Genome editing by targeted nonhomologous DNA insertion using retroviral integrase-Cas9 fusion protein

Abstract

The present invention provides fusion proteins for editing genomic material, as well as related nucleic acids, systems and methods, wherein the fusion protein comprises a retroviral integrase and a Cas protein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 748,703, filed on October 22, 2018, which is incorporated herein by reference in its entirety. Background of the Invention

[0004] CRISPR-Cas9 greatly improves the ability to quickly change mammalian genomes for basic research and clinical applications. CRISPR-Cas9 uses guide RNA to guide Cas9 to specific DNA target sequences, where it induces double-stranded DNA cleavage and triggers cell repair pathways to introduce frameshift mutations or insert donor sequences by homology-directed repair (HDR). Despite these major advances, the efficiency of targeted delivery of large DNA sequences to use CRISPR-Cas9-mediated HDR for genome editing is still low, requiring donor templates containing significant flanking homology regions and inducing p53 DNA damage pathways (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). In short, these greatly limit the efficiency of CRISPR-Cas9 genome editing. Therefore, improved integrated genome editing is needed.

[0005] In contrast, the lentiviral enzyme integrase (IN) is necessary and sufficient to catalyze the insertion of the large lentiviral genome into host cell DNA by a method that does not require target sequence homology. IN-mediated lentiviral DNA insertion occurs with little DNA target sequence specificity, in part due to nonspecific binding of its C-terminal domain to DNA (Lutzke and Plasterk 1998, J Virol 72: 4841-48).

[0006] Current limitations of gene therapy technologies prevent the treatment of most human monogenic diseases. CRISPR-Cas9 gene editing has been a new focus for developing therapeutic approaches to correct deleterious mutations in mammalian genomes. This remains a significant challenge due to the numerous patient-specific mutations within the human genome that can cause disease and disorders. CRISPR guide RNAs designed to target exon-intron boundaries may allow exon skipping strategies to target these sets of mutations, however, the efficacy of these strategies remains to be tested and are not applicable to all patients.

[0007] Transgenic expression of many genes can prevent and reverse disease outcomes in animal models, however the large size of some genes greatly exceeds the size limits of traditional gene editing methods such as CRISPR-Cas9 or traditional viral gene therapy approaches such as AAV (~4.9kb limit), preventing their use in human gene therapy. Approaches using smaller engineered genes delivered by AAV are currently in clinical trials, however whether these strategies provide long-term recovery and are only applicable to patients with specific mutations remains to be determined.

[0008] In contrast, lentiviral vectors are able to deliver large genes and allow permanent correction by integration into the host genome. However, the random nature of current lentiviral integration is likely to cause off-target mutations and diseases, which prevents it from being used in clinical applications (Milone et al., 2018, Leukemia 23: 1529-41). The lentiviral sequence is inserted into the host genome by the virally encoded enzyme integrase (IN), which utilizes the non-specific DNA binding domain required for genome integration (Andrake et al., 2015, Annu Rev Virol 2: 241-64).

[0009] Therefore, there is a need for improved editing of genomic materials. The present invention satisfies this need. Summary of the invention

[0010] In one aspect, the present invention provides a fusion protein. In one embodiment, the fusion protein comprises a retroviral integrase (IN) or a fragment thereof having a first amino acid sequence; a CRISPR-associated (Cas) protein having a second amino acid sequence; and a nuclear localization signal (NLS) having a third amino acid sequence.

[0011] In one embodiment, the retroviral IN is selected from the group consisting of: human immunodeficiency virus (HIV) IN, Rous sarcoma virus (RSV) IN, mouse mammary tumor virus (MMTV) IN, Moloney murine leukemia virus (MoLV) IN, bovine leukemia virus (BLV) IN, human T-lymphotropic virus (HTLV) IN, avian sarcoma leukemia virus (ASLV) IN, feline leukemia virus (FLV) IN, xenotropic murine leukemia virus-related virus (XMLV) IN, simian immunodeficiency virus (SIV) IN, feline immunodeficiency virus (FIV) IN, equine infectious anemia virus (EIAV) IN, prototype foamy virus (PFV) IN, simian foamy virus (SFV) IN, human foamy virus (HFV) IN, walleye cutaneous sarcoma virus (WDSV) IN and bovine immunodeficiency virus (BIV) IN.

[0012] 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 comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 1-40. In one embodiment, the retroviral IN comprises a sequence of one of SEQ ID NOs: 1-40.

[0013] In one embodiment, the Cas protein is selected from the group consisting of: Cas9, Cas13 and Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas). In one embodiment, the Cas protein comprises a sequence that is at least 95% identical to one of SEQ ID NOs: 41-46. In one embodiment, the Cas protein comprises the sequence of one of SEQ ID NOs: 41-46.

[0014] In one embodiment, the NLS is a retrotransposon NLS. In one embodiment, the retrotransposon NLS is a Ty1 or Ty2 NLS. In one embodiment, the NLS is a Ty1-like NLS. In one embodiment, the NLS comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 47-56, 254-257, and 275-887. In one embodiment, the NLS comprises a sequence that is one of SEQ ID NOs: 47-56, 254-257, and 275-887.

[0015] In one embodiment, the fusion protein comprises a sequence that is at least 70% identical to one of SEQ ID NOs: 57-98. In one embodiment, the fusion protein comprises the sequence of one of SEQ ID NOs: 57-98.

[0016] In one aspect, the invention provides a nucleic acid encoding a fusion protein of the invention. In one embodiment, the nucleic acid comprises a sequence at least 70% identical to one of SEQ ID NOs: 155-196. In one embodiment, the nucleic acid comprises a sequence selected from SEQ ID NOs: 155-196.

[0017] In one aspect, the present invention provides a method for editing genetic material. In one embodiment, the method comprises administering to the genetic material: (a) a fusion protein of the present invention or a nucleic acid molecule encoding a fusion protein of the present invention, (b) a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genetic material, and (c) a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the method for editing genetic material is an in vitro method. In one embodiment, the method for editing genetic material is an in vivo method.

[0018] In one aspect, the present invention provides a system for editing genetic material. In one embodiment, the system comprises in one or more vectors: (a) a nucleic acid sequence encoding a fusion protein of the present invention, (b) a nucleic acid sequence encoding a CRISPR-Cas system guide RNA, and (c) a nucleic acid sequence encoding a donor template nucleic acid, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the fusion protein comprises a retroviral integrase (IN) or a fragment thereof; a CRISPR-associated (Cas) protein and a nuclear localization signal (NLS). In one embodiment, the nucleic acid is located on the same vector. In one embodiment, the nucleic acid is located on different vectors.

[0019] In one embodiment, the CRISPR-Cas system guide RNA substantially hybridizes to a target DNA sequence in the gene. In one embodiment, the U3 sequence and the U5 sequence are specific for the retroviral IN.

[0020] In aspect, the present invention provides a system for delivering genome editing components. In one embodiment, the system comprises: (a) a packaging plasmid comprising a sequence encoding a gag-pol polyprotein, the gag-pol polyprotein comprising an integrase fused to a catalytically dead Cas (dCas) protein; (b) a transfer plasmid comprising a sequence encoding a donor sequence, a 5'LTR and a 3'LTR; and (c) an envelope plasmid comprising a nucleic acid sequence encoding an envelope protein. In one embodiment, the packaging plasmid further comprises a sequence encoding a guide RNA sequence.

[0021] In one embodiment, the system comprises: (a) a packaging plasmid comprising a sequence encoding a gag-pol polyprotein; (b) a transfer plasmid comprising a sequence encoding a donor sequence, a 5'LTR and a 3'LTR; (c) an envelope plasmid comprising a nucleic acid sequence encoding an envelope protein; and (d) a VPR-IN-dCas plasmid comprising a nucleic acid sequence encoding a fusion protein comprising a VPR, an integrase and a catalytically dead Cas (dCas). In one embodiment, the VPR-IN-dCas plasmid further comprises a sequence encoding a guide RNA sequence.

[0022] In one embodiment, the system comprises (a) a packaging plasmid comprising a nucleic acid sequence encoding a gag-pol polyprotein; (b) a transfer plasmid comprising a nucleic acid sequence encoding a guide RNA, a fusion protein comprising an integrase and a catalytically dead Cas, a 5' LTR and a 3' LTR; and (c) an envelope plasmid comprising a nucleic acid sequence encoding an envelope protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0024] Figure 1 ,include Figure 1 A to Figure 1 C, depicts experimental results demonstrating enhanced nuclear localization of retroviral integrase-dCas9 fusion proteins for editing mammalian genomic DNA. Figure 1 A. Schematic diagram depicting the IN-dCas9 fusion protein. Figure 1 B depicts the nuclear localization of IN-dCas9 fusion protein. Figure 1 C depicts experimental results demonstrating the enzymatic activity of the INΔC-dCas9 fusion protein incorporating an IRES-mCherry template targeting the 3'UTRE of EF1-α in HEK293 cells.

[0025] Figure 2 A schematic depicting nucleic acid editing technology showing that the fusion of viral integrase (IN) with CRISPR-dCas9 allows the integration of large DNA sequences in a target-specific manner. This approach allows the safe and permanent delivery of large genetic sequences that normally exceed the limitations of non-integrating AAV vectors.

[0026] Figure 3Depicts 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.

[0027] Figure 4 Schematic diagram depicting CRISPER-Cas9-mediated homology-directed repair and retroviral integrase-mediated random DNA integration.

[0028] Figure 5 Schematic diagram depicting integrase-Cas genome editing.

[0029] Figure 6 Schematic diagram depicting the donor vector, generation of blunt-ended templates, and generation of 3'-processed templates.

[0030] Figure 7 Experimental design depicting co-transfection of the INsrt template and the IN-dCas9 vector targeting the amilCP sequence into Cos7 cells.

[0031] Figure 8 Depicts the experimental design for knocking in the IGR-mCherry-2A-puromycin-pA cassette into the human HEK293 cell line using paired guide RNAs specific for the 3'UTR of the human EF1-α locus, and images of mCherry-positive cells 48 hours after transfection.

[0032] Fig. 9 Schematic diagram depicting demonstration of targeted editing.

[0033] Fig.10 Depicted is a schematic showing multiplex genome editing for generating floxed alleles.

[0034] Fig.11 ,include Fig.11 A to Fig.11 C, depicts experimental results demonstrating the efficiency of the Ty1 NLS-like sequence for nuclear localization of the INΔC-Cas9 fusion protein. Fig.11 A depicts the detection of INΔC-dCas9 fusion proteins containing the C-terminal canonical SV40, Ty1, or Ty2 NLS expressed in Cos-7 cells using anti-FLAG antibody. Fig.11 B depicts Ty1 NLS-like sequences isolated from yeast proteins that either provide robust nuclear localization (MAK11) or no significant localization activity (INO4 and STH1). Fig.11 C depicts the sequences of Ty1, Ty2 and Ty1 NLS-like sequence. Ty1 and Ty2 are highly conserved in length and residue composition. Scale bar = 10 μm.

[0035] Fig.12 ,include Fig.12 A to Fig.12 C, depicts experimental results demonstrating that Ty1 NLS enhances Cas9 DNA editing in mammalian cells. Fig.12 A depicts a diagram of the px330 CRISPR-Cas9 expression plasmid encoding hU6 driven single guide RNA (sgRNA) and CAG driven Cas9 protein containing an N-terminal 3x FLAG tag, SV40 NLS and a C-terminal NPM NLS. The Ty1 NLS was cloned to replace the NPM NLS in px330 (px330-Ty1). Fig.12 B depicts the generation of a frameshift-activated luciferase reporter gene in which an upstream 20 nt target sequence (ts) interrupts open reading from the downstream luciferase open reading frame. Frameshift induced by non-homologous end joining (NHEJ) reconstructs the downstream reporter gene and allows luciferase expression. Fig.12 C depicts that co-expression of a frameshift-responsive luciferase reporter and px330 containing a single guide RNA specific for the target sequence produced approximately 20-fold activation of luciferase activity relative to non-targeting sgRNA. Co-expression of px330-Ty1 produced approximately 44% enhancement relative to px330.

[0036] Fig.13 ,include Fig.13 A to Fig.13 E, Depicts the genome targeting strategy for editing. Integration of the DNA donor sequence can be targeted to different genomic locations depending on the desired application. Fig.13 A depicts that delivery of a DNA donor sequence carrying a gene cassette can be targeted to an intergenic 'safe harbor' locus to prevent disruption of adjacent or essential gene expression. Fig.13 B depicts that delivery of a DNA donor sequence carrying a gene cassette can be targeted to a non-essential 'safe harbor' locus to prevent disruption of adjacent or essential gene expression. Fig.13 C depicts that integration of a DNA sequence encoding a splice acceptor sequence (SA) can be delivered to an intronic region of a gene (eg, a disease gene locus), which will allow expression of the integrated sequence and prevent expression of downstream sequences. Fig.13 D depicts that integration of a DNA sequence encoding a splice acceptor sequence (SA) can be delivered to an intronic region of a gene (eg, a disease gene locus), which will allow expression of the integrated sequence and prevent expression of downstream sequences. Fig.13 E depicts that integration 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.

[0037] Fig.14Diagram depicting the life cycle of a lentivirus. Lentiviruses (a subclass of retroviruses) are single-stranded RNA viruses that integrate a permanent double-stranded DNA (dsDNA) copy of their proviral genome into the host cell DNA. After viral transduction, the lentiviral RNA genome is replicated as blunt-ended dsDNA by the virally encoded reverse transcriptase (RT) and inserted into the host genome by integrase I (IN). The lentiviral genome is flanked by short (approximately 20 base pairs) sequence motifs at its U3 and U5 ends that are required for IN to carry out proviral genome integration. IN-mediated retroviral DNA insertion occurs with little DNA target sequence specificity and can integrate into active gene loci, which can disrupt normal gene function and potentially cause human disease.

[0038] Fig.15 ,include Fig.15 A to Fig.15 E, Depicts genome editing in mammalian cells. Fusion of lentiviral integrase to dCas9 allows targeted nonhomologous insertion of donor DNA sequences containing short viral ends. Fig.15 A depicts a diagram of a mammalian expression vector encoding a human U6-driven single guide RNA (sgRNA) and integrase-dCas9 fusion protein. Fig.15 B depicts a diagram showing a dsDNA donor template containing an IGR IRES-mCherry-2A-puromycin (puro) cassette flanked by U3 / U5 viral motifs. Fig.15 C Schematic depicting the integrase-Cas9-mediated integration of this donor template into a CMV-eGFP reporter gene stably expressed in COS-7 cells. Fig.15 D depicts a schematic showing that integrase-Cas9-mediated integration of this donor template into a CMV-eGFP reporter transgene stably expressed in COS-7 results in disruption of eGFP expression while allowing for mCherry expression. Fig.15 E depicts experimental results demonstrating loss of eGFP expression and gain of mCherry expression in edited COS-7 cells.

[0039] Fig.16 ,include Fig.16 A to Fig.16 C, Depiction of the traditional lentiviral gene delivery system. Fig.16 A depicts a diagram of the lentiviral genome encoding viral proteins between flanking long terminal repeats (LTRs). Fig.16 B and Fig.16C depicts a schematic diagram, which demonstrates that the lentiviral genome has been used as a robust gene delivery tool. Slow virus particles can be used for packaging, delivery and stable expression of donor transgenic sequences. For lentiviral vector gene expression systems, viral polyproteins are removed from the viral genome and expressed using a separate mammalian expression plasmid. The donor DNA sequence can then be cloned to replace the viral polyprotein between the flanking LTR sequences. The co-transfection of these vectors in mammalian packaging cells allows the formation of slow virus particles that can deliver and integrate the encoded donor sequence, but does not require the coding information of other viral proteins necessary for integrase and subsequent viral propagation. Slow virus particles are natural vectors for delivering both viral proteins (such as integrase and reverse transcriptase) and dsDNA donor sequences, which contain the necessary viral end sequences required for integrase-mediated insertion into mammalian cells. Fig.16 B depicts the production of lentiviral vectors. Fig.16 C depicts transduction of lentiviral particles that deliver and stably express donor transgene sequences.

[0040] Fig.17 ,include Fig.17 A to Fig.17 C, Depicts targeted lentiviral integration. For the purpose of targeted lentiviral donor template integration for genome editing in mammalian cells, existing lentiviral delivery systems can be modified to incorporate editing components. Fig.17 A depicts an approach in which dCas9 is fused directly to integrase (or integrase lacking its C-terminal non-specific DNA binding domain) within a lentiviral packaging plasmid (e.g., psPax2) encoding the gag-pol polyprotein. Fig.17 B depicts the translation of the modified gag-pol polyprotein into a polyprotein along with other viral components, loaded with guide RNA and packaged into lentiviral particles. For this approach, the IN-dCas9 fusion protein retains the sequence required for protease cleavage (PR) and is therefore typically cleaved from the gag-pol polyprotein during particle maturation. Transduction of mammalian cells to produce viral proteins involves delivery of the IN-dCas9 fusion protein, sgRNA, and lentiviral donor sequence. Fig.17 C depicts that after lentiviral transduction, the ssRNA genome is reverse transcribed by reverse transcriptase to produce a dsDNA sequence containing the correct viral terminal sequences (U3 and U5), which is integrated into the mammalian genome through the IN-dCas9 fusion protein.

[0041] Fig.18 ,include Fig.18 A to Fig.18 C, Depicts targeted lentiviral integration via fusion to viral proteins. Fig.18A depicts IN-dCas9 expression and packaging as N-terminal and C-terminal fusions with viral proteins (e.g., viral protein R, VPR) as a method to achieve targeted lentiviral gene integration. A viral protease cleavage sequence is included between the VPR and the IN-dCas9 fusion protein, so that after maturation, IN-dCas9 will be released from the VPR. Fig.18 B depicts co-transfection of packaging cells with lentiviral components to produce viral particles containing VPR-IN-dCas9 protein and sgRNA. The packaging plasmid required for viral particle formation (e.g., psPax2) contains a mutation in the integrase to inhibit its catalytic activity in the packaging plasmid background, thereby preventing non-integrase-Cas9-mediated integration. Fig.18 C depicts that after viral transduction, the IN-dCas9 protein is delivered as a protein and mediates integration of the lentiviral donor sequence. The benefit of delivering the IN-dCas9 fusion and sgRNA as a riboprotein is that it is only transiently expressed in the target cells.

