Peptides and nanoparticles for intracellular delivery of genome-editing molecules

Abstract

To provide peptides and nanoparticles for intracellular delivery of genome-editing molecules.SOLUTION: The present application provides complexes and nanoparticles comprising cell- penetrating peptide that are useful for stabilizing and / or delivering into a cell one or more molecules of a genome-editing system, such as CRISPR-associated proteins and nucleic acids. In some embodiments, there is provided a genome-editing complex for modifying a target polynucleotide comprising a cell-penetrating peptide associated with an RGEN / gRNA complex comprising an RNA-guided endonuclease (RGEN) and a guide RNA (gRNA). The gRNA comprises a guide sequence complementary to a target sequence in the target polynucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 342,823, filed May 27, 2016, U.S. Provisional Application No. 62 / 394,140, filed September 13, 2016, and U.S. Provisional Application No. 62 / 477,357, filed March 27, 2017, all of which are incorporated by reference in their entireties.

[0002] The present invention relates to peptide-containing complexes / nanoparticles that are useful for stabilizing and / or delivering one or more molecules of a genome editing system, such as CRISPR-associated proteins and nucleic acids, into cells. Submitting a sequence listing as an ASCII text file

[0003] The following submission in an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (Filename: 737372000840SeqList.txt, Date Recorded: May 26, 2017, Size: 48KB). [Background technology]

[0004] CRISPR interference technology has numerous potential applications, including altering the germline of humans, animals, and other organisms, as well as modifying the genes of food crops. By delivering a CRISPR-associated nuclease, such as Cas9 or Cpf1, and an appropriate guide RNA into a cell, the genome of an organism can be cut at almost any desired location (Ledford, 2003). , H. (2015). Nature. Vol. 522 (p. 7554); Zetsche et al. (2015). Cell. Vol. 163 (No. 3): pp. 759-771). CRISPR has been shown to be effective in many different species. CRISPR tools are being used in conjunction with specific endonuclease enzymes for genome editing and gene regulation in mammalian cells (Mali et al., (2013). Nature Methods. 10(10):957-63). CRISPR tools are being used for double-strand break formation using non-homologous end joining (NHEJ)-mediated mutagenesis (Wang et al., (2013). Cell. 153:910-918), nicking using NHEJ-mediated mutagenesis (Cheng et al., (2014). FEBS Lett. 588:3954-3958), knock-in using homology-directed repair (HDR) (Inui et al., (2014). Sci. Rep. 4:5396), and genome rearrangement (Maddalo Nuclease-deficient Cas nucleases have been used for a variety of genome modifications, including silencing and activating gene expression (Ledford, H. (2016). Nature. 531:156-159). Nuclease-deficient Cas nucleases can be used to block the binding of transcriptional activators to prevent the expression of specific genes, to deliver transcriptional activators to stimulate the expression of specific genes, or to deliver epigenetic modifiers to target sequences to alter histone methylation or acetylation (Ledford, H. (2016). Nature. 531:156-159). The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Ledford, H. (2015). Nature. Volume 522 (7554 pages) [Non-patent document 2] Zetsche et al. (2015). Cell. 163(3):759-771 [Non-patent document 3] Mali et al. (2013). Nature Methods. Vol. 10(10): 957-63 [Non-patent document 4] Wang et al. (2013). Cell. 153:910-918 [Non-patent document 5] Cheng et al. (2014). FEBS Lett. 588:3954-3958 [Non-patent document 6] Inui et al. (2014). Sci. Rep. 4:5396 [Non-Patent Document 7] Maddalo et al. (2014) Nature. 516:423-427 [Non-patent document 8] Ledford, H. (2016). Nature. 531:156-159 Summary of the Invention [Means for solving the problem]

[0006] The present application provides complexes and nanoparticles comprising cell membrane-permeable peptides that are useful for stabilizing and / or delivering one or more molecules of a genome editing system, such as CRISPR-associated proteins and nucleic acids, to cells.

[0007] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, comprising a cell membrane-permeable peptide associated with an RGEN / gRNA complex comprising an RNA-guided endonuclease (RGEN) and a guide RNA (gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide.

[0008] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide and one or more molecules of a genome editing system that targets the target polynucleotide, wherein the cell membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the one or more genome editing system molecules are selected from the group consisting of: a) a guide RNA (gRNA) comprising an RNA-guided endonuclease (RGEN) and a guide sequence complementary to a target sequence in the target polynucleotide; b) a guide DNA (gDNA) comprising an DNA-guided endonuclease (DGEN) and a guide sequence complementary to a target sequence in the target polynucleotide; c) a zinc finger protein (ZFP) that recognizes a target sequence in the target polynucleotide; d) a transcription activator-like effector nuclease (TALEN) that recognizes a target sequence in the target polynucleotide; e) a homing endonuclease that recognizes a target sequence in the target polynucleotide; and f) an integrase that recognizes a recombination site in the target polynucleotide.

[0009] In some embodiments, according to any of the genome editing complexes described above, the cell membrane-permeable peptide is a VEPEP-3 peptide. In some embodiments, the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14. In some embodiments, the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 75 or 76.

[0010] In some embodiments, the cell membrane-permeable peptide is a VEPEP-6 peptide. In some embodiments, the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 40. In some embodiments, the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 77.

[0011] In some embodiments, according to any of the genome editing complexes described above, the cell membrane-permeable peptide is a VEPEP-9 peptide. In some embodiments, the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52. In some embodiments, the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 78.

[0012] In some embodiments, according to any of the genome editing complexes described above, the cell membrane-permeable peptide is an ADGN-100 peptide. In some embodiments, the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 70. In some embodiments, the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 79 or 80.

[0013] In some embodiments, according to any of the genome editing complexes described above, the cell membrane-permeable peptide further comprises one or more moieties covalently linked to the N-terminus of the cell membrane-permeable peptide, wherein the one or more moieties are selected from the group consisting of acetyl, a fatty acid, cholesterol, polyethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, and a targeting molecule. In some embodiments, the cell membrane-permeable peptide comprises an acetyl group covalently linked to its N-terminus. In some embodiments, the cell membrane-permeable peptide further comprises one or more moieties covalently linked to the C-terminus of the cell membrane-permeable peptide, wherein the one or more moieties are selected from the group consisting of cysteamide, cysteine, thiol, amide, optionally substituted nitrilotriacetic acid, carboxyl, optionally substituted linear or branched C1-C6 alkyl, primary or secondary amine, osidic derivative, lipid, phospholipid, fatty acid, cholesterol, polyethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, and a targeting molecule. In some embodiments, the cell membrane-penetrating peptide comprises a cysteamide group covalently linked to its C-terminus.

[0014] In some embodiments, according to any of the genome editing complexes described above, at least a portion of the cell membrane-permeable peptides in the genome editing complex are linked to the targeting moiety by a linkage. In some embodiments, the linkage is a covalent bond.

[0015] In some embodiments, according to any of the above genome editing complexes comprising a gRNA, the gRNA is a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises a specificity-determining CRISPR RNA (crRNA) fused to a complementary trans-activating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence.

[0016] In some embodiments, according to any of the above genome editing complexes comprising RGEN, the RGEN is Cas9.

[0017] In some embodiments, in any of the above genome editing complexes comprising RGEN and gRNA, the molar ratio of RGEN to gRNA is between about 1:10 and about 10:1.

[0018] In some embodiments, according to any of the above genome editing complexes containing RGEN, the molar ratio of the cell membrane-permeable peptide to RGEN is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of the cell membrane-permeable peptide to RGEN is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)).

[0019] In some embodiments, according to any of the above genome editing complexes comprising a gRNA, the genome editing complex further comprises one or more additional gRNAs comprising different guide sequences. In some embodiments, the target sequence is present in a target gene, and one or more additional gRNAs each comprise a guide sequence complementary to a sequence in the target gene. In some embodiments, the target sequence is present in a first target gene, and one or more additional gRNAs each comprise a guide sequence complementary to a sequence present in one or more additional target genes.

[0020] In some embodiments, according to any of the above genome editing complexes, one or more genome editing system molecules comprise a ZFP. In some embodiments, according to any of the above genome editing complexes, one or more genome editing system molecules comprise a TALEN. In some embodiments, according to any of the above genome editing complexes, one or more genome editing system molecules comprise a homing endonuclease.

[0021] In some embodiments, according to any of the genome editing complexes described above, the genome editing complex further comprises a donor nucleic acid for introducing a modification into a target polynucleotide, the donor nucleic acid comprising a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification. In some embodiments, the modification is an addition, deletion, or substitution of one or more nucleotides in the target polynucleotide. In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is an insertion of a heterologous nucleic acid into the target polynucleotide. In some embodiments, the donor nucleic acid is a double-stranded DNA comprising a heterologous nucleic acid flanked by 5' and 3' homology arms, the 5' and 3' homology arms being homologous to sequences flanking the region of the target polynucleotide to be modified. In some embodiments, the genome editing complex further comprises one or more additional donor nucleic acids for introducing a modification into one or more additional target polynucleotides. In some embodiments, each of the one or more additional donor nucleic acids comprises a sequence corresponding to a portion of one of the one or more additional target polynucleotides to be modified to include the modification.

[0022] In some embodiments, according to any of the genome editing complexes described above, the one or more genome editing system molecules comprise an integrase. In some embodiments, the genome editing complex further comprises a donor nucleic acid for insertion into a target polynucleotide, wherein the integrase can recognize a recombination site in the donor nucleic acid and mediate recombination between the recombination site in the target polynucleotide and the recombination site in the donor nucleic acid to insert the donor nucleic acid into the target polynucleotide.

[0023] In some embodiments, according to any of the above genome editing complexes, the average diameter of the genome editing complex is between about 10 nm and about 300 nm.

[0024] In some embodiments, a nanoparticle is provided comprising a core containing a genome editing complex according to any of the above embodiments. In some embodiments, the core comprises one or more additional genome editing complexes according to any of the above embodiments. In some embodiments, the core further comprises a donor nucleic acid for introducing a modification into a target polynucleotide, the donor nucleic acid comprising a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the donor nucleic acid being complexed with a second cell membrane-permeable peptide. In some embodiments, the second cell membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the second cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-70 and 75-80. In some embodiments, the modification is an addition, deletion, or substitution of one or more nucleotides in the target polynucleotide. In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is an insertion of a heterologous nucleic acid into the target polynucleotide. In some embodiments, the donor nucleic acid is a double-stranded DNA comprising a heterologous nucleic acid flanked by a 5' homology arm and a 3' homology arm, the 5' homology arm and the 3' homology arm being homologous to the sequence flanking the region of the target polynucleotide to be modified. In some embodiments, the core further comprises one or more additional donor nucleic acids for introducing a modification into one or more additional target polynucleotides, each of the one or more additional donor nucleic acids comprising a sequence corresponding to the portion to be modified to include the modification for one of the one or more additional target polynucleotides, each of the one or more additional donor nucleic acids being complexed with a cell membrane-permeable peptide. In some embodiments, at least a portion of the cell membrane-permeable peptide in the nanoparticle is linked to the targeting moiety by a linkage. In some embodiments, the core is coated with a shell comprising a peripheral cell membrane-permeable peptide. In some embodiments, at least a portion of the peripheral cell membrane-permeable peptide in the shell is linked to the targeting moiety by a linkage. In some embodiments, the linkage is a covalent bond.In some embodiments, the peripheral cell membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the peripheral cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-70 and 75-80. In some embodiments, the nanoparticles have an average diameter between about 10 nm and about 400 nm. In some embodiments, the nanoparticles have an average diameter between about 50 nm and about 300 nm. In some embodiments, the nanoparticles have an average diameter between about 80 nm and about 200 nm.

[0025] In some embodiments, a pharmaceutical composition is provided comprising a genome editing complex according to any of the above embodiments or a nanoparticle according to any of the above embodiments and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for intravenous, intratumoral, intraarterial, topical, intraocular, ophthalmic, intraportal, intracranial, intracerebral, intraventricular, intrathecal, intravesical, intradermal, subcutaneous, intramuscular, intranasal, intratracheal, pulmonary, intracavity, or oral administration. In some embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition further comprises an expression complex comprising a third cell-membrane-permeable peptide and a nucleic acid molecule encoding an exogenous protein. In some embodiments, the exogenous protein is a recombinant receptor that can be expressed on the surface of a cell. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the third cell-membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the third cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-70 and 75-80.

[0026] In some embodiments, a method for preparing a genome editing complex according to any of the above embodiments is provided, comprising combining a cell membrane-permeable peptide with an RGEN / gRNA complex, thereby forming a genome editing complex. In some embodiments, the cell membrane-permeable peptide is combined with the RGEN / gRNA complex at a ratio of about 1:1 to about 80:1. In some embodiments, the cell membrane-permeable peptide is combined with the RGEN / gRNA complex at a ratio of about 5:1 to about 20:1.

[0027] In some embodiments, a method for preparing a genome editing complex according to any of the above embodiments is provided, comprising combining a cell membrane-permeable peptide with one or more genome editing system molecules, thereby forming a genome editing complex. In some embodiments, the genome editing system molecule comprises RGEN and gRNA, and the cell membrane-permeable peptide and RGEN are combined at a ratio of about 1:1 to about 80:1, respectively, and the RGEN and gRNA are combined at a ratio of about 10:1 to about 1:10, respectively. In some embodiments, the cell membrane-permeable peptide and RGEN are combined at a ratio of about 5:1 to about 20:1, respectively. In some embodiments, the RGEN and gRNA are combined at a ratio of about 1:1.

[0028] In some embodiments, a method of delivering one or more molecules of a genome editing system to a cell is provided, comprising contacting the cell with a genome editing complex according to any of the above embodiments or a nanoparticle according to any of the above embodiments, wherein the genome editing complex or nanoparticle comprises one or more genome editing system molecules. In some embodiments, contacting the cell with the genome editing complex nanoparticle occurs in vivo. In some embodiments, contacting the cell with the genome editing complex nanoparticle occurs ex vivo. In some embodiments, contacting the cell with the genome editing complex nanoparticle occurs in vitro. In some embodiments, the cell is a granulocyte, mast cell, monocyte, dendritic cell, B cell, T cell, natural killer cell, fibroblast, or hepatocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is a fibroblast. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a lung progenitor cell. In some embodiments, the cell is a neuronal cell. In some embodiments, the genome editing system targets a sequence in a gene selected from the group consisting of PD-1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, CTLA-4, TIGIT, 4-1BB, OX40, CD27, TIM-1, CD28, HVEM, GITR, and ICOS. In some embodiments, the method further comprises contacting the cell with an expression complex comprising a fourth cell-membrane-permeable peptide and a nucleic acid molecule encoding an exogenous protein. In some embodiments, the exogenous protein is a recombinant receptor capable of being expressed on the surface of the cell. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the fourth cell-membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the fourth cell-membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-70 and 75-80.

[0029] In some embodiments, a method of modifying a target polynucleotide in a cell is provided, comprising contacting the cell with a genome editing complex according to any of the above embodiments or a nanoparticle according to any of the above embodiments, wherein the genome editing complex or nanoparticle comprises one or more molecules of a genome editing system that targets a sequence within the target polynucleotide.

[0030] In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition according to any of the above embodiments. In some embodiments, the disease is selected from the group consisting of cancer, diabetes, inflammatory diseases, fibrosis, viral infection diseases, genetic diseases, eye diseases, liver diseases, lung diseases, kidney diseases, and aging and degenerative diseases. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulates expression of one or more proteins selected from the group consisting of growth factors and cytokines, cell surface receptors, signaling molecules and kinases, transcription factors and other transcription regulators, regulators of protein expression and modification, and regulators of apoptosis and metastasis. In some embodiments, the cancer is cancer of the liver, lung, or kidney. In some embodiments, the cancer is a hematological malignancy, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that modulates expression of one or more proteins selected from the group consisting of growth factors and cytokines, cell surface receptors, signaling molecules and kinases, transcription factors and other transcriptional regulators, regulators of protein expression and modification, and regulators of apoptosis and metastasis.

[0031] In some embodiments, according to any of the methods of treating a disease described above, the disease is a viral infectious disease, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulate the expression of one or more proteins involved in the onset and / or progression of the viral infectious disease.

[0032] In some embodiments, according to any of the methods of treating a disease described above, the disease is a genetic disease and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulate the expression of one or more proteins involved in the onset and / or progression of the genetic disease.

[0033] In some embodiments, according to any of the methods of treating a disease described above, the disease is an aging or degenerative disease, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulates the expression of one or more proteins involved in the onset and / or progression of the aging or degenerative disease.

[0034] In some embodiments, according to any of the above methods of treating a disease, the disease is a fibrotic or inflammatory disease, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulates the expression of two or more proteins involved in the development and / or progression of the fibrotic or inflammatory disease.

[0035] In some embodiments, according to any of the methods of treating a disease described above, the individual is a human.

