terminal non-specific DNA binding domain (INΔC)) into a lentiviral packaging plasmid (e.g., psPax2) that encodes the gag-pol polyprotein (Figure 17A). In this approach, the modified gag-pol polyprotein is translated as a polyprotein along with other viral components and packaged into lentiviral particles loaded with guide RNA (Figure 4B). The integrase-dCas9 fusion protein retains sequences required for protease cleavage (PR), which allows it to be successfully cleaved from the gag-pol polyprotein during particle maturation. Transduction of mammalian cells delivers viral proteins including the IN-dCas9 fusion protein, sgRNA, and lentiviral donor sequences. Reverse transcription of the ssRNA genome by reverse transcriptase generates a dsDNA sequence containing the correct viral terminal sequences (U3 and U5), which is then integrated into the mammalian genome by the IN-dCas9 fusion protein.

[0354] The second approach is to generate N- and C-terminal fusions of integrase-dCas9 with HIV viral protein R (VPR) (Figure 18A). VPR is efficiently packaged into lentiviral particles as an accessory protein and has been used to package heterologous proteins (e.g., GFP) into lentiviral particles. A viral protease cleavage sequence is included between VPR and the IN-dCas9 fusion protein, which allows IN-dCas9 to be released from VPR after maturation (Figure 18A). Co-transfection of packaging cells with lentiviral components generates viral particles containing VPR-IN-dCas9 protein and sgRNA. The packaging plasmid (e.g., psPax2) required for viral particle formation contains a mutation in integrase that inhibits its catalytic activity, thereby inhibiting non-mediated integration (Figure 18B). Upon viral transduction, the integrase-dCas9 protein is delivered, which mediates the integration of the lentiviral donor sequence (Figure 18C). The advantage of delivering the IN-dCas9 fusion and sgRNA as a riboprotein is that it is only expressed transiently in the target cell.

[0355] The third method is to directly introduce the integrase-dCas9 fusion protein and sgRNA expression cassette into a lentiviral transfer plasmid or other viral vector (such as AAV) (Figure 19A). The transfer plasmid containing the IN-dCas9 fusion protein and sgRNA is co-transfected with the packaging and envelope plasmids required to generate lentiviral particles. When using lentivirus, the packaging plasmid contains a catalytic mutation in the integrase to inhibit non-specific integration (Figure 19B). Upon transduction of mammalian cells, expression of the IN-dCas9 fusion protein and sgRNA generates a component that can target its own viral donor vector (self-integration) for targeted integration (Figure 19C). This method is used for targeted gene disruption or as a gene drive. Alternatively, co-transduction with additional lentiviral particles encoding the donor sequence serves as an integrated donor template (Figure 19). In this approach, prevention of self-integration of the own viral coding sequence is achieved by using integrase enzymes from different members of the retroviridae family and their corresponding transfer plasmids. For example, an HIV lentiviral particle encoding an FIV IN-dCas9 fusion protein is utilized to incorporate an FIV donor template encoded within the FIV lentiviral particle (Figure 20).

[0356] Single-locus, constitutively active, ubiquitous ROSA26 mGFP / + Generation of reporter mouse lines The ROSA26mT / mG reporter mouse line (Jackson Labs, stock no. 007576) contains a floxed membrane-localized tdTO (mT) fluorescent reporter cassette that, upon recombination with CRE recombinase, removes the mT reporter and expresses the membrane-localized eGFP (mG) reporter. To generate a single locus, the in vivo GFP reporter line, ROSA26mT / mG mice, was crossed with universal CAG-CRE recombinase mice to generate ROSA26mG reporter mice that constitutively and ubiquitously express heterozygous ROSA26 mG / + Isolation of mouse embryonic fibroblasts (MEFs) from mice revealed strong membrane GFP expression in all cells in culture (Figure 21). A similar strategy is utilized to generate a ubiquitous, constitutively active nuclear GFP reporter by recombination of the ROSA26nT / nG mouse line (Jackson Labs, stock no. 023035).

[0357] Packaging of components into lentiviral particles for targeted integration into the ROSA-mGFP locus. ROSA26 mG / + For targeted integration of the IRES-tdTO sequence into the GFP coding sequence in MEFs, lentiviral particles were generated in a packaging cell line (Lenti-X 293T, Clontech). Lentiviral particles were generated by co-transfection of a lentiviral transfer plasmid (Lenti-IRES-tdTO) encoding an IRES-tdTO fluorescent reporter between second generation SIN lentiviral LTRs, an expression vector encoding a pantropic envelope protein (VSV-G), an expression plasmid encoding an inverted pair of guide RNAs targeting GFP, and a packing plasmid (INΔC-dCas9-psPax2) encoding an INΔC-dCas9 fusion in the context of the Gag-Pol lentiviral polyprotein in the psPax2 packing plasmid. Lentiviral particles were collected from the supernatant and filtered using a 0.45 μm PES filter.