[0042] Fig.19 ,include Fig.19 A to Fig.19 C, depicts targeted lentiviral integration via incorporation into a transfer plasmid. Fig.19 A depicts that expressing IN-dCas9 fusion protein and / or guide RNA from a viral transfer plasmid (or other viral vectors such as AAV) is one method to achieve targeted lentiviral gene integration. Fig.19 B depicts In this method, a transfer plasmid containing the IN-dCas9 fusion protein and sgRNA is co-transfected with the packaging plasmid and envelope plasmid required to produce lentiviral particles. If lentivirus is used, the packaging plasmid contains a catalytic mutation in the integrase to inhibit nonspecific integration. Fig.19 C depicts the expression of IN-dCas9 fusion protein and sgRNA after transduction of mammalian cells, resulting in a component capable of targeting its own viral donor vector for targeted integration (self-integration). This approach is useful for targeted gene disruption or as a gene drive.

[0043] Fig. 20 ,include Fig. 20 A to Fig. 20 D, Depicts co-delivery of lentiviral donor sequences. Fig. 20 A depicts co-transduction with lentiviral particles encoding donor DNA sequences that can serve as donor templates for integration. Fig. 20 B and Fig. 20 C depicts a method for preventing self-integration of one's own viral coding sequences by using integrases from different retroviral family members and their corresponding transfer plasmids. Fig. 20 B depicts the production of HIV lentiviral particles encoding IN(FIV)-dCas9 fusion protein. Fig. 20 C depicts the production of FIV lentiviral particles containing the FIV transfer plasmid. Fig. 20 D depicts the use of HIV lentiviral particles encoding the IN(FIV)-dCas9 fusion protein to integrate the FIV donor template encoded in the FIV lentiviral particles.

[0044] Fig.21 Delineation of targeted lentiviral integration in primary mammalian cells. This data demonstrates lentiviral packaging, delivery, and targeted integration of a lentiviral donor template encoding an IRES-tdTO cassette into ROSA26 of mouse embryonic fibroblasts. mG / + Two days later, ubiquitous red fluorescent protein expression was detectable in MEFs transduced with lentivirus encoding the IRES-tdTO reporter gene, but GFP fluorescence was retained. Notably, seven days after transduction, tdTO red fluorescent cells were detectable in the culture, which was in the ROSA26 mG / + Lack of green fluorescence in primary cells.

[0045] Fig. 22 Depicting targeted lentiviral integration in a mammalian stable cell line. This data demonstrates 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.

[0046] Fig.23 ,include Fig.23 A to Fig.23 C, depicts the DNA binding domain for targeted integration of lentiviral particles. Replacing the nonspecific 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 TALEN) can be used as fusions with viral integrase for targeted integration. Using a similar lentiviral production method, TALEN targeting a specific sequence replaces dCas9 in the previous packaging strategy. Fig.23 A depicts TALENs packaged and delivered as fusions with integrase in the context of the gag-pol polyprotein. Fig.23 B depicts TALEN packaged and delivered as a fusion with integrase (as a fusion with a viral protein). Fig.23 C depicts TALEN packaged and delivered as a fusion with an integrase encoded within a transfer plasmid.

[0047] Fig.24 ,include Fig.24 A to Fig.24 C, depicts experimental results demonstrating that Ty1 NLS enhances Cas9 DNA editing in mammalian cells. Fig.24 A depicts a diagram of the px330 CRISPR-Cas9 expression plasmid encoding hU6 driven single guide RNA (sgRNA) and CAG driven Cas9 protein containing an N-terminal 3x FLAG tag, SV40 NLS and a C-terminal NPM NLS. The Ty1 NLS was cloned to replace the NPM NLS in px330 (px330-Ty1). Fig.24 B depicts the results demonstrating the generation of a frameshift-activated luciferase reporter gene in which an upstream 20nt target sequence (ts) interrupts open reading from the downstream luciferase open reading frame. Frameshift induced by non-homologous end joining (NHEJ) reconstructs the downstream reporter gene and allows luciferase expression. Fig.24 C depicts results demonstrating that co-expression of a frameshift-responsive luciferase reporter and px330 containing a single guide RNA specific for the target sequence produced approximately 20-fold activation of luciferase activity relative to non-targeting sgRNA. Co-expression of px330-Ty1 produced approximately 44% enhancement relative to px330.

[0048] Fig.25 Depicted is a schematic demonstrating that TALENs can be used to direct retroviral integrase-mediated integration of a donor DNA template.

[0049] Fig.26 Schematic diagram depicting the plasmid DNA integration assay.

[0050] Fig. 27 Depicts experimental data demonstrating that TALEN pairs separated by 16 bp produced approximately 6-fold more chloramphenicol-resistant colonies, while TALEN pairs separated by 28 bp were similar to non-targeted integrases.

[0051] Fig.29 ,include Fig.29 A to Fig.29 C, depicts the experimental results. Fig.29 A depicts expression of the amilCP chromoprotein in E. coli producing purple E. coli (white arrows). Integrase-Cas mediated integration of a donor sequence containing viral ends disrupts amilCP expression (orange arrows) (growth on kanamycin plates). Fig.29 B depicts the integration of the Insrt IGR-CAT donor template with blunt ends (ScaI cleaved) or 3' processed mimic (FauI cleaved) ends into the pCRII-amilCP reporter gene in mammalian cells. Interestingly, as a fusion with dCas9, the deletion of the C-terminal non-specific DNA binding domain does not inhibit integrase-Cas mediated integration. The use of 3' processed mimic ends showed an increase of CAT-resistant clones by about 2 times. Fig.29 C depicts the evaluation of integrase mutations for integrase-Cas mediated integration in plasmid DNA. Using dual guide RNA targeted integration of the IGR-CAT donor template into amilCP, mutations that inhibit dimerization (E85G and E85F) do not disrupt integrase-Cas mediated integration. However, the IN E87G mutation cannot be rescued by paired targeting sgRNA. Interestingly, the tandem INΔC fusion with dCas9 (tdINΔC-dCas9) showed an approximately 2-fold enhancement of integration. DETAILED DESCRIPTION

[0052] The present invention relates to fusion proteins, nucleic acids encoding fusion proteins, systems and methods for editing genetic material. In one embodiment, the present invention relates to retroviral integrase (IN)-CRISPR-associated (Cas) fusion proteins and nucleic acid molecules encoding retroviral IN-Cas fusion proteins. In one embodiment, the IN-Cas fusion protein further comprises a nuclear localization signal (NLS).

[0053] The fusion proteins, nucleic acid molecules, systems and methods of the present invention have the ability to deliver donor DNA sequences to target genomic locations. In addition, the present invention eliminates the need for homology arms and relies on targeting by guide RNA, thereby greatly simplifying editing genetic material.

[0054] In one aspect, the present invention provides an IN-Cas fusion protein. In one embodiment, the fusion protein comprises a retroviral IN or a fragment thereof having a first amino acid sequence; a Cas protein having a second amino acid sequence; and an NLS having a third amino acid sequence.

[0055] In one aspect, the present invention provides a nucleic acid molecule encoding an IN-Cas fusion protein. In one embodiment, the nucleic acid molecule comprises a first nucleic acid sequence encoding a retroviral IN or a fragment thereof; a second nucleic acid sequence encoding a Cas protein; and a third nucleic acid sequence encoding an NLS.

[0056] In one embodiment, the retroviral IN can be a human immunodeficiency virus (HIV) IN, a Rous sarcoma virus (RSV) IN, a mouse mammary tumor virus (MMTV) IN, a Moloney murine leukemia virus (MoLV) IN, a bovine leukemia virus (BLV) IN, a human T-lymphotropic virus (HTLV) IN, an avian sarcoma leukemia virus (ASLV) IN, a feline leukemia virus (FLV) IN, a heterotropic murine leukemia virus-related virus (XMLV) IN, a simian immunodeficiency virus (SIV) IN, a feline immunodeficiency virus (FIV) IN, an equine infectious anemia virus (EIAV) IN, a prototype foamy virus (PFV) IN, a simian foamy virus (SFV) IN, a human foamy virus (HFV) IN, a walleye cutaneous sarcoma virus (WDSV) IN, or a bovine immunodeficiency virus (BIV) IN. In one embodiment, the Cas protein is Cas9 or Cpf1. In one embodiment, the NLS is a retrotransposon NLS, such as a Ty1 NLS. In one embodiment, the retrotransposon NLS increases nuclear localization.

[0057] In one aspect, the present invention provides a system for editing genetic material. In one embodiment, the system comprises in one or more vectors: a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a retroviral IN or a fragment thereof, a Cas protein, and an NLS; a nucleic acid sequence encoding a CRISPR-Cas system guide RNA; and a nucleic acid sequence encoding a donor template nucleic acid, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence, and a donor template sequence.

[0058] In one aspect, the present invention provides a method for editing genetic material. In one embodiment, the method comprises administering a nucleic acid molecule of the present invention; 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, and a donor template sequence.

[0059] definition

[0060] Unless defined otherwise, 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 invention belongs.

[0061] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

[0062] Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (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), which are provided in this document.

[0063] The nomenclature used herein and the laboratory procedures utilized in analytical chemistry and organic syntheses described below are those well known and commonly employed in the art. Standard techniques or their modifications were used for chemical syntheses and chemical analyses.

[0064] The terms "a", "an", "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0065] As used herein, "about" when referring to a measurable value (such as an amount, a duration, etc.) is meant to encompass deviations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such deviations are appropriate for performing the disclosed methods.

[0066] "Antisense" refers in particular to a nucleic acid sequence of a non-coding strand of a double-stranded DNA molecule encoding a protein, or a sequence substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule encoding a protein. An antisense sequence need not be complementary only to the coding portion of the coding strand of a DNA molecule. An antisense sequence may be complementary to a specified regulatory sequence on the coding strand of a DNA molecule encoding a protein, and the regulatory sequence controls the expression of the coding sequence.

[0067] "Disease" is a health condition in an animal wherein the animal is unable to maintain homeostasis and wherein the animal's health will continue to deteriorate if the disease is not ameliorated.

[0068] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis but the animal's state of health is less favorable than it would be in the absence of the disorder. If untreated, the disorder will not necessarily result in a further decline in the animal's state of health.

[0069] A disease or condition is "reduced" if the signs or symptoms of the disease or condition are reduced in severity, the frequency with which the patient experiences such signs or symptoms, or both.

[0070] "Encoding" refers to the intrinsic properties and resulting biological properties of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA or mRNA) that serves as a template for synthesizing other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence in a biological process. Therefore, a gene encodes a protein if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence table) and the non-coding strand (used as a transcription template for a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

[0071] The terms "patient", "subject", "individual" and the like are used interchangeably herein and refer to any animal or cell thereof that is 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.

[0072] The term "specifically binds" as used herein with respect to antibodies refers to antibodies that recognize a specific antigen but do not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to the antigen from one or more species. However, this cross-species reactivity itself does not 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 the antigen. However, this cross-reactivity itself does not change the classification of the antibody as specific.

[0073] In some cases, the term "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical substance to refer to the interaction being dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, rather than binding to proteins broadly. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0074] 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 an mRNA molecule produced by transcription of the gene.

[0075] The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that match the anticodon region of the transfer RNA molecule or encode a stop codon during translation of the mRNA molecule. Thus, the coding region may 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 export signal sequence).

[0076] As used herein with respect to nucleic acid, "complementarity" refers to the broad concept of sequence complementarity between regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that the adenine residues of the first nucleic acid region can form specific hydrogen bonds ("base pairing") with the residues of the second nucleic acid region, and if the residues of the second nucleic acid region are thymine or uracil, the second nucleic acid region is antiparallel to the first region. Similarly, it is known that the cytosine residues of the first nucleic acid chain can base pair with the residues of the second nucleic acid chain, and if the residues of the second nucleic acid chain are guanine, the second nucleic acid chain is antiparallel to the first chain. If at least one nucleotide residue in the first region can base pair with the residues in the second region when the two regions are arranged in an antiparallel manner, the first region of the nucleic acid is complementary to the second region of the same or different nucleic acids. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first portion and the second portion are arranged in an antiparallel manner, 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 the nucleotide residues in the second portion. In one embodiment, all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0077] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0078] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0079] The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated in the production of, for example, antisense molecules, siRNA, ribozymes, etc. An expression vector may contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possible translation of an operably linked coding sequence in a specific host organism. In addition to control sequences for transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0080] The term "wild type" as used herein is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene, or characteristic that distinguishes it from mutant or variant forms as it occurs in nature.

[0081] The term "homology" refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705). This software matches similar sequences by assigning values ​​to the degree of homology of different substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0082] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in its normal environment in a living organism is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural environment is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment (e.g., a host cell).

[0083] When used in relation to nucleic acids, the term "isolated" as in "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, isolated nucleic acids exist in a form or environment different from that found in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) are found in the state in which they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome close to adjacent genes; an RNA sequence (e.g., a specific mRNA sequence encoding a specific protein) is found in a cell as a mixture with many other mRNAs encoding a variety of proteins. However, for example, an isolated nucleic acid includes such a nucleic acid in a cell that normally expresses the nucleic acid, wherein the nucleic acid is in a chromosomal position different from that of a natural cell, or is otherwise flanked by a different nucleic acid sequence 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 contains at least the sense or coding strand (ie, the oligonucleotide may be single-stranded), but may contain both the sense and antisense strands (ie, the oligonucleotide may be double-stranded).

[0084] The term "isolated" when used in reference to a polypeptide, as in "isolated protein" or "isolated polypeptide," refers to a polypeptide that has been identified and separated from at least one contaminant with which it is normally associated in its source. Thus, an isolated polypeptide exists in a form or environment different from that found in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) are found in the state in which they exist in nature.

[0085] "Nucleic acid" refers to any nucleic acid, whether it is composed of deoxyribonucleosides or ribonucleosides, and whether it is composed of phosphodiester linkages or modified linkages, such as phosphotriesters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamide esters, carbamates, thioethers, bridged phosphoramidates, bridged methylenephosphonates, phosphorothioates, methylphosphonates, phosphorodithioates, bridged phosphorothioates or sulfone linkages and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases that are different from five biologically existing bases (adenine, guanine, thymine, cytosine and uracil). The term "nucleic acid" generally refers to large polynucleotides.

[0086] Conventional notation is used herein to describe polynucleotide sequences: the left end of a single-stranded polynucleotide sequence is the 5'-end; the left direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction.

[0087] The direction in which nucleotides are added 5' to 3' to the nascent RNA transcript is called the transcription direction. The DNA strand with the same sequence as the mRNA is called the "coding strand"; the sequence on the DNA strand that is 5' to a reference point on the DNA is called the "upstream sequence"; the sequence on the DNA strand that is 3' to a reference point on the DNA is called the "downstream sequence".

[0088] "Expression cassette" refers to a nucleic acid molecule comprising a coding sequence operably linked to promoter / regulatory sequences necessary for the transcription and, optionally, translation of the coding sequence.

[0089] As used herein, the term "operably linked" refers to the linkage 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 subsequent protein molecule is produced. The term also refers to the linkage of amino acid encoding sequences in such a way that a functional (e.g., enzymatically active, capable of binding to a binding partner, capable of inhibition, etc.) protein or polypeptide is produced.

[0090] As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for expressing a gene product that is operably linked to a promoter / regulatory factor sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for expressing a gene product. The promoter / regulatory sequence may be, for example, a promoter / regulatory sequence that expresses a gene product in an inducible manner.

[0091] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence hybridizes primarily with the target sequence and does not substantially hybridize with non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with 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, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0092] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stable via hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson / Crick base pairing, Hoogstein binding or in any other sequence-specific manner. The complex may comprise two chains that form a duplex structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. Hybridization reactions may constitute steps in a broader process, such as the initiation of PCR or the cleavage of polynucleotides by enzymes. A sequence that is capable of hybridizing with a given sequence is referred to as the "complementary sequence" of a given sequence.

[0093] 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 produced substantially only in the presence of an inducer corresponding to the promoter.

[0094] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0095] The term "polypeptide" as used herein is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include but are not limited to all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes, using common cloning techniques and PCR, etc.) and by synthetic means.

[0096] In the context of the present invention, the following abbreviations for ubiquitous nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0097] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting 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 on the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the terms refer to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0098] The term "RNA" as used herein is defined as ribonucleic acid.

[0099] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally linked together. The amplified or assembled recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform a suitable host cell.

[0100] A recombinant polynucleotide may also serve a non-coding function (eg, promoter, origin of replication, ribosome binding site, etc.).

[0101] The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by the use of recombinant DNA methodologies.

[0102] As used herein, "transcription activator-like effector nuclease (TALEN)" is an artificial restriction enzyme produced by fusing the TAL effector DNA binding domain to the DNA cleavage domain. These reagents can achieve efficient, programmable and specific DNA cleavage, and represent a powerful tool for in situ editing of genetic material. Transcription activator-like effectors (TALEs) can be quickly engineered to bind to almost any DNA sequence. As used herein, the term TALEN is extensive and includes monomeric TALENs that can cleave double-stranded DNA without the help of another TALEN. The term TALEN can also be used to refer to one or two members of a pair of TALENs, which are engineered to work together to cleave DNA at the same site. The co-acting TALENs can be referred to as left-TALEN and right-TALEN, which reference the handedness of 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 herein by reference in their entirety).

[0103] As used herein, "variant" is a nucleic acid sequence or peptide sequence whose sequence is different from a reference nucleic acid sequence or peptide sequence, but retains the basic biological properties of the reference molecule. The changes in the sequence of nucleic acid variants may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may produce amino acid substitutions, additions, deletions, fusions and truncations. The changes in the sequence of peptide variants are usually limited or conservative, so that the sequences of the reference polypeptide and variant are generally closely similar and identical in many regions. Variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The variant of nucleic acid or peptide may be naturally occurring, such as an allelic variant, or may be a variant not known to be naturally occurring. The non-naturally occurring variants of nucleic acids and peptides may be prepared by mutagenesis techniques or by direct synthesis.

[0104] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Various vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic compounds or amphipathic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that help transfer 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.

[0105] Range: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that the description in range form is merely for convenience and brevity, and should not be interpreted as a hard limit to the scope of the invention. Therefore, the description of a range should be considered to have all possible sub-ranges disclosed as well as individual numerical values ​​within the range. For example, a description of a range such as 1 to 6 should be considered to have sub-ranges disclosed as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies to any width range.