[0036] In some embodiments, a kit is provided that includes a composition comprising a genome editing complex according to any of the above embodiments and / or a nanoparticle according to any of the above embodiments. In certain embodiments, for example, the following items are provided: (Item 1) A genome editing complex for modifying a target polynucleotide, comprising a cell membrane-permeable peptide and one or more molecules of a genome editing system, wherein the cell membrane-permeable peptide is selected from the group consisting of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, and an ADGN-100 peptide, and the one or more molecules of the genome editing system are: a) an RNA-guided endonuclease (RGEN) and / or a guide RNA (gRNA) comprising a guide sequence complementary to a target sequence within the target polynucleotide; b) one or both of a DNA-guided endonuclease (DGEN) and a guide DNA (gDNA) comprising a guide sequence complementary to a target sequence within the target polynucleotide; c) a zinc finger protein (ZFP) that recognizes a target sequence within the target polynucleotide; d) a transcription activator-like effector nuclease (TALEN) that recognizes a target sequence within the target polynucleotide; e) a homing endonuclease that recognizes a target sequence within the target polynucleotide; and f) an integrase that recognizes a recombination site within the target polynucleotide A genome editing complex selected from the group consisting of: (Item 2) The genome editing complex described in item 1, wherein the cell membrane-permeable peptide is a VEPEP-3 peptide. (Item 3) 3. The genome editing complex according to item 2, wherein the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14. (Item 4) The genome editing complex described in Item 2, wherein the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 75 or 76. (Item 5) The genome editing complex described in item 1, wherein the cell membrane-permeable peptide is a VEPEP-6 peptide. (Item 6) 6. The genome editing complex described in Item 5, wherein the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 40. (Item 7) The genome editing complex described in Item 5, wherein the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 77. (Item 8) The genome editing complex described in item 1, wherein the cell membrane-permeable peptide is a VEPEP-9 peptide. (Item 9) 9. The genome editing complex described in Item 8, wherein the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 52. (Item 10) The genome editing complex described in Item 8, wherein the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 78. (Item 11) The genome editing complex described in item 1, wherein the cell membrane-permeable peptide is an ADGN-100 peptide. (Item 12) Item 12. The genome editing complex described in Item 11, wherein the cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 70. (Item 13) The genome editing complex described in Item 11, wherein the cell membrane-permeable peptide comprises the amino acid sequence of SEQ ID NO: 79 or 80. (Item 14) 14. The genome editing complex of any one of items 1 to 13, wherein the cell membrane-permeable peptide further comprises one or more moieties covalently linked to the N-terminus of the cell membrane-permeable peptide, and the one or more moieties are selected from the group consisting of acetyl, fatty acids, cholesterol, polyethylene glycol, nuclear localization signals, nuclear export signals, antibodies, polysaccharides, and targeting molecules. (Item 15) Item 15. The genome editing complex of item 14, wherein the cell membrane-permeable peptide comprises an acetyl group covalently linked to its N-terminus. (Item 16) 16. The genome editing complex of any one of items 1 to 15, wherein the cell membrane-permeable peptide further comprises one or more moieties covalently linked to the C-terminus of the cell membrane-permeable peptide, and the one or more moieties are selected from the group consisting of cysteamide, cysteine, thiol, amide, optionally substituted nitrilotriacetic acid, carboxyl, optionally substituted linear or branched C1-C6 alkyl, primary or secondary amine, oside derivative, lipid, phospholipid, fatty acid, cholesterol, polyethylene glycol, nuclear localization signal, nuclear export signal, antibody, polysaccharide, and targeting molecule. (Item 17) Item 17. The genome editing complex of Item 16, wherein the cell membrane-permeable peptide comprises a cysteamide group covalently linked to its C-terminus. (Item 18) 18. A genome editing complex described in any one of items 1 to 17, wherein at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety by a linkage. (Item 19) The genome editing complex described in Item 18, wherein the linkage is a covalent bond. (Item 20) 20. A genome editing complex according to any one of items 1 to 19, comprising one or both of RGEN and gRNA. (Item 21) 21. The genome editing complex of item 20, comprising RGEN and gRNA. (Item 22) 22. The genome editing complex of item 20 or 21, wherein the gRNA is a single guide RNA (sgRNA). (Item 23) 23. The genome editing complex of any one of items 20 to 22, wherein the RGEN is Cas9. (Item 24) 24. A genome editing complex according to any one of items 20 to 23, wherein the molar ratio of RGEN to gRNA is between about 1:10 and about 10:1. (Item 25) 25. The genome editing complex of any one of items 20 to 24, wherein the molar ratio of the cell membrane-permeable peptide to the RGEN is between about 1:1 and about 80:1. (Item 26) 26. The genome editing complex of item 25, wherein the molar ratio of the cell membrane-permeable peptide to the RGEN is between about 5:1 and about 20:1. (Item 27) 27. The genome editing complex of any one of items 20 to 26, further comprising one or more additional gRNAs comprising different guide sequences. (Item 28) 28. The genome editing complex of Item 27, wherein the target sequence is present within a target gene and the one or more additional gRNAs each comprise a guide sequence complementary to a sequence within the target gene. (Item 29) 28. The genome editing complex of Item 27, wherein the target sequence is present in a first target gene and the one or more additional gRNAs each comprise a guide sequence complementary to a sequence present in one or more additional target genes. (Item 30) 20. A genome editing complex according to any one of items 1 to 19, comprising one or both of a DGEN and gDNA. (Item 31) 32. The genome editing complex of any one of claims 20, comprising a DGEN and gDNA. 20. The genome editing complex of any one of items 1 to 19, comprising a ZFP. (Item 33) 20. The genome editing complex of any one of items 1 to 19, comprising a TALEN. (Item 34) 20. A genome editing complex described in any one of items 1 to 19, comprising a homing nuclease. (Item 35) 35. The genome editing complex of any one of items 20 to 34, further comprising a donor nucleic acid for introducing a modification into the target polynucleotide, the donor nucleic acid comprising a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification. (Item 36) 36. The genome editing complex of Item 35, wherein the modification is an addition, deletion or substitution of one or more nucleotides in the target polynucleotide. (Item 37) 37. The genome editing complex of item 35 or 36, wherein the donor nucleic acid is a single-stranded DNA oligonucleotide. (Item 38) 36. The genome editing complex of Item 35, wherein the modification is the insertion of a heterologous nucleic acid into the target polynucleotide. (Item 39) 39. The genome editing complex of claim 35 or 38, wherein the donor nucleic acid is a double-stranded DNA comprising a heterologous nucleic acid flanked by a 5' homology arm and a 3' homology arm, and the 5' homology arm and the 3' homology arm are homologous to sequences adjacent to the region of the target polynucleotide to be modified. (Item 40) 40. The genome editing complex of any one of items 35 to 39, further comprising one or more additional donor nucleic acids for introducing modifications into one or more additional target polynucleotides. (Item 41) 20. The genome editing complex of any one of items 1 to 19, comprising an integrase. (Item 42) 42. The genome editing complex of Item 41, further comprising a donor nucleic acid for insertion into the target polynucleotide, wherein the integrase can recognize a recombination site in the donor nucleic acid and mediate recombination between the recombination site in the target polynucleotide and the recombination site in the donor nucleic acid to insert the donor nucleic acid into the target polynucleotide. (Item 43) 43. The genome editing complex of any one of items 1 to 42, wherein the average diameter of the genome editing complex is between about 10 nm and about 300 nm. (Item 44) A nanoparticle comprising a core containing the genome editing complex described in any one of items 1 to 43. (Item 45) The nanoparticle described in Item 44, wherein the core further comprises one or more additional genome editing complexes described in any one of items 1 to 43. (Item 46) 46. The nanoparticle of item 44 or 45, wherein the core further comprises a donor nucleic acid for introducing a modification into the target polynucleotide, the donor nucleic acid comprising a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, and the donor nucleic acid is complexed with a second cell membrane-penetrating peptide. (Item 47) 47. The nanoparticles according to item 46, wherein the second cell membrane-penetrating peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide and ADGN-100 peptide. (Item 48) Item 48. The nanoparticle according to item 47, wherein the second cell membrane-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 70 and 75 to 80. (Item 49) 49. The nanoparticle of any one of items 46 to 48, wherein the modification is an addition, deletion or substitution of one or more nucleotides in the target polynucleotide. (Item 50) 50. The nanoparticle of item 49, wherein the donor nucleic acid is a single-stranded DNA oligonucleotide. (Item 51) 49. The nanoparticle of any one of items 46 to 48, wherein the modification is an insertion of a heterologous nucleic acid into the target polynucleotide. (Item 52) 52. The nanoparticle of claim 51, wherein the donor nucleic acid is a double-stranded DNA comprising a heterologous nucleic acid flanked by a 5' homology arm and a 3' homology arm, and the 5' homology arm and the 3' homology arm are homologous to sequences adjacent to the region of the target polynucleotide to be modified. (Item 53) 53. The nanoparticle of any one of items 46 to 52, wherein the core further comprises one or more additional donor nucleic acids for introducing modifications into one or more additional target polynucleotides. (Item 54) 54. The nanoparticle of any one of items 44 to 53, wherein at least a portion of the cell membrane-penetrating peptide in the nanoparticle is linked to a targeting moiety by a linkage. (Item 55) 54. The nanoparticle according to any one of items 44 to 53, wherein the core is coated with a shell comprising a peripheral cell membrane-penetrating peptide. (Item 56) 56. The nanoparticles according to item 55, wherein the peripheral cell membrane-penetrating peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide and ADGN-100 peptide. (Item 57) 57. The nanoparticle according to item 56, wherein the peripheral cell membrane-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 70 and 75 to 80. (Item 58) 58. The nanoparticle of any one of items 55 to 57, wherein at least a portion of the peripheral cell membrane-penetrating peptide within the shell is linked to a targeting moiety by a linkage. (Item 59) 59. The nanoparticle of claim 54 or 58, wherein the linkage is a covalent bond. (Item 60) 60. The nanoparticles according to any one of items 44 to 59, wherein the average diameter of the nanoparticles is between about 10 nm and about 400 nm. (Item 61) A pharmaceutical composition comprising a genome editing complex described in any one of items 1 to 43 or a nanoparticle described in any one of items 44 to 60, and a pharmaceutically acceptable carrier. (Item 62) 62. The pharmaceutical composition of item 61, further comprising an expression complex comprising a third cell membrane-permeable peptide and a nucleic acid molecule encoding an exogenous protein. (Item 63) 63. The pharmaceutical composition of item 62, wherein the foreign protein is a recombinant receptor capable of being expressed on the surface of a cell. (Item 64) 64. The pharmaceutical composition of item 63, wherein the recombinant receptor is a chimeric antigen receptor (CAR). (Item 65) 65. The pharmaceutical composition of any one of items 62 to 64, wherein the third cell membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide and ADGN-100 peptide. (Item 66) 66. The pharmaceutical composition according to item 65, wherein the third cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 70 and 75 to 80. (Item 67) A method for preparing a genome editing complex described in any one of items 1 to 19, comprising a step of combining the cell membrane-permeable peptide with the one or more genome editing system molecules, thereby forming the genome editing complex. (Item 68) Item 67, wherein the one or more genome editing system molecules comprise RGEN and gRNA, the cell membrane-permeable peptide and the RGEN are combined at a ratio of about 1: 1 to about 80: 1, and the RGEN and the gRNA are combined at a ratio of about 10: 1 to about 1: 10. (Item 69) Item 69. The method of item 68, wherein the cell membrane-permeable peptide and the RGEN are combined in a ratio of about 5:1 to about 20:1, respectively. (Item 70) 69. The method of claim 68, wherein the RGEN and the gRNA are combined in a ratio of about 1:1. (Item 71) A method for delivering one or more molecules of a genome editing system to a cell, comprising contacting the cell with a genome editing complex described in any one of items 1 to 43 or a nanoparticle described in any one of items 44 to 60, wherein the genome editing complex or the nanoparticle comprises the one or more molecules of the genome editing system. (Item 72) 72. The method of claim 71, wherein the step of contacting the cell with the genome editing complex or nanoparticle is performed in vivo. (Item 73) 72. The method of claim 71, wherein the step of contacting the cell with the genome editing complex or nanoparticle is performed ex vivo. (Item 74) 72. The method of claim 71, wherein the step of contacting the cell with the genome editing complex or nanoparticle is carried out in vitro. (Item 75) 75. The method of any one of paragraphs 71 to 74, wherein the cell is a granulocyte, mast cell, monocyte, dendritic cell, B cell, T cell, natural killer cell, fibroblast, hepatocyte, lung progenitor cell, or neuronal cell. (Item 76) 76. The method of item 75, wherein the cell is a T cell. (Item 77) 77. The method of paragraph 75 or 76, wherein the genome editing system targets a sequence in a gene selected from the group consisting of PD-1, PD-L1, PD-L2, TIM-3, BTLA, VISTA, LAG-3, CTLA-4, TIGIT, 4-1BB, OX40, CD27, TIM-1, CD28, HVEM, GITR, and ICOS. (Item 78) 78. The method of any one of items 71 to 77, further comprising contacting the cell with an expression complex comprising a fourth cell membrane-permeable peptide and a nucleic acid molecule encoding an exogenous protein. (Item 79) 79. The method of claim 78, wherein the foreign protein is a recombinant receptor capable of being expressed on the surface of a cell. (Item 80) 80. The method of claim 79, wherein the recombinant receptor is a chimeric antigen receptor (CAR). (Item 81) 81. The method of any one of items 78 to 80, wherein the fourth cell membrane-permeable peptide is selected from the group consisting of VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide and ADGN-100 peptide. (Item 82) Item 82. The method according to Item 81, wherein the fourth cell membrane-permeable peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 70 and 75 to 80. (Item 83) A method for modifying a target polynucleotide in a cell, comprising contacting the cell with a genome editing complex described in any one of items 1 to 43 or a nanoparticle described in any one of items 44 to 60, wherein the genome editing complex or the nanoparticle comprises one or more molecules of a genome editing system that targets a sequence within the target polynucleotide. (Item 84) 67. A method for treating a disease in an individual, comprising administering to said individual an effective amount of the pharmaceutical composition of any one of items 61 to 66. (Item 85) 85. The method of item 84, wherein the disease is selected from the group consisting of cancer, diabetes, inflammatory diseases, fibrosis, viral infectious diseases, genetic diseases, eye diseases, liver diseases, lung diseases, kidney diseases, aging and degenerative diseases, and diseases characterized by abnormal cholesterol levels. (Item 86) Item 86. The method of item 85, wherein the disease is cancer. (Item 87) 87. The method of item 86, wherein the cancer is a solid tumor, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that modulates expression of one or more proteins selected from the group consisting of growth factors and cytokines, cell surface receptors, signaling molecules and kinases, transcription factors and other transcription regulators, regulators of protein expression and modification, tumor suppressors, and regulators of apoptosis and metastasis. (Item 88) 88. The method of item 87, wherein the cancer is liver, lung, or kidney cancer. (Item 89) 87. The method of claim 86, wherein the cancer is a hematological malignancy, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that modulates expression of one or more proteins selected from the group consisting of growth factors and cytokines, cell surface receptors, signaling molecules and kinases, transcription factors and other transcription regulators, regulators of protein expression and modification, tumor suppressors, and regulators of apoptosis and metastasis. (Item 90) 86. The method of claim 85, wherein the disease is a viral infectious disease and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulates the expression of one or more proteins involved in the onset and / or progression of the viral infectious disease. (Item 91) 86. The method of claim 85, wherein the disease is a genetic disease and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulate the expression of one or more proteins involved in the onset and / or progression of the genetic disease. (Item 92) 86. The method of claim 85, wherein the disease is an aging or degenerative disease, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulates the expression of one or more proteins involved in the onset and / or progression of the aging or degenerative disease. (Item 93) 86. The method of claim 85, wherein the disease is a fibrotic or inflammatory disease, and the pharmaceutical composition comprises a genome editing complex or nanoparticle comprising one or more molecules of a genome editing system that regulates the expression of two or more proteins involved in the onset and / or progression of the fibrotic or inflammatory disease. (Item 94) 94. The method of any one of items 84 to 93, wherein the individual is a human. (Item 95) A kit comprising a composition comprising a genome editing complex described in any one of items 1 to 43 and / or a nanoparticle described in any one of items 44 to 60. [Brief explanation of the drawings]

[0037] [Figure 1A] Figure 1A shows the binding titration curve of CAS9 to CPPs monitored by CY-5 fluorescence. A fixed concentration of 5 nM fluorescently labeled CAS9 was titrated with increasing concentrations of VEPEP-3b (SEQ ID NO: 76), VEPEP-6 (SEQ ID NO: 77), VEPEP-9 (SEQ ID NO: 78), CADY (SEQ ID NO: 81), and ADGN-100b (SEQ ID NO: 80) peptides. The dissociation constant was calculated from data fitting using a quadratic equation.

[0038] [Figure 1B] Figure 1B shows the binding titration curve of CAS9:gRNA to CPPs monitored by CY-5 fluorescence. A fixed concentration of 5 nM fluorescently labeled CAS9:gRNA was titrated with increasing concentrations of VEPEP-3b (SEQ ID NO: 76), VEPEP-6 (SEQ ID NO: 77), VEPEP-9 (SEQ ID NO: 78), CADY (SEQ ID NO: 81), and ADGN-100b (SEQ ID NO: 80) peptides. The dissociation constant was calculated from data fitting using a quadratic equation.

[0039] [Figure 2A]Figure 2A shows the gene disruption frequency of the EGFP reporter gene in U2OS cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a 1:5 molar ratio, as well as with lipofectamine 2000 and RNAiMAX.

[0040] [Figure 2B] Figure 2B shows the gene disruption frequency of the EGFP reporter gene in U2OS cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a 1:10 molar ratio, as well as with lipofectamine 2000 and RNAiMAX.

[0041] [Figure 2C] Figure 2C shows the gene disruption frequency of the EGFP reporter gene in U2OS cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a molar ratio of 1:20, as well as with lipofectamine 2000 and RNAiMAX.

[0042] [Figure 2D] Figure 2D shows the dose-response of gene disruption of the EGFP reporter gene in U2OS using different peptides. Lipofectamine 2000 and RNAiMAX were used as controls. The values in parentheses indicate the molar ratio of peptide to CAS9:gRNA complex.

[0043] [Figure 3A]Figure 3A shows the gene disruption frequency of the EGFP reporter gene in HEK cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a 1:5 molar ratio, as well as with lipofectamine 2000 and RNAiMAX.

[0044] [Figure 3B] Figure 3B shows the gene disruption frequency of the EGFP reporter gene in HEK cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a 1:10 molar ratio, as well as with lipofectamine 2000 and RNAiMAX.

[0045] [Figure 3C] Figure 3C shows the gene disruption frequency of the EGFP reporter gene in HEK cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a molar ratio of 1:20, as well as with lipofectamine 2000 and RNAiMAX.

[0046] [Figure 3D] Figure 3D shows the dose-response of gene disruption of the EGFP reporter gene in HEK cells using different peptides. Lipofectamine 2000 and RNAiMAX were used as controls. The values in parentheses indicate the molar ratio of peptide to CAS9:gRNA complex.

[0047] [Figure 4A]Figure 4A shows the frequency of gene disruption of the EGFP reporter gene in JURKAT T cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a molar ratio of 1:50, as well as with lipofectamine 2000 and RNAiMAX.

[0048] [Figure 4B] Figure 4B shows the gene disruption frequency of the EGFP reporter gene in JURKAT T cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a 1:10 molar ratio, as well as with lipofectamine 2000 and RNAiMAX.

[0049] [Figure 4C] Figure 4C shows the gene disruption frequency of the EGFP reporter gene in JURKAT T cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with different peptides at a 1:20 molar ratio, as well as with lipofectamine 2000 and RNAiMAX.

[0050] [Figure 4D] Figure 4D shows the dose-response of gene disruption of the EGFP reporter gene in JURKAT T cells using different peptides. Lipofectamine 2000 and RNAiMAX were used as controls. The values in parentheses indicate the molar ratio of peptide to CAS9:gRNA complex.

[0051] [Figure 5A]Figure 5A shows the frequency of gene disruption of the EGFP reporter gene in U2OS cells from delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with VEPEP-3 and ADGN-100 variant sequences at a molar ratio of 1:20, respectively. RNAiMAX was used as a control delivery system.

[0052] [Figure 5B] Figure 5B shows the frequency of gene disruption of the EGFP reporter gene in JURKAT T cells following delivery of CAS9:sgRNA, as quantified by flow cytometry. 10 nM, 25 nM, and 50 nM CAS9:gRNA complexes were combined with VEPEP-3 and ADGN-100 variant sequences at a molar ratio of 1:20, respectively. RNAiMAX was used as a control delivery system.

[0053] [Figure 6A] Figure 6A shows the toxicity of ADGN-100a / CAS9 / gRNA and VEPEP-3b / CAS9 / gRNA complexes to U2OS cells as measured by MTT assay. Peptides were compared to CAS9 / gRNA complexed with Lipofectamine 2000 and RNAiMAX. Values in parentheses indicate the molar ratio of peptide to CAS9:gRNA complex.