[0358] Targeted lentiviral integration in mammalian cells Using Incriptr-modified lentiviral particles, ROSA26 in culture medium mG / + MEFs were transduced. After 2 days, ubiquitous red fluorescent protein expression was detectable in MEFs transduced with lentivirus encoding the IRES-tdTO reporter, while GFP fluorescence was retained. This initial widespread expression is likely due to translation of the lentiviral IRES-tdTO-encoded viral RNA, indicating that lentiviral packaging was not inhibited by modification of the packaging plasmid (Figure 21). In conventional lentiviral transduction, lentiviral transgene expression is not maintained in the absence of viral integration. Of note, 7 days after transduction, ROSA26 mG / + In primary cells (Figure 21) or when targeting the previously described CMV-GFP COS-7 stable cell line (Figure 22), tdTO red fluorescent cells, this time lacking green fluorescence, were detectable in culture. These data indicate that fusing integrase (lacking the C-terminal DNA binding domain) to catalytically dead Cas9 in the context of the Gag-Pol lentiviral polyprotein allows lentiviral packaging, delivery and targeting of lentiviral encoded donor sequences in mammalian cells. Furthermore, these data suggest that expression of guide RNA in lentiviral packaging cells is sufficient for incorporation into lentiviral particles, which may occur through a strong interaction with dCas9. Alternative approaches to deliver guide RNA into lentiviral particles may improve targeted integration, such as through constitutive expression of guide RNA(s) in the transfer plasmid.

[0359] Alternative DNA-binding domains for targeted integration of lentiviral particles. This data shows that replacing the non-specific DNA binding domain of integrase with the programmable DNA binding domain of dCas9 allows targeted integration of dsDNA donor templates for delivery of lentiviral-encoded donor sequences or through delivery in lentiviral particles. The CRISPR-Cas system is two-component and relies on both Cas protein and small guide RNA for targeting. In some cases, it may be beneficial to utilize single-component DNA targeting proteins such as TALENs for delivery via lentiviral particles, since they are targeted only by the encoded protein. Using a similar lentivirus production approach, replacing dCas9 in the previous packaging strategy with TALENs targeting given sequences (e.g., eGFP and safe harbor loci) allows lentivirus packaging and targeting without the need for delivery of guide RNA (Figure 23). For example, TALENs are packaged and delivered as fusions to integrase, either as the gag-pol polyprotein (Figure 23A), as IN-TALENs as fusions to proteins introduced into viruses such as VPR (Figure 23B), or as IN-TALENs delivered within a transfer plasmid (Figure 23C).

[0360] Example 9: Improved CRISPR-Cas9 DNA editing using Ty1 NLS The CRISPR-Cas DNA cleavage system is derived from bacteria, and the Cas proteins are large and lack an endogenous mammalian nuclear localization signal (NLS), preventing their efficient nuclear localization in mammalian cells.

[0361] To determine whether Ty1 improves CRISPR-Cas9 editing, we modified an existing CRISPR-Cas9 expression plasmid (px330) by replacing the C-terminal NPM NLS with the non-classical Ty1 NLS (px330-Ty1) (Figure 24A). We then created a frameshift-responsive luciferase reporter that encodes an out-of-frame luciferase coding sequence downstream of the target sequence (ts) (Figure 24B). In this reporter assay, breaks near the target sequence and incomplete repair by the cellular non-homologous end joining (NHEJ) pathway can induce nucleotide insertions or deletions that can reconstitute the luciferase coding sequence and result in luciferase expression.

[0362] Co-expression of a luciferase reporter with a vector encoding Cas9 containing the NPM NLS and a single guide RNA specific for a 20-nucleotide target sequence resulted in an approximately 20-fold increase in luciferase activity over background compared to non-targeting guide RNA (Figure 24C). Notably, expression of Cas9 containing the Ty1 NLS significantly enhanced reporter activity (approximately 44%) in COS-7 cells compared to Cas9 containing the NPM NLS (Figure 24C).