[0106] Fusion Protein

[0107] In one aspect, the present invention is based on the development of novel fusions of editing proteins that are effectively delivered to the nucleus. In one aspect, the present invention provides a fusion protein comprising an editing protein and a nuclear localization signal (NLS) having a second amino acid sequence.

[0108] In one embodiment, editing proteins include, but are not limited to, CRISPR-associated (Cas) proteins, transcription activator-like effector-based nuclease (TALEN) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA binding domains.

[0109] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, 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, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both chains to cleave within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is Cas9. In one embodiment, the Cas protein is catalytically defective (dCas).

[0110] 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: 41-46. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs: 41-46.

[0111] 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, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmic protein (NPM2), nucleolar phosphatase (NPM1) or monkey vims 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 256. 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% identical to one of SEQ ID NOs: 47-56 and 254-257. In one embodiment, the NLS protein comprises the sequence of one of SEQ ID NOs: 47-56 and 254-257.

[0112] 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 and KKR motifs. 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 between 20 and 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: 275-887. In one embodiment, the Ty1-like NLS protein comprises the sequence of one of SEQ ID NOs: 275-887.

[0113] 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: 249-250. In one embodiment, the fusion protein comprises the sequence of one of SEQ ID NOs: 249-250.

[0114] In one aspect, the present invention is based 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 DNA integration activity of viral integrase and the programmable DNA targeting ability of catalytic death Cas. Therefore, since this fusion protein does not rely on cellular pathways for DNA insertion or requires a cellular energy source (such as ATP), this enzyme can work in many environments, such as from in vitro to prokaryotic cells, to fission or non-division eukaryotic cells. In addition, because integrase does not require homology regions for insertion, it is only specific for each integrase family. Small terminal motif sequences, these fusion protein editors can use a single DNA donor template for multiple genome integration (if guided by multiple guide RNAs).

[0115] Therefore, in one aspect, the present invention provides a fusion protein comprising a CRISPR-associated (Cas) protein having a first amino acid sequence, a nuclear localization signal (NLS) having a second amino acid sequence, and a retroviral integrase (IN) or a fragment or variant thereof having a third amino acid sequence.

[0116] In one embodiment, the retroviral IN is a human immunodeficiency virus (HIV) IN, a Rous sarcoma virus (RSV) IN, a mouse mammary tumor virus (MMTV) IN, a Moloney murine leukemia virus (MoLV) IN, a bovine leukemia virus (BLV) IN, a human T-lymphotropic virus (HTLV) IN, an avian sarcoma leukemia virus (ASLV) IN, a feline leukemia virus (FLV) IN, a xenotropic murine leukemia virus-related virus (XMLV) IN, a simian immunodeficiency virus (SIV) IN, a feline immunodeficiency virus (FIV) IN, an equine infectious anemia virus (EIAV) IN, a prototype foamy virus (PFV) IN, a simian foamy virus (SFV) IN, a human foamy virus (HFV) IN, a walleye cutaneous sarcoma virus (WDSV) IN, or a bovine immunodeficiency virus (BIV) IN.

[0117] 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.

[0118] In one embodiment, the retroviral IN is HIV IN. In one embodiment, HIV IN comprises one or more amino acid substitutions, wherein the substitutions improve catalytic activity, improve solubility or increase interaction with one or more host cell cofactors. In one embodiment, 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, HIV IN comprises amino acid substitutions F185K and C280S. In one embodiment, HIV IN comprises amino acid substitutions T97A and Y134R. In one embodiment, HIV IN comprises amino acid substitutions G140S and Q148H.

[0119] 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, a fragment of an integrase retains at least one activity of a full-length integrase. Retroviral integrase functions and fragments 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.

[0120] 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: 1-40. In one embodiment, the retroviral IN comprises a sequence of one of SEQ ID NOs: 1-40.

[0121] In some embodiments, the CRISPR-Cas domain comprises a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, 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, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRERSpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both strands to cleave within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0122] 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: 41-46. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs: 41-46.

[0123] 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, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmic protein (NPM2), nucleolar phosphatase (NPM1) or monkey vims 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 256. 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% identical to one of SEQ ID NOs: 47-56 and 254-257. In one embodiment, the NLS protein comprises the sequence of one of SEQ ID NOs: 47-56 and 254-257.

[0124] 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 and KKR motifs. 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 between 20 and 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: 275-887. In one embodiment, the Ty1-like NLS protein comprises the sequence of one of SEQ ID NOs: 275-887.

[0125] 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: 249-250. In one embodiment, the fusion protein comprises the sequence of one of SEQ ID NOs: 249-250.

[0126] In one embodiment, the NLS comprises a combination of two different NLSs. For example, in one embodiment, the NLS comprises an NLS derived from Ty1 and an NLS derived from SV40. In one embodiment, the NLS is an NLS derived from Ty1 or Ty1, an NLS derived from Ty2 or Ty2, or an NLS derived from MAK11 or MAK11. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 256.

[0127] 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.

[0128] In one embodiment, the NLS comprises 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% identical to one of SEQ ID NOs: 47-56, 254-257, and 275-887, and 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% identical to one of SEQ ID NOs: NO: 47-56, 254-257 and 275-887 are at least 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 a second sequence. In one embodiment, the first sequence and the second sequence are identical. In one embodiment, the first sequence and the second sequence are different.

[0129] In one embodiment, the fusion protein comprises a 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%, 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: 57-98. In one embodiment, the fusion protein comprises the sequence of one of SEQ ID NOs: 57-98.

[0130] The peptides of the present invention can be prepared using chemical methods. For example, the peptides can be synthesized by solid phase techniques (Roberge JY et al. (1995) Science 269: 202-204), cleaved from resin, and purified by preparative high performance liquid chromatography. Automatic synthesis can be achieved, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0131] The present invention should also be interpreted as including any form of peptides having substantial homology to the fusion proteins disclosed herein. In one embodiment, a "substantially homologous" peptide has about 50% homology, about 70% homology, about 80% homology, about 90% homology, about 95% homology, or about 99% homology to the amino acid sequence of the fusion proteins disclosed herein.

[0132] The peptides may alternatively be prepared by recombinant means or by cleavage from longer polypeptides. The composition of the peptides may be confirmed by amino acid analysis or sequencing.

[0133] Variants of peptides according to the present invention may be: (i) peptides in which one or more of the amino acid residues are substituted with conservative or non-conservative amino acid residues and such substituted amino acid residues may or may not be residues encoded by the genetic code, (ii) peptides in which there are one or more modified amino acid residues (e.g., residues modified by connecting substituents), (iii) peptides in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptide and / or (v) peptides in which the peptide is fused to another peptide (such as a leader sequence or secretory sequence or a sequence used for purification (e.g., His-tag) or for detection (e.g., Sv5 epitope tag). The fragments include peptides generated by proteolytic cleavage of the original sequence (including multi-site proteolysis). Variants may be post-translationally modified or chemically modified. Such variants should be within the scope of those skilled in the art according to the teachings herein.

[0134] As known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitute with the sequence of the second polypeptide. Variants are defined as peptide sequences that include different from the original sequence. In one embodiment, the variant is different from the original sequence in less than 40% of the residues in each target segment, different from the original sequence in less than 25% of the residues in each target segment, different from the original sequence in less than 10% of the residues in each target segment, or different from the original sequence in only some residues in each target segment, and is sufficiently homologous to the original sequence at the same time, to retain the functionality of the original sequence and / or stimulate stem cells to differentiate into the ability of osteoblast lineage. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods widely 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., NCBINLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)].

[0135] The peptide of the present invention can be post-translationally modified. For example, post-translation modifications falling within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events need to introduce additional biological machinery. For example, processing events (such as signal peptide cleavage and core glycosylation) are tested by adding canine microsomal membranes or African clawed frog egg extracts (U.S. Patent number 6,103,489) to standard translation reactions.

[0136] The peptides of the invention may comprise unnatural amino acids formed by post-translational modification or by the introduction of unnatural amino acids during translation. Various methods are available for the introduction of unnatural amino acids during protein translation.

[0137] The peptides or proteins of the present invention can be phosphorylated using conventional methods, such as the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992).

[0138] Cyclic derivatives of the peptides of the present invention are also part of the present invention. Cyclization can make the peptide present a conformation that is more favorable for association with other molecules. Cyclization can be achieved 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 the amino group of one component and the carboxyl group of another component. Cyclization can also be achieved using azobenzene-containing amino acids, as described by Ulysse, L., et al., J.Am.Chem.Soc.1995,117,8466-8467. The components forming the bond can be side chains of amino acids, non-amino acid components, or a combination of the two. In one embodiment of the present invention, the cyclic peptide may contain a β-turn in the right position. The β-turn can be introduced into the peptide of the present invention by adding the amino acid Pro-Gly at the right position.

[0139] It may be desirable to produce a more flexible cyclic peptide than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide can be prepared by introducing cysteines at the right and left positions of the peptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to deform the β-sheet and the β-turn. Due to the length of the disulfide bonds in the β-sheet portion and the smaller number of hydrogen bonds, the peptide is more flexible. The relative flexibility of the cyclic peptide can be determined by molecular dynamics simulation.

[0140] The present invention also relates to peptides fused to a target protein and / or capable of directing a chimeric protein to a desired cellular component or cell type or tissue targeting domain or integrated into the target protein and / or targeting domain, the peptide comprising an IN-Cas9 peptide. The chimeric protein may also contain additional amino acid sequences or domains. The chimeric protein is recombinant because the various components come from different sources and because such components do not exist together in nature (i.e., are heterologous).

[0141] In one embodiment, the targeting domain can be a transmembrane domain, a membrane binding domain, or a sequence that directs the protein to associate, for example, with a vesicle or with a 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 a cell surface ligand or antibody to a cell surface antigen of the target tissue. The targeting domain can target the peptides of the invention to cellular components.

[0142] The peptide of the present invention 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 adopt solid phase or liquid phase synthesis methods (see, for example, about solid phase synthesis technology JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and RBM Merrifield, The Peptides: Analysis Synthesis, Biology edited E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; and about classical solution synthesis M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, editors, The Peptides: Analysis, Synthesis, Biology, suprs, Vol. 1). By way of example, the peptides of the invention can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry, wherein phosphothreonine is directly incorporated as an N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0143] The N-terminal or C-terminal fusion protein comprising a peptide or chimeric protein of the present invention conjugated to other molecules can be prepared by recombinant technology by fusing the N-terminal or C-terminal of the peptide or chimeric protein with a sequence of a selected protein or a selective marker having a desired biological function. The resulting fusion protein contains an IN-Cas9 peptide fused to a selected protein or marker protein as described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0144] The peptides of the present invention can be developed using biological expression systems. Using these systems allows large libraries of random peptide sequences to be generated and these libraries to be screened for peptide sequences that bind to specific proteins. Libraries can be generated by cloning synthetic DNA encoding random peptide sequences into appropriate expression vectors (see, Christian 1992, J. Mol. Biol. 227: 711; Devlin, 1990 Science 249: 404; Cwirla 1990, Proc. Natl. Acad, Sci. USA, 87: 6378). Libraries can also be constructed by parallel synthesis of overlapping peptides (see U.S. Patent No. 4,708,871).

[0145] The peptides and chimeric proteins of the present invention can be converted into pharmaceutical salts by reacting with inorganic acids, such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., 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.

[0146] Nucleic Acids

[0147] In one embodiment, the present invention is a nucleic acid molecule encoding a fusion protein. In one embodiment, the nucleic acid molecule comprises a first nucleic acid sequence encoding an editing protein; and a second nucleic acid sequence encoding a nuclear localization signal (NLS).

[0148] In one embodiment, the editing protein includes but is not limited to CRISPR-associated (Cas) proteins, transcription activator-like effector-based nuclease (TALEN) proteins, zinc finger nuclease (ZFN) proteins, and proteins with DNA binding domains. In one embodiment, the editing protein is a Cas protein.

[0149] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, 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, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, SpCas9, StCas9, NmCas9, SaCas9, CjCas9, CjCas9, AsCpf1, LbCpf1, FnCpf1, VRER SpCas9, VQR SpCas9, xCas9 3.7, homologs thereof, orthologs thereof, or modified versions thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs the cleavage of one or both strands of a nucleic acid molecule at a target sequence position (e.g., within the target sequence and / or within the complement of the target sequence). In some embodiments, the Cas protein directs one or both chains to cleave within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is Cas9. In one embodiment, the Cas protein is catalytically defective (dCas).

[0150] In one embodiment, the first 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%, 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: 41-46. In one embodiment, the first nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding one of SEQ ID NOs: 41-46.

[0151] In one embodiment, the first 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%, 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: 139-144. In one embodiment, the first nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 139-144.

[0152] In one embodiment, the second nucleic acid sequence encodes a nuclear localization signal (NLS). 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, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmic protein (NPM2), nucleolar phosphatase (NPM1) or monkey vims40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO:51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 256.

[0153] 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 and KKR motifs. 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 between 20 and 40 amino acids.

[0154] In one embodiment, the retrotransposon NLS increases nuclear localization. In one embodiment, the retrotransposon NLS increases nuclear localization significantly more than a non-retrotransposon NLS.

[0155] In one embodiment, the second nucleic acid sequence encoding the NLS 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%, 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 nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding one of SEQ ID NOs: 47-56, 254-257, and 275-887.

[0156] In one embodiment, the second nucleic acid sequence encoding the NLS 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: 145-154. In one embodiment, the second nucleic acid sequence encoding the NLS comprises a nucleic acid sequence of one of SEQ ID NOs: 145-154.

[0157] In one embodiment, the nucleic acid molecule 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: 249-250. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising the sequence of one of SEQ ID NOs: 249-250.

[0158] In one embodiment, the nucleic acid molecule comprises: a first nucleic acid sequence encoding an editing protein; a second nucleic acid sequence encoding a nuclear localization signal (NLS); and a third nucleic acid sequence encoding a retroviral integrase (IN) or a fragment thereof.

[0159] In one embodiment, the retroviral IN is a human immunodeficiency virus (HIV) IN, a Rous sarcoma virus (RSV) IN, a mouse mammary tumor virus (MMTV) IN, a Moloney murine leukemia virus (MoLV) IN, a bovine leukemia virus (BLV) IN, a human T-lymphotropic virus (HTLV) IN, an avian sarcoma leukemia virus (ASLV) IN, a feline leukemia virus (FLV) IN, a xenotropic murine leukemia virus-related virus (XMLV) IN, a simian immunodeficiency virus (SIV) IN, a feline immunodeficiency virus (FIV) IN, an equine infectious anemia virus (EIAV) IN, a prototype foamy virus (PFV) IN, a simian foamy virus (SFV) IN, a human foamy virus (HFV) IN, a walleye cutaneous sarcoma virus (WDSV) IN, or a bovine immunodeficiency virus (BIV) IN.

[0160] In one embodiment, the retroviral IN is HIV IN. In one embodiment, HIV IN comprises one or more amino acid substitutions, wherein the substitutions improve catalytic activity, improve solubility or increase interaction with one or more host cell cofactors. In one embodiment, 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, HIV IN comprises amino acid substitutions F185K and C280S. In one embodiment, HIV IN comprises amino acid substitutions T97A and Y134R. In one embodiment, HIV IN comprises amino acid substitutions G140S and Q148H.

[0161] 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, a fragment of an integrase retains at least one activity of a full-length integrase. Retroviral integrase functions and fragments 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.

[0162] In one embodiment, the third nucleic acid sequence encoding a 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%, 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: 1-40. In one embodiment, the third nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding one of SEQ ID NOs: 1-40.

[0163] In one embodiment, the third nucleic acid sequence encoding a 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: 99-138. In one embodiment, the third nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence of one of SEQ ID NOs: 99-138.

[0164] In one embodiment, the editing protein includes but is not limited to CRISPR-associated (Cas) proteins, nucleases (TALEN) proteins based on transcription activator-like effectors, zinc finger nucleases (ZFN) proteins, and DNA-binding proteins. In one embodiment, the editing protein is a Cas protein. In one embodiment, the Cas protein is Cas9, Cas13, or Cpf1. In one embodiment, the Cas protein is a catalytically defective (dCas).

[0165] In one embodiment, the first nucleic acid sequence encodes a Cas protein. In one embodiment, the first 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%, 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: 41-46. In one embodiment, the first nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding one of SEQ ID NOs: 41-46.

[0166] In one embodiment, the first 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%, 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: 139-144. In one embodiment, the first nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 139-144.

[0167] In one embodiment, the second nucleic acid sequence encodes a nuclear localization signal (NLS). 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, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein, nucleoplasmic protein (NPM2), nucleolar phosphatase (NPM1) or monkey vims40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO:51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO: 256.

[0168] In one embodiment, the retrotransposon NLS increases nuclear localization. In one embodiment, the retrotransposon NLS increases nuclear localization significantly more than a non-retrotransposon NLS.

[0169] In one embodiment, the second nucleic acid sequence encoding the NLS 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%, 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 275-87. In one embodiment, the second nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding one of SEQ ID NOs: 47-56, 254-257, and 275-887.

[0170] In one embodiment, the second nucleic acid sequence encoding the NLS 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: 145-154. In one embodiment, the second nucleic acid sequence encoding the NLS comprises a nucleic acid sequence of one of SEQ ID NOs: 145-154.

[0171] In one embodiment, the nucleic acid molecule 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: 57-98. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising the sequence of one of SEQ ID NOs: 57-98.

[0172] 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: 155-196. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence of one of SEQ ID NOs: 155-196.

[0173] The isolated nucleic acid sequence encoding the fusion protein can be obtained using any of the many recombinant methods known in the art, such as by screening a library from cells expressing the gene, by obtaining the gene from a vector known to contain the gene, or by isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the target gene can be produced synthetically rather than cloned.

[0174] The nucleic acid of separation can comprise any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition comprises the DNA molecule of separation, including, for example, the cDNA molecule of separation encoding the fusion protein of the present invention. In one embodiment, the composition comprises the RNA molecule of separation encoding the fusion protein of the present invention or its functional fragment.