[0054] [Figure 6B] Figure 6B shows the toxicity of ADGN-100a / CAS9 / gRNA and VEPEP-3b / CAS9 / gRNA complexes to HEK cells as measured by MTT assay. The peptides were compared with Lipofectamine 2000 and RNAiMAX. The values in parentheses indicate the molar ratio of peptide to CAS9:gRNA complex.

[0055] [Figure 6C]Figure 6C shows the toxicity of ADGN-100a / CAS9 / gRNA and VEPEP-3b / CAS9 / gRNA complexes to JURKAT cells as measured by MTT assay. The peptides were compared with Lipofectamine 2000 and RNAiMAX. The values in parentheses indicate the molar ratio of peptide to CAS9:gRNA complex.

[0056] [Figure 7A] Figure 7A shows EMX1 gene editing in U2OS cells. Cells were transfected with 25 nM CAS9 / gRNA associated with either VEPEP-3b, ADGN-100a, VEPEP-9, or RNAiMAX. Cell samples were analyzed by the T7EI method, and indel results were determined by quantifying the amount of uncut versus cut DNA using a Fragment Analyzer System and are reported in Table 3.

[0057] [Figure 7B] Figure 7B shows EMX1 gene editing in K562 cells. Cells were transfected with 25 nM CAS9 / gRNA associated with either VEPEP-b3, ADGN-100a, VEPEP-9, or RNAiMAX. Cell samples were analyzed by the T7EI method, and insertion / deletion outcomes were determined by quantifying the amount of uncut versus cut DNA using a Fragment Analyzer System, as reported in Table 3.

[0058] [Figure 7C] Figure 7C shows EMX1 gene editing in JURKAT cells. Cells were transfected with 25 nM CAS9 / gRNA associated with either VEPEP-3b, ADGN-100a, VEPEP-9, or RNAiMAX. Cell samples were analyzed by the T7EI method, and insertion / deletion outcomes were determined by quantifying the amount of uncut versus cut DNA using a Fragment Analyzer System, as reported in Table 3.

[0059] [Figure 8A] Figure 8A shows EMX1 gene editing in EGFP-U2OS cells. Cells were transfected with 25 nM CAS9 / gRNA associated with either VEPEP-3b, ADGN-100a, VEPEP-9, or RNAiMAX. Cell samples were analyzed by the T7EI method, and indel results were determined by quantifying the amount of uncut versus cut DNA using a Fragment Analyzer System, as reported in Table 4.

[0060] [Figure 8B] Figure 8B shows EMX1 gene editing in EGFP-JURKAT cells. Cells were transfected with 25 nM CAS9 / gRNA associated with either VEPEP-3b, ADGN-100a, VEPEP-9, or RNAiMAX. Cell samples were analyzed by the T7EI method, and indel results were determined by quantifying the amount of uncut versus cut DNA using a Fragment Analyzer System, as reported in Table 4.

[0061] [Figure 8C] Figure 8C shows the gene disruption frequency of the EGFP reporter gene in both U2OS and JURKAT T cells from delivery of CAS9:sgRNA, quantified by flow cytometry. 25 nM of CAS9:gRNA complex was combined with VEPEP-3b, VEPEP-9, and ADGN-100a at a molar ratio of 1:20, respectively, as well as with lipofectamine 2000 and RNAiMAX.

[0062] [Figure 9A] Figure 9A shows the analysis of HDR insertion efficiency of HindIII and NheI restriction enzyme sites in the EMX1 gene in U2OS cells. The percentage of HDR editing was calculated using an RFLP assay by NheI digestion of the EMX1 PCR amplicon.

[0063] [Figure 9B]Figure 9B shows the analysis of HDR insertion efficiency of HindIII and NheI restriction enzyme sites in the EMX1 gene in JURKAT cells. The percentage of HDR editing was calculated using RFLP assay by NheI digestion of the EMX1 PCR amplicon.

[0064] [Figure 10] Figure 10 shows the gene disruption frequency of the EGFP reporter gene in U2OS cells from co-delivery of a CAS9 expression plasmid and sgRNA using the ADGN-100a peptide. The CAS9 expression plasmid and sgRNA were combined with ADGN-100a at a molar ratio of 10:1 or 20:1 (indicated by the values in parentheses) of the CAS9 expression plasmid and sgRNA, respectively, and lipofectamine 2000.

[0065] [Figure 11] Figure 11 shows the transfection efficiency of ADGN-100a / plasmid complexes. Primary T cells were transfected with ADGN-100a particles containing a plasmid encoding an anti-CD19 CAR at two concentrations (2 μg and 5 μg). On day 4, expression of the anti-CD19 CAR was assessed by flow cytometry using a protein L assay. Data were normalized to untransfected cells and compared to cells incubated with free plasmid.

[0066] [Figure 12] Figure 12 shows the viability of primary T cells after transfection with ADGN-100a / plasmid complexes. Primary T cells were transfected with ADGN-100a particles containing a plasmid encoding an anti-CD19 CAR at two concentrations (2 μg and 5 μg). On day 4, cell viability was quantified by flow cytometry using 7-AAD. Data were normalized to untransfected cells and compared to T cells incubated with free plasmid.

[0067] [Figure 13] Figure 13 shows the transfection efficiency of ADGN-100a / plasmid complexes used in combination with peptide / RGEN / gRNA complexes. Primary T cells were transfected with ADGN-100a particles (5 μg) containing a plasmid encoding an anti-CD19 CAR and peptide particles containing a gRNA / CAS9 complex. On day 6, anti-CD19 CAR expression was assessed by flow cytometry using a protein L assay. Data were normalized to untransfected cells and compared to cells incubated with free plasmid.

[0068] [Figure 14] Figure 14 shows the toxicity assessment of ADGN-100a / CAS9 / gRNA and VEPEP-3a / CAS9 / gRNA complexes on JURKAT and K562 cells as measured by MTT assay. Delivery of CAS9 / gRNA complexes by RNAiMAX was included for comparison.

[0069] [Figure 15] Figure 15 shows the viability of primary T cells after transfection with ADGN-100a or VEPEP-3a particles containing CAS9 / gRNA complexes. T cells were transfected with ADGN-100a or VEPEP-3a particles containing CAS9 / gRNA particles at two concentrations (5 μg / 10 μg CAS9 / gRNA and 2.5 μg / 5 μg CAS9 / gRNA). After 48 hours, cell viability was quantified by flow cytometry using 7-AAD. Data were normalized to untransfected cells and compared to T cells incubated with free CAS9 / gRNA.

[0070] [Figure 16]Figures 16A and 16B show the frequency of CAS9 / gRNA-mediated gene disruption of the EMX1 and HPRT endogenous genes in primary human fibroblasts (Figure 16A) or primary human hepatocytes (Figure 16B), as quantified by the T7E1 assay. CAS9 / gRNA complexes (10 nM, 20 nM, or 50 nM) were delivered by either ADGN-100a (20:1 molar ratio of peptide to complex) or RNAiMAX.

[0071] [Figure 17] Figures 17A and 17B show toxicity assessment of ADGN-100a / CAS9 / gRNA complexes on human primary fibroblasts (Figure 17A) or human primary hepatocytes (Figure 17B) as measured by MTT assay. Peptide-mediated delivery was compared to RNAiMAX delivery.

[0072] [Figure 18] FIG. 18 shows Western blot analysis and quantification of CAS9 protein expression in U2OS cells transfected with CAS9 mRNA (0.2 μg or 0.5 μg) using either ADGN-100a or Lipofectamine 2000.

[0073] [Figure 19] Figure 19 shows the frequency of gene disruption of the EGFP reporter gene in U2OS cells by ADGN-100a-mediated delivery of CAS9 / gRNA or CAS9 mRNA / gRNA, as quantified by flow cytometry. CAS9 / gRNA (2.5 μM / 5 μM) or CAS9 mRNA / gRNA (0.5 μg / 5 μg) complexes were associated with ADGN-100a at a molar ratio of 20:1 (peptide to complex) for peptide-mediated delivery and compared with delivery by lipofectamine 2000 or RNAiMAX.

[0074] [Figure 20]Figures 20A and 20B show quantification of CAS9 protein expression by Western blot (Figure 20A) or ELISA (Figure 20B) in different tissues after in vivo administration of CAS9 mRNA (10 μg) associated with ADGN-100a peptide at a 20:1 molar ratio of peptide to mRNA. Tissues were homogenized, and CAS9 expression was analyzed by Western blot or ELISA on protein extracts using an antibody against CRISPR / Cas9.

[0075] [Figure 21] Figure 21 shows whole animal fluorescence imaging for luciferase expression 72 hours after injection of free ADGN-100a peptide (2 animals / group), free CAS9 mRNA (2 animals / group), or CAS9 mRNA / gRNA / ADGN-100a (3 animals per group).

[0076] [Figure 22] Figure 22 shows the in vivo gene disruption frequency of the luciferase reporter gene in the liver resulting from ADGN-100a-mediated delivery of mRNA CAS9 / sgRNA, as quantified by fluorescence imaging. Mice were given a single intravenous injection of free ADGN-100a peptide, free CAS9 mRNA, or CAS9 mRNA / gRNA / ADGN-100a. Three animals per group (referred to as M1, M2, and M3) were included.

[0077] [Figure 23] Figure 23 shows ELISA analysis of CAS9 protein expression in different tissues after in vivo administration of CAS9 mRNA / gRNA (10 μg) associated with ADGN-100a peptide at a molar ratio of peptide to mRNA / gRNA of 20:1. Tissues were homogenized, and CAS9 expression was analyzed by ELISA on protein extracts using an antibody against CRISPR / Cas9.

[0078] [Figure 24]Figure 24 shows FACS analysis of β2 microglobulin gene editing in T cells. T cells were transfected with CAS9 / gRNA (5 μg / 10 μg CAS9 / gRNA) by association with either ADGN-100a or VEPEP-3a or by electroporation. Free CAS9 / gRNA and untreated conditions were included as controls. β2 microglobulin expression was assessed by FACS 72 hours later.

[0079] [Figure 25] Figures 25A and 25B show the toxicity analysis of CPP / CAS9 / gRNA complexes to T cells. T cells isolated from PBMCs were treated with CAS9 / gRNA complexes associated with ADGN-100a or VEPEP-3b, or electroporated with CAS9 / gRNA complexes, and toxicity was assessed by MTT assay after 48 hours (Figure 25A) and by monitoring activation damage-induced cell death (DICD) after 72 hours (Figure 25B). Untreated, free CAS9 / gRNA, free ADGN-100a, and free VEPEP-3b conditions were included as controls.

[0080] [Figure 26] Figure 26 shows gene disruption of the β-catenin gene in undifferentiated and differentiated mouse muscle myoblasts (C2C12) by VEPEP-3b- and ADGN-100a-mediated delivery of CAS9 / gRNA, as quantified by PCR gel analysis. Cells were transfected with CAS9 / gRNA (2.5 μM / 5 μM) complexes associated with (+) or not associated with (-) CPP at a molar ratio of 20:1 (peptide to complex), and β-catenin expression was compared to delivery by RNAiMAX. β-Actin expression was included as a loading control.

[0081] [Figure 27]Figure 27 shows the gene disruption efficiency of the HPRT gene in undifferentiated and differentiated mouse muscle myoblasts (C2C12) by VEPEP-3b-mediated and ADGN-100a-mediated delivery of CAS9 / gRNA. Cell samples were analyzed by the T7EI method, and indel results were determined by quantifying the amount of uncut versus cut DNA using a Fragment Analyzer System. Delivery by RNAiMAX was included as a control.

[0082] [Figure 28] Figures 28A-28D show quantification of CAS9 protein expression by ELISA in different cell types transfected with CAS9 mRNA (0.5 μg) using ADGN-100a, VEPEP-6, VEPEP-9, or VEPEP-3a peptides, or RNAiMAX. Figure 28A shows results for U2OS cells. Figure 28B shows results for HEPG2 cells. Figure 28C shows results for primary human fibroblasts. Figure 28D shows results for K562 cells.

[0083] [Figure 29] Figures 29A-29D show the frequency of CAS9 mRNA / gRNA-mediated gene disruption of the endogenous EMX1 gene in U2OS cells (Figure 29A), HEPG2 cells (Figure 29B), primary human fibroblasts (Figure 29C), or K562 cells (Figure 29D), as quantified by T7E1 assay. CAS9 mRNA / gRNA (0.5 μg / 2.5 μg) was delivered with ADGN-100a, VEPEP-6, VEPEP-9, or VEPEP-3a peptides (20:1 molar ratio of CPP to cargo), or by RNAiMAX.

[0084] [Figure 30] Figure 30 shows the toxicity assessment of CPP / CAS9 mRNA / gRNA complexes on U2OS, HEPG2, and K562 cell lines, and human primary fibroblasts, as measured by MTT assay. Peptide-mediated delivery was compared to RNAiMAX delivery.

[0085] [Figure 31] Figures 31A-31D show the frequency of gene disruption of the endogenous EMX1 gene mediated by CAS9 protein / gRNA in U2OS cells (Figure 31A), HEPG2 cells (Figure 31B), primary human fibroblasts (Figure 31C), or K562 cells (Figure 31D), as quantified by the T7E1 assay. Preloaded CAS9 / gRNA complexes (2.5 μg / 5 μg CAS9 / gRNA) were mixed with CPP peptide at a molar ratio of 20:1 CPP to CAS9 / gRNA complex for peptide-mediated delivery, and compared with delivery by lipofectamine 2000 or RNAiMAX.

[0086] [Figure 32] Figures 32A-32C show quantification of CAS9 protein expression by ELISA in different cell types transfected with CAS9 expression plasmid (0.5 μg) using ADGN-100a, VEPEP-6, VEPEP-9, or VEPEP-3a peptides, or using lipofectamine 2000. Figure 32A shows results for U2OS cells. Figure 32B shows results for HEPG2 cells. Figure 32C shows results for primary human fibroblasts.

[0087] [Figure 33] Figures 33A-33C show the frequency of gene disruption of the endogenous EMX1 gene in U2OS cells (Figure 33A), HEPG2 cells (Figure 33B), or primary human fibroblasts (Figure 33C) mediated by a CAS9 expression plasmid and gRNA, as quantified by the T7E1 assay. CAS9 expression plasmid (0.5 μg) and gRNA (5 μg) were mixed with CPP peptide at a 20:1 molar ratio of CPP to nucleic acid (plasmid + gRNA). Peptide-mediated delivery of the CAS9 expression plasmid / gRNA was compared to delivery by lipofectamine 2000 or RNAiMAX.

[0088] [Figure 34]Figure 34 shows the evaluation of nonspecific cytokine induction associated with in vivo administration of CPP / mRNA complexes. Mice were given a single intravenous injection of CAS9 mRNA / ADGN-100a, CAS9 mRNA / VEPEP-6, or CAS9 mRNA / VEPEP-9. Serum cytokine levels were assessed at different time points using the Cytokine Mouse Magnetic 20-Plex Panel. Controls included administration of LPS or free mRNA. For each time point, the bars correspond, from left to right, to TNF-α, GM-CSF, IFN-γ, IL1α, IL2, IL5, IL6, IL10, IL12 (p40 / p70), IL13, IL17, IL1β, VEGF, FGF, MIP-1α, and FGF, respectively.

[0089] [Figure 35] Figures 35A-35C show quantification of CAS9 protein expression by ELISA in various tissues 3 days (Figure 35A), 6 days (Figure 35B), and 10 days (Figure 35C) after in vivo administration of CAS9 mRNA (5 μg) associated with ADGN-100a, VEPEP-6, or VEPEP-9 peptides at a CPP to mRNA molar ratio of 20:1. Tissues were homogenized, and CAS9 expression was analyzed by ELISA on protein extracts using an antibody against CRISPR / Cas9.

[0090] [Figure 36] Figure 36 shows quantification of serum PCSK9 protein levels at different time points after in vivo administration of CAS9 mRNA (5 μg) / gRNA targeting PCSK9 exon 1 associated with ADGN-100a, VEPEP-6, or VEPEP-9 peptides at a CPP to nucleic acid (mRNA + gRNA) molar ratio of 20:1. Serum levels of PCSK9 were determined by ELISA using the Mouse Proprotein Convertase 9 / PCSK9 Quantikine ELISA Kit (MPC-900, R&D Systems).

[0091] [Figure 37] Figure 37 shows quantification of total serum cholesterol levels at different time points after in vivo administration of CAS9 mRNA (5 μg) / gRNA targeting PCSK9 exon 1 associated with ADGN-100a, VEPEP-6, or VEPEP-9 peptides at a CPP to nucleic acid (mRNA + gRNA) molar ratio of 20:1. Total serum cholesterol levels were measured using Infinity Cholesterol Reagent (Thermo Fisher).

[0092] [Figure 38] Figure 38 shows the assessment of nonspecific cytokine induction associated with in vivo administration of VEPEP-3a / CAS9 protein complexes. Mice were given a single intravenous injection of CAS9 / VEPEP-3a. Serum cytokine levels were assessed at different time points using the Cytokine Mouse Magnetic 20-Plex Panel. Controls included administration of free cas9 protein or LPS. For each time point, the bars correspond to TNF-α, GM-CSF, IFN-γ, IL1α, IL2, IL5, IL6, IL10, IL12 (p40 / p70), IL13, IL17, IL1β, VEGF, FGF, MIP-1α, and FGF, respectively, from left to right.

[0093] [Figure 39] Figures 39A and 39B show quantification of CAS9 protein expression determined by ELISA in primary human T cells. Figure 39A corresponds to T cells transfected with ADGN-100a, VEPEP-6, VEPEP-9, or VEPEP-3a peptide, or CAS9 mRNA (0.5 μg) using RNAiMAX. Figure 39B corresponds to T cells transfected with ADGN-100a, VEPEP-6, VEPEP-9, or VEPEP-3a peptide, or CAS9 expression plasmid (0.5 μg) using lipofectamine 2000.