[0363] Example 10: Non-homologous DNA integration by integrase-TALEN fusion proteins Transcription activator-like effector nucleases (TALENs) are well-studied programmable DNA-binding proteins constructed by assembling individual nucleotide targeting domains in tandem (Reyon et al., 2012). In a similar approach demonstrated for Cas-IN fusions, TALENs can be utilized to induce retroviral integrase-mediated integration of donor DNA templates (Figure 25). To generate TALEN-integrase fusion proteins, mammalian expression vectors were constructed to obtain TALEN targeting repeats from the previously described TALEN expression vectors to generate either IN-TALEN or TALEN-IN fusions. Each fusion protein introduced TALEN repeats separated by a linker sequence between HIV integrase lacking the 3xFLAG epitope, Ty1 NLS, and C-terminal nonspecific DNA binding domain (INΔC). In some cases, IN mutations can be introduced to alter IN activity, dimerization, interaction with cellular proteins, resistance to dimerization inhibitors, or tandem copies of INΔC (tdINΔC). For example, an E85G mutation can be introduced to prevent obligate dimer formation.

[0364] The TALEN pair targeting eGFP was previously described and validated for targeting efficiency (Reyon et al., 2012; available from Addgene). The TALEN pair (ClaI / BamHI fragment) was subcloned to generate TALEN-IN fusion proteins targeting eGFP with 16-bp or 28-bp spacers.

[0365] Using a plasmid DNA integration assay (Figure 26), a TALEN-IN pair targeting eGFP, a linear double-stranded DNA donor sequence encoding an IGR-CAT resistance gene, and amilCP bacterial expression reporter were co-transfected into mammalian COS-7 cells. Two days after transfection, the edited plasmids were recovered from the mammalian cells, transformed into e. coli, and selected on chloramphenicol plates. Interestingly, the TALEN pair separated by 16 bp gave rise to approximately six-fold more chloramphenicol-resistant colonies, while the TALEN pair separated by 28 bp was similar to the non-targeted integrase (Figure 27). These data suggest that the proximity of the TALEN pair is important for targeting and integration, a feature previously reported for TALEN-FokI-mediated dsDNA cleavage.

[0366] Example 11: Construction and in vitro validation of Cas-IN fusion targeting safe harbor sites for delivery and expression in mouse and human cells Human frataxin lentiviral vectors are generated under the control of a ubiquitous promoter (EF1a), cardiac-specific (cardiac troponin T, cTnT), brain-specific (hSYN1), or novel promoters useful for expression of FXN in key therapeutically beneficial tissues, such as the brain (CNS and PNS), heart, and skeletal muscle (EF1a2). A second vector encoding FXN with a fluorescent protein and a selection marker is generated to aid in selection and validation of expression and delivery. These lentiviral vectors can function as both gene transfer plasmids for both conventional lentiviral transduction as well as Cas-IN fusion mediated gene targeting (Figure 30).

[0367] Paired single CRISPR guide RNAs (for Cas-IN fusion) and TALEN pairs (Cas-TALENs) are designed and validated for Cas-IN or Cas-TALEN fusion-mediated targeting of the mouse safe harbor site (ROSA26 GFP) or the human Surf harbor site (AAVS1).

[0368] The safe harbor portion will be validated first in mammalian plasmid-based integration assays (editing and recovering plasmids in mammalian cells for clonal analysis) and then in direct genome editing in mouse and human cell lines.

[0369] Cas-IN targeted clones will be isolated for whole genome sequencing to identify / compare different guide RNAs and TALENs versus on-target / off-target alignment and efficiency for mouse and human safe harbor sites.

[0370] Example 12: Repair of Friedreich's ataxia using Cas-IN or Cas-TALEN fusion-mediated frataxin gene therapy Design and validation of lentiviral particles suitable for delivering and expressing FXN in tissues of therapeutic benefit to FA As described in Example 11, promoters driving expression of reporter genes in tissues affected by FA were identified in WT mice in vivo for specificity and expression levels across different tissues using RT-PCR, immunohistochemistry and Western blot.

[0371] The well-characterized envelope spike VSV-G will be used in pseudotyped lentiviral vectors for broad delivery and expression in vivo in cell types. The spike envelope protein from SARS-CoV-2 will also be tested for tissue-specific transduction.

[0372] Evaluation of Cas-IN fusion genes targeting FXN in a mouse model of FA Mice containing a conditional floxed allele (Jackson Labs) will be crossed with tissue-specific Cre reporter mice to knock out FXN expression in the heart. Echocardiography, histological and genetic analyses will be performed to verify the disease phenotype.

[0373] Lentiviral particles encoding the FXN gene transfer cassette and Cas-IN enzyme protein were delivered via systemic injection to 6-day-old neonates. Saline and conventional lentivirus encoding WT integrase with the same FXN expression cassette were injected as controls. Noninvasive echocardiography was performed at 4, 6 and 10 weeks after injection to determine cardiac output, including ejection fraction, fractional shortening and combustion chamber volume. Hearts were isolated and subjected to histological, gene expression and sequencing analyses.