[0175] Nucleic acid molecules of the present invention can be modified to improve stability in serum or in a growth medium for cell culture. Modifications can be added to enhance the stability, functionality and / or specificity of nucleic acid molecules of the present invention and to minimize immunostimulatory properties. For example, in order to enhance stability, 3'-residues can be stabilized for degradation, for example, they can be selected so that they consist of purine nucleotides, specifically adenosine or guanosine nucleotides. Alternatively, replacing pyrimidine nucleotides with modified analogs, for example replacing uridine with 2'-deoxythymidine is tolerable and does not affect the function of the molecule.

[0176] In one embodiment of the invention, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0177] The limiting examples of nucleotide analogs include sugar-modified and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to include at least one of nitrogen or sulfur heteroatoms. In exemplary backbone-modified ribonucleotides, the phosphate groups connected to adjacent ribonucleotides are replaced by modified groups (e.g., modified groups of thiophosphate groups). In exemplary sugar-modified ribonucleotides, 2'OH-groups are replaced by groups selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0178] Other examples of modifications are ribonucleotides modified with nucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8 position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. It should be noted that the above modifications may be combined.

[0179] In some cases, nucleic acid molecules include at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modifications of one or more nucleotides. In certain embodiments, nucleic acid molecules of the present invention may have enhanced resistance to nucleases. For increased nuclease resistance, nucleic acid molecules may include, for example, 2'-modified ribose units and / or thiophosphate linkages. For example, 2' hydroxyl (OH) can be modified or replaced using a variety of different "oxy" or "deoxy" substituents. For enhanced core base resistance, nucleic acid molecules of the present invention may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate linkages. Comprising locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene-bridged nucleic acid) and certain core base modifications (such as 2-amino-A, 2-thio (e.g., 2-thio-U), G- clamp modifications) may also increase binding affinity to the target.

[0180] In one embodiment, the nucleic acid molecule comprises 2'-modified nucleotides, 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-methylacetamido (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.

[0181] In certain embodiments, nucleic acid molecules of the invention have one or more of the following properties:

[0182] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA and polymers modified, for example, to improve efficacy RNA and DNA and nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of nucleic acid (i.e., sugar, base and phosphate moiety) are identical or substantially identical to the components present in nature or to the components naturally present in the human body. Rare or unusual but naturally occurring RNA is referred to as modified RNA in the art, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or unusual RNA (often referred to as modified RNA) is usually the result of post-transcriptional modification and is within the term unmodified RNA as used herein. Modified RNA as used herein refers to one or more of the components of nucleic acid (i.e., sugar, base and phosphate moiety) that are different from the components present in nature, or different from the components present in the human body. Although they are referred to as "modified RNA", because of modification, they certainly include molecules that are not strictly RNA. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct (e.g., an uncharged mimetic of the ribose phosphate backbone) that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with the ribose phosphate backbone.

[0183] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobases.

[0184] The present invention also includes a vector into which the isolated nucleic acid of the present invention is inserted. There are many suitable vectors that can be used in the present invention in the art.

[0185] In short, the expression of the natural or synthetic nucleic acid encoding the fusion protein of the present invention is usually achieved by operably connecting the nucleic acid encoding the fusion protein of the present invention or its part to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication in eukaryotic cells and is optionally integrated into eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0186] The vectors of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0187] The isolated nucleic acid of the present invention can be cloned into various types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0188] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, and is described in, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site and one or more selective markers (such as WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193) that function in at least one organism.

[0189] Delivery systems and methods

[0190] In one aspect, the present invention relates to the development of novel lentiviral packaging and delivery systems. Lentiviral particles deliver viral enzymes as proteins. In this way, the lifespan of lentiviral enzymes is short, thereby limiting the possibility of off-target editing due to long-term expression throughout the entire life cycle of the cell. Considering that their required activity is only required for a short period of time, it is advantageous to incorporate editing components or traditional CRISPR-Cas editing components as proteins into lentiviral particles. Therefore, in one embodiment, the present invention provides a lentiviral delivery system, as well as a method for delivering a composition of the present invention, editing genetic material, and using a lentiviral delivery system for nucleic acid delivery.

[0191] 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.

[0192] 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. Therefore, 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 have specificity for the integrase in the Insctriptr packaging plasmid.

[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 envelope protein. In one embodiment, the envelope protein can be selected based on the desired cell type.

[0194] In one embodiment, packaging plasmids, transfer plasmids, and envelope plasmids are introduced into cells. In one embodiment, cells transcribe and translate nucleic acid sequences encoding modified gag-pol proteins to produce modified gag-pol proteins. In one embodiment, cells transcribe nucleic acid sequences encoding sgRNA. In one embodiment, sgRNA binds to integrase-Cas fusion proteins. In one embodiment, cells transcribe and translate nucleic acid sequences encoding envelope proteins to produce envelope proteins. In one embodiment, cells transcribe donor sequences to provide donor sequence RNA molecules. In one embodiment, modified gag-pol proteins bound to sgRNA, envelope polyproteins, and donor sequence RNA are packaged into viral particles. In one embodiment, viral particles are collected from cell culture media. In one embodiment, viral particles transduce target cells, wherein sgRNA binds to target regions of cell DNA, thereby targeting IN-Cas9 fusion proteins, and integrase catalyzes the integration of donor sequences into cell DNA.

[0195] 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.

[0196] 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. Therefore, 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 to the integrase in the VPR-IN-dCas packaging plasmid.

[0197] 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 can be selected based on the desired cell type.

[0198] In one embodiment, the VPR-IN-dCas plasmid comprises a nucleic acid sequence encoding a fusion protein comprising 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 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 VPR, an integrase, and a dCas protein. In one embodiment, the fusion protein comprises a protease cleavage site between VPR and an integrase. In one embodiment, the VPR-IN-dCas plasmid packaging plasmid further comprises a sequence encoding an sgRNA sequence.

[0199] In one embodiment, a packaging plasmid, a transfer plasmid, an envelope plasmid, and a VPR-IN-dCas plasmid are introduced into a 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 a VPR-integrase-dCas fusion protein.

[0200] In one embodiment, the gag-pol protein, the envelope polyprotein, the donor sequence RNA, and the VPR-integrase-dCas9 protein bound to the sgRNA are packaged into viral particles. In one embodiment, the viral particles are collected from the cell culture medium. In one embodiment, the VPR is cleaved from the fusion protein in the viral particles via a protease site to provide an IN-dCas fusion protein. In one embodiment, the viral particles transduce the target cells, wherein the sgRNA binds to the target region of the cell DNA, thereby targeting the IN-dCas fusion protein, and the integrase catalyzes the integration of the donor sequence into the cell DNA.

[0201] 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.

[0202] In one embodiment, the transfer plasmid comprises a nucleic acid encoding sgRNA and a nucleic acid sequence encoding a fusion protein, and the fusion protein comprises 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, 3'LTR is a self-inactivating (SIN) LTR. Therefore, in one embodiment, 5'LTR comprises a U3 sequence, an R sequence, and a U5 sequence, and 3'LTR comprises an R sequence and a U5 sequence but does not comprise a U3 sequence. In one embodiment, 5'LTR and 3'LTR are specific to 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, 5'LTR and 3'LTR are located on the flanks of the sequence encoding the fusion protein and the sequence encoding the sgRNA.

[0203] 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 can be selected based on the desired cell type.

[0204] In one embodiment, the packaging plasmid, transfer plasmid, and envelope plasmid are introduced into the cell. In one embodiment, the cell transcribes and translates the nucleic acid sequence encoding the gag-pol protein to produce the gag-pol polyprotein. In one embodiment, the cell transcribes and translates the nucleic acid sequence encoding the envelope protein to produce the envelope protein. In one embodiment, the cell transcribes the nucleic acid sequence encoding the sgRNA. In one embodiment, the cell transcribes the nucleic acid sequence encoding the fusion protein.

[0205] In one embodiment, the gag-pol protein, the envelope polyprotein, the donor sequence RNA, and the VPR-integrase-dCas9 protein bound to the sgRNA are packaged into viral particles. In one embodiment, the viral particles are collected from the cell culture medium. In one embodiment, the viral particles transduce the target cells, wherein the virus is reverse translated, and the cells express the fusion protein and the sgRNA. In one embodiment, the sgRNA binds to the Cas protein of the fusion protein and another viral DNA transcript, wherein the integrase catalyzes its own integration. In one embodiment, the sgRNA binds to the Cas protein of the fusion protein and the target region of the cell DNA, thereby destroying the target gene.

[0206] 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.

[0207] 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 can be selected based on the desired cell type.

[0208] In one embodiment, the transfer plasmid comprises a nucleic acid encoding an sgRNA and a nucleic acid sequence encoding a fusion protein, the 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 from a different type of lentivirus compared to the gag-pol polyproteins 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, different lentiviral species are used to prevent self-integration.

[0209] 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. Therefore, 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 are located on the flanks of the sequence encoding the fusion protein and the sequence encoding the sgRNA.

[0210] In one embodiment, the transfer plasmid comprises a donor sequence. The donor sequence can be any nucleic acid sequence to be delivered to genome. In one embodiment, the transfer plasmid comprises a 5' long terminal repeat (LTR) sequence and a 3' LTR sequence. In one embodiment, 3' LTR is a self-inactivating (SIN) LTR. Therefore, in one embodiment, 5' LTR comprises a U3 sequence, an R sequence and a U5 sequence, and 3' LTR comprises an R sequence and a U5 sequence but does not comprise a U3 sequence. In one embodiment, 5' LTR and 3' LTR have specificity to the integrase in the Inscrtipter transfer plasmid.

[0211] 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 the nucleic acid sequence encoding the gag-pol protein to produce the gag-pol polyprotein. In one embodiment, the cell transcribes and translates the nucleic acid sequence encoding the envelope protein to produce the envelope protein. In one embodiment, the cell transcribes the nucleic acid sequence encoding the sgRNA. In one embodiment, the cell transcribes the nucleic acid sequence encoding the fusion protein. In one embodiment, the gag-pol protein, the envelope polyprotein, the gRNA, and the fusion protein RNA are packaged into the first virus particle. In one embodiment, the first virus particle is collected from the cell culture medium.

[0212] In one embodiment, a second packaging plasmid, a transfer plasmid, and a 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 collected from the cell culture medium.

[0213] In one embodiment, the first packaging plasmid, transfer plasmid, first envelope plasmid, second packaging plasmid, transfer plasmid and second envelope plasmid are introduced into the same cell. In one embodiment, the first packaging plasmid, transfer plasmid, first envelope plasmid and the second packaging plasmid, transfer plasmid and second envelope plasmid are introduced into different cells.

[0214] In one embodiment, the first viral particle and the second viral particle transduce the target cell. In one embodiment, the virus reverse translates, and the cell expresses the fusion protein and sgRNA, wherein 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, which binds to the integrase of the fusion protein. In one embodiment, the sgRNA binds to the target region of the cell DNA, thereby targeting the IN-dCas fusion protein, and the integrase catalyzes the integration of the donor DNA sequence into the cell DNA.

[0215] In addition, a variety of other virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0216] For example, vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its progeny in daughter cells. Lentiviruses have the added advantage over those derived from oncogenic retroviruses (such as murine leukemia viruses) because lentiviruses can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0217] 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, and the adeno-associated virus serotype includes but is 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 treating various diseases. AAV vectors have a variety of features that make them ideally suitable for gene therapy, including lack of pathogenicity, minimum immunogenicity and the ability to transduce post-mitotic cells in a stable and efficient manner. The expression of the specific gene contained in the AAV vector can be specifically targeted to one or more types of cells by selecting an appropriate combination of AAV serotype, promoter and delivery method.

[0218] AAV vectors may have one or more of the AAV wild-type genes, preferably the rep and / or cap genes, completely or partially deleted, but retain functional flanking ITR sequences. Despite their high homology, different serotypes have tropisms for different tissues. The receptor for AAV1 is unknown; however, it is known that AAV1 transduces skeletal and cardiac muscle more efficiently than AAV2. Since most studies have been done with pseudotyped vectors, in which vector DNA flanked by AAV2 ITRs is packaged into capsids of alternative serotypes, it is clear that the biological differences are related to the capsid rather than the genome. Recent evidence suggests that DNA expression cassettes packaged in AAV 1 capsids are at least 1 log 10 more efficient at transducing cardiomyocytes than DNA expression cassettes packaged in 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).

[0219] The desired AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3 and hypervariable regions; rep proteins, including rep 78, rep 68, rep 52 and rep 40; and sequences encoding these proteins. These fragments can be easily used 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, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be produced by any suitable technique using a combination of selected AAV sequences (e.g., fragments of vp1 capsid proteins) and heterologous sequences, which can be obtained from different selected AAV serotypes, non-continuous parts of the same AAV serotype, non-AAV viral sources, or non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Thus, exemplary AAVs or artificial AAVs suitable for expressing one or more proteins include 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. WO2005 / 033321), AAV2 / 6 (U.S. Pat. No. 6,156,303), and AAVrh8 (International Patent Publication No. WO2003 / 042397), among others.

[0220] In certain embodiments, the vector further comprises conventional control elements, which are operably connected to the transgene in a manner that allows it to be transcribed, translated and / or expressed in cells transfected with a plasmid vector or infected with a virus produced by the present invention. As used herein, an "operably connected" sequence includes both an expression control sequence adjacent to the target gene and an expression control sequence that acts in trans or at a distance to control the target gene. Expression control sequences include: appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product when necessary. A variety of expression control sequences (including natural, constitutive, inducible and / or tissue-specific promoters) are known in the art and can be utilized.

[0221] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these promoters are located in the 30-110bp region upstream of the start site, but many promoters have recently been shown to also contain functional elements downstream of the start site. The intervals between promoter elements are often flexible, so that promoter function is retained when elements are reversed or moved relative to each other. In the thymidine kinase (tk) promoter, before the activity begins to decline, the intervals between promoter elements can be increased to 50bp apart. Depending on the promoter, it seems that individual elements can act collaboratively or independently to activate transcription.

[0222] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a powerful constitutive promoter sequence that is capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may 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 (Epstein-Barr) virus immediate early promoter, Rous (Rous) sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters may also be considered as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to it when such expression is desired, or turn off such expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0223] The enhancer sequence present on the vector also regulates the expression of the gene contained therein. Generally, the enhancer is combined with a protein factor to enhance the transcription of the gene. The enhancer can be located upstream or downstream of the gene it regulates. The enhancer can also be tissue-specific to enhance the transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention contains one or more enhancers to strengthen the transcription of the gene present in the vector.

[0224] In order to evaluate the expression of fusion protein of the present invention, the expression vector to be introduced into the cell may also contain a selective marker gene or a reporter gene or both, so as to help identify and select expressing cells from a cell population attempted to be transfected or infected by a viral vector. In other aspects, the selective marker may be carried on a separate DNA and used in a co-transfection procedure. Both the selective marker and the reporter gene may be flanked by appropriate regulator sequences to enable expression in host cells. Useful selective markers include, for example, antibiotic resistance genes, such as neo, etc.

[0225] Reporter gene is used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter gene is a gene that is not present in a recipient organism or tissue or is not expressed by the recipient organism or tissue and encodes a polypeptide that is expressed by some properties (e.g., enzymatic activity) that are easy to detect. At the appropriate time after DNA has been introduced into the recipient cell, the expression of reporter gene is measured. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression system is well known, and known technology can be used to prepare or commercially obtain. In general, the construct with the minimum 5' flanking region of the highest level expression of display reporter gene is identified as promoter. Such promoter region can be connected to reporter gene, and is used to assess the ability of promoter-driven transcription.

[0226] Methods for introducing genes into cells and expressing genes are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells (e.g., mammals, bacteria, yeast or insect cells) by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means.

[0227] 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 exogenous 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.

[0228] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0229] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems used as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

[0230] In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used for introducing nucleic acids into a host cell (in vitro, in vitro or in vivo). In another aspect, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule associated with both the liposome and the oligonucleotide, embedded in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids in the form of a suspension, contained in micelles or compounded with micelles, or otherwise associated with lipids. The composition associated with lipids, lipid / DNA or lipid / expression vectors is not limited to any specific structure in the solution. For example, they may be present in a double-layer structure, exist in the form of micelles, or have a "collapsed" structure. They may also be only dispersed in the solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances, and they can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0231] Lipids suitable for use are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dihexadecyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids 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 covering a variety of monolayer and multilayer lipid vehicles that are formed by generating closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They are formed spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components rearrange themselves before forming a closed structure, and water and dissolved solutes are embedded between lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions having different structures in solution compared with normal vesicle structures are also contemplated. For example, lipids can present micellar structures or exist only in the form of non-uniform lipid molecule aggregates. Lipofectamine-nucleic acid complexes are also considered.

[0232] Regardless of the method used to introduce the exogenous nucleic acid into the host cell, various assays can be performed to confirm that the recombinant DNA sequence is present in the host cell. Such assays include, for example, "molecular biology" 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 droplet polypeptides, such as by immunological means (ELISA and Western blotting) or by assays described herein to identify agents falling within the scope of the present invention.

[0233] system

[0234] In one aspect, the present invention provides a system for editing genetic material such as nucleic acid molecules, genomes or genes. In one embodiment, the system comprises in one or more vectors: a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises 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, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence and a donor template sequence. In one embodiment, the CRISPR-Cas system guide RNA substantially hybridizes with a target DNA sequence in the gene.

[0235] In one embodiment, the system comprises in one or more vectors: a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises 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, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence, and a donor template sequence. In one embodiment, the first CRISPR-Cas system guide RNA is substantially hybridized with the first DNA sequence, and the second CRISPR-Cas system guide RNA is substantially hybridized with the second DNA sequence. In one embodiment, the first DNA sequence and the second DNA sequence are located on the flanks of the target insertion region. In one embodiment, the system catalyzes the insertion of the donor template nucleic acid into the target insertion region.

[0236] In one embodiment, the system comprises in one or more vectors: a nucleic acid sequence encoding a first fusion protein, wherein the first fusion protein comprises 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, wherein the second fusion protein comprises 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, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence, and a donor template sequence.

[0237] 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 HIV IN or a fragment thereof, dCas9 protein and NLS; and the second fusion protein comprises BIV IN or a fragment thereof, Cpf1 Cas protein and NLS.

[0238] 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 HIV IN or a fragment thereof, a dCas9 protein, and an NLS; the second fusion protein comprises BIV IN or a fragment thereof, a Cpf1 Cas protein, and an NLS; the U3 sequence is specific for HIV IN, and the U5 sequence is specific for BIV IN.

[0239] 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 a donor template nucleic acid into the target insertion region.