[0094] [Figure 40]Figures 40A-40D show the frequency of gene disruption of the endogenous EMX1 gene in human primary T cells mediated by RGEN / gRNA, as quantified by the T7E1 assay. Figure 40A corresponds to T cells transfected with CAS9 mRNA / gRNA (0.5 μg / 2.5 μg) using ADGN-100a, VEPEP-6, or VEPEP-9 (20:1 molar ratio of CPP to cargo) or by RNAiMAX. Figure 40B corresponds to T cells transfected with pre-loaded CAS9 / gRNA complexes (2.5 μg / 5 μg of CAS9 / gRNA). The complexes were mixed with ADGN-100a, VEPEP-6, VEPEP-9, or VEPEP-3a (20:1 molar ratio of CPP to complex) and compared to delivery by RNAiMAX. Figure 40C corresponds to T cells transfected with CAS9 expression plasmid (0.5 μg) and gRNA (5 μg) using ADGN-100a, VEPEP-6, or VEPEP-9 (20:1 molar ratio of CPP to cargo) or by lipofectamine 2000 / RNAiMAX. Figure 40D corresponds to toxicity assessment of CPP in complex with CAS9 mRNA, CAS9 protein, or CAS9 expression plasmid (CAS9 pls) associated with or not associated with gRNA in human primary T cells measured by MTT assay. Peptide-mediated delivery was compared to delivery by RNAiMAX or Lipofectamine 2000. DETAILED DESCRIPTION OF THE INVENTION

[0095] For genome editing technologies (e.g., designer nuclease-based genome modification, e.g., CRISPR) to be therapeutically applicable, genome editing system molecules (e.g., CRISPR system molecules) must be efficiently delivered to their targets inside cells. Adeno-associated virus particles have been frequently used as gene delivery agents, but due to safety issues and limited loading capacity, viral carriers are not ideal delivery vehicles (Swiech et al., Nat. Biotechnol. 33:102-106, 2015). Non-viral delivery of genome editing system molecules has been proposed. Delivery methods include lipid-based vectors, lipid nanoparticles, polymeric vectors, polyethyleneimine, and poly(L-lysine), to name a few; however, in vivo application of genome editing (e.g., by using CRISPR tools) remains a challenge due to limitations of currently used delivery methods (Li et al., 2015). Human Gene Therapy, Vol. 26(No. 7): pp. 452-462; Wang et al. (2016). Proceedings of the National Academy of Sciences, Vol. 113(No. 11): pp. 2868- (p. 2873). Therefore, there is a need for improved, simple, and efficient delivery methods for genome editing system molecules (e.g., CRISPR system molecules).

[0096] The present application provides complexes and nanoparticles comprising a cell-penetrating peptide (CPP) and one or more molecules of a genome editing system, wherein the CPP is suitable for stabilizing and / or delivering the one or more genome editing system molecules to a cell. The complexes and nanoparticles can include multiple genome editing system molecules, some of which may already be complexed prior to association with the CPP. The complexes and nanoparticles can include the entire genome editing system (e.g., the complexes and nanoparticles can include sufficient molecules to effect the genome modification for which the genome editing system is designed when delivered to a cell). Cell-penetrating peptide technology is less complex, less toxic, and easier to use than viral vectors or chemical transfection. Genome editing system molecules are intended to include nucleic acids encoding the genome editing system molecules. For example, the genome editing system molecules may include, for example, a) an enzyme and RNA, b) RNA and a nucleic acid encoding the enzyme, c) an enzyme and RNA, or d) a nucleic acid encoding both the enzyme and RNA. In some embodiments, the genome editing system comprises a designer nuclease (or a nucleic acid, e.g., an mRNA or DNA plasmid, encoding the designer nuclease), such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a homing endonuclease (e.g., ARC Nuclease™), or a nucleic acid-guided endonuclease (NGEN), such as an RNA-guided endonuclease (RGEN, e.g., Cas9) or a DNA-guided endonuclease (DGEN). In some embodiments, the genome editing system further comprises a guide nucleic acid (gNA) (or a nucleic acid, e.g., an mRNA or DNA plasmid encoding the guide nucleic acid), such as a guide RNA (gRNA) or a guide DNA (gDNA).In some embodiments, the genome editing system is a clustered regularly interspaced short palindromic repeat (CRISPR) system (e.g., CRISPR). In some embodiments, the genome editing system comprises a ZFN. In some embodiments, the genome editing system comprises a TALEN. In some embodiments, the genome editing system comprises a homing endonuclease. In some embodiments, the genome editing system comprises an integrase (or a nucleic acid, e.g., an mRNA or DNA plasmid, encoding the integrase). In some embodiments, the genome editing system further comprises a donor nucleic acid comprising a recombination site recognized by the integrase.

[0097] Thus, in one aspect, the present application provides novel genome editing complexes and nanoparticles, which are described in more detail further below.

[0098] In another aspect, a method is provided for delivering a genome editing system molecule to a cell using a cell membrane-permeable peptide.

[0099] Pharmaceutical compositions comprising the cell membrane-permeable peptide and one or more molecules of the genome editing system (e.g., in the form of complexes and nanoparticles) and their use for treating diseases are also provided. definition

[0100] In an embodiment of the present invention, the term "single guide RNA" or "sgRNA" refers to a polynucleotide sequence comprising a guide sequence, a tracr sequence, and a tracr mate sequence. The term "guide sequence" refers to a sequence of approximately 20 bp within the guide RNA that specifies the target site. The term "tracr mate sequence" may also be used synonymously with the term "direct repeat."

[0101] The term "wild-type" as used herein is a term of the art understood by those skilled in the art and means the typical form of an organism, strain, gene or trait occurring in nature, as distinguished from mutant or variant forms.

[0102] The term "variant" as used herein should be taken to mean exhibiting the quality of having a pattern that deviates from that found in nature.

[0103] The terms "non-naturally occurring" and "modified" are used interchangeably and indicate the involvement of human intervention. These terms, when referring to a nucleic acid molecule or polypeptide, mean that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated, as found in nature.

[0104] "Complementary" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either through traditional Watson-Crick base pairing or other non-traditional methods. The percent complementarity refers to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with another nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 would be 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all of the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in another nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 nucleotides or more, or refers to two nucleic acids that hybridize under stringent conditions.

[0105] As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on several factors. Generally, the longer the sequence, the higher the temperature at which the sequence hybridizes to its target sequence. Non-limiting examples of stringent conditions include those described in Tijssen et al. (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assay. ". Elsevier, NY.

[0106] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can also constitute a step in a larger process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence that can hybridize to a given sequence is referred to as the "complement" of that given sequence.

[0107] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a template DNA (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and encoded polypeptide are sometimes collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0108] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also encompassed are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0109] The terms "therapeutic agent," "therapeutic agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that has some beneficial effect when administered to a subject. Beneficial effects include enabling a diagnosis to be made; ameliorating a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally suppressing a disease, symptom, disorder, or condition.

[0110] As used herein, "treatment" or "treating" refers to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. A therapeutic benefit refers to any treatment-related improvement of, or any treatment-related effect on, one or more diseases, conditions, or symptoms being treated.

[0111] The term "effective amount" or "therapeutically effective amount" refers to the amount of a drug that is sufficient to produce beneficial or desired results.The therapeutically effective amount may vary depending on one or more of the subject and condition being treated, the subject's weight and age, the severity of the condition, the method of administration, etc., and those skilled in the art can easily determine the therapeutically effective amount.This term also applies to the dose that will obtain an image for detection by any one of the imaging methods described herein.The specific dose may vary depending on one or more of the specific drug selected, the administration regimen that will be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue that will be imaged, and the physical delivery system that will be carried.

[0112] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0113] Reference herein to "about" a value or parameter includes (and states) embodiments relating to that value or parameter per se. For example, a statement referring to "about X" includes a statement of "X."

[0114] The compositions and methods of the present invention may comprise, consist of, or consist essentially of the essential elements and limitations of the present invention described herein, as well as any additional or optional components, ingredients, or limitations described herein or otherwise useful.

[0115] Unless otherwise noted, terminology is used according to conventional usage. Complexes and Nanoparticles

[0116] In some aspects, the present invention provides complexes and nanoparticles comprising cell membrane-permeable peptides for delivering one or more molecules of a genome editing system to cells. In some embodiments, the cell membrane-permeable peptide is complexed with a genome editing nuclease (or a nucleic acid encoding the genome editing nuclease, for example, an mRNA or DNA plasmid). In some embodiments, the genome editing nuclease is a ZFN, a TALEN, a homing endonuclease, an RGEN (e.g., Cas9), or a DGEN. In some embodiments, the genome editing nuclease is a ZFN. In some embodiments, the genome editing nuclease is a TALEN. In some embodiments, the genome editing nuclease is a nucleic acid-guided nuclease (e.g., an RGEN or a DGEN), and the cell membrane-permeable peptide is complexed with the nucleic acid-guided nuclease (or a nucleic acid encoding the nucleic acid-guided nuclease) combined with (e.g., complexed with) a guide sequence (or a nucleic acid encoding the guide sequence) to form a genome editing complex or nanoparticle that can be delivered to a cell. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide and a ZFN or TALEN, or a nucleic acid encoding a ZFN or TALEN. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide, an RGEN, or a nucleic acid encoding an RGEN, and a guide RNA (gRNA), or a nucleic acid encoding a gRNA. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide, a DGEN, or a nucleic acid encoding a DGEN, and a guide DNA (gDNA), or a nucleic acid encoding a gDNA. In some embodiments, the cell membrane-permeable peptide and the genome editing nuclease are covalently linked. In some embodiments, the RGEN and the gRNA, or the DGEN and the gDNA, have already formed a complex before associating with the cell membrane-permeable peptide to form the genome editing complex. In some embodiments, the cell membrane-permeable peptide forms a complex with a genome editing integrase (or a nucleic acid encoding a genome editing integrase). In some embodiments, the genome editing integrase is a bacteriophage integrase.In some embodiments, the cell membrane-permeable peptide is complexed with a genome editing integrase (or a nucleic acid encoding the genome editing integrase) combined with (e.g., complexed with) a donor nucleic acid comprising a recombination site recognized by the integrase to form a genome editing complex or nanoparticle that can be delivered to a cell. The cell membrane-permeable peptide can form a stable complex and nanoparticle with genome editing system molecules (e.g., a Cas nuclease and / or a guide RNA, or a nucleic acid encoding a Cas nuclease and / or a guide RNA). In some embodiments, the genome editing complex or nanoparticle comprises the entire genome editing system (e.g., delivery of the molecules in the genome editing complex or nanoparticle is sufficient to effect genome editing in a cell).

[0117] In some aspects, the present invention provides complexes and nanoparticles comprising a cell membrane-permeable peptide for delivering ZFPs or ZFNs to host cells. In some embodiments, the cell membrane-permeable peptide is complexed with a ZFP or ZFN (or a nucleic acid encoding a ZFP or ZFN) to form a genome editing complex or nanoparticle that can be delivered to a cell. The cell membrane-permeable peptide can form a stable complex and nanoparticle with a ZFP or ZFN or a nucleic acid encoding a ZFP or ZFN. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide and a ZFP or ZFN or a nucleic acid encoding a ZFP or ZFN. In some embodiments, the cell membrane-permeable peptide and the ZFP or ZFN are covalently linked.

[0118] In some aspects, the present invention provides complexes and nanoparticles comprising a cell membrane-permeable peptide for delivering a TALE or TALEN to a host cell. In some embodiments, the cell membrane-permeable peptide is complexed with a TALE or TALEN (or a nucleic acid encoding a TALE or TALEN) to form a genome editing complex or nanoparticle that can be delivered to a cell. The cell membrane-permeable peptide can form a stable complex and nanoparticle with the TALE or TALEN, or a nucleic acid encoding a TALE or TALEN. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide and a TALE or TALEN, or a nucleic acid encoding a TALE or TALEN. In some embodiments, the cell membrane-permeable peptide and the TALE or TALEN are covalently linked.

[0119] In some aspects, the present invention provides complexes and nanoparticles comprising cell membrane-permeable peptides for delivering one or more CRISPR system molecules to host cells. In some embodiments, the cell membrane-permeable peptide is complexed with a CRISPR nuclease (e.g., Cas9) or a nucleic acid encoding a CRISPR nuclease, optionally combined (e.g., complexed) with a guide sequence (or a nucleic acid encoding a guide sequence), to form a genome editing complex or nanoparticle that can be delivered to cells. The cell membrane-permeable peptide can form a stable complex and nanoparticle with a CRISPR system molecule (e.g., Cas nuclease and / or guide RNA) and / or a nucleic acid encoding one or more of the CRISPR system molecules. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide and a) an RGEN (e.g., Cas9) or a nucleic acid encoding an RGEN, and / or b) a gRNA or a nucleic acid encoding a gRNA. In some embodiments, the cell membrane-permeable peptide and the RGEN are covalently linked. In some embodiments, RGEN and gRNA have already formed a complex before being combined with cell membrane-permeable peptide or nanoparticle to form genome editing complex.In some embodiments, genome editing complex or nanoparticle comprises the entire CRISPR system.For example, in some embodiments, genome editing complex or nanoparticle comprises cell membrane-permeable peptide, RGEN (e.g., Cas9) or the nucleic acid encoding RGEN, and gRNA or the nucleic acid encoding gRNA.

[0120] In some aspects, the present invention uses a DNA-guided genome editing system comprising Natronobacterium gregoryi Argonaute (NgAgo) and guide DNA (gDNA), as described in Gao et al. (2016). Nature Biotechnology.

[0003] Provided are complexes and nanoparticles comprising a cell membrane-permeable peptide for delivering one or more molecules of a DGEN system, such as a system comprising a DGEN system, to a host cell. In some embodiments, the cell membrane-permeable peptide is complexed with a DGEN (or a nucleic acid encoding a DGEN) in combination with (e.g., complexed with) a guide sequence (or a nucleic acid encoding a guide sequence) to form a genome editing complex or nanoparticle that can be delivered to a cell. The cell membrane-permeable peptide can form a stable complex and nanoparticle with a DGEN system molecule (e.g., NgAgo nuclease and / or guide DNA) and / or a nucleic acid encoding one or more of the DGEN system molecules. In some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide, a DGEN or a nucleic acid encoding a DGEN, and gDNA or a nucleic acid encoding gDNA. In some embodiments, the cell membrane-permeable peptide and the DGEN are covalently linked. In some embodiments, the DGEN and gDNA are already complexed before association with the cell membrane-permeable peptide to form a genome editing complex. In some embodiments, the genome editing complex or nanoparticle comprises the entire DGEN system. For example, in some embodiments, the genome editing complex or nanoparticle comprises a cell membrane-permeable peptide, a DGEN or a nucleic acid encoding a DGEN, and gDNA or a nucleic acid encoding a gDNA.

[0121] In some aspects, the present invention provides complexes and nanoparticles comprising a cell membrane-permeable peptide for delivering integrase to a host cell. In some embodiments, the cell membrane-permeable peptide is complexed with integrase (or a nucleic acid encoding integrase) to form a genome editing complex or nanoparticle that can be delivered to a cell. In some embodiments, the cell membrane-permeable peptide is complexed with integrase (or a nucleic acid encoding integrase) combined with (e.g., complexed with) a donor nucleic acid comprising a recombination site recognized by integrase to form a genome editing complex or nanoparticle that can be delivered to a cell. The cell membrane-permeable peptide can form stable complexes and nanoparticles with integrase or a nucleic acid encoding integrase. In some embodiments, the genome editing complex or nanoparticle comprises the cell membrane-permeable peptide and integrase or a nucleic acid encoding integrase. In some embodiments, the cell membrane-permeable peptide and integrase are covalently linked. In some embodiments, the integrase and donor nucleic acid are already complexed prior to association with the cell membrane-permeable peptide to form the genome editing complex or nanoparticle. Cell membrane-penetrating peptides

[0122] The cell membrane-permeable peptides in the genome editing complexes or nanoparticles of the present invention can form stable complexes and nanoparticles with various molecules of genome editing systems, such as nucleases (e.g., ZFNs, TALENs, and CRISPR-associated nucleases (e.g., Cas9 and Cpf1)), integrases (e.g., bacteriophage integrases, such as ΦC31), and nucleic acids (e.g., guide RNAs, guide DNAs, and donor nucleic acids). Any cell membrane-permeable peptide in any genome editing complex or nanoparticle described herein may comprise or consist of any cell membrane-permeable peptide sequence described in this section.

[0123] In some embodiments, the genome editing complex or nanoparticle described herein comprises a cell membrane-permeable peptide selected from the group consisting of CADY, PEP-1, MPG, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell membrane-permeable peptide is present in the genome editing complex. In some embodiments, the cell membrane-permeable peptide is present in the genome editing complex present in the core of the nanoparticle. In some embodiments, the cell membrane-permeable peptide is present in the core of the nanoparticle. In some embodiments, the cell membrane-permeable peptide is present in the core of the nanoparticle and associated with a ZFN, TALEN, homing endonuclease, RGEN (e.g., Cas9), DGEN, or integrase. In some embodiments, the cell membrane-permeable peptide is present in the core of the nanoparticle and associated with a gRNA or gDNA. In some embodiments, the cell membrane-permeable peptide is present in the core of the nanoparticle and associated with an RGEN / gRNA complex or a DGEN / gDNA complex. In some embodiments, the cell membrane-permeable peptide is present in the core of the nanoparticle and associated with the donor nucleic acid. In some embodiments, the cell membrane-permeable peptide is present in the middle layer of the nanoparticle. In some embodiments, the cell membrane-permeable peptide is present in the surface layer of the nanoparticle. In some embodiments, the cell membrane-permeable peptide is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond.WO2014 / 053879 discloses a VEPEP-3 peptide, WO2014 / 053881 discloses a VEPEP-4 peptide, WO2014 / 053882 discloses a VEPEP-5 peptide, WO2012 / 137150 discloses a VEPEP-6 peptide, WO2014 / 053880 discloses a VEPEP-9 peptide, WO2016 / 102687 discloses an ADGN-100 peptide, U.S. Patent Application Publication No. 2010 / 0099626 discloses a CADY peptide, and U.S. Patent No. 7,514,530 discloses an MPG peptide, the disclosures of which are incorporated herein by reference in their entireties.

[0124] In some embodiments, the genome editing complex or nanoparticle described herein has the amino acid sequence X1X2X3X4X5X2X3X4X6X7X3X8X9X 10 X 11 X 12 X 13 (SEQ ID NO: 1), wherein X1 is beta-A or S, X2 is (independently of each other) K, R or L, X3 is (independently of each other) F or W, X4 is (independently of each other) F, W or Y, X5 is E, R or S, X6 is R, T or S, X7 is E, R or S, X8 is absent, F or W, X9 is P or R, and X 10 is R or L, and X 11 is K, W or R, and X 12 is R or F, and X 13 is R or K. In some embodiments, the VEPEP-3 peptide has the amino acid sequence X1X2WX4EX2WX4X6X7X3PRX 11 RX 13(SEQ ID NO: 2), wherein X1 is beta-A or S, X2 is K, R or L, X3 is F or W, X4 is F, W or Y, X5 is E, R or S, X6 is R, T or S, X7 is E, R or S, X8 is absent, F or W, X9 is P or R, and X 10 is R or L, and X 11 is K, W or R, and X 12 is R or F, and X 13 is R or K. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1KWFERWFREWPRKRR (SEQ ID NO: 3), X1KWWERWWREWPRKRR (SEQ ID NO: 4), X1KWWERWWREWPRKRK (SEQ ID NO: 5), X1RWWEKWWTRWPRKRK (SEQ ID NO: 6), or X1RWYEKWYTEFPRRRR (SEQ ID NO: 7), where X1 is beta-A or S. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-7, in which the cell membrane-penetrating peptide is modified by substituting an unnatural amino acid at amino acid position 10, adding an unnatural amino acid between the amino acids at positions 2 and 3, and adding a hydrocarbon linkage between the two unnatural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1KX 14 WWERWWRX 14 WPRKRK (SEQ ID NO: 8), in which X1 is beta-A or S, and X 14 is an unnatural amino acid and there is a hydrocarbon linkage between the two unnatural amino acids. In some embodiments, the VEPEP-3 peptide has the amino acid sequence X1X2X3WX5X 10 X3WX6X7WX8X9X 10 WX 12 X1 is beta-A or S, X2 is K, R or L, X3 is F or W, X5 is R or S, X6 is R or S, X7 is R or S, X8 is F or W, X9 is R or P, and X 10 is L or R, and X12 is R or F. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWRLWWRSWFRLWRR (SEQ ID NO: 10), X1LWWRRWWSRWWPRWRR (SEQ ID NO: 11), X1LWWSRWWRSWFRLWFR (SEQ ID NO: 12), or X1KFWSRFWRSWFRLWRR (SEQ ID NO: 13), where X1 is beta-A or S. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 and 9-13, in which the cell membrane-penetrating peptide is modified by substitution of the amino acid at position 5 or 12 with a non-natural amino acid and addition of a hydrocarbon linkage between the two non-natural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWX 14 LWWRSWX 14 RLWRR (SEQ ID NO: 14), in which X1 is beta-alanine or serine, and X 14is an unnatural amino acid, and there is a hydrocarbon linkage between the two unnatural amino acids. In some embodiments, the VEPEP-3 peptide is present in the genome editing complex. In some embodiments, the VEPEP-3 peptide is present in the genome editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and associated with a ZFN, TALEN, homing endonuclease, RGEN (e.g., Cas9), DGEN, or integrase. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and associated with a gRNA or gDNA. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and associated with an RGEN / gRNA complex or a DGEN / gDNA complex. In some embodiments, the VEPEP-3 peptide is present in the core of a nanoparticle and associated with a donor nucleic acid. In some embodiments, the VEPEP-3 peptide is present in the middle layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-3 peptide is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond.