[0374] Example 13: Promoters for tissue-specific expression in heart, skeletal muscle, CNS and PNS The promoter sequence of eukaryotic translation elongation factor 1alpha1 (EEF1a1 / EF1a) is the most commonly used promoter to drive continuous, robust, and widespread gene expression in viral transgene therapy (Uetsuki et al. 1989; Kim et al. 1990). The small core promoter of EF1a consists of only 213 base pairs (bp) and maintains similar promoter activity, and its small size is beneficial for packaging restrictive vectors such as AAV.

[0375] Tissue-specific promoters are commonly used to drive gene expression in a subset of tissues to reduce efficacy and exogenous expression in undesired tissues. They are often specific to a single tissue type, such as heart, skeletal muscle, liver, or CNS. However, to target tissues affected by neuromuscular diseases, it is necessary to express the transgene in all affected tissues, such as heart and skeletal muscle and central nervous tissue as well as peripheral nervous tissue. Interestingly, EEF1A2 (EF1A2), a paralog of EF1A1, is robustly and specifically expressed in heart, skeletal muscle, and nervous tissues, such as brain and motor neurons (Lee et al. 1992). This promoter sequence, or the smaller Colla promoter sequence, can also be used to drive strong and continuous gene expression in these tissues to treat neuromuscular diseases.

[0376] In addition, selective mutations of transcription factor enhancer binding sequences also function to selectively drive expression in lean tissues or cell types.

[0377] Example 14: Fusion of integrase to compact catalytically dead Cas14 (dCas14) for Cas-IN mediated gene integration The large size of spCas9 hampers the efficiency with which it can be delivered using viral vectors. Recently, programmable miniature CRISPR-Cas14 (CRISPR-Cas12f) RNA-guided nucleases ranging in size from 400 to 700 amino acids have been discovered in archaea (Harrington et al. 2018; Karvelis et al. 2020). The small but easily programmable nature of the CRISPR-Cas14 system could be beneficial as a fusion to lentiviral integrase (IN) in non-homologous gene integration. Similar to CRISPR-Cas9, CRISPR-Cas14 uses two RNAs, tracrRNA and CRISPR RNA cRNA, to guide target recognition and cleavage. Fusion of these two RNAs to a single RNA (sgRNA) has been shown to be functional for target cleavage in vitro and in bacteria. However, tracrRNA contains a stretch of consecutive 5Ts that act as a termination sequence recognized by Pol III promoters, which are commonly used to drive guide RNA expression in mammalian cells, and this sequence would likely prevent guide RNA expression in lactating animal cells (Figure 31).

[0378] To quantify the activity of CRISPR-Cas14 in mammalian cells, a frameshift-activated luciferase reporter was generated, which contains a validated Cas14 target sequence with a 5' PAM upstream of an out-of-frame luciferase open reading frame. In this assay, cleavage of the target sequence in mammalian cells leads to incomplete NHEJ repair of the FAR reporter and, in some cases, reframing and expression of the luciferase reporter. As expected, expression of WT Cas14 sgRNA failed to activate the luciferase reporter when co-expressed with active Cas14 fused to a strong Ty1 nuclear localization signal. However, mutation of residues to break up the internal Pol III terminating T-stretch or truncation of the sgRNA to remove the T-stretch leads to detectable activity of the luciferase reporter in mammalian cells (Figure 32). These data demonstrate that Cas14 sgRNAs with specific mutations that disrupt premature termination can be used for CRISPR-Cas14 DNA targeting in mammalian cells.

[0379] The disclosures of any and all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0380] While the present invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the present invention may be devised without departing from the true spirit and scope of the invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. 1. A pharmaceutical composition for treating Friedreich's ataxia in a subject comprising a fusion protein comprising a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein, and a nuclear localization signal (NLS), or a nucleic acid molecule comprising one or more nucleic acids encoding a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein, and a nuclear localization signal (NLS), A guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genome of the subject; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence, the donor template sequence comprising a nucleic acid sequence encoding frataxin; The pharmaceutical composition is administered in combination with

2. 2. The pharmaceutical composition of claim 1, wherein the retrovirus IN is selected from the group consisting of human immunodeficiency virus (HIV), rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), moloney murine leukemia virus (MoLV), bovine leukemia virus (BLV), human T-lymphotropic virus (HTLV), avian sarcoma leukemia virus (ASLV), feline leukemia virus (FLV), xenotropic murine leukemia virus-related virus (XMLV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), prototype foamy virus (PFV), simian foamy virus (SFV), human foamy virus (HFV), walleye cutaneous sarcoma virus (WDSV), and bovine immunodeficiency virus (BIV).