[0240] In one embodiment, the system comprises a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises 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, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence, and a donor template sequence.

[0241] 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 on the same or different vectors.

[0242] 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: 57-98. In one embodiment, the nucleic acid sequence encoding the fusion protein encodes a fusion protein comprising the sequence of one of SEQ ID NOs: 57-98.

[0243] 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: 155-196. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a nucleic acid sequence of one of SEQ ID NOs: 155-196.

[0244] In one embodiment, the U3 sequence and the U5 sequence are specific for a retroviral IN. For example, in one embodiment, the retroviral IN is HIV 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: 197, and the U5 sequence comprises a sequence that is 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: 197. NO:198 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 the sequence.

[0245] In one embodiment, the retroviral IN is a 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: 199, and the U5 sequence comprises a sequence that is 95% identical to SEQ ID NO: 200.

[0246] In one embodiment, the retroviral IN is a 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: 201, and the U5 sequence comprises a sequence that is 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: 201. NO:202 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 the sequence.

[0247] In one embodiment, the retroviral IN is an 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: 203, and the U5 sequence comprises a sequence that is 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. NO:204 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 the sequence.

[0248] In one embodiment, the retroviral IN is a 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: 205, and the U5 sequence comprises a sequence that is 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: 205. NO:206 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 the sequence.

[0249] 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: 207, and the U5 sequence comprises a sequence that is 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: 207. NO:208 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 the sequence.

[0250] In one embodiment, the retroviral IN is a 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: 209, and the U5 sequence comprises a sequence that is 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: 209. NO:210 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 a sequence.

[0251] In one embodiment, the retroviral IN is a 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: 211, and the U5 sequence comprises a sequence that is 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. NO:212 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 the sequence.

[0252] In one embodiment, the retroviral IN is a 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: 213, and the U5 sequence comprises a sequence that is 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: 214. NO:214 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 the sequence.

[0253] In one embodiment, the retroviral IN is a 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: 215, and the U5 sequence comprises a sequence that is 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: 215. NO:216 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 the sequence.

[0254] 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: 217, and the U5 sequence comprises a sequence that is 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: 217. NO:218 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 the sequence.

[0255] In one embodiment, IN is TY1 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: 219, 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: 219. NO:220 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 a sequence.

[0256] In one embodiment, the IN is an InsF IN, and the U3 sequence is an IS3 IRL sequence, and the U5 sequence is an IS3 IRR sequence. In one embodiment, the IN is an InsF 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: 221, and the U5 sequence comprises a sequence that is 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: 221. NO:222 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 the sequence.

[0257] Systems and vectors can be designed to express 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, the recombinant expression vector can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and a T7 polymerase.

[0258] The vector can be introduced into a prokaryotic organism and propagated therein. In some embodiments, the prokaryotic organism is used to amplify the vector to be introduced into a eukaryotic cell or as a copy of the intermediate vector in the vector to be introduced into a eukaryotic cell (for example, amplification plasmid as part of a viral vector packaging system). In some embodiments, the prokaryotic organism is used to amplify multiple copies of the vector 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. The expression of proteins in prokaryotes is most often carried out in Escherichia coli with a vector containing a constitutive or inducible promoter that directs the expression of fusion proteins or non-fusion proteins. The fusion vector adds many amino acids to the protein encoded therein, such as being added to the amino terminus of the recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing the expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) helping to purify the recombinant protein by acting as a ligand in affinity purification. Often, in a fusion expression vector, a protein cleavage site is introduced at the junction of the fusion part and the recombinant protein so that the recombinant protein can be separated from the fusion part after the fusion protein is purified. Such enzymes and their homologous 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 to the target recombinant protein, respectively.

[0259] 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 INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0260] In some embodiments, the vector 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 (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.).

[0261] In some embodiments, the vector uses a baculovirus expression vector to drive protein expression in insect cells. Baculovirus vectors that can be used to express proteins 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).

[0262] In some embodiments, a mammalian expression vector is used, and the vector is capable of driving one or more sequences to be expressed in mammalian cells. 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 function of the expression vector is generally provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, simian virus 40, and other promoters disclosed herein and known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, see, for example, Sambrook, et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 16 and 17.

[0263] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (eg, using tissue-specific regulatory elements to express the nucleic acid). 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), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters for T-cell receptors (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, such as the mouse hox promoter (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546).

[0264] In some embodiments, the regulatory element is operably connected 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 spaced short palindromic repeats), also known as SPIDR (SPacer-spaced direct repeats), constitutes a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus includes a unique class of interspersed short sequence repeats (SSRs) identified in Escherichia 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 also been identified in Halobacterium thaliana, 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 generally different from other SSRs in the structure of the repeat sequences, which are called short regularly interspaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

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

[2000] ). In general, the repeat sequences are short elements that exist in clusters and are regularly spaced apart by unique insert sequences of essentially constant length (Mojica et al.,

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

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

[2002] ; and Mojica et al.,

[2005] ), including but not limited to Pyrophora, Pyrococcus, Sulfolobus, Archaeoglobaceae, Halocarcula, Methanobacterium, Methanogenococcus, Methanosarcina, Methanopyrus, Pyrococcus, Acidophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Acidophilus, and Streptomyces. quifrx), Porphromonas, Chlorobacter, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photorhabdus, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0265] As used herein, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence may comprise 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 within an organelle of a eukaryotic cell, such as a mitochondria or a chloroplast. A sequence or template that can be used to recombine into a targeted locus comprising a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In aspects of the present invention, an exogenous template polynucleotide may be referred to as an editing template. In one aspect of the present invention, recombination is homologous recombination.

[0266] The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the cell genome. Exemplary target sequences include those unique in the target genome. For example, for Streptococcus pyogenes Cas9, the unique target sequence in the genome may include a Cas9 target site in the form of MMMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNNNXGG (N is A, G, T or C; and X can be any substance) appears only once in the genome. The unique target sequence in the genome may include a Streptococcus pyogenes Cas9 target site in the form of MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T or C; and X can be any substance) appears only once in the genome. For S. thermophilus CRISPR1Cas9, a unique target sequence in a genome may comprise a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 1), wherein NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 2) (N is A, G, T, or C; X can be anything; and W is A or T) occurs only once in the genome. A unique target sequence in a C genome may comprise a S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 3), wherein NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 4) (N is A, G, T, or C; X can be anything; and W is A or T) occurs only once in the genome. For S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) occurs only once in the genome. A unique target sequence in a genome may include a S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) occurs only once in the genome. In each of these sequences, "M" can be A, G, T, or C, and need not be considered when identifying a sequence as unique.

[0267] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, which was developed by the Institute of Theoretical Chemistry of the University of Vienna, using a centroid structure prediction algorithm (see, for example, ARGruber et al., 2008, Cell 106 (1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62).

[0268] In general, a tracr mate sequence includes any sequence that has sufficient complementarity to a tracr sequence to promote one or more of the following: (1) excision of a guide sequence flanking the tracr mate sequence in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises a tracr mate sequence hybridized to the tracr sequence. In general, the degree of complementarity is about the optimal alignment of the tracr mate sequence and the tracr sequence along the length of the shorter of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm, and can further take into account secondary structures, such as self-complementarity within the tracr sequence or 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 or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more. In some embodiments, the length of the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides. In some embodiments, the tracr sequence and the tracr pairing sequence are contained in a single transcript so that hybridization between the two produces a transcript having a secondary structure (such as a hairpin). In one embodiment, the length of the loop-forming sequence for the hairpin structure is four nucleotides. In one embodiment, the loop-forming sequence for the hairpin structure has the sequence GAAA. However, longer or shorter loop sequences can be used, and alternative sequences can also be used. The sequence may include a nucleotide triplet (e.g., AAA) and additional nucleotides (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In one embodiment of the invention, 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 another embodiment of the invention, the transcript has up to five hairpins. In some embodiments, a single transcript further comprises a transcription termination sequence; in some embodiments, this is a polyT sequence, eg, six T nucleotides.

[0269] Methods for editing and delivering nucleic acids

[0270] In one embodiment, the invention provides methods 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.

[0271] 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.

[0272] In one embodiment, the present invention provides a method for delivering a nucleic acid sequence to genetic material. In one embodiment, the method includes administering to the gene: a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to the 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 includes administering to the genetic material: a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to the 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.

[0273] In one embodiment, the method comprises administering to the cell: a nucleic acid molecule encoding a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to the 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 the cell: a fusion protein; a guide nucleic acid comprising a targeting nucleotide sequence complementary to the target region in the gene; and a donor template nucleic acid comprising a U3 sequence, a U5 sequence, and a donor template sequence.

[0274] 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 a cell. In one embodiment, the method of editing genetic material is a method of editing a nucleic acid.

[0275] In one embodiment, the present invention 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 the cell: 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. In one embodiment, the method comprises administering to the cell: 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.

[0276] Targeted delivery of large DNA sequences for genome editing using CRISPR-Cas9-mediated HDR is still inefficient. However, the present invention provides a method for inserting a large donor template sequence into a target sequence preparation in a cell. In one embodiment, the method inserts a donor template sequence of at least 1 kb or greater, at least 2 kb or greater, at least 3 kb or greater, at least 4 kb or greater, at least 5 kb or greater, at least 6 kb or greater, at least 7 kb or greater, at least 8 kb or greater, at least 9 kb or greater, at least 10 kb or greater, at least 11 kb or greater, at least 12 kb or greater, at least 13 kb or greater, at least 14 kb or greater, at least 15 kb or greater, at least 16 kb or greater, at least 17 kb or greater, or at least 18 kb or greater. In one embodiment, the method comprises administering to the cell: 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.

[0277] In one embodiment, the target sequence is located within a gene. In one embodiment, the donor template sequence disrupts the sequence of the gene, thereby inhibiting or reducing the expression of the gene. In one embodiment, the target sequence has a mutation, and the donor template sequence inserts the corrected sequence into the target sequence, thereby correcting the gene mutation. In one embodiment, the donor template sequence is a gene sequence, and insertion of the donor template sequence into the target sequence in a cell allows expression of the gene.

[0278] In one embodiment, the donor template sequence is inserted into the safe harbor site. Therefore, in one embodiment, the guide nucleic acid comprises a nucleotide sequence complementary to the safe harbor region in the gene. The safe harbor region allows the expression of therapeutic genes without affecting the expression of adjacent genes. The safe harbor region may include an intergenic region away from adjacent genes such as H11, or an intergenic region 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.

[0279] In one embodiment, the donor template sequence is inserted into the 3' untranslated region (UTR), thereby allowing expression of the donor template sequence to be controlled by the promoter of other genes.

[0280] In one embodiment, the nucleic acid molecule comprises a first nucleic acid sequence encoding a retroviral integrase (IN) or a fragment thereof; a second nucleic acid sequence encoding a CRISPR-associated (Cas) protein; and a third nucleic acid sequence encoding a nuclear localization signal (NLS).

[0281] In one embodiment, the retroviral IN is a human immunodeficiency virus (HIV) IN, a Rous sarcoma virus (RSV) IN, a mouse mammary tumor virus (MMTV) IN, a Moloney murine leukemia virus (MoLV) IN, a bovine leukemia virus (BLV) IN, a human T-lymphotropic virus (HTLV) IN, an avian sarcoma leukemia virus (ASLV) IN, a feline leukemia virus (FLV) IN, a xenotropic murine leukemia virus-related virus (XMLV) IN, a simian immunodeficiency virus (SIV) IN, a feline immunodeficiency virus (FIV) IN, an equine infectious anemia virus (EIAV) IN, a prototype foamy virus (PFV) IN, a simian foamy virus (SFV) IN, a human foamy virus (HFV) IN, a walleye cutaneous sarcoma virus (WDSV) IN, or a bovine immunodeficiency virus (BIV) IN.

[0282] In one embodiment, the retroviral IN is HIV IN. In one embodiment, HIV IN comprises one or more amino acid substitutions, wherein the substitutions improve catalytic activity, improve solubility or increase interaction with one or more host cell cofactors. In one embodiment, 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, HIV IN comprises amino acid substitutions F185K and C280S. In one embodiment, HIV IN comprises amino acid substitutions T97A and Y134R. In one embodiment, HIV IN comprises amino acid substitutions G140S and Q148H.

[0283] In one embodiment, the retroviral IN segment comprises an IN N-terminal domain (NTD) and an IN catalytic core domain (CCD). In one embodiment, the retroviral IN segment comprises an IN CCD and an IN C-terminal domain (CTD). In one embodiment, the retroviral IN segment comprises an IN NTD. In one embodiment, the retroviral IN segment comprises an IN CCD. In one embodiment, the retroviral IN segment comprises an IN CTD.

[0284] In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding a sequence at least 95% identical to one of SEQ ID NOs: 1-40. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding a sequence at least 96% identical to one of SEQ ID NOs: 1-40. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding a sequence at least 97% identical to one of SEQ ID NOs: 1-40. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding a sequence at least 98% identical to one of SEQ ID NOs: 1-40. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding a sequence at least 99% identical to one of SEQ ID NOs: 1-40. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence encoding one of SEQ ID NOs: 1-40.

[0285] In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence that is at least 95% identical to one of SEQ ID NOs: 99-138. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence that is at least 96% identical to one of SEQ ID NOs: 99-138. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence that is at least 97% identical to one of SEQ ID NOs: 99-138. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence that is at least 98% identical to one of SEQ ID NOs: 99-138. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 99-138. In one embodiment, the first nucleic acid sequence encoding a retroviral IN comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 99-138.

[0286] In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0287] In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding a sequence at least 95% identical to one of SEQ ID NOs: 41-46. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding a sequence at least 96% identical to one of SEQ ID NOs: 41-46. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding a sequence at least 97% identical to one of SEQ ID NOs: 41-46. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding a sequence at least 98% identical to one of SEQ ID NOs: 41-46. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding a sequence at least 99% identical to one of SEQ ID NOs: 41-46. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence encoding one of SEQ ID NOs: 41-46.

[0288] In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 95% identical to one of SEQ ID NOs: 139-144. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 96% identical to one of SEQ ID NOs: 139-144. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 97% identical to one of SEQ ID NOs: 139-144. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 98% identical to one of SEQ ID NOs: 139-144. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 139-144. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 139-144. In one embodiment, the second nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence that is one of SEQ ID NOs: 139-144.

[0289] 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, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein or simian vims 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO:256.

[0290] In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding a sequence at least 95% identical to one of SEQ ID NOs: 47-56. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding a sequence at least 96% identical to one of SEQ ID NOs: 47-56. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding a sequence at least 97% identical to one of SEQ ID NOs: 47-56. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding a sequence at least 98% identical to one of SEQ ID NOs: 47-56. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding a sequence at least 99% identical to one of SEQ ID NOs: 47-56. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence encoding one of SEQ ID NOs: 47-56.

[0291] In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 95% identical to one of SEQ ID NOs: 145-154. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 96% identical to one of SEQ ID NOs: 145-154. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 97% identical to one of SEQ ID NOs: 145-154. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 98% identical to one of SEQ ID NOs: 145-154. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 145-154. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 145-154. In one embodiment, the third nucleic acid sequence encoding the NLS comprises a nucleic acid sequence that is one of SEQ ID NOs: 145-154.

[0292] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a sequence that is at least 95% identical to one of SEQ ID NOs: 57-98. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a sequence that is at least 96% identical to one of SEQ ID NOs: 57-98. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a sequence that is at least 97% identical to one of SEQ ID NOs: 57-98. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a sequence that is at least 98% identical to one of SEQ ID NOs: 57-98. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a sequence that is at least 99% identical to one of SEQ ID NOs: 57-98. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a sequence of one of SEQ ID NOs: 57-98.

[0293] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 95% identical to one of SEQ ID NOs: 155-196. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 96% identical to one of SEQ ID NOs: 155-196. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 97% identical to one of SEQ ID NOs: 155-196. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 98% identical to one of SEQ ID NOs: 155-196. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 155-196. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence that is at least 99% identical to one of SEQ ID NOs: 155-196.

[0294] In one embodiment, the U3 sequence and the U5 sequence are specific for a retroviral IN.

[0295] 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 becoming ill 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 to achieve cleavage of the target polynucleotide within the gene. In one embodiment, the IN-Cas9 fusion protein is complexed with a guide nucleic acid, which hybridizes with a target sequence within a target polynucleotide. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding a donor template nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding a guide nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding a guide nucleic acid and a nucleic acid sequence encoding a 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, which hybridizes with 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.

[0296] 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 a gene. In some embodiments, the mutation results in one or more amino acid changes in protein expression from a gene comprising a target sequence.

[0297] In one embodiment, IN-mediated DNA sequence integration can occur in either direction of the target DNA sequence. In one embodiment, different combinations of Cas and IN retroviral class proteins are used to facilitate directional editing. For example, in one embodiment, a fusion of IN from a retroviral class is bound to a first catalytic dead Cas, allowing the use of Cas-specific guide RNAs to bind to a specific target sequence. In one embodiment, the donor sequence comprises HIV and BIV LTR sequences. Therefore, in one embodiment, the sequence is integrated with the target DNA in a single orientation.

[0298] In one embodiment, flanking LoxP (Floxed) sequences are incorporated around the target gene. The inclusion of floxed sequences allows CRE-mediated recombination and conditional mutagenesis. The current method of using CRISPR-Cas9 to produce floxed alleles is inefficient. The most widely used method is to use two guide RNAs to induce DNA cleavage at the flanking target sequence, and use homology-directed repair to insert a ssDNA template containing a LoxP sequence. However, when double sgRNA is used to induce cleavage, the most favorable reaction is the deletion of the inserted sequence, resulting in full gene deletion. Therefore, in one embodiment, the use of integrase-Cas-mediated gene insertion increases the efficiency of tandem insertion of DNA sequences. In one embodiment, the integration of a sequence containing a reverse LoxP sequence allows recombination of the flanking LoxP sequence because IN-mediated integration can occur in either direction.

[0299] Treatment and use

[0300] The present invention provides methods for treating a disease or condition, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition, and / or genetically modifying to produce a desired phenotypic outcome. For example, in one embodiment, the methods of the present invention treat a disease or condition in a mammal, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition. In one embodiment, the methods of the present invention treat a disease or condition in a plant, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition. In one embodiment, the methods of the present invention treat a disease or condition in a yeast organism, alleviating the symptoms of the disease or condition, and / or reducing the risk of developing the disease or condition.