[0125] In some embodiments, the genome editing complex or nanoparticle described herein comprises a VEPEP-6 cell membrane-permeable peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LX2RALWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 15), X1LX2LARWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 16), and X1LX2ARLWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 17), wherein X1 is beta-A or S, X2 is F or W, X3 is L, W, C, or I, X4 is S, A, N, or T, X5 is L or W, X6 is W or R, X7 is K or R, X8 is A or absent, and X9 is R or S. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6X7X8 (SEQ ID NO: 18), in which X1 is beta-A or S, X2 is F or W, X3 is L, W, C or I, X4 is S, A, N or T, X5 is L or W, X6 is W or R, X7 is K or R, and X8 is A or absent. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence X1LX2RALWRLX3RX4LWRLX5X6KX7 (sequence number 19), wherein X1 is beta-A or S, X2 is F or W, X3 is L or W, X4 is S, A or N, X5 is L or W, X6 is W or R, and X7 is A or absent.In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LFRALWRLLRX2LWRLLWX3 (SEQ ID NO: 20), X1LWRALWRLWRX2LWRLLWX3A (SEQ ID NO: 21), X1LWRALWRLX4RX2LWRLWRX3A (SEQ ID NO: 22), X1LWRALWRLWRX2LWRLWRX3A (SEQ ID NO: 23), X1LWRALWRLX5RALWRLLWX3A (SEQ ID NO: 24), and X1LWRALWRLX4RNLWRLLWX3A (SEQ ID NO: 25), wherein X1 is beta-A or S, X2 is S or T, X3 is K or R, X4 is L, C or I, and X5 is L or I. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of Ac-X1LFRALWRLLRSLWRLLWK-cysteamide (SEQ ID NO:26), Ac-X1LWRALWRLWRSLWRLLWKA-cysteamide (SEQ ID NO:27), Ac-X1LWRALWRLLRSLWRLWRKA-cysteamide (SEQ ID NO:28), Ac-X1LWRALWRLWRSLWRLWRKA-cysteamide (SEQ ID NO:29), Ac-X1LWRALWRLLRALWRLLWKA-cysteamide (SEQ ID NO:30), and Ac-X1LWRALWRLLRNLWRLLWKA-cysteamide (SEQ ID NO:31), wherein X1 is beta-A or S. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs:15-31, further comprising a hydrocarbon linkage between the two residues at positions 8 and 12. In some embodiments, the VEPEP-6 peptide comprises Ac-X1LFRALWR. S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 32), Ac-X1LFLARWR S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 33), Ac-X1LFRALWS S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 34), Ac-X1LFLARWS S LLRS SLWRLLWK-cysteamide (SEQ ID NO: 35), Ac-X1LFRALWRLLR S SLWS S LLWK-cysteamide (SEQ ID NO: 36), Ac-X1LFLARWRLLR S SLWS S LLWK-cysteamide (SEQ ID NO: 37), Ac-X1LFRALWRLLS S SLWS S LLWK-cysteamide (SEQ ID NO: 38), Ac-X1LFLARWRLLS S SLWS S LLWK-cysteamide (SEQ ID NO: 39), and Ac-X1LFAR S LWRLLRS S LWRLLWK-cysteamide (SEQ ID NO: 40), wherein X1 is beta-A or S, and the residue followed by the subscript "S" is the residue linked by the hydrocarbon linkage. In some embodiments, the VEPEP-6 peptide is present in the genome editing complex. In some embodiments, the VEPEP-6 peptide is present in the genome editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and associated with a ZFN, TALEN, homing endonuclease, RGEN (e.g., Cas9), DGEN, or integrase. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and associated with a gRNA or gDNA. In some embodiments, the VEPEP-6 peptide is present in the core of a nanoparticle and associated with an RGEN / gRNA complex or a DGEN / gDNA complex. In some embodiments, the VEPEP-6 peptide is present in the core of the nanoparticle and associated with the donor nucleic acid. In some embodiments, the VEPEP-6 peptide is present in the middle layer of the nanoparticle. In some embodiments, the VEPEP-6 peptide is present in the surface layer of the nanoparticle. In some embodiments, the VEPEP-6 peptide is linked to a targeting moiety. In some embodiments, the link is a covalent bond.

[0126] In some embodiments, the genome editing complex or nanoparticle described herein has the amino acid sequence X1X2X3WWX4X5WAX6X3X7X8X9X 10 X 11 X 12 WX 13 X1 is beta-A or S, X2 is L or absent, X3 is R or absent, X4 is L, R or G, X5 is R, W or S, X6 is S, P or T, X7 is W or P, X8 is F, A or R, X9 is S, L, P or R, and X 10 is R or S, and X 11 is either W or absent and X 12 is A, R, or absent, and X 13 is W or F, and if X3 is absent, then X2, X 11 and X 12 In some embodiments, the VEPEP-9 peptide has the amino acid sequence X1X2RWWLRWAX6RWX8X9X 10 WX 12 WX 13 X1 is beta-A or S, X2 is L or absent, X6 is S or P, X8 is F or A, X9 is S, L or P, and X 10 is R or S, and X 12 is A or R, and X 13is W or F. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1LRWWLRWASRWFSRWAWWR (SEQ ID NO:43), X1LRWWLRWASRWASRWAWFR (SEQ ID NO:44), X1RWWLRWASRWALSWRWWR (SEQ ID NO:45), X1RWWLRWASRWFLSWRWWR (SEQ ID NO:46), X1RWWLRWAPRWFPSWRWWR (SEQ ID NO:47), and X1RWWLRWASRWAPSWRWWR (SEQ ID NO:48), wherein X1 is beta-A or S. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1WWX4X5WAX6X7X8RX 10 X1 is beta-A or S, X4 is R or G, X5 is W or S, X6 is S, T or P, X7 is W or P, X8 is A or R, and X 10is S or R. In some embodiments, the VEPEP-9 peptide comprises an amino acid sequence selected from the group consisting of X1WWRWWASWARSWWR (SEQ ID NO: 50), X1WWGSWATPRRRWWR (SEQ ID NO: 51), and X1WWRWWAPWARSWWR (SEQ ID NO: 52), wherein X1 in these sequences is beta-A or S. In some embodiments, the VEPEP-9 peptide is present in a genome editing complex. In some embodiments, the VEPEP-9 peptide is present in a genome editing complex in the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and associated with a ZFN, TALEN, homing endonuclease, RGEN (e.g., Cas9), DGEN, or integrase. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and associated with a gRNA or gDNA. In some embodiments, the VEPEP-9 peptide is present in the core of the nanoparticle and associated with the RGEN / gRNA complex or the DGEN / gDNA complex. In some embodiments, the VEPEP-9 peptide is present in the core of the nanoparticle and associated with the donor nucleic acid. In some embodiments, the VEPEP-9 peptide is present in the middle layer of the nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the surface layer of the nanoparticle. In some embodiments, the VEPEP-9 peptide is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond.

[0127] In some embodiments, the genome editing complex or nanoparticle described herein comprises an ADGN-100 cell membrane-permeable peptide comprising the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 53), where X1 is any amino acid or absent, and X2 through X8 are any amino acids. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 54), where X1 is βA, S, or absent, X2 is A or V, X3 is or L, X4 is W or Y, X5 is V or S, X6 is R, V, or A, X7 is S or L, and X8 is W or Y. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence KWRSAGWRWRLWRVRSWSR (SEQ ID NO: 55), KWRSALYRWRLWRVRSWSR (SEQ ID NO: 56), KWRSALYRWRLWRSRSWSR (SEQ ID NO: 57), or KWRSALYRWRLWRSALYSR (SEQ ID NO: 58). In some embodiments, the ADGN-100 peptide comprises two residues separated by three or six residues linked by a hydrocarbon linkage. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence KWRSAGWRWRLWRVRSWSR (SEQ ID NO: 58). S AGWR S WRLWRVRSWSR (SEQ ID NO: 59), KWR S SAGWRWR S LWRVRSWSR (SEQ ID NO: 60), KWRSAGWR S WRLWRVR S SWSR (SEQ ID NO: 61), KWRS S ALYR S WRLWRSRSWSR (SEQ ID NO: 62), KWR S SALYRWR S LWRSRSWSR (SEQ ID NO: 63), KWRSALYR S WRLWRSR S SWSR (SEQ ID NO: 64), KWRSALYRWR S LWRS S RSWSR (SEQ ID NO: 65), KWRSALYRWRLWRS SRSWS S R (SEQ ID NO: 66), KWR S SALYRWR S LWRSALYSR (SEQ ID NO: 67), KWRS S ALYR S WRLWRSALYSR (SEQ ID NO: 68), KWRSALYRWR S LWRS S ALYSR (SEQ ID NO: 69), or KWRSALYRWRLWRS S ALYS S R (SEQ ID NO: 70), and residues in these sequences marked with the subscript "S" are linked by a hydrocarbon linkage. In some embodiments, the ADGN-100 peptide is present in a genome editing complex. In some embodiments, the ADGN-100 peptide is present in a genome editing complex in the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and associated with a ZFN, TALEN, homing endonuclease, RGEN (e.g., Cas9), DGEN, or integrase. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and associated with a gRNA or gDNA. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and associated with an RGEN / gRNA complex or a DGEN / gDNA complex. In some embodiments, the ADGN-100 peptide is present in the core of a nanoparticle and associated with a donor nucleic acid. In some embodiments, the ADGN-100 peptide is present in the intermediate layer of a nanoparticle. In some embodiments, the ADGN-100 peptide is present in the surface layer of the nanoparticle. In some embodiments, the ADGN-100 peptide is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond.

[0128] In some embodiments, a CPP described herein (e.g., a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide) further comprises one or more moieties linked to the N-terminus of the CPP. In some embodiments, the one or more moieties are covalently linked to the N-terminus of the CPP. In some embodiments, the one or more moieties are selected from the group consisting of an acetyl group, a stearyl group, a fatty acid, cholesterol, polyethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody or antibody fragment thereof, a peptide, a polysaccharide, and a targeting molecule. In some embodiments, the one or more moieties are an acetyl group and / or a stearyl group. In some embodiments, the CPP comprises an acetyl group and / or a stearyl group linked to its N-terminus. In some embodiments, the CPP comprises an acetyl group and / or a stearyl group linked to its N-terminus. In some embodiments, the CPP comprises a stearyl group linked to its N-terminus. In some embodiments, the CPP comprises an acetyl group and / or a stearyl group covalently linked to its N-terminus. In some embodiments, the CPP comprises an acetyl group covalently linked to its N-terminus, hi some embodiments, the CPP comprises a stearyl group covalently linked to its N-terminus.

[0129] In some embodiments, a CPP described herein (e.g., a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide) further comprises one or more moieties linked to the C-terminus of the CPP. In some embodiments, the one or more moieties are covalently linked to the C-terminus of the CPP. In some embodiments, the one or more moieties are selected from the group consisting of a cysteamide group, cysteine, a thiol, an amide, nitrilotriacetic acid, a carboxyl group, a linear or branched C1-C6 alkyl group, a primary or secondary amine, an oside derivative, a lipid, a phospholipid, a fatty acid, cholesterol, polyethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody or antibody fragment thereof, a peptide, a polysaccharide, and a targeting molecule. In some embodiments, the one or more moieties are cysteamide groups. In some embodiments, a CPP comprises a cysteamide group linked to its C-terminus. In some embodiments, a CPP comprises a cysteamide group covalently linked to its C-terminus.

[0130] In some embodiments, a CPP described herein (e.g., a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide) is stapled. As used herein, "stapled" refers to a chemical linkage between two residues in a peptide. In some embodiments, a CPP comprising a chemical linkage between two amino acids of the peptide is stapled. In some embodiments, the two amino acids linked by the chemical linkage are separated by three or six amino acids. In some embodiments, the two amino acids linked by the chemical linkage are separated by three amino acids. In some embodiments, the two amino acids linked by the chemical linkage are separated by six amino acids. In some embodiments, each of the two amino acids linked by the chemical linkage is R or S. In some embodiments, each of the two amino acids linked by the chemical linkage is R. In some embodiments, each of the two amino acids linked by the chemical linkage is S. In some embodiments, one of the two amino acids linked by the chemical linkage is R and the other is S. In some embodiments, the chemical linkage is a hydrocarbon linkage. Complex

[0131] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide and one or more molecules of a genome editing system (including a nucleic acid encoding one or more genome editing system molecules). In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptide) and a genome editing nuclease (or a nucleic acid encoding a genome editing nuclease). In some embodiments, the genome editing nuclease is a ZFN, TALEN, homing endonuclease, RGEN (e.g., Cas9), or DGEN. In some embodiments, the genome editing complex further comprises a gRNA or gDNA (or a nucleic acid encoding a gRNA or gDNA), wherein the gRNA or gDNA comprises a guide sequence complementary to a target sequence in the target polynucleotide. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide associated with a pre-formed complex comprising an RGEN and a gRNA or a pre-formed complex comprising a DGEN and a gDNA. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide and an integrase (or a nucleic acid encoding the integrase). In some embodiments, the integrase is a bacteriophage integrase (e.g., ΦC31). In some embodiments, the genome editing complex further comprises a donor nucleic acid comprising a recombination site recognized by the integrase. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the molar ratio of the cell membrane-permeable peptide to at least one of the one or more genome editing system molecules (e.g., all of the one or more genome editing system molecules) in the genome editing complex is between about 1:1 and about 80:1 (e.g., about any of 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, or 80:1 (including any range between these ratios)).In some embodiments, the molar ratio of the cell membrane-permeable peptide to at least one of the one or more genome editing system molecules (e.g., all of the one or more genome editing system molecules) in the genome editing complex is between about 5:1 and about 20:1 (e.g., about any of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween)). In some embodiments, the CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the genome editing complex further comprises one or more additional gRNAs or dDNAs comprising different guide sequences. In some embodiments, the genome editing complex further comprises a donor nucleic acid for introducing a specific modification into the target polynucleotide. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the genome editing complex comprises a whole genome editing system.

[0132] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., a VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide) and a ZFN or TALEN (or one or more nucleic acids encoding the ZFN or TALEN). In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the molar ratio of the cell membrane-permeable peptide to the ZFN or TALEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of the cell membrane-penetrating peptide to the ZFN or TALEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about any of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, the CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the genome editing complex further comprises a donor nucleic acid for introducing specific modifications into a target polynucleotide. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by resulting in a modified target gene that expresses an inactive product.In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product.

[0133] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., a VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide) and an integrase (or one or more nucleic acids encoding the integrase). In some embodiments, the integrase is a bacteriophage integrase (e.g., ΦC31). In some embodiments, the genome editing complex further comprises a donor nucleic acid comprising a recombination site recognized by the integrase, the donor nucleic acid for introducing a specific modification into the target polynucleotide. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the molar ratio of the cell membrane-permeable peptide to the integrase in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of the cell membrane-permeable peptide to the integrase in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, CPPs include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by resulting in a modified target gene that expresses an inactive product.In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product.

[0134] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide), and one or both of RGEN (or a nucleic acid encoding RGEN) and gRNA (or a nucleic acid encoding gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide associated with a preformed complex comprising RGEN and gRNA. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a complementary transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)).In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, the CPP includes, but is not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the genome editing complex further includes one or more additional gRNAs containing different guide sequences. In some embodiments, the genome editing complex further comprises a donor nucleic acid for introducing a specific modification into the target polynucleotide. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product.

[0135] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide), and one or both of a DGEN (or a nucleic acid encoding the DGEN) and a gDNA (or a nucleic acid encoding the gDNA), wherein the gDNA comprises a guide sequence complementary to a target sequence in the target polynucleotide. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide associated with a preformed complex comprising the DGEN and the gDNA. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the DGEN is NgAgo. In some embodiments, the molar ratio of DGEN to gDNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of DGEN to gDNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of cell membrane-permeable peptide to DGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, and 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of cell membrane-permeable peptide to DGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., any of about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)).In some embodiments, CPPs include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the genome editing complex further comprises one or more additional gDNAs comprising different guide sequences. In some embodiments, the genome editing complex further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product.

[0136] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide) associated with a preformed RGEN / gRNA complex comprising RGEN and gRNA, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a complementary transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)).In some embodiments, CPPs include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the genome editing complex further comprises one or more additional gRNAs comprising different guide sequences. In some embodiments, the genome editing complex further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product.

[0137] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide), a ZFN or TALEN, and a donor nucleic acid, wherein the ZFN or TALEN cleaves a target sequence within the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide to be modified to include the modification. In some embodiments, the modification is an addition, deletion, or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is an insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, such as a plasmid. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region comprising a portion adjacent to the 5' side of the modification in the target polynucleotide, and a 3' homology arm that is homologous to a 3' target homology region comprising a portion adjacent to the 3' side of the modification in the target polynucleotide. In some embodiments, the 5' target homology region and / or the 3' target homology region overlaps with at least about one nucleotide in the target sequence (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more). In some embodiments, the 5' target homology region and / or the 3' target homology region is within about 1000 nucleotides (e.g., within about 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the molar ratio of cell membrane-permeable peptide to ZFN or TALEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)).In some embodiments, the molar ratio of cell membrane-penetrating peptide to ZFN or TALEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about any of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, the genome editing complex comprises a TALEN. In some embodiments, CPPs include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the genome editing complex further comprises one or more additional donor nucleic acids containing different modifications. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by decreasing expression of the target gene or by resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product.