3. The pharmaceutical composition of claim 1, wherein the IN comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 9-48.

4. 2. The pharmaceutical composition of claim 1, wherein the Cas protein is selected from the group consisting of Cas9, Cas14 and Cpf1.

5. 2. The pharmaceutical composition of claim 1, wherein the Cas protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 1-8.

6. The pharmaceutical composition of claim 1, wherein the Cas protein is catalytically dead (dCas).

7. 7. The pharmaceutical composition of claim 6, wherein the Cas protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 2, 4, 6 and 8.

8. The pharmaceutical composition of claim 1 , wherein the NLS is a Ty1 NLS or a Ty2 NLS.

9. 2. The pharmaceutical composition of claim 1, wherein the NLS comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 53 or 54 and 361-973.

10. The pharmaceutical composition of claim 1 , wherein the target region in the genome of the subject is a safe harbor site.

11. The pharmaceutical composition of claim 1 , wherein the donor template sequence encodes a protein that is at least 70% identical to SEQ ID NO:

357.

12. 2. The pharmaceutical composition of claim 1, wherein the donor template sequence comprises a sequence that is at least 70% identical to SEQ ID NO:

358.

13. 2. The pharmaceutical composition of claim 1, wherein the IN is HIV IN and the donor template sequence comprises a U3 sequence of SEQ ID NO:258, a U5 sequence of SEQ ID NO:259, or both.

14. A fusion protein comprising: a) a CRISPR-associated (Cas) 14 protein, and b) the fusion protein comprising a nuclear localization signal (NLS).

15. The fusion protein of claim 14, wherein the Cas14 protein comprises a sequence that is at least 70% identical to SEQ ID NO: 7 or 8.

16. The fusion protein of claim 14 , wherein the NLS is a Ty1 NLS or a Ty2 NLS.

17. The fusion protein of claim 14, wherein the NLS comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 53 or 54 and 361-973.

18. The fusion protein of claim 14, wherein the fusion protein comprises a sequence that is at least 70% identical to SEQ ID NO:

145.

19. The fusion protein of claim 14 , further comprising a retroviral integrase (IN) or a fragment thereof.

20. The fusion protein of claim 19, wherein the IN comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 9-48.

21. The fusion protein of claim 19, wherein the Cas14 protein is catalytically dead (dCas).

22. 20. The fusion protein of claim 19, wherein the fusion protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 63-102 and 146.

23. A nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein according to any one of claims 14 to 22.

24. 1. A method for editing genetic material, comprising: a) a fusion protein according to any one of claims 14 to 22 or a nucleic acid molecule encoding the same; b) a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region within the genetic material; and c) a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence; to the genetic material.

25. 25. The method of claim 24, wherein the guide nucleic acid comprises tracrRNA and CRISPR RNA (crRNA).

26. 26. The method of claim 25, wherein the tracrRNA comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 336-339.

27. 1. A system for editing genetic material comprising: a) a nucleic acid sequence encoding a fusion protein comprising a CRISPR-associated (Cas) 14 protein and a nuclear localization signal (NLS); b) a nucleic acid sequence encoding a CRISPR-Cas system guide RNA; and c) a nucleic acid sequence encoding a donor template nucleic acid, the donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence; The above system, comprising:

28. The system of claim 27, wherein the Cas14 protein comprises a sequence that is at least 70% identical to SEQ ID NO: 7 or 8.

29. 28. The system of claim 27, wherein the NLS is a Ty1 NLS or a Ty2 NLS.

30. 28. The system of claim 27, wherein the NLS comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 53 or 54, and 361-973.

31. The system of claim 27, wherein the Cas14 protein is catalytically dead (dCas).

32. 28. The system of claim 27, wherein the fusion protein further comprises a retroviral integrase (IN) or a fragment thereof.

33. The system of claim 32, wherein the IN comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 9-48.

34. The system of claim 32, wherein the Cas14 protein is catalytically dead (dCas).

35. The system of claim 32, wherein the fusion protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 63-102 and 146.

36. 28. The system of claim 27, wherein the fusion protein comprises a sequence that is at least 70% identical to SEQ ID NO:

145.

37. 37. The system of any one of claims 27 to 36, wherein the guide RNA comprises tracrRNA and CRISPR RNA (crRNA).

38. 28. The system of claim 27, wherein the tracrRNA comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 336-339.

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