[0301] In one embodiment, the disease or condition is caused by one or more mutations in a genomic locus. Therefore, in one embodiment, the disease or condition can be treated, alleviated, or the risk can be reduced by introducing a nucleic acid sequence corresponding to a wild-type sequence in a region with the one or more mutations and / or introducing an element that prevents or reduces the expression of a genomic sequence with the one or more mutations. Therefore, in one embodiment, the method includes manipulating a target sequence within a coding, non-coding, or regulatory element of a genomic locus in the target sequence.

[0302] 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 (mutation in dystrophin), limb-girdle muscular dystrophy type 2B (LGMD2B), and Miyoshi myopathy (mutation in Dysferlin), cystic fibrosis (mutation in CFTR), Wilson's disease (mutation in ATP7B), and Stargardt's macular degeneration (mutation in ABCA4).

[0303] The present invention also provides methods for regulating the expression of genes or genetic material. For example, in one embodiment, the methods of the present invention provide delivery of genetic material to impart phenotypes in cells or organisms. For example, in one embodiment, the methods provide resistance to pathogens. In one embodiment, the methods provide regulation of metabolic pathways. In one embodiment, the methods provide the production and use of materials in organisms. For example, in one embodiment, the methods produce materials such as biologics, drugs, and biofuels in organisms such as eukaryotes, yeasts, bacteria, or plants.

[0304] In one embodiment, the method includes administering a fusion protein or 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. In one embodiment, the method further includes administering a donor template sequence.

[0305] In one embodiment, the target sequence is located within a gene. In one embodiment, the donor template sequence disrupts the sequence of the gene, thereby inhibiting or reducing the expression of the gene. In one embodiment, the target sequence has a mutation, and the donor template sequence inserts the corrected sequence into the target sequence, thereby correcting the gene mutation. In one embodiment, the donor template sequence is a gene sequence, and insertion of the donor template sequence into the target sequence in a cell allows expression of the gene.

[0306] In one embodiment, the fusion protein comprises a CRISPR-associated (Cas) protein and a nuclear localization signal (NLS). In one embodiment, the fusion protein comprises a Cas protein, an NLS and a retroviral integrase (IN) or a fragment thereof.

[0307] In one embodiment, the retroviral IN is a human immunodeficiency virus (HIV) IN, a Rous sarcoma virus (RSV) IN, a mouse mammary tumor virus (MMTV) IN, a Moloney murine leukemia virus (MoLV) IN, a bovine leukemia virus (BLV) IN, a human T-lymphotropic virus (HTLV) IN, an avian sarcoma leukemia virus (ASLV) IN, a feline leukemia virus (FLV) IN, a xenotropic murine leukemia virus-related virus (XMLV) IN, a simian immunodeficiency virus (SIV) IN, a feline immunodeficiency virus (FIV) IN, an equine infectious anemia virus (EIAV) IN, a prototype foamy virus (PFV) IN, a simian foamy virus (SFV) IN, a human foamy virus (HFV) IN, a walleye cutaneous sarcoma virus (WDSV) IN, or a bovine immunodeficiency virus (BIV) IN.

[0308] In one embodiment, the retroviral IN is HIV IN. In one embodiment, HIV IN comprises one or more amino acid substitutions, wherein the substitutions improve catalytic activity, improve solubility or increase interaction with one or more host cell cofactors. In one embodiment, 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, HIV IN comprises amino acid substitutions F185K and C280S. In one embodiment, HIV IN comprises amino acid substitutions T97A and Y134R. In one embodiment, HIV IN comprises amino acid substitutions G140S and Q148H.

[0309] In one embodiment, the retroviral IN segment comprises an IN N-terminal domain (NTD) and an IN catalytic core domain (CCD). In one embodiment, the retroviral IN segment comprises an IN CCD and an IN C-terminal domain (CTD). In one embodiment, the retroviral IN segment comprises an IN NTD. In one embodiment, the retroviral IN segment comprises an IN CCD. In one embodiment, the retroviral IN segment comprises an IN CTD.

[0310] 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: 1-40. In one embodiment, the retroviral IN comprises a sequence of one of SEQ ID NOs: 1-40.

[0311] In one embodiment, the nucleic acid 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 SEQ ID NOs: 99-138. In one embodiment, the nucleic acid encoding the retroviral IN comprises the nucleic acid sequence of one of SEQ ID NOs: 99-138.

[0312] In one embodiment, the Cas protein is Cas9, Cas13 or Cpf1. In one embodiment, the Cas protein is catalytically defective (dCas).

[0313] 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: 41-46. In one embodiment, the Cas protein comprises a sequence of one of SEQ ID NOs: 41-46.

[0314] 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%, 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: 139-144. In one embodiment, the nucleic acid sequence encoding the Cas protein comprises a nucleic acid sequence of one of SEQ ID NOs: 139-144.

[0315] 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, polyoma virus large T protein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, adenovirus El a or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or mammalian nuclear lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx protein or simian vims 40 ("SV40") T antigen. In one embodiment, the NLS is Ty1 or Ty1 derived NLS, Ty2 or Ty2 derived NLS or MAK11 or MAK11 derived NLS. In one embodiment, the Ty1 NLS comprises the amino acid sequence of SEQ ID NO: 51. In one embodiment, the Ty2 NLS comprises the amino acid sequence of SEQ ID NO: 254. In one embodiment, the MAK11 NLS comprises the amino acid sequence of SEQ ID NO:256.

[0316] In one embodiment, the NLS comprises a nucleic acid sequence that encodes 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: 47-56, 254-256, and 275-887. In one embodiment, the NLS comprises a nucleic acid sequence that encodes one of SEQ ID NOs: 47-56, 254-256, and 275-887.

[0317] 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: 145-154. In one embodiment, the nucleic acid sequence encoding the NLS comprises a nucleic acid sequence of one of SEQ ID NOs: 145-154.

[0318] 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: 57-98. In one embodiment, the fusion protein comprises the sequence of one of SEQ ID NOs: 57-98.

[0319] In one embodiment, the U3 sequence and the U5 sequence are specific for a retroviral IN.

[0320] 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 becoming ill 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 to achieve cleavage of the target polynucleotide within the gene. In one embodiment, the IN-Cas9 fusion protein is complexed with a guide nucleic acid, which hybridizes with a target sequence within a target polynucleotide. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding a donor template nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding a guide nucleic acid. In one embodiment, the IN-Cas9 fusion protein is complexed with a nucleic acid sequence encoding a guide nucleic acid and a nucleic acid sequence encoding a 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, which hybridizes with 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.

[0321] 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 a gene. In some embodiments, the mutation results in one or more amino acid changes in protein expression from a gene comprising a target sequence.

[0322] Experimental Examples

[0323] The present invention can be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Therefore, the present invention should not be interpreted in any way as being limited to the following examples, but should be interpreted as covering any and all variations that become apparent due to the teachings provided herein.

[0324] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the present invention and practice the claimed methods. Therefore, the following working examples particularly point out certain embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0325] Example 1: Amplification of Retroviral Integrase-dCas9 Fusion Protein for Editing Mammalian Genomic DNA Strong nuclear localization

[0326] Effective CRISPR-Cas9 editing of mammalian genomic DNA requires nuclear localization of Cas9, a large bacterial RNA-guided endonuclease that usually works in prokaryotic cells that lack a nuclear membrane. Studies have shown that effective nuclear localization of Cas9 in mammalian cells requires the addition of at least two mammalian nuclear localization signals, one at the N-terminus and one at the C-terminus (Cong et al., 2013, Science 339:819-23).

[0327] To facilitate nuclear localization of the retroviral integrase-dCas9 fusion protein for editing, an N-terminal SV40 NLS ( Figure 1 A). Surprisingly, only a small fraction of the IN-dCas9 fusion protein was nuclear localized when expressed in mammalian cells, as detected using a FLAG antibody that recognizes the C-terminal 3xFLAG epitope on dCas9 ( Figure 1 B). Interestingly, while the full-length IN-dCas9 fusion protein produced cytoplasmic aggregates, deletion of the C-terminal domain of the integrase (INΔC) resulted in greater solubility and increased nuclear localization ( Figure 1 B).

[0328] Fusion of retroviral integrase to dCas9 appears to greatly reduce its ability to localize to the nucleus. To further enhance nuclear localization of integrase-Cas9 fusion proteins, a number of different mammalian nuclear localization sequences were tested for their ability to direct nuclear import of IN-dCas9 ( Figure 1 B). Multimerization of 3 copies of the SV40 NLS (3xSV40) had no apparent effect on the extent of nuclear localization of either IN-dCas9 or INΔC-dCas9. However, addition of a bipartite NLS from nucleoplasmin (NPM) provided increased nuclear localization of the INΔC-dCas9 fusion protein, but not the full-length IN fusion protein. The combination of the 3xSV40 and NPM NLS appeared similar to NPM alone.

[0329] Interestingly, yeast LTR-retrotransposons (e.g., Ty1) are the evolutionary ancestors of retroviruses, and replicate their genomes by reverse transcription of RNA intermediates in the cytoplasm (Curcio et al., 2015, Microbiol Spectr3: MDNA3-0053-2014). LTR-retrotransposons contain integrases, which are necessary for the insertion of the retrotransposon genome. In contrast to the higher eukaryotic organisms that undergo open mitosis during cell division, yeast undergoes closed mitosis, whereby its nuclear envelope remains intact. Therefore, for Ty1 biogenesis, the nuclear import of the integrase / retrotransposon genome complex requires active nuclear import. Therefore, in contrast to mammalian integrases, Ty1 integrase contains the large C-terminal dyad NLS required for retrotransposition (Moore et al., 1998, Mol Cell Biol 18: 1105-14). Interestingly, the results presented here demonstrate that fusion of the Ty1 NLS to the C-terminus of two IN-dCas9 fusion proteins can provide robust nuclear localization in mammalian cells ( Figure 1 B).

[0330] Increased nuclear localization of the INΔC-dCas9 fusion protein significantly enhanced 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-α in HEK293 cells ( Figure 1 C) Utilization of a robust Ty1 NLS may further allow editing in non-dividing cells that maintain a constant nuclear envelope (e.g., in vivo therapeutic applications).

[0331] Example 2: Integrated gene editing method for correction of muscular dystrophy

[0332] As demonstrated elsewhere herein, the fusion of lentiviral integrase with CRISPR-Cas9 allows sequence-specific integration of large DNA sequences into genomic DNA. This approach can be used to deliver therapeutically beneficial genes to non-pathogenic genomic locations (safe harbors) to permanently correct human genetic diseases ( Figure 2 ). This technology allows sequence-specific integration of large DNA donor sequences containing short viral terminal motifs.

[0333] The main advantage of the gene therapy method of the present invention is the ability to deliver donor DNA sequences to targeted genomic locations. In addition, this method eliminates the need for homology arms and relies on targeting by guide RNA, greatly simplifying genome editing. Therefore, once a specific reporter gene donor sequence is generated, it can be directed to any location (or multiple locations) for a variety of applications.

[0334] The fusion of lentiviral integrase and 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, a donor vector containing the IGR IRES sequence followed by the mCherry-2a-puromycin gene and the SV40 polyadenylation sequence was generated ( Figure 3 ). Next, sgRNAs targeting stable human CMV-eGFP stable cell lines in COS-7 cells were designed. The hCMV-eGFP stable transgene provides a heterologous target sequence that can be used to determine editing at robustly expressed, but non-essentially expressed loci. The donor mCherry-2a-puro template was purified and co-transfected into GFP stable cells along with sgRNA and IN-dCas9 and cultured for 48 hours. After 48 hours, mCherry-positive cells were visible in the culture and replaced the GFP-positive signal ( Figure 3 ).

[0335] Efficacy and fidelity of integrase-Cas-mediated integration of human dystrophin into the mammalian genome.

[0336] Integrase-Cas mediated gene delivery directs sequence-specific integration of large DNA sequences into mammalian genomic DNA. Integrase-Cas was used to deliver the human dystrophin gene to a safe harbor location under the control of the human α-skeletal actin (HSA) promoter in cultured cells using CRISPR guide RNAs specific for human AAVS1 and mouse ROSA26 genomic DNA. Correct targeting of dystrophin was assessed using PCR-based genotyping.

[0337] Integrase-Cas-mediated dystrophin gene therapy works in a mouse model of Duchenne muscular dystrophy Restore muscle function.

[0338] The efficacy of human dystrophin delivery mediated by Inscritpr was determined in the MDX mouse strain, the most commonly used mouse model of muscular dystrophy. After systemic delivery, anti-dystrophin antibodies were used to quantify and measure the level of dystrophin expression in limb skeletal muscle, heart and diaphragm during the time course of 2, 4 and 6 months. The alleviation of DMD disease pathogenesis was assessed by quantitative serum creatine kinase (CK) (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.

[0339] Histological analysis of gene expression.

[0340] At 8 weeks of age, the left hindlimb quadriceps, heart, and diaphragm were harvested, weighed, fixed in 4% formaldehyde in PBS, and processed using conventional methods for paraffin histology. Mdx / y The percentage of myofibers expressing the HSA-dystrophin / GFP fusion protein was determined using an anti-GFP antibody in WT and WT mice. Right hindlimb muscles were snap-frozen in liquid nitrogen for subsequent PCR-based genotyping, gene expression analysis by RT-PCR, and protein expression analysis by Western blotting.

[0341] Integrase-Cas-mediated delivery mitigates disease pathogenesis in a mouse model of Duchenne muscular dystrophy.

[0342] Hematoxylin and eosin (H&E), von Kossa, and Masson's trichrome staining of transverse histological sections were used to identify myofibers containing central nuclei, mineralization, and endomysial fibrosis, respectively. Quantitative comparisons and statistical analyses were used to compare the ratio of myofibers to central nuclei, or to compare the areas of mineralization or fibrosis stained in quadriceps femoris limb muscles. At least three different section planes from three different mice of each genotype were compared for each muscle. mdx / y (These mice exhibit a less severe phenotype) have a reduced ratio of myofibers to central nuclei, and a smaller total area of ​​fibrosis and mineralization.

[0343] Serum creatine kinase (CK) measurement.

[0344] Serum CK is a relevant marker of skeletal muscle damage and is a diagnostic marker in patients with DMD. CK was measured in the above animal cohorts using a non-lethal procedure at weeks 2, 4, 6, and 8. Briefly, blood was harvested from the periorbital plexus directly into a microhematocrit tube, allowed to clot for 30 minutes at room temperature, and then centrifuged at 1,700 × g for 10 minutes. mdx / y Treated mice that exhibited a less severe phenotype had significantly reduced serum CK levels compared to the KO.

[0345] Example 3: Genome Editing—Targeted Non-homologous DNA Integration

[0346] The data presented here demonstrate that optimized integrase-Cas can achieve efficient editing of mammalian genomes.

[0347] Optimized editing

[0348] To optimize IN-mediated integration, we determined whether amino acid mutations that enhance integrase catalytic activity, solubility, or interaction with host cell cofactors enhance editing. In addition, we assessed the efficiency and fidelity of IN proteins isolated from seven unique classes of retroviruses.

[0349] In order to quantify and characterize the integration mediated by IN-dCas9 in mammalian cells, a plasmid-based reporter gene system was used, which utilizes a blue chromoprotein (amilCP) from Acropora multiporus, which produces dark blue colonies when expressed in Escherichia coli. The destruction of the amilCP open reading frame eliminates blue protein expression, which can be used as a direct readout of targeting fidelity. In addition, a donor template encoding a chloramphenicol antibiotic resistance gene was produced, which was flanked by U3 and U5 retroviral end sequences from HIV. The integration of this donor template confers resistance to chloramphenicol, which can be used to monitor DNA integration mediated by integrase-Cas. In this reporter gene assay, an expression plasmid containing IN-dCas9 fusion protein, sgRNA targeting amilCP, and a donor template was co-transfected into mammalian COS-7 cells together with a bacterial amilCP reporter gene. After 48 hours, total plasmid DNA was recovered using column purification and transformed into Escherichia coli. IN-dCas9 is sufficient to integrate chloramphenicol encoding template DNA into the amilCP reporter plasmid, thereby disrupting amilCP expression and conferring resistance to chloramphenicol. This rapid assay, which allows quantification and clonal sequence analysis of individual integration events, can be used to optimize editing.

[0350] Enhanced integrase activity: Although most mutations in IN abolish its activity, decades of research have identified several mutations that enhance IN integration by increasing IN catalytic activity (D116N), dimerization (E85F), solubility (F185K / C280S), and interaction with host cell proteins (K71R). We used a plasmid-based reporter assay with IN-dCas9 fusion proteins containing activating IN mutations to determine whether this enhanced activity.

[0351] Modification of integrase activity by host cell proteins : Although IN is the only protein that is necessary and sufficient for integration of proviral DNA in vitro, interactions with host cell proteins can greatly alter IN-mediated DNA integration18. Notably, LEDGF / p75, VBP1, and SNF5 are well-characterized HIV IN-interacting proteins that promote IN-mediated integration. These factors were expressed using a plasmid reporter gene assay to determine whether they enhance donor template integration.

[0352] Compare and contrast integrants from different retroviral classes:Although all IN enzymes from retroviral classes contain a conserved core catalytic D,D(35)E residue, they vary greatly in genome size, complexity, U3 and U5 terminal sequences, and DNA ligation efficiency. To determine the editing efficiency of different retroviral INs, model examples from each retroviral class were cloned as fusions with dCas9, including α (RSV), β (MMTV), γ (MoLV), δ (BLV), ε (WDSV), and foamy virus (HFV). Donor plasmids containing their respective U3 and U5 terminal motifs were generated. Protein expression was verified by western blotting, and nuclear localization was verified using immunocytochemistry using a FLAG antibody to detect the 3xFLAG epitope located at the C-terminus of dCas9.

[0353] Efficiency of editing mammalian genomic DNA

[0354] The efficiency and fidelity of editing mammalian genomic DNA was determined using a stable CMV-driven GFP reporter cell line and donor templates containing RFP and puromycin selection cassettes were generated. Integration events were quantified and clonally characterized to determine the efficacy and fidelity of the method as a novel genome editing technology.