[0138] In some embodiments, a genome editing complex for introducing an exogenous nucleic acid into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., a VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptide), an integrase, and a donor nucleic acid comprising the exogenous nucleic acid, wherein the integrase is capable of mediating recombination between a first recombination site in the target polynucleotide and a second recombination site in the donor nucleic acid. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the molar ratio of the cell membrane-permeable peptide to the integrase in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, or 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of cell membrane-permeable peptide to integrase in the genome editing complex is between about 5:1 and about 20:1 (e.g., about any of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, the CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the genome editing complex further includes one or more additional donor nucleic acids comprising a different exogenous nucleic acid. In some embodiments, the exogenous nucleic acid is inserted into the coding sequence of the target polynucleotide. In some embodiments, the exogenous nucleic acid is inserted into a non-coding sequence of the target polynucleotide. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by resulting in a modified target gene that expresses an inactive product.In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product. In some embodiments, the target polynucleotide is modified to express a product of an exogenous nucleic acid (e.g., a protein, e.g., an exogenous protein).

[0139] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptide) and one or more of RGEN (or a nucleic acid encoding RGEN), gRNA (or a nucleic acid encoding gRNA) and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence in the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide to be modified to include the modification. In some embodiments, the genome editing complex comprises a) a pre-formed complex comprising RGEN and gRNA and b) a cell membrane-permeable peptide associated with the donor nucleic acid. In some embodiments, the modification is the addition, deletion or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is the insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, such as a plasmid. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' target homology region and / or the 3' target homology region overlap with at least about one nucleotide (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) in the target sequence. In some embodiments, the 5' target homology region and / or the 3' target homology region is within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, at least a portion of the cell membrane-permeable peptides in the genome editing complex are linked to a targeting moiety. In some embodiments, the linkage is a covalent bond.In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a supplemental transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range therebetween)). In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of the cell membrane-permeable peptide to RGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of the cell membrane-permeable peptide to RGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, the CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the genome editing complex further comprises one or more additional gRNAs comprising different guide sequences. In some embodiments, the genome editing complex further comprises one or more additional donor nucleic acids comprising different modifications. In some embodiments, the target polynucleotide has a modified coding sequence.In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product.

[0140] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptide), RGEN (or a nucleic acid encoding RGEN), gRNA (or a nucleic acid encoding gRNA) and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to the target sequence in the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide to be modified to include the modification, wherein the modification is the addition, deletion or substitution of one or more nucleotides in the target nucleic acid. In some embodiments, the genome editing complex comprises a) a pre-formed complex comprising RGEN and gRNA and b) a cell membrane-permeable peptide associated with the donor nucleic acid. In some embodiments, the modification is an addition, deletion, or substitution of between about 1 and about 50 nucleotides in the target polynucleotide (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence.In some embodiments, the 5' homology arm and the 3' homology arm are each individually between about 20 and about 150 nucleotides in length (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 nucleotides, including any range therebetween). In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a supplemental transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)).In some embodiments, the CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the genome editing complex further includes one or more additional gRNAs comprising different guide sequences. In some embodiments, the genome editing complex further includes one or more additional donor nucleic acids comprising different modifications. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product.

[0141] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptide), RGEN (or a nucleic acid encoding RGEN), gRNA (or a nucleic acid encoding gRNA) and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to the target sequence in the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide that is modified to include the modification, and the modification is the insertion of a heterologous nucleic acid into the target nucleic acid. In some embodiments, the genome editing complex comprises a) a pre-formed complex comprising RGEN and gRNA and b) a cell membrane-permeable peptide associated with the donor nucleic acid. In some embodiments, the modification is an insertion of a heterologous nucleic acid that is greater than about 50 nucleotides in length (e.g., greater than about any of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a double-stranded DNA molecule. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that comprises a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that comprises a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence.In some embodiments, the 5' homology arm and the 3' homology arm are each individually longer than about 300 nucleotides in length (e.g., longer than about 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides or more, including any range therebetween). In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to the targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a supplemental transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)).In some embodiments, the CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the genome editing complex further includes one or more additional gRNAs comprising different guide sequences. In some embodiments, the genome editing complex further includes one or more additional donor nucleic acids comprising different modifications. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product.

[0142] In some embodiments, a genome editing complex for modifying one or more target polynucleotides is provided, the genome editing complex comprising a cell membrane-permeable peptide (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptide), RGEN (or a nucleic acid encoding RGEN), and multiple gRNAs (or one or more nucleic acids encoding multiple gRNAs), each of the multiple gRNAs comprising a different guide sequence complementary to a target sequence in one of one or more target polynucleotides. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide associated with a preformed complex comprising RGEN and multiple gRNAs. In some embodiments, at least a portion of the cell membrane-permeable peptide in the genome editing complex is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, each of the multiple gRNAs is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to an auxiliary transactivating crRNA (tracrRNA). In some embodiments, each of the multiple gRNAs is an sgRNA comprising a gRNA guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the genome editing complex is about 1:1. In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)).In some embodiments, the molar ratio of cell membrane-permeable peptide to RGEN in the genome editing complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range between these ratios)). In some embodiments, the CPP includes, but is not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the genome editing complex further comprises one or more donor nucleic acids, each of which individually comprises a sequence for introducing a modification into one of one or more target polynucleotides. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product.

[0143] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide associated with a genome editing enzyme (or a nucleic acid encoding the genome editing enzyme), such as a nuclease or integrase, wherein the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing enzyme is a nuclease selected from ZFN, TALEN, homing endonuclease, RGEN, or DGEN. In some embodiments, the genome editing enzyme is Cas9. In some embodiments, the genome editing complex further comprises a gRNA or gDNA (or a nucleic acid encoding the gRNA or gDNA). In some embodiments, the genome editing enzyme is a TALEN. In some embodiments, the genome editing enzyme is an integrase. In some embodiments, the genome editing complex further comprises a donor nucleic acid for introducing a specific modification into the target polynucleotide.

[0144] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide associated with a preformed RGEN / gRNA complex comprising RGEN and gRNA, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the RGEN is Cas9.

[0145] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, an RGEN (or a nucleic acid encoding an RGEN), and a gRNA (or a nucleic acid encoding the gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the RGEN is Cas9.

[0146] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, an RGEN (or a nucleic acid encoding an RGEN), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid comprises a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a) a pre-formed complex comprising RGEN and gRNA, and b) a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the modification is the addition, deletion, or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is the insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, such as a plasmid. The donor nucleic acid comprises a 5' homology arm that is homologous to the 5' target homology region comprising the portion adjacent to the 5' side of the modification of the target polynucleotide, and a 3' homology arm that is homologous to the 3' target homology region comprising the portion adjacent to the 3' side of the modification of the target polynucleotide. In some embodiments, the 5' target homology region and / or the 3' target homology region overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence.In some embodiments, the 5' target homology region and / or the 3' target homology region is within about 1000 nucleotides (e.g., within about 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the RGEN is Cas9.

[0147] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, an RGEN (or a nucleic acid encoding an RGEN), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid comprises a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being an addition, deletion, or substitution of one or more nucleotides in the target nucleic acid, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a) a preformed complex comprising RGEN and gRNA, and b) a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the modification is an addition, deletion, or substitution of between about 1 and about 50 nucleotides in the target polynucleotide (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. The donor nucleic acid comprises a 5' homology arm homologous to a 5' target homology region comprising a portion adjacent to the 5' side of the modification in the target polynucleotide, and a 3' homology arm homologous to a 3' target homology region comprising a portion adjacent to the 3' side of the modification in the target polynucleotide.In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually between about 20 and about 150 nucleotides in length (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 nucleotides in length, including any range therebetween). In some embodiments, the RGEN is Cas9.

[0148] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, an RGEN (or a nucleic acid encoding an RGEN), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid comprises a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being insertion of a heterologous nucleic acid into the target nucleic acid, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises any one of the amino acid sequences of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a) a pre-formed complex comprising RGEN and gRNA, and b) a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the modification is the insertion of a heterologous nucleic acid longer than about 50 nucleotides in length (e.g., longer than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 nucleotides or more (including any range therebetween). In some embodiments, the donor nucleic acid is a double-stranded DNA molecule. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification.In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually greater than about 300 nucleotides in length (e.g., greater than about 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides, including any range therebetween). In some embodiments, the RGEN is Cas9.

[0149] In some embodiments, a genome editing complex for modifying one or more target polynucleotides is provided, the genome editing complex comprising a cell membrane-permeable peptide, RGEN (or a nucleic acid encoding RGEN), and multiple gRNAs (or one or more nucleic acids encoding multiple gRNAs), each of the multiple gRNAs individually comprising a different guide sequence complementary to a target sequence in one of the one or more target polynucleotides, and the cell membrane-permeable peptide comprising the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide associated with a pre-formed complex comprising RGEN and multiple gRNAs. In some embodiments, the RGEN is Cas9.

[0150] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide associated with a preformed Cas9 / gRNA complex comprising Cas9 and a gRNA, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80.

[0151] In some embodiments, a genome editing complex for modifying a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), and a gRNA (or a nucleic acid encoding the gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80.

[0152] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid comprises a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a) a preformed complex comprising Cas9 and a gRNA and b) a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the modification is an addition, deletion, or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is an insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, e.g., a plasmid. The donor nucleic acid comprises a 5' homology arm homologous to a 5' target homology region comprising a portion adjacent to the 5' side of the modification in the target polynucleotide, and a 3' homology arm homologous to a 3' target homology region comprising a portion adjacent to the 3' side of the modification in the target polynucleotide. In some embodiments, the 5' target homology region and / or the 3' target homology region overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence.In some embodiments, the 5' target homology region and / or the 3' target homology region are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence.

[0153] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid comprises a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being an addition, deletion, or substitution of one or more nucleotides in the target nucleic acid, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a) a preformed complex comprising Cas9 and a gRNA and b) a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the modification is an addition, deletion, or substitution of between about 1 and about 50 nucleotides in the target polynucleotide (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. The donor nucleic acid comprises a 5' homology arm homologous to a 5' target homology region comprising a portion of the target polynucleotide adjacent to the 5' side of the modification and a 3' homology arm homologous to a 3' target homology region comprising a portion of the target polynucleotide adjacent to the 3' side of the modification.In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually between about 20 and about 150 nucleotides in length (e.g., any of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 nucleotides, including any range between these values).

[0154] In some embodiments, a genome editing complex for introducing a modification into a target polynucleotide is provided, the genome editing complex comprising a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid, wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid comprises a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being insertion of a heterologous nucleic acid into the target nucleic acid, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a) a preformed complex comprising Cas9 and a gRNA, and b) a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the modification is the insertion of a heterologous nucleic acid longer than about 50 nucleotides in length (e.g., longer than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 nucleotides, or more, including any range therebetween). In some embodiments, the donor nucleic acid is a double-stranded DNA molecule. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification.In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually greater than about 300 nucleotides in length (e.g., greater than about any of 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides, including any range between these values).

[0155] In some embodiments, a genome editing complex for modifying one or more target polynucleotides is provided, the genome editing complex comprising a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), and multiple gRNAs (or one or more nucleic acids encoding the multiple gRNAs), each of the multiple gRNAs individually comprising a different guide sequence complementary to a target sequence in one of the one or more target polynucleotides, and the cell membrane-permeable peptide comprising the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing complex comprises a cell membrane-permeable peptide associated with a preformed complex comprising Cas9 and multiple gRNAs.

[0156] In some embodiments, the guide sequence of a gRNA contained in a genome editing complex according to any of the embodiments described herein is complementary to a target sequence in a gene encoding a protein involved in regulating the immune response, including immune checkpoint regulators and proteins involved in antigen presentation. In some embodiments, the guide sequence of a gRNA contained in a genome editing complex according to any of the embodiments described herein is complementary to a target sequence in a gene encoding a protein involved in regulating cholesterol transport and / or metabolism. In some embodiments, the guide sequence may be selected from, but is not limited to, PD-1, PD-L1, PD-L2, TIM-1, TIM-3, TIM-4, BTLA, VISTA, LAG-3, CTLA-4, TIGIT, 4-1BB, OX40, CD27, CD28, HVEM, GITR, ICOS, CD40, CD80, CD86, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, CD160, gp49B, PIR-B, KIR family receptors, SIRP alpha (CD47), CD48, 2B 4 (CD244), B7.1, B7.2, ILT-2, ILT-4, A2aR, Toll-like receptors TLR-2, 3, 4, 6, 7, 8, and 9, granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF, CD40L, FLT-3 ligand, cytokines such as IL-1, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, and IL-35, FasL, TGF-β, indoleamine-2,3-dioxygenase (IDO), major histocompatibility complex (MHC) proteins (including beta-2 microglobulin (β2M)), low-density lipoprotein It is complementary to target sequences within genes encoding proteins, including the low-density lipoprotein (LDL) receptor (LDLR), apolipoprotein B (ApoB), low-density lipoprotein receptor adaptor protein 1 (LDLRAP1), and proprotein convertase subtilisin kexin type 9 (PCSK9).

[0157] In some embodiments, according to any of the genome editing complexes described herein, the genome editing complex further comprises a protein other than the genome editing system molecule, or a nucleic acid molecule (e.g., a DNA plasmid or mRNA) encoding the protein.

[0158] In some embodiments, the average size (diameter) of the genome editing complexes described herein is between about 10 nm and about 10 microns, including, for example, between about 30 nm and about 1 micron, between about 50 nm and about 250 nm, between about 50 nm and about 180 nm, and between about 150 nm and about 200 nm. In some embodiments, the genome editing complex is between about 10 nm and about 400 nm. In some embodiments, the genome editing complex is between about 20 nm and about 400 nm. In some embodiments, the genome editing complex is between about 30 nm and about 200 nm. In some embodiments, the genome editing complex is between about 40 nm and about 200 nm. In some embodiments, the genome editing complex is between about 40 nm and about 150 nm. In some embodiments, the genome editing complex is between about 40 nm and about 100 nm. In some embodiments, the genome editing complex is substantially non-toxic.

[0159] In some embodiments, the targeting moiety of the genome editing complex described herein targets the genome editing complex to a tissue or specific cell type. In some embodiments, the tissue is a tissue in need of treatment. In some embodiments, the targeting moiety targets the genome editing complex to a tissue or cell that can be treated with the genome editing system. nanoparticles

[0160] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising one or more genome editing complexes described herein. In some embodiments, the nanoparticle core comprises multiple genome editing complexes. In some embodiments, the nanoparticle core comprises multiple genome editing complexes present in a predetermined ratio. In some embodiments, the predetermined ratio is selected to enable the most effective use of the nanoparticle in any of the methods described in more detail below. In some embodiments, the nanoparticle core further comprises one or more additional cell membrane-permeable peptides, one or more additional genome editing nucleases, one or more additional gRNAs, and / or one or more additional donor nucleic acids. In some embodiments, the one or more additional cell membrane-permeable peptides do not comprise cell membrane-permeable peptides found in any of the one or more genome editing complexes. In some embodiments, the one or more additional genome editing nucleases do not comprise genome editing nucleases found in any of the one or more genome editing complexes. In some embodiments, the one or more additional gRNAs do not comprise gRNAs found in any of the one or more genome editing complexes. In some embodiments, the one or more additional donor nucleic acids do not comprise donor nucleic acids found in any of the one or more genome editing complexes. In some embodiments, the one or more additional cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (for example, VEPEP-3, VEPEP-6 or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides and Pep-2 peptides.In some embodiments, at least a portion of the one or more additional cell membrane-permeable peptides is linked to a targeting moiety.In some embodiments, the link is a covalent bond.