[0355] Generation of cell-based reporter assays: In order to quantify the integration events at this locus, a donor template containing an IRES-RFP-2A-puromycin box and a guide RNA targeting the GFP coding sequence was used. After the donor box was inserted into the CMV-GFP locus, RFP expression replaced GFP expression and provided resistance to the antibiotic puromycin. FACS sorting was used to quantify the efficiency and fidelity of Inscripr editing to determine the percentage of cells that were RFP+ / GFP- (targeted integration) after transfection and culture for 48 hours. Puromycin was used to select clonal integration events, and PCR primers were used to characterize the events with the sequence between the amplified GFP locus and the donor box.

[0356] Editing at multiple endogenous loci: Integrase-Cas was used to knock in the RFP-2A puromycin cassette using sgRNAs specific for the CMV-GFP locus as well as the 3'UTR of the human EF1-α locus in the HEK293 human cell line. Targeting the 3'UTR allowed expression of the IRES-dependent vector without disrupting normal gene expression. After clonal selection using puromycin, PCR genotyping was used to determine the percentage of clones that had integrated the donor template at both loci.

[0357] Example 4: Generation and Characterization of Incriptr

[0358] Generation of functional IN-dCas9 fusion proteins.

[0359] To generate a functional IN-dCas9 fusion protein for use in mammalian cells, the full-length retroviral IN from HIV-1 (amino acids 1148–1435 of the gag-pol polyprotein) was cloned into the N-terminus of human codon-optimized dCas9, separated by a 15-amino acid flexible linker [(GGGGS)3)] ( Figure 6 ). The SV40 nuclear localization signal (NLS) is included at the N-terminus of IN, which together with the C-terminal SV40 NLS on dCas9 provides 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 containing only the N-terminus and catalytic core domain of IN (amino acids 1148-1369) was generated as an N-terminal fusion to dCas9 ( Figure 6 ).

[0360] Generation of reporter genes for monitoring editing of plasmid DNA.

[0361] To quantify and characterize IN-dCas9-mediated integration in mammalian cells, a plasmid-based reporter gene assay was designed that utilizes a blue chromoprotein from Acropora polyporus (amilCP), which produces dark blue colonies when expressed in Escherichia coli ( Figure 6 Disruption of the amilCP open reading frame abolished blue protein expression, which can be used as a direct readout of targeting fidelity and as a target DNA for integrase-Cas-mediated integration. Single guide RNA (sgRNA) target sequences separated by 16 bp spacer sequences were designed with a 'PAM-out' orientation to promote efficient dimerization of N-terminal dCas9 fusion proteins at the target DNA ( Figure 4 ).

[0362] Generation of viral end donor sequences for integrase-Cas mediated integration.

[0363] To construct a targeting vector that could be used to generate donor sequences for integrase-Cas mediated integration, 30 base pairs containing the U3 and U5 HIV termini were subcloned into pCRII ( Figure 6 To facilitate subcloning of donor sequences, a multiple cloning site containing 9 unique restriction enzymes was included between U3 and U5. Since U3 and U5 share the same 3 nucleotides at their ends (ACT and AGT, respectively), additional half-site sequences were included to create a ScaI restriction site at each end 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 cuts and leaves two 5' nucleotide overhangs, thereby mimicking a 3' pre-processed viral end with an exposed CA dinucleotide ( Figure 6 To facilitate gel purification and separation of the Faul-digested template from the plasmid backbone, multiple site-directed mutagenesis was used to remove the six Faul sites present in the pCR II plasmid backbone.

[0364] Protocol: Preparation of INsrt donor template for transfection

[0365] 1) Set up restriction digest of INsrt plasmid DNA

[0366] 2) Restriction digestion reaction

[0367] 3) Gel purification of donor template from backbone DNA

[0368] 4) Elute the donor DNA for transfection.

[0369] Integrase-Cas mediated integration of donor sequences into plasmid DNA in mammalian cells.

[0370] To allow positive selection for cooperative IN-dCas9-mediated integration, an INsrt donor vector was designed that carries the chloramphenicol resistance gene (CAT), which is absent from the reporter gene in the expression plasmid ( Figure 7 ). The CAT gene contains an IGR IRES from Plautia stali enterovirus (PSIV) in front of it, which can initiate translation in prokaryotes and eukaryotic cells to facilitate translation of multiple integration sites. The template containing the chloramphenicol resistance gene and viral ends was digested with ScaI (blunt ends) or FauI (processed ends) and gel purified from the plasmid backbone DNA. Co-transfection of INsrt template, 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 Escherichia coli. Chloramphenicol-resistant clones were observed for both full-length IN and INDC-dCas9 fusion proteins. Sequencing of the plasmid showed that the IG3-CAT plasmid sequence had been integrated into the amilCP reporter gene. Interestingly, the donor sequence digested with FauI (which simulates the pre-3' processing of the viral DNA end) produced twice the chloramphenicol-resistant clones compared to the flat-end template digested with ScaI. Integrase-Cas-mediated integration contains the hallmarks of HIV IN lentiviral integration, including 5 base pair repeats of the host DNA flanking the integration site. Interestingly, the integration site does not appear between the two sgRNA target sites, but appears on either side of the amilCP target sequence.

[0371] Insrt IGR-CAT donor templates with blunt ends (ScaI cleaved) or 3' processed mimic (FauI cleaved) ends were integrated into the pCRII-amilCP reporter gene in mammalian cells. Interestingly, as a fusion with dCas9, the deletion of the C-terminal nonspecific DNA binding domain did not inhibit integrase-Cas mediated integration. The use of mimic 3' processed ends showed an increase of CAT-resistant clones by about 2 times. ( Fig.29 B) Using dual-guide RNA targeted integration into amilCP using the IGR-CAT donor template, mutations that inhibit dimerization (E85G and E85F) do not disrupt integrase-Cas mediated integration. However, the IN E87G mutation cannot be rescued by paired targeting sgRNAs. Interestingly, a tandem INΔC fusion with dCas9 (tdINΔC-dCas9) showed an approximately 2-fold enhancement of integration ( Fig.29 C).

[0372] Protocol: Integrase Cas-mediated integration of donor sequences into plasmid DNA in mammalian cells

[0373] 1) Co-transfect the polycistronic sgRNA and IN-dCas9 plasmid, bacterial amilCP reporter gene plasmid and INsrt donor template into mammalian (e.g. Cos7) cells.

[0374] a. Set up the transfection reaction immediately before plating cells.

[0375] b. Harvesting, plating and transfecting cells

[0376] 2) Recovery of plasmid DNA from transfected cells:

[0377] 3) The recovered plasmid DNA was transformed into chemically competent Escherichia coli.

[0378] Generation of a CMV-GFP stable mammalian cell line for integrase-Cas mediated integration into genomic DNA.

[0379] A stable GFP reporter cell line was generated 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 robust gene expression that can be targeted for disruption without compromising normal cell viability, which can otherwise occur when targeting essential host genes.

[0380] Integrase Cas mediated integration of donor sequences into mammalian genomic DNA.

[0381] To quantify integration events at the CMV-GFP locus, a donor template containing an IGR-mCherry-2A-puromycin-pA cassette and a pair of guide RNAs targeting the GFP coding sequence ( Figure 3 Integration of the donor cassette into the CMV-GFP locus will drive mCherry expression and disrupt GFP expression, and provide resistance to the antibiotic puromycin. After transfection and 48 hours of culture, mCherry-positive cells were observed, some of which still contained weak but detectable levels of GFP expression ( Figure 3 ).

[0382] Integrase-Cas mediated integration of donor sequences at endogenous loci.

[0383] A targeting strategy was designed and a guide RNA specific for the 3'UTR of the human EF1-α locus was selected to knock-in the IGR-mCherry-2A-puromycin-pA cassette into the human HEK293 cell line ( Figure 8 ). The 3'UTR was targeted to allow expression of the IGR-mCherry cassette without disrupting the open reading frame for EF1-α expression. After transfection and 48 h of culture, mCherry-positive cells were observed in the culture ( Figure 8 ).

[0384] Scheme: Integrase Cas-mediated integration of donor sequences into mammalian genomic DNA.

[0385] 1) Use Fugene6 or Lipofectamine2000 to co-transfect plasmids encoding sgRNA, IN-dCas9 and INsrt donor template into mammalian cells (COS7, HEK293, etc.) at a ratio of 1:1:1.

[0386] a. Harvesting, plating and transfecting cells.

[0387] 2) Antibiotic selection of integrated sequences

[0388] a. Wash the cells with 10 ml of medium containing antibiotic selection and plate

[0389] b. Cultivate the cells and then generate clones.

[0390] Directed editing.

[0391] IN-mediated DNA sequence integration can occur in either direction of the target DNA sequence. Different combinations of Cas and IN retroviral class proteins are used to provide the ability to promote directional editing. For example, a fusion of IN from BIV (bovine immunodeficiency virus or other HIV-related viruses) fused to the catalytic dead LbCpf1 (LbCpf1) allows the use of Cpf1-specific guide RNAs to bind to specific target sequences. The use of donor sequences containing both HIV and BIV terminal sequences locks the binding to the target DNA into a single orientation. ( Fig. 9 ).

[0392] Multiplex genome editing for generating floxed alleles.

[0393] The incorporation of flanking LoxP (Floxed) sequences around the target gene allows CRE-mediated recombination and conditional mutagenesis. Current methods for generating Floxed alleles using CRISPR-Cas9 are inefficient. The most widely used method is to use two guide RNAs to induce DNA cleavage at the flanking target sequence and use homology-directed repair to insert a ssDNA template containing the LoxP sequence. However, when dual sgRNAs are used to induce cleavage, the most favorable reaction is the deletion of the inserted sequence, resulting in full gene deletion. If IN-mediated strand transfer with host DNA does not allow efficient deletion of the inserted sequence, then the use of integrase-Cas-mediated gene insertion may provide an alternative and more efficient method for tandem insertion of DNA sequences. Since IN-mediated integration can occur in either direction, the integration of sequences containing reverse LoxP sequences allows recombination of flanking LoxP sequences ( Fig.10 ).

[0394] Example 5: Identification and activity of Ty1 NLS-like sequences

[0395] The integrase from the yeast Ty1 retrotransposon contains a non-classical bipartite nuclear localization signal consisting of a tandem KKR motif separated by a larger linker sequence. Previous studies in yeast have demonstrated the necessity of these basic motifs for nuclear localization and Ty1 translocation (Kenna et al., 1998, Mol Cell Biol 18, 1115-1124; Moore et al., 1998, Mol Cell Biol 18, 1105-1114). Ty1 translocation is absolutely dependent on the presence of Ty1 NLS, and interestingly, the classical NLS is not sufficient to summarize the Ty1 NLS activity required for translocation. Interestingly, other yeast proteins share this tandem KKR motif, which can play the role of NLS in view of the fact that many of these proteins are nuclear localized (Kenna et al., 1998, Mol Cell Biol 18, 1115-1124).

[0396] As shown in Example 1, yeast Ty1 NLS provides robust nuclear localization of Cas proteins and Cas-fusion proteins in mammalian cells. To determine whether this activity is a unique feature of Ty1 NLS, we tested whether the closely related NLS from Ty2 integrase and other yeast Ty1 NLS-like motifs are sufficient to localize integrase-dCas9 fusion proteins (INΔC-Cas9) to the nucleus in mammalian cells. Interestingly, Ty2 NLS, which is highly conserved with Ty1 NLS, is equally effective as Ty1 NLS in nuclear localization ( Fig.11 Fusions of three different Ty1 NLS-like sequences identified in yeast that differ from the Ty1 / Ty2 NLS sequences (Kenna et al., 1998) showed either robust NLS activity (MAK11) or no significant NLS activity (INO4 and STH1). The MAK11 sequence is derived from a yeast nuclear protein that also appeared at the C-terminus of further screened proteins, indicating that this sequence does function as an NLS. Using the motif KKR N20-40 All proteins in KKR's SWISS-PROT protein sequence database, which identified a large number of potential Ty1 NLS-like sequences (SEQ ID NO: 275-887) in multiple species. These data suggest that other Ty1 NLS-like sequences may have robust NLS activity and may be useful for the localization of proteins (including Cas and Cas-fusion proteins) in dividing and non-dividing eukaryotic cells.

[0397] Example 6: Enhancing CRISPR-Cas9 DNA Editing with Ty1 NLS

[0398] The CRISPR-Cas DNA cleavage system is derived from bacteria, and the Cas protein is both large and lacks intrinsic mammalian nuclear localization signals (NLS), thereby preventing their effective nuclear localization in mammalian cells. It has been previously shown that it is necessary to add two classical nuclear localization signals (N-terminal SV40 and C-terminal nucleoplasmin (NPM) dichotomous NLS) to perform effective nuclear localization and editing of DNA by CRISPR-Cas9 in mammalian cells (Cong et al., 2013, Science 339, 819-823). Since the non-classical yeast retrotransposon Ty1 NLS is used to locate the robustness of Cas fusion proteins in mammalian cells (Example 1), it is tested whether Ty1 NLS can also enhance the editing efficiency of traditional CRISPR-Cas9 in mammalian cells.

[0399] To determine whether Ty1 enhances CRISPR-Cas9 editing, an existing CRISPR-Cas9 expression plasmid (px330) was modified by replacing the C-terminal NPM NLS with a non-canonical Ty1 NLS (px330-Ty1) ( Fig.12 A). Next, a frameshift-responsive luciferase reporter gene was generated, which encodes an out-of-frame luciferase coding sequence ( Fig.12 B) For this reporter gene assay, cleavage near the target sequence and incomplete repair by the cellular non-homologous end joining (NHEJ) pathway can induce nucleotide insertions or deletions that may reconstitute the luciferase coding sequence and result in luciferase expression.

[0400] Co-expression of a luciferase reporter gene with a vector encoding Cas9 containing an NPM NLS and a single guide RNA specific for a 20-nucleotide target sequence ( Fig.12 C). Notably, expression of Cas9 containing the Ty1 NLS produced a significant enhancement (approximately 44%) of reporter gene activity in COS-7 cells compared to Cas9 containing the NPM NLS ( Fig.12 C).

[0401] Example 7: Genome Targeting Strategy for Editing

[0402] Targeted integration of DNA donor sequences using integrase-DNA binding fusion proteins can be targeted to different locations within the genome depending on the desired results. For example, a therapeutic DNA donor sequence consisting of a gene expression cassette (e.g., a promoter, a gene sequence, and a transcription terminator) can be targeted to a 'safe harbor' position (for a review and list of safe harbor sites in the human genome, see Pellenz et al., 2019, Hum Gene Ther 30, 814-828), which will allow expression of therapeutic genes without affecting neighboring gene expression. These may include intergenic regions away from neighboring genes such as H11, or intergenic regions within 'non-essential' genes such as CCR5, hROSA26, or AAVS1 ( Fig.13 A and 13b).

[0403] To restore expression of a disease-causing gene mutation, it may be advantageous to target the integration of a therapeutic gene sequence into an endogenous disease gene locus, since this locus is already defective and the temporal and spatial expression of this locus is under endogenous regulatory control. In one iteration, a DNA donor sequence encoding a therapeutic gene containing a splice acceptor can be integrated into the first intron of the endogenous gene locus so that splicing will 1) allow expression of the introduced gene sequence, and 2) prevent downstream expression of the mutated sequence (due to termination from the integrated poly(A) sequence or LTR sequence) ( Fig.13 C). If targeting downstream introns, smaller DNA donor sequences can be delivered or expressed ( Fig.13 D).

[0404] Targeted insertion of a DNA donor sequence containing an IRES sequence into the 3' untranslated region (3'UTR) of a gene may be beneficial because this approach will allow expression at the same temporal and spatial level as the targeted locus and will be less likely to disrupt the targeted gene locus ( Fig.13 E).

[0405] Example 8: Targeted lentiviral integration into mammalian genomes using CRISPR-CAS

[0406] The data presented here demonstrate three different approaches for the delivery and targeted integration of lentiviral donor sequences into the mammalian genome.

[0407] Lentivirus life cycle

[0408] Lentiviruses are single-stranded RNA viruses that integrate a permanent double-stranded DNA (dsDNA) copy of their proviral genome into the host cell DNA ( Fig.14). The lentiviral genome is flanked by long terminal repeats (LTRs) that control viral gene transcription and contain short (about 20 base pairs) sequence motifs required for proviral genome integration at its U3 and U5 ends. After viral infection, the lentiviral RNA genome is replicated as flat-ended dsDNA by the virally encoded reverse transcriptase (RT) and inserted into the host genome by the integrase (IN). IN consists of three functional domains that are critical for IN activity, including a C-terminal domain (CTD) that binds non-specifically to DNA. IN-mediated retroviral DNA insertion occurs with little DNA target sequence specificity and can be integrated into active gene loci, which can disrupt normal gene function and potentially cause human disease. Despite the benefits of having a large sequence carrying capacity, this limits the use of lentiviral vectors for gene therapy.

[0409] Genome editing

[0410] CRISPR-Cas9 allows programmable DNA targeting by utilizing a short single guide RNA to recognize and bind DNA. Catalytically inactive Cas9 (dCas9) retains the ability to target DNA and has recently been repurposed as a programmable DNA binding platform for various applications in genome interrogation and regulation. As shown in Example 1, a fusion of a lentiviral integrase to dCas9 is sufficient to insert a donor DNA sequence containing short viral ends into a target sequence using a CRISPR guide RNA in mammalian cells ( Fig.15 To monitor integrase-Cas mediated integration in mammalian cells, a donor vector containing the IGR IRES sequence followed by the mCherry-2a-puromycin gene and the SV40 polyadenylation sequence was generated ( Fig.15 B). sgRNA targeting stable human CMV-eGFP stable cell line in COS-7 cells was designed ( Fig.15 C and 15D). The hCMV-eGFP stable transgene provides a heterologous target sequence that can be used to determine editing at robustly expressed, but non-essentially expressed loci. The donor mCherry-2a-puro template was purified and co-transfected into GFP stable cells along with sgRNA and IN-dCas9 and cultured for 48 hours. After 48 hours, mCherry-positive cells were visible in the culture and replaced the GFP-positive signal ( Fig.15 E). Incorporation of the editing components (integrase-CRISPR-Cas9 fusion) into lentiviral particles allows targeted and easily programmable integration of the lentiviral genome into the host DNA, eliminating the main limitation of lentiviral gene therapy (i.e., non-specific lentiviral integration). This approach is useful for both basic research and therapeutic applications.

[0411] Lentiviral gene delivery system

[0412] Lentiviral vectors have been used as a robust gene delivery tool for research applications ( Fig.16 The structural and enzymatic proteins of lentivirus are transcribed and translated into a large polyprotein (gag-pol and envelope) ( Fig.16 A). Once incorporated into the budding virus particle, the polyproteins are processed into individual proteins by viral proteases. For lentiviral gene expression systems, these polyproteins are removed from the viral genome and expressed using separate mammalian expression plasmids ( Fig.16 B). The donor DNA sequence can then be cloned to replace the viral polyprotein between the flanking LTR sequences. Co-transfection of these vectors in mammalian cells allows the formation of lentiviral particles capable of delivering and integrating the encoded donor sequence, but without the coding information for integrase and other viral proteins necessary for subsequent viral propagation ( Fig.16 B). Lentiviral particles are the natural vector for delivering both viral proteins (e.g., integrase and reverse transcriptase) and dsDNA donor sequences, containing the necessary viral terminal sequences required for integrase-mediated insertion into mammalian cells ( Fig.16 C).

[0413] The integrase-dCas9 fusion protein was packaged into lentiviral particles.

[0414] For the purpose of targeted lentiviral donor template integration for genome editing in mammalian cells, existing lentiviral delivery systems can be modified to incorporate editing components ( Figure 17-20 ). Here we describe three different approaches for the delivery and targeted integration of lentiviral donor sequences into the mammalian genome.

[0415] The first approach is to directly incorporate dCas9 as a fusion to the integrase (or an integrase lacking its C-terminal nonspecific DNA binding domain, INΔC) within a lentiviral packaging plasmid encoding the gag-pol polyprotein (e.g., psPax2) ( Fig.17 A). In this method, the modified gag-pol polyprotein is translated into a polyprotein along with other viral components, loaded with guide RNA and packaged into lentiviral particles ( Figure 4B). The integrase-dCas9 fusion protein retains the sequences required for protease cleavage (PR) and is therefore typically cleaved from the gag-pol polyprotein during particle maturation. Transduction of mammalian cells produces viral proteins, including delivery of IN-dCas9 fusion protein, sgRNA, and lentiviral donor sequences. Reverse transcription of the ssRNA genome by reverse transcriptase produces a dsDNA sequence containing the correct viral end sequences (U3 and U5), which is then integrated into the mammalian genome by the IN-dCas9 fusion protein.

[0416] The second approach was to generate N-terminal and C-terminal fusions of integrase-dCas9 with HIV viral protein R (VPR) ( Fig.18 A). VPR is efficiently packaged into lentiviral particles as an auxiliary 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 IN-dCas9 fusion protein, so after maturation, IN-dCas9 is released from VPR ( Fig.18 A). Co-transfection of packaging cells with lentiviral components produces viral particles containing VPR-IN-dCas9 protein and sgRNA. The packaging plasmid required for viral particle formation (e.g., psPax2) contains a mutation in the integrase to inhibit its catalytic activity, thereby preventing non-mediated integration ( Fig.18 B) After viral transduction, the integrase-dCas9 protein is delivered and mediates the integration of the lentiviral donor sequence ( Fig.18 C) A benefit of delivering IN-dCas9 fusion and sgRNA as a riboprotein is that it is only transiently expressed in the target cells.

[0417] The third approach is to directly incorporate the integrase-dCas9 fusion protein and sgRNA expression cassette into a lentiviral transfer plasmid or other viral vector (such as AAV) ( Fig.19 A). Co-transfect the transfer plasmid containing the IN-dCas9 fusion protein and sgRNA with the packaging and envelope plasmids required to produce lentiviral particles. If using lentivirus, the packaging plasmid contains a catalytic mutation in the integrase to inhibit nonspecific integration ( Fig.19 B). Following transduction of mammalian cells, expression of the IN-dCas9 fusion protein and sgRNA produces a component capable of targeting its own viral donor vector for targeted integration (self-integration) ( Fig.19 C). This approach is used for targeted gene disruption or as a gene drive. Alternatively, co-transduction with additional lentiviral particles encoding donor sequences serves as a donor template for integration ( Fig.19). This approach can be used to prevent the self-integration of one's own viral coding sequences by using integrases from different retroviral family members and their corresponding transfer plasmids. For example, HIV lentiviral particles encoding the FIV IN-dCas9 fusion protein can be used to integrate the FIV donor template encoded in the FIV lentiviral particles ( Fig. 20 ).

[0418] Generation of a single locus, constitutively active ubiquitous ROSA26 mGFP / + Reporter gene mouse strains

[0419] The ROSA26 mT / mG reporter mouse strain (Jackson Labs, Stock #007576) contains a floxed membrane-localized tdTO(mT) fluorescent reporter cassette that upon recombination with CRE recombinase removes the mT reporter gene and allows expression of the membrane-localized eGFP(mG) reporter gene. To generate a single locus in vivo GFP reporter line, ROSA26 mT / mG mice were crossed with universal CAG-CRE recombinase mice to generate constitutively and ubiquitously expressing ROSA26 mG reporter mice. mG / + Isolation of mouse embryonic fibroblasts (MEFs) revealed robust membrane GFP expression in all cells in culture ( Fig.21 A similar strategy was used to generate a ubiquitous and constitutively active nuclear GFP reporter gene by recombining the ROSA26 nT / nG mouse strain (Jackson Labs, Stock #023035).

[0420] The components were packaged into lentiviral particles for targeted integration into the ROSA-mGFP locus.

[0421] To target the IRES-tdTO sequence to ROSA26 mG / + Lentiviral particles were generated in a packaging cell line (Lenti-X 293T, Clontech) in the presence of a GFP coding sequence in MEFs. Lentiviral particles were generated by co-transfection of a lentiviral transfer plasmid (Lenti-IRES-tdTO) encoding an IRES-tdTO fluorescent reporter gene between the second generation SIN lentiviral LTRs, an expression vector encoding a pantropic envelope protein (VSV-G), an expression plasmid encoding a reverse pair of guide RNAs targeting GFP, and a packaging plasmid encoding an INΔC-dCas9 fusion in the context of the Gag-Pol lentiviral polyprotein in the psPax2 packaging plasmid (INΔC-dCas9-psPax2). Lentiviral particles were harvested from the supernatant and filtered using a 0.45 μm PES filter.

[0422] Targeted Lentiviral Integration in Mammalian Cells

[0423] Use of Incriptr modified lentiviral particles to transduce ROSA26 in culture mG / + MEFs. Two days later, ubiquitous red fluorescent protein expression was detectable in MEFs transduced with lentivirus encoding the IRES-tdTO reporter, but GFP fluorescence was retained. This initial widespread expression is likely due to translation of the viral RNA encoded by the lentiviral IRES-tdTO and demonstrates that lentiviral packaging is not inhibited by modifications in the packaging plasmid ( Fig.21 ). For conventional lentiviral transduction, lentiviral transgene expression cannot be maintained in the absence of viral integration. Notably, 7 days after transduction, tdTO red fluorescent cells were detectable in the culture, which were now in ROSA26 mG / + In primary cells ( Fig.21 ) or lack of green fluorescence when targeted into the previously described CMV-GFP COS-7 stable cell line ( Fig. 22 ). These data demonstrate that fusions of integrase (lacking the C-terminal DNA binding domain) with catalytically dead Cas9 in the context of the Gag-Pol lentiviral polyprotein allow lentiviral packaging, delivery, and targeting of lentivirally encoded donor sequences in mammalian cells. In addition, these data suggest that expression of guide RNA in lentiviral packaging cells is sufficient for incorporation into lentiviral particles, which may occur through strong interactions with dCas9. Alternative methods of delivering guide RNA into lentiviral particles may enhance targeted integration, such as by constitutively expressing the guide RNA in a transfer plasmid.

[0424] Alternative DNA binding domains for targeted integration into lentiviral particles.

[0425] This data demonstrates that replacing the nonspecific DNA binding domain of integrase with the programmable DNA binding domain of dCas9 allows targeted integration of dsDNA donor templates, or delivery via lentiviral particles for delivery of lentivirally encoded donor sequences. The CRISPR-Cas system is a two-component system that relies on both Cas proteins and small guide RNAs for targeting. In some cases, it may be beneficial to use single-component DNA targeting proteins (such as TALEN) for delivery via lentiviral particles because these proteins are only targeted by the encoded proteins. Using a similar lentiviral production method, replacing dCas9 in the previous packaging strategy with TALEN targeting a given sequence (e.g., eGFP or a safe harbor locus) allows lentiviral packaging and targeting without the need to deliver guide RNAs ( Fig.23 For example, TALENs are packaged and delivered as fusions with integrase in the context of the gag-pol polyprotein ( Fig.23A), IN-TALENs are packaged and delivered as fusions with viral incorporation proteins such as VPR ( Fig.23 B), or IN-TALEN delivered within a transfer plasmid ( Fig.23 C).

[0426] Example 9: Enhancing CRISPR-Cas9 DNA Editing with Ty1 NLS

[0427] The CRISPR-Cas DNA cleavage system is derived from bacteria, and the Cas proteins are both large and lack an intrinsic mammalian nuclear localization signal (NLS), preventing their efficient nuclear localization in mammalian cells.

[0428] To determine whether Ty1 enhances CRISPR-Cas9 editing, an existing CRISPR-Cas9 expression plasmid (px330) was modified by replacing the C-terminal NPM NLS with a non-canonical Ty1 NLS (px330-Ty1) ( Fig.24 A). Next, a frameshift-responsive luciferase reporter gene was generated, which encodes an out-of-frame luciferase coding sequence ( Fig.24 B) For this reporter gene assay, cleavage near the target sequence and incomplete repair by the cellular non-homologous end joining (NHEJ) pathway can induce nucleotide insertions or deletions that may reconstitute the luciferase coding sequence and result in luciferase expression.

[0429] Co-expression of a luciferase reporter gene with a vector encoding Cas9 containing an NPM NLS and a single guide RNA specific for a 20-nucleotide target sequence ( Fig.24 C). Notably, expression of Cas9 containing the Ty1 NLS produced a significant enhancement (approximately 44%) of reporter gene activity in COS-7 cells compared to Cas9 containing the NPM NLS ( Fig.24 C).

[0430] Example 10: Non-homologous DNA integration using integrase-TALEN fusion proteins

[0431] Transcription activator-like effector nucleases (TALENs) are well-studied programmable DNA-binding proteins constructed by the tandem assembly of separate nucleotide targeting domains (Reyon et al., 2012). In a similar approach demonstrated for Inscriptr, TALENs can be used to direct retroviral integrase-mediated integration of a donor DNA template ( Fig.25). To produce TALEN-integrase fusion proteins, mammalian expression vectors were constructed to accept TALEN targeting repeat sequences from previously described TALEN expression vectors to produce IN-TALEN or TALEN-IN fusions. Each fusion protein incorporates a 3xFLAG epitope, a Ty1 NLS, and a TALEN repeat sequence, separated by a linker sequence between HIV integrase (INΔC) lacking a C-terminal nonspecific DNA binding domain. In some cases, IN mutations may be incorporated to alter IN activity, dimerization, interaction with cellular proteins, resistance to dimerization inhibitors, or tandem copies of INΔC (tdINΔC). For example, an E85G mutation may be incorporated to inhibit obligate dimer formation.

[0432] TALEN pairs targeting eGFP have been described previously and the targeting efficiency has been verified (Reyon et al., 2012; available from Addgene). TALEN pairs (ClaI / BamHI fragments) were subcloned to generate TALEN-IN fusion proteins for eGFP with spacers of 16 bp or 28 bp in length.

[0433] Using plasmid DNA integration assay ( Fig.26 ), co-transfection of TALEN-IN pairs targeting eGFP, co-transfection of linear double-stranded DNA donor sequences encoding IGR-CAT resistance genes and amilCP bacterial expression reporter genes into mammalian COS-7 cells. Two days after transfection, the edited plasmids were recovered from mammalian cells and transformed into Escherichia coli and selected on chloramphenicol plates. Interestingly, TALEN pairs separated by 16 bp produced about 6 times more chloramphenicol-resistant colonies, while TALEN pairs separated by 28 bp were similar to non-targeted integrases ( Fig. 27 ). These data suggest that the proximity of the TALEN pair is important for targeting and integration, a feature that has been previously reported for TALEN-FokI-mediated dsDNA cleavage.

[0434] Implementation 11: Sequence Listing

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[0456] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0457] Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be designed by others skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. A fusion protein, comprising: a) a retroviral integrase (IN) or a fragment thereof having a first amino acid sequence; b) a CRISPR-associated (Cas) protein having a second amino acid sequence; and c) a retrotransposon nuclear localization signal (NLS) having a third amino acid sequence, wherein the retrotransposon nuclear localization signal is the Ty1 NLS shown in SEQ ID NO: 51 or SEQ ID NO: 56, wherein the retroviral integrase is represented by a sequence selected from SEQ ID NOs: 1-40, And wherein the CRISPR-associated protein is represented by a sequence selected from SEQ ID NOs: 41-46.

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

3. The fusion protein of claim 1, wherein the retroviral integrase fragment comprises an integrase N-terminal domain (NTD) and an integrase catalytic core domain (CCD).

4. The fusion protein of claim 1, wherein the CRISPR-associated protein is selected from the group consisting of Cas9, Cas13 and Cpf1.

5. The fusion protein of claim 1, wherein the CRISPR-associated protein is catalytically defective (dCas). The fusion protein of claim 1 , wherein the fusion protein is represented by a sequence selected from SEQ ID NOs: 57-98.

7. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 6.

8. The nucleic acid molecule of claim 7, wherein the nucleic acid is represented by a sequence selected from the group consisting of SEQ ID NOs: 155-196.

9. A method of editing genetic material, wherein the method of editing genetic material is for non-therapeutic use, and the method comprises administering to the genetic material: a) the fusion protein according to any one of claims 1 to 6 or the nucleic acid molecule according to any one of claims 7 to 8; b) a guide nucleic acid comprising a targeting nucleotide sequence complementary to a target region in the genetic material; and c) a donor template nucleic acid, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence and a donor template sequence.

10. Use of the fusion protein of any one of claims 1-6 or the nucleic acid molecule, guide nucleic acid and donor template nucleic acid of any one of claims 7-8 in editing genetic material in vivo, wherein the guide nucleic acid comprises a targeting nucleotide sequence complementary to a target region in the genetic material, and the donor template nucleic acid comprises a U3 sequence, a U5 sequence and a donor template sequence, wherein the use is for non-therapeutic purposes.

11. A system for editing genetic material, the system comprising in one or more vectors: a) a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a retroviral integrase (IN) or a fragment thereof, a CRISPR-associated (Cas) protein and a retrotransposon nuclear localization signal (NLS), wherein the retrotransposon nuclear localization signal is the Ty1 NLS shown in SEQ ID NO: 51 or SEQ ID NO: 56; b) a nucleic acid sequence encoding a guide RNA for the CRISPR-Cas system; and c) a nucleic acid sequence encoding a donor template nucleic acid, wherein the donor template nucleic acid comprises a U3 sequence, a U5 sequence and a donor template sequence, wherein the retroviral integrase is represented by a sequence selected from SEQ ID NOs: 1-40, And wherein the CRISPR-associated protein is represented by a sequence selected from SEQ ID NOs: 41-46.

12. The system of claim 11, wherein the nucleic acids of a), b) and c) are on the same or different vectors.

13. The system of claim 11, wherein the fusion protein is represented by a sequence selected from SEQ ID NOs: 57-98.

14. The system of claim 11, wherein the CRISPR-Cas system guide RNA substantially hybridizes to a target DNA sequence in the genetic material.

15. The system of claim 11, wherein the U3 sequence and the U5 sequence are specific for the retroviral integrase.

16. A system for delivering genome editing components, the system comprising: a) a packaging plasmid comprising a sequence encoding a gag-pol polyprotein, wherein the gag-pol polyprotein comprises an integrase fused to a catalytically dead Cas (dCas) protein and a retrotransposon nuclear localization signal, wherein the retrotransposon nuclear localization signal is a Ty1 NLS set forth in SEQ ID NO: 51 or SEQ ID NO: 56; b) a transfer plasmid comprising a sequence encoding a donor sequence, a 5' LTR and a 3' LTR; and c) an envelope plasmid, said envelope plasmid comprising a nucleic acid sequence encoding an envelope protein, wherein the integrase is represented by a sequence selected from SEQ ID NOs: 1-40, And wherein the catalytic death Cas protein is represented by a sequence selected from SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:

46.

17. The system of claim 16, wherein the packaging plasmid further comprises a sequence encoding a guide RNA sequence.

18. A system for delivering genome editing components, the system comprising: a) a packaging plasmid, the packaging plasmid comprising a sequence encoding a gag-pol polyprotein; b) a transfer plasmid comprising a sequence encoding a donor sequence, a 5' LTR and a 3' LTR; c) an envelope plasmid, said envelope plasmid comprising a nucleic acid sequence encoding an envelope protein; and d) a VPR-IN-dCas plasmid, the VPR-IN-dCas plasmid comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising VPR, integrase and catalytic dead Cas (dCas) and a retrotransposon nuclear localization signal, wherein the retrotransposon nuclear localization signal is a Ty1 NLS shown in SEQ ID NO: 51 or SEQ ID NO: 56, wherein the integrase is represented by a sequence selected from SEQ ID NOs: 1-40, And wherein the catalytic death Cas is represented by a sequence selected from SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:

46.

19. The system of claim 18, wherein the VPR-IN-dCas plasmid further comprises a sequence encoding a guide RNA sequence.

20. A system for delivering genome editing components, the system comprising: a) a packaging plasmid, the packaging plasmid comprising a nucleic acid sequence encoding a gag-pol polyprotein; b) a transfer plasmid comprising a nucleic acid sequence encoding a guide RNA, a nucleic acid sequence encoding a fusion protein comprising an integrase and a catalytically dead Cas and a retrotransposon nuclear localization signal, and a 5'LTR and a 3'LTR, wherein the retrotransposon nuclear localization signal is a Ty1 NLS shown in SEQ ID NO: 51 or SEQ ID NO: 56; and c) an envelope plasmid, said envelope plasmid comprising a nucleic acid sequence encoding an envelope protein, wherein the integrase is represented by a sequence selected from SEQ ID NOs: 1-40, And wherein the catalytic death Cas is represented by a sequence selected from SEQ ID NO:42, 44, 46.

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