[0161] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptides) and a genome editing enzyme, such as a nuclease or integrase (or a nucleic acid encoding the genome editing enzyme). In some embodiments, at least a portion of the one or more cell membrane-permeable peptides in the nanoparticles are linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the genome editing enzyme associated with the cell membrane-permeable peptide is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, or 80:1 (including any range between these ratios)). In some embodiments, the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the genome editing enzyme associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about any of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween)). In some embodiments, the genome editing enzyme is a nuclease selected from ZFN, TALEN, homing endonuclease, RGEN, or DGEN. In some embodiments, the genome editing enzyme is Cas9. In some embodiments, the nanoparticle core further comprises one or more gRNAs or gDNAs (or one or more nucleic acids encoding one or more gRNAs or gDNAs). In some embodiments, the genome editing enzyme is a TALEN. In some embodiments, the genome editing enzyme is an integrase. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing a specific modification into a target polynucleotide. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence.In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0162] In some embodiments, nanoparticles for modifying target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptides), RGEN (or a nucleic acid encoding RGEN), and gRNA (or a nucleic acid encoding gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence in the target polynucleotide. In some embodiments, the nanoparticle core comprises one of one or more cell membrane-permeable peptides associated with a preformed complex comprising RGEN and gRNA (RGEN / gRNA complex). In some embodiments, the nanoparticle core comprises a first complex comprising one of one or more cell membrane-permeable peptides associated with RGEN, and a second complex comprising one of one or more cell membrane-permeable peptides associated with gRNA. In some embodiments, at least a portion of the one or more cell membrane-permeable peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a complementary transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range therebetween)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1.In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, and / or b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)). In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, and / or b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween). In some embodiments, the nanoparticle core further comprises one or more additional gRNAs containing different guide sequences. In some embodiments, at least one of the one or more additional gRNAs is present in a preformed RGEN / gRNA complex. In some embodiments, at least one of the one or more additional gRNAs is present in a second complex. In some embodiments, at least one of the one or more additional gRNAs is present in an additional complex containing one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a pre-formed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in the first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex containing one or more cell membrane-permeable peptides. In some embodiments, the target polynucleotide is modified in its coding sequence. In some embodiments, the target polynucleotide is modified in its non-coding sequence.In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0163] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptides), a DGEN (or a nucleic acid encoding the DGEN), and gDNA (or a nucleic acid encoding the gDNA), wherein the gDNA comprises a guide sequence complementary to a target sequence in the target polynucleotide. In some embodiments, the nanoparticle core comprises one of the one or more cell membrane-permeable peptides associated with a preformed complex comprising DGEN and gDNA (DGEN / gDNA complex). In some embodiments, the nanoparticle core comprises a first complex comprising one of the one or more cell membrane-permeable peptides associated with DGEN, and a second complex comprising one of the one or more cell membrane-permeable peptides associated with gDNA. In some embodiments, at least a portion of the one or more cell membrane-permeable peptides in the nanoparticle are linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the DGEN is NgAgo. In some embodiments, the molar ratio of DGEN to gDNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of DGEN to gDNA in the nanoparticles is about 1:1. In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the DGEN associated with the cell membrane-permeable peptide, and / or b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gDNA associated with the cell membrane-permeable peptide is between about 1:1 and about 80:1 (e.g., about 1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1 (including any range between these ratios)).In some embodiments, the molar ratio of a) the cell membrane-penetrating peptide in the complex present in the nanoparticle to the DGEN associated with the cell membrane-penetrating peptide, and / or b) the molar ratio of the cell membrane-penetrating peptide in the complex present in the nanoparticle to the gDNA associated with the cell membrane-penetrating peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween)). In some embodiments, the nanoparticle core further comprises one or more additional gDNAs comprising different guide sequences. In some embodiments, at least one of the one or more additional gDNAs is present in a preformed DGEN / gDNA complex. In some embodiments, at least one of the one or more additional gDNAs is present in a second complex. In some embodiments, at least one of the one or more additional gDNAs is present in an additional complex comprising one of one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing a specific modification into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a preformed DGEN / gDNA complex. In some embodiments, the donor nucleic acid is present in a first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex comprising one of one or more cell membrane-permeable peptides. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product.In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0164] In some embodiments, nanoparticles for modifying target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptides) and a preformed complex comprising RGEN and gRNA (RGEN / gRNA complex), wherein at least one of the one or more cell membrane-permeable peptides is associated with the preformed RGEN / gRNA complex, and the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide. In some embodiments, at least a portion of the one or more cell membrane-permeable peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a complementary transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1. In some embodiments, the molar ratio of the cell membrane-permeable peptide to the RGEN in the preformed RGEN / gRNA complex associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)).In some embodiments, the molar ratio of the cell membrane-permeable peptide to the RGEN in the preformed RGEN / gRNA complex associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween)). In some embodiments, the nanoparticle core further comprises one or more additional gRNAs comprising a different guide sequence. In some embodiments, at least one of the one or more additional gRNAs is present in the preformed RGEN / gRNA complex. In some embodiments, at least one of the one or more additional gRNAs is present in a second complex. In some embodiments, at least one of the one or more additional gRNAs is present in an additional complex comprising one of the one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a pre-formed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in a first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex containing one or more cell membrane-permeable peptides. In some embodiments, the target polynucleotide is modified in its coding sequence. In some embodiments, the target polynucleotide is modified in its non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by causing the modified target gene to express an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by causing the modified target gene to express an active target gene product.In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0165] In some embodiments, nanoparticles for modifying target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9, or ADGN-100 peptides), a first complex comprising one or more cell membrane-permeable peptides associated with RGEN (or a nucleic acid encoding RGEN), and a second complex comprising one or more cell membrane-permeable peptides associated with gRNA (or a nucleic acid encoding gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide. In some embodiments, at least a portion of the one or more cell membrane-permeable peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a complementary transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1. In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the first complex to the RGEN associated with the cell membrane-permeable peptide, and / or b) the molar ratio of the cell membrane-permeable peptide in the second complex to the gRNA associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)).In some embodiments, a) the molar ratio of the cell membrane-permeable peptide to the RGEN associated with the cell membrane-permeable peptide in the first complex, and / or b) the molar ratio of the cell membrane-permeable peptide to the gRNA associated with the cell membrane-permeable peptide in the second complex is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween). In some embodiments, the nanoparticle core further comprises one or more additional gRNAs containing different guide sequences. In some embodiments, at least one of the one or more additional gRNAs is present in the preformed RGEN / gRNA complex. In some embodiments, at least one of the one or more additional gRNAs is present in the second complex. In some embodiments, at least one of the one or more additional gRNAs is present in an additional complex containing one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing a specific modification into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a preformed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in the first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex containing one or more cell membrane-permeable peptides. In some embodiments, the target polynucleotide is modified in its coding sequence. In some embodiments, the target polynucleotide is modified in its non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or by resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or by resulting in a modified target gene that expresses an active target gene product.In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0166] In some embodiments, nanoparticles for modifying target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptides), RGEN (or a nucleic acid encoding RGEN), gRNA (or a nucleic acid encoding gRNA), and a donor nucleic acid for introducing specific modifications into target polynucleotides, wherein the gRNA comprises a guide sequence complementary to the target sequence in the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide to be modified to include the modification. In some embodiments, the nanoparticle core comprises one of one or more cell membrane-permeable peptides associated with a pre-formed complex (RGEN / gRNA complex) comprising RGEN and gRNA. In some embodiments, the donor nucleic acid is present in a ternary complex comprising one of one or more cell membrane-permeable peptides, a pre-formed RGEN / gRNA complex, and the donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising one or more cell membrane-permeable peptides associated with RGEN, and a second complex comprising one or more cell membrane-permeable peptides associated with gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex. In some embodiments, the modification is the addition, deletion, or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is the insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, such as a plasmid. The donor nucleic acid comprises a 5' homology arm that is homologous to the 5' target homology region comprising the portion adjacent to the 5' side of the modification of the target polynucleotide, and a 3' homology arm that is homologous to the 3' target homology region comprising the portion adjacent to the 3' side of the modification of the target polynucleotide.In some embodiments, the 5' target homology region and / or the 3' target homology region overlap with at least about one nucleotide in the target sequence (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more). In some embodiments, the 5' target homology region and / or the 3' target homology region are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, at least a portion of the one or more cell membrane-penetrating peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a supplemental trans-activating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range therebetween)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1.In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide, and / or c) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the donor nucleic acid associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)). In some embodiments, the molar ratio of a) the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide, and / or c) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the donor nucleic acid associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween). In some embodiments, the nanoparticle core further comprises one or more additional gRNAs containing different guide sequences. In some embodiments, at least one of the one or more additional gRNAs is present in a preformed RGEN / gRNA complex. In some embodiments, at least one of the one or more additional gRNAs is present in a second complex. In some embodiments, at least one of the one or more additional gRNAs is present in an additional complex comprising one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a preformed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in the first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex comprising one or more cell membrane-permeable peptides.In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0167] In some embodiments, nanoparticles for modifying target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptides), RGEN (or a nucleic acid encoding RGEN), gRNA (or a nucleic acid encoding gRNA), and a donor nucleic acid for introducing specific modifications into target polynucleotides, wherein the gRNA comprises a guide sequence complementary to the target sequence in the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide to be modified to include the modification, wherein the modification is the addition, deletion, or substitution of one or more nucleotides in the target nucleic acid. In some embodiments, the nanoparticle core comprises one of one or more cell membrane-permeable peptides associated with a pre-formed complex (RGEN / gRNA complex) comprising RGEN and gRNA. In some embodiments, the donor nucleic acid is present in a ternary complex comprising one of one or more cell membrane-permeable peptides, a pre-formed RGEN / gRNA complex, and the donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising one of one or more cell membrane-permeable peptides associated with RGEN, and a second complex comprising one of one or more cell membrane-permeable peptides associated with gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex. In some embodiments, the modification is an addition, deletion, or substitution of between about 1 and about 50 nucleotides in the target polynucleotide (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification.In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually between about 20 and about 150 nucleotides in length (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 nucleotides, including any range therebetween). In some embodiments, at least a portion of the one or more cell membrane-penetrating peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a supplemental transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1.In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide, and / or c) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the donor nucleic acid associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)). In some embodiments, the molar ratio of a) the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide, and / or c) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the donor nucleic acid associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween). In some embodiments, the nanoparticle core further comprises one or more additional gRNAs containing different guide sequences. In some embodiments, at least one of the one or more additional gRNAs is present in a preformed RGEN / gRNA complex. In some embodiments, at least one of the one or more additional gRNAs is present in a second complex. In some embodiments, at least one of the one or more additional gRNAs is present in an additional complex comprising one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a preformed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in the first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex comprising one or more cell membrane-permeable peptides.In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0168] In some embodiments, nanoparticles for modifying target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptides), RGEN (or a nucleic acid encoding RGEN), gRNA (or a nucleic acid encoding gRNA), and a donor nucleic acid for introducing specific modifications into target polynucleotides, wherein the gRNA comprises a guide sequence complementary to the target sequence in the target polynucleotide, and the donor nucleic acid comprises a sequence corresponding to the portion of the target polynucleotide that is modified to include the modification, and the modification is the insertion of a heterologous nucleic acid into the target nucleic acid. In some embodiments, the nanoparticle core comprises one of one or more cell membrane-permeable peptides associated with a pre-formed complex (RGEN / gRNA complex) comprising RGEN and gRNA. In some embodiments, the donor nucleic acid is present in a ternary complex comprising one of one or more cell membrane-permeable peptides, a pre-formed RGEN / gRNA complex, and the donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising one of one or more cell membrane-permeable peptides associated with RGEN, and a second complex comprising one of one or more cell membrane-permeable peptides associated with gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex. In some embodiments, the modification is the insertion of a heterologous nucleic acid having a length of more than about 50 nucleotides (e.g., about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 nucleotides or more (including any range between these values)). In some embodiments, the donor nucleic acid is a double-stranded DNA molecule. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification.In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually longer than about 300 nucleotides in length (e.g., longer than about 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides, including any range therebetween). In some embodiments, at least a portion of the one or more cell membrane-penetrating peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to a complementary transactivating crRNA (tracrRNA). In some embodiments, the gRNA is an sgRNA comprising a guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1. In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1.In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide, and / or c) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the donor nucleic acid associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)). In some embodiments, the molar ratio of a) the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide, and / or c) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the donor nucleic acid associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween). In some embodiments, the nanoparticle core further comprises one or more additional gRNAs containing different guide sequences. In some embodiments, at least one of the one or more additional gRNAs is present in a preformed RGEN / gRNA complex. In some embodiments, at least one of the one or more additional gRNAs is present in a second complex. In some embodiments, at least one of the one or more additional gRNAs is present in an additional complex comprising one or more cell membrane-permeable peptides. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a preformed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in the first or second complex. In some embodiments, the donor nucleic acid is present in an additional complex comprising one or more cell membrane-permeable peptides.In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence. In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0169] In some embodiments, nanoparticles for modifying one or more target polynucleotides are provided, the nanoparticles comprising a core comprising one or more cell membrane-permeable peptides (e.g., VEPEP-3, VEPEP-6, VEPEP-9 or ADGN-100 peptide), RGEN (or a nucleic acid encoding RGEN), and a plurality of gRNAs (or one or more nucleic acids encoding a plurality of gRNAs), wherein each of the plurality of gRNAs comprises a different guide sequence complementary to a target sequence in one of one or more target polynucleotides. In some embodiments, the nanoparticle core comprises one of one or more cell membrane-permeable peptides associated with a pre-formed complex (RGEN / gRNA complex) comprising RGEN and at least one of a plurality of gRNAs. In some embodiments, the pre-formed RGEN / gRNA complex comprises each of a plurality of gRNAs. In some embodiments, the nanoparticle core comprises a first complex comprising one of one or more cell membrane-permeable peptides associated with RGEN, and one or more complexes each comprising one of one or more cell membrane-permeable peptides associated with at least one of a plurality of gRNAs. In some embodiments, the one or more complexes are second complexes each containing a plurality of gRNAs. In some embodiments, at least a portion of the one or more cell membrane-penetrating peptides in the nanoparticle is linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, each of the multiple gRNAs is a single guide RNA (sgRNA) comprising a specificity-determining CRISPR RNA (crRNA) fused to an auxiliary transactivating crRNA (tracrRNA). In some embodiments, each of the multiple gRNAs is an sgRNA comprising a gRNA guide sequence, a tracr mate sequence, a tracr sequence, and a tail sequence. In some embodiments, the RGEN is Cas9 or Cpf1.In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is between about 10:1 and about 1:10 (e.g., about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10 (including any range between these ratios)). In some embodiments, the molar ratio of RGEN to gRNA in the nanoparticles is about 1:1. In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, and / or b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide is between about 1: 1 and about 80: 1 (e.g., about 1: 1, 5: 1, 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1 (including any range between these ratios)). In some embodiments, a) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the RGEN associated with the cell membrane-permeable peptide, and / or b) the molar ratio of the cell membrane-permeable peptide in the complex present in the nanoparticle to the gRNA associated with the cell membrane-permeable peptide is between about 5:1 and about 20:1 (e.g., about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1 (including any range therebetween). In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing specific modifications into the target polynucleotide. In some embodiments, the donor nucleic acid is present in a preformed RGEN / gRNA complex. In some embodiments, the donor nucleic acid is present in the first complex or one or more complexes. In some embodiments, the donor nucleic acid is present in an additional complex containing one or more cell membrane-permeable peptides. In some embodiments, the target polynucleotide is modified in a coding sequence. In some embodiments, the target polynucleotide is modified in a non-coding sequence.In some embodiments, the target polynucleotide is modified to inactivate the target gene, such as by reducing expression of the target gene or resulting in a modified target gene that expresses an inactive product. In some embodiments, the target polynucleotide is modified to activate the target gene, such as by increasing expression of the target gene or resulting in a modified target gene that expresses an active target gene product. In some embodiments, the one or more cell membrane-permeable peptides include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.

[0170] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising a cell membrane-permeable peptide and a genome editing enzyme (or a nucleic acid encoding the genome editing enzyme), wherein the cell membrane-permeable peptide is associated with the genome editing enzyme, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the genome editing enzyme is a nuclease selected from ZFN, TALEN, homing endonuclease, RGEN, or DGEN. In some embodiments, the genome editing enzyme is Cas9. In some embodiments, the nanoparticle core further comprises a gRNA or gDNA (or a nucleic acid encoding the gRNA or gDNA). In some embodiments, the genome editing enzyme is a TALEN. In some embodiments, the genome editing enzyme is an integrase. In some embodiments, the nanoparticle core further comprises a donor nucleic acid for introducing a specific modification into a target polynucleotide.

[0171] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, RGEN (or a nucleic acid encoding RGEN), and gRNA (or a nucleic acid encoding the gRNA), wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide includes the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a preformed complex (RGEN / gRNA complex) comprising RGEN and gRNA.In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with RGEN, and a second complex comprising a cell membrane-permeable peptide associated with gRNA.In some embodiments, the RGEN is Cas9.

[0172] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising a cell membrane-permeable peptide and a preformed complex comprising RGEN and gRNA (RGEN / gRNA complex), wherein the cell membrane-permeable peptide is associated with the preformed RGEN / gRNA complex, the gRNA comprises a guide sequence complementary to a target sequence in the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the RGEN is Cas9.

[0173] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, a first complex comprising the cell membrane-permeable peptide associated with RGEN (or a nucleic acid encoding RGEN), and a second complex comprising the cell membrane-permeable peptide associated with gRNA (or a nucleic acid encoding the gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the RGEN is Cas9.

[0174] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, RGEN (or a nucleic acid encoding RGEN), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid for introducing a specific modification into a target polynucleotide, wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid includes a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a preformed complex (RGEN / gRNA complex) comprising RGEN and gRNA, and another complex comprising a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the donor nucleic acid is present in a ternary complex comprising a cell membrane-permeable peptide, a preformed RGEN / gRNA complex, and a donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with RGEN, and a second complex comprising a cell membrane-permeable peptide associated with gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex. In some embodiments, the modification is an addition, deletion, or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is the insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, for example, a plasmid.The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' target homology region and / or the 3' target homology region overlap with at least about one nucleotide (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) in the target sequence. In some embodiments, the 5' target homology region and / or the 3' target homology region is within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the RGEN is Cas9.

[0175] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, RGEN (or a nucleic acid encoding RGEN), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid for introducing a specific modification into the target polynucleotide, wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, and the donor nucleic acid includes a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, wherein the modification is an addition, deletion, or substitution of one or more nucleotides in the target nucleic acid, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a preformed complex (RGEN / gRNA complex) comprising RGEN and gRNA, and another complex comprising a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the donor nucleic acid is present in a ternary complex comprising a cell membrane-permeable peptide, a preformed RGEN / gRNA complex, and a donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with RGEN, and a second complex comprising a cell membrane-permeable peptide associated with gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex.In some embodiments, the modification is an addition, deletion, or substitution of between about 1 and about 50 nucleotides in the target polynucleotide (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually between about 20 and about 150 nucleotides in length (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 nucleotides in length, including any range therebetween). In some embodiments, the RGEN is Cas9.

[0176] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, RGEN (or a nucleic acid encoding RGEN), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid for introducing a specific modification into the target polynucleotide, wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid includes a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being insertion of a heterologous nucleic acid into the target nucleic acid, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a preformed complex (RGEN / gRNA complex) comprising RGEN and gRNA, and another complex comprising a cell membrane-permeable peptide associated with a donor nucleic acid. In some embodiments, the donor nucleic acid is present in a ternary complex comprising a cell membrane-permeable peptide, a preformed RGEN / gRNA complex, and a donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with RGEN, and a second complex comprising a cell membrane-permeable peptide associated with gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex.In some embodiments, the modification is an insertion of a heterologous nucleic acid that is greater than about 50 nucleotides in length (e.g., greater than about any of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a double-stranded DNA molecule. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that comprises a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that comprises a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually greater than about 300 nucleotides in length (e.g., greater than about 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides, including any range therebetween). In some embodiments, the RGEN is Cas9.

[0177] In some embodiments, nanoparticles for modifying one or more target polynucleotides are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, RGEN (or a nucleic acid encoding RGEN), and multiple gRNAs (or one or more nucleic acids encoding multiple gRNAs), each of the multiple gRNAs individually including a different guide sequence complementary to a target sequence in one of the one or more target polynucleotides, and the cell membrane-permeable peptide including the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide includes the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a pre-formed complex (RGEN / gRNA complex) comprising RGEN and at least one of a plurality of gRNAs. In some embodiments, the pre-formed RGEN / gRNA complex comprises each of a plurality of gRNAs. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with RGEN, and one or more complexes, each of which comprises a cell membrane-permeable peptide associated with at least one of a plurality of gRNAs. In some embodiments, the one or more complexes are second complexes comprising each of a plurality of gRNAs. In some embodiments, the RGEN is Cas9.

[0178] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), and a gRNA (or a nucleic acid encoding the gRNA), wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide includes the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a preformed complex (Cas9 / gRNA complex) comprising Cas9 and gRNA. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with RGEN, and a second complex comprising a cell membrane-permeable peptide associated with gRNA.

[0179] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising a cell membrane-permeable peptide and a preformed complex (Cas9 / gRNA complex) comprising Cas9 and a gRNA, wherein the cell membrane-permeable peptide is associated with the preformed Cas9 / gRNA complex, the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 53-70, 79, and 80.

[0180] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core comprising a cell membrane-permeable peptide, a first complex comprising the cell membrane-permeable peptide associated with Cas9 (or a nucleic acid encoding Cas9), and a second complex comprising the cell membrane-permeable peptide associated with a gRNA (or a nucleic acid encoding the gRNA), wherein the gRNA comprises a guide sequence complementary to a target sequence within the target polynucleotide, and the cell membrane-permeable peptide comprises the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOS: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80.

[0181] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid for introducing a specific modification into a target polynucleotide, wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid includes a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOS: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOS: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOS: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-penetrating peptide associated with a preformed complex (Cas9 / gRNA complex) comprising Cas9 and gRNA, and another complex comprising a cell membrane-penetrating peptide associated with a donor nucleic acid. In some embodiments, the donor nucleic acid is present in a ternary complex comprising a cell membrane-penetrating peptide, a preformed Cas9 / gRNA complex, and a donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-penetrating peptide associated with Cas9 and a second complex comprising a cell membrane-penetrating peptide associated with a gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex. In some embodiments, the modification is an addition, deletion, or substitution of one or more nucleotides in the target polynucleotide, and the donor nucleic acid is a single-stranded DNA oligonucleotide. In some embodiments, the modification is the insertion of a heterologous nucleic acid into the target polynucleotide, and the donor nucleic acid is a double-stranded DNA molecule, for example, a plasmid.The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' target homology region and / or the 3' target homology region overlap with at least about one nucleotide (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) in the target sequence. In some embodiments, the 5' target homology region and / or the 3' target homology region is within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence.

[0182] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid for introducing a specific modification into the target polynucleotide, wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid includes a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being an addition, deletion, or substitution of one or more nucleotides in the target nucleic acid, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-penetrating peptide associated with a preformed complex (Cas9 / gRNA complex) comprising Cas9 and gRNA, and another complex comprising a cell membrane-penetrating peptide associated with a donor nucleic acid. In some embodiments, the donor nucleic acid is present in a ternary complex comprising a cell membrane-penetrating peptide, a preformed Cas9 / gRNA complex, and a donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-penetrating peptide associated with Cas9 and a second complex comprising a cell membrane-penetrating peptide associated with a gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex.In some embodiments, the modification is an addition, deletion, or substitution of between about 1 and about 50 nucleotides in the target polynucleotide (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a single-stranded DNA oligonucleotide. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that includes a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that includes a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually between about 20 and about 150 nucleotides in length (e.g., any of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 nucleotides, including any range between these values).

[0183] In some embodiments, nanoparticles for modifying a target polynucleotide are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), a gRNA (or a nucleic acid encoding the gRNA), and a donor nucleic acid for introducing a specific modification into the target polynucleotide, wherein the gRNA includes a guide sequence complementary to a target sequence within the target polynucleotide, the donor nucleic acid includes a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification, the modification being insertion of a heterologous nucleic acid into the target nucleic acid, and the cell membrane-permeable peptide includes the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide includes the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide includes the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide includes the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-penetrating peptide associated with a preformed complex (Cas9 / gRNA complex) comprising Cas9 and gRNA, and another complex comprising a cell membrane-penetrating peptide associated with a donor nucleic acid. In some embodiments, the donor nucleic acid is present in a ternary complex comprising a cell membrane-penetrating peptide, a preformed Cas9 / gRNA complex, and a donor nucleic acid. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-penetrating peptide associated with Cas9 and a second complex comprising a cell membrane-penetrating peptide associated with a gRNA. In some embodiments, the donor nucleic acid is present in the first complex. In some embodiments, the donor nucleic acid is present in the second complex.In some embodiments, the modification is an insertion of a heterologous nucleic acid that is greater than about 50 nucleotides in length (e.g., greater than about any of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more nucleotides, including any range therebetween). In some embodiments, the donor nucleic acid is a double-stranded DNA molecule. The donor nucleic acid comprises a 5' homology arm that is homologous to a 5' target homology region that comprises a portion of the target polynucleotide adjacent to the 5' side of the modification, and a 3' homology arm that is homologous to a 3' target homology region that comprises a portion of the target polynucleotide adjacent to the 3' side of the modification. In some embodiments, the 5' and / or 3' target homology regions overlap with at least about 1 (e.g., at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more) nucleotides in the target sequence. In some embodiments, the 5' and / or 3' target homology regions are within about 1000 nucleotides (e.g., within about any of 1000, 500, 400, 300, 200, 100, 75, 50, 25, 20, 15, 10, 5, or 1 nucleotide) of the target sequence. In some embodiments, the 5' homology arm and the 3' homology arm are each individually greater than about 300 nucleotides in length (e.g., greater than about any of 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides, including any range between these values).

[0184] In some embodiments, nanoparticles for modifying one or more target polynucleotides are provided, the nanoparticles comprising a core including a cell membrane-permeable peptide, Cas9 (or a nucleic acid encoding Cas9), and multiple gRNAs (or one or more nucleic acids encoding the multiple gRNAs), each of the multiple gRNAs individually including a different guide sequence complementary to a target sequence in one of the one or more target polynucleotides, and the cell membrane-permeable peptide comprising the amino acid sequence of a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-14, 75, and 76. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 15-40, and 77. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 41-52, and 78. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-70, 79, and 80. In some embodiments, the nanoparticle core comprises a cell membrane-permeable peptide associated with a preformed complex (Cas9 / gRNA complex) comprising Cas9 and at least one of the multiple gRNAs. In some embodiments, the preformed Cas9 / gRNA complex comprises each of the multiple gRNAs. In some embodiments, the nanoparticle core comprises a first complex comprising a cell membrane-permeable peptide associated with Cas9, and one or more complexes, each comprising a cell membrane-permeable peptide associated with at least one of the multiple gRNAs. In some embodiments, the one or more complexes are second complexes comprising each of the multiple gRNAs.

[0185] In some embodiments, the nanoparticle further comprises a surface layer comprising peripheral CPPs surrounding the core. In some embodiments, the peripheral CPPs are the same as the CPPs in the core. In some embodiments, the peripheral CPPs are different from any CPPs in the core. In some embodiments, the peripheral CPPs include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (e.g., VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the peripheral CPPs are VEPEP-3 peptides, VEPEP-6 peptides, VEPEP-9 peptides, or ADGN-100 peptides. In some embodiments, at least a portion of the peripheral cell membrane-penetrating peptides in the surface layer are linked to a targeting moiety. In some embodiments, the linkage is a covalent bond. In some embodiments, the nanoparticle further comprises an intermediate layer between the core and the surface layer of the nanoparticle. In some embodiments, the intermediate layer comprises an intermediate CPP. In some embodiments, the intermediate CPP is the same as the CPP in the core. In some embodiments, the intermediate CPP is different from any CPP in the core. In some embodiments, the intermediate CPP includes, but is not limited to, a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a VEPEP peptide (e.g., a VEPEP-3, VEPEP-6, or VEPEP-9 peptide), an ADGN-100 peptide, a Pep-1 peptide, and a Pep-2 peptide. In some embodiments, the intermediate CPP is a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide.

[0186] In some embodiments, the guide sequence of the gRNA or gDNA contained in the nanoparticle according to any of the embodiments described herein is complementary to a target sequence in a gene encoding a protein involved in regulating the immune response, including immune checkpoint regulators and proteins involved in antigen presentation. In some embodiments, the guide sequence of the gRNA or gDNA contained in the nanoparticle according to any of the embodiments described herein is complementary to a target sequence in a gene encoding a protein involved in regulating cholesterol transport and / or metabolism. In some embodiments, the guide sequence may be selected from, but is not limited to, PD-1, PD-L1, PD-L2, TIM-1, TIM-3, TIM-4, BTLA, VISTA, LAG-3, CTLA-4, TIGIT, 4-1BB, OX40, CD27, CD28, HVEM, GITR, ICOS, CD40, CD80, CD86, B7-H2, B7-H3, B7-H4, B7-H6, 2B4, CD160, gp49B, PIR-B, KIR family receptors, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, A2aR, Toll-like receptors It is complementary to target sequences within genes encoding proteins, including TLR-2, 3, 4, 6, 7, 8 and 9, granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF, CD40L, FLT-3 ligand, cytokines such as IL-1, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21 and IL-35, FasL, TGF-β, indoleamine-2,3-dioxygenase (IDO), major histocompatibility complex (MHC) proteins (including beta 2-microglobulin (β2M)), LDLR, ApoB, LDLRAP1, PCSK9.

[0187] In some embodiments, according to any of the nanoparticles described herein, the nanoparticle further comprises a protein other than the genome editing system molecule, or a nucleic acid molecule (e.g., a DNA plasmid or mRNA) encoding the protein.

[0188] In some embodiments, according to any of the nanoparticles described herein, the average size (diameter) of the nanoparticles is about 10 nm to about 400 nm, including, for example, about 50 nm to about 200 nm, about 60 nm to about 180 nm, about 80 nm to about 140 nm, and about 90 nm to about 120 nm. In some embodiments, the average size (diameter) of the nanoparticles is about 1000 nanometers (nm) or less, for example, about 900, 800, 700, 600, 500, 400, 300, 200, or 100 nm or less. In some embodiments, the average diameter or average diameter of the nanoparticles is about 200 nm or less. In some embodiments, the average diameter or average diameter of the nanoparticles is about 150 nm or less. In some embodiments, the average diameter or average diameter of the nanoparticles is about 100 nm or less. In some embodiments, the average diameter or average diameter of the nanoparticles is about 10 nm to about 400 nm. In some embodiments, the nanoparticles have an average diameter or mean diameter of about 20 nm to about 400 nm. In some embodiments, the nanoparticles have an average diameter or mean diameter of about 30 nm to about 200 nm. In some embodiments, the nanoparticles have an average diameter or mean diameter of about 40 nm to about 200 nm. In some embodiments, the nanoparticles have an average diameter or mean diameter of about 40 nm to about 150 nm. In some embodiments, the nanoparticles have an average diameter or mean diameter of about 40 nm to about 100 nm. In some embodiments, the nanoparticles are sterile-filterable.

[0189] In some embodiments, the zeta potential of the nanoparticles is about -30 mV to about 60 mV (e.g., about -30, -25, -20, -10, -5, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 mV, including any range therebetween). In some embodiments, the zeta potential of the nanoparticles is about -30 mV to about 30 mV, including, for example, about -25 mV to about 25 mV, about -20 mV to about 20 mV, about -15 mV to about 15 mV, about -10 mV to about 10 mV, and about -5 mV to about 10 mV. In some embodiments, the polydispersity index (PI) of the nanoparticles is about 0.05 to about 0.6 (e.g., about any of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, and 0.6, including any range therebetween). In some embodiments, the nanoparticles are substantially non-toxic. cargo

[0190] In some embodiments, the genome editing system molecule (e.g., RGEN) of the genome editing complex or nanoparticle described herein is a protein or polypeptide. For example, in some embodiments, the genome editing complex or nanoparticle described herein comprises an RGEN (e.g., Cas9). In some embodiments, the protein or polypeptide is between about 10 kDa and about 200 kDa (e.g., about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 kDa, including any range therebetween). In some embodiments, the genome editing complex or nanoparticle comprises multiple proteins or polypeptides, each of the multiple proteins or polypeptides being between about 10 kDa and about 200 kDa (e.g., any of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 kDa (including any range between these values)).

[0191] In some embodiments, the genome editing system molecule (e.g., gRNA) of the genome editing complex or nanoparticle described herein is a nucleic acid. In some embodiments, the nucleic acid is between about 20 nt and about 20 kb (e.g., about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any range therebetween). For example, in some embodiments, a genome editing complex or nanoparticle described herein comprises a gRNA (e.g., a Cas9 gRNA). In some embodiments, the gRNA is between about 20 nt and about 200 nt (e.g., about any of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 nt (including any range therebetween)). In some embodiments, the nucleic acid is DNA, e.g., a DNA plasmid encoding a genome editing system molecule. In some embodiments, the DNA plasmid comprises an expression cassette for expressing the genome editing system molecule. In some embodiments, the DNA plasmid is between about 1 kb and about 20 kb (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any ranges therebetween). In some embodiments, the nucleic acid is RNA, e.g., mRNA encoding a genome editing system molecule. In some embodiments, the mRNA is between about 100 nt and about 10 kb (e.g., about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 kb, including any ranges therebetween). In some embodiments, the genome editing complex or nanoparticle comprises multiple nucleic acids, e.g., any of the nucleic acids described herein.For example, in some embodiments, the genome editing complex or nanoparticle comprises a gRNA and a nucleic acid encoding a genome editing system molecule (e.g., a DNA plasmid or mRNA encoding the genome editing system molecule). In some embodiments, the genome editing complex or nanoparticle comprises a nucleic acid encoding multiple genome editing system molecules (e.g., one or more DNA plasmids encoding multiple genome editing system molecules, or multiple mRNAs encoding multiple genome editing system molecules).

[0192] In some embodiments, the genome editing system molecule (e.g., RGEN or gRNA) of the genome editing complex or nanoparticle described herein is replaced with a nucleic acid encoding the genome editing system molecule. For example, in some embodiments, the genome editing complex or nanoparticle described herein comprises a nucleic acid encoding RGEN and / or a nucleic acid encoding gRNA. In some embodiments, the nucleic acid is DNA, e.g., a DNA plasmid encoding the genome editing system molecule. In some embodiments, the DNA plasmid comprises an expression cassette for expressing the genome editing system molecule. In some embodiments, the DNA plasmid is between about 1 kb and about 20 kb (e.g., any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 kb, including any range therebetween). In some embodiments, the nucleic acid is RNA, e.g., an mRNA encoding the genome editing system molecule. In some embodiments, the mRNA is between about 100 nt and about 10 kb (e.g., any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 kb, including any ranges between these values).

[0193] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may also include modified nucleotides, such as methylated nucleotides, and their analogs. As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA and RNA. DNA may be in the form of, for example, an antisense molecule, plasmid DNA, precondensed DNA, PCR product, vector (PAC, BAC, YAC, artificial chromosome), expression cassette, chimeric sequence, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, RNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, including, for example, locked nucleic acids (LNA), unlocked nucleic acids (UNA), and zip nucleic acids (ZNA), which may be synthetic, naturally occurring, or non-naturally occurring, and have similar binding specificity to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence expressly encompasses not only the sequence explicitly stated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences.Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chern., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). "Nucleotide" means a nucleotide. Nucleotides contain the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other by phosphate groups. "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymidine, guanine, cytosine, uracil, inosine, as well as natural analogs and synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. As used herein, "oligonucleotide" generally refers to a short, generally synthetic, polynucleotide, generally less than about 200 nucleotides in length, although this is not necessarily the case. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides applies equally and completely to oligonucleotides.

[0194] In some embodiments, nucleic acid is single-stranded oligonucleotide.In some embodiments, nucleic acid is double-stranded oligonucleotide.Nucleic acid described herein can be, but is not necessarily, in the case of antisense oligonucleotide, RNAi, siRNA, shRNA, iRNA, antagomir, the length of any range of 200 nucleotides or less, or in the case of plasmid DNA, the length of 1000 kilobases or less.

[0195] In some embodiments, the nucleic acid is a plasmid DNA or DNA fragment (e.g., a DNA fragment of about 1000 bp or less in length). In addition, the plasmid DNA or DNA fragment may be hypermethylated or hypomethylated. In some embodiments, the plasmid DNA or DNA fragment may encode one or more genes and contain regulatory elements necessary for expression of the one or more genes. In some embodiments, the plasmid DNA or DNA fragment may include one or more genes encoding a selectable marker that allows maintenance of the plasmid DNA or DNA fragment in a suitable host cell. qualification

[0196] In some embodiments, the genome editing complex or nanoparticle described herein comprises a targeting moiety, which is a ligand capable of cell-specific targeting and / or nuclear targeting. The cell membrane surface receptor and / or cell surface marker is a molecule or structure that can bind to the ligand with high affinity and preferably high specificity. The cell membrane surface receptor and / or cell surface marker is preferably specific to a particular cell, i.e., is found primarily in one cell type but not in another cell type (e.g., galactosyl residues that target the asialoglycoprotein receptor on the surface of hepatocytes). The cell membrane surface receptor facilitates cell targeting and internalization of the ligand (e.g., targeting moiety) and binding molecule (e.g., the complex or nanoparticle of the present invention) into the target cell. Numerous ligand moieties / ligand binding partners that can be used in the present invention have been widely described in the literature. Such ligand moieties can confer the ability of the complex or nanoparticle of the present invention to bind to a given binding partner molecule or to a class of binding partner molecules localized on the surface of at least one target cell. Suitable binding partner molecules include, but are not limited to, polypeptides selected from the group consisting of cell-specific markers, tissue-specific markers, cellular receptors, viral antigens, antigenic epitopes, and tumor-associated markers. The binding partner molecule may further comprise, for example, one or more sugars, lipids, glycolipids, antibody molecules or fragments of antibody molecules, or aptamers. According to the present invention, the ligand moiety may be, for example, all or a portion of a lipid, glycolipid, hormone, sugar, polymer (e.g., PEG, polylysine, PET), oligonucleotide, vitamin, antigen, all or a portion of a lectin, all or a portion of a polypeptide such as JTS1 (WO94 / 40958), an antibody or a fragment thereof, or a combination thereof. In some embodiments, the ligand moiety used in the present invention is a peptide or polypeptide having a minimum length of 7 amino acids. The ligand moiety is a naturally occurring polypeptide or a polypeptide derived from a naturally occurring polypeptide."Derived from" means containing (a) one or more modifications to the native sequence (e.g., addition, deletion, and / or substitution of one or more residues), (b) amino acid analogs, including non-naturally occurring amino acids, (c) substituted linkages, or (d) other modifications known in the art. Polypeptides useful as ligand moieties include variants and chimeric polypeptides obtained by fusing sequences from various sources, such as, for example, a humanized antibody combining the variable region of a murine antibody and the constant region of a human immunoglobulin. In addition, such polypeptides can have linear or cyclized structures (e.g., by flanking both ends of the polypeptide ligand with cysteine residues). In addition, polypeptides used as ligand moieties can include modifications of their original structure by substitution or addition of chemical moieties (e.g., glycosylation, alkylation, acetylation, amidation, phosphorylation, addition of sulfhydryl groups, etc.). The present invention further contemplates modifications that render the ligand moiety detectable. For this purpose, modification with a detectable moiety (i.e., a scintigraphic, radioactive or fluorescent moiety, or a dye label, etc.) is contemplated. Such detectable labels may be attached to the ligand moiety by any conventional technique and may be used for diagnostic purposes (e.g., imaging of tumor cells). In some embodiments, the binding partner molecule is an antigen (e.g., a target cell-specific antigen, a disease-specific antigen, an antigen specifically expressed on the surface of modified target cells), and the ligand moiety is an antibody, a fragment thereof, or a minimal recognition unit (e.g., a fragment that still exhibits antigen specificity), such as those described in detail in immunology manuals (see, e.g., Immunology, 3rd ed., 1993, Roitt, Brostoff, and Male, eds. Gambli, Mosby). The ligand moiety may also be a monoclonal antibody. Many monoclonal antibodies that bind to many of these antigens are known, and antibodies against most antigens can be prepared using techniques known in the art for monoclonal antibody technology. The ligand moiety may be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., an ScFv).In some embodiments, the ligand moiety is selected from an antibody fragment rather than a whole antibody. Effective functions of a whole antibody, such as complement binding, are eliminated. ScFv and dAb antibody fragments may be expressed as fusions with one or more other polypeptides. The minimal recognition unit may be derived from the sequence of one or more of the complementarity-determining regions (CDRs) of an Fv fragment. Whole antibodies and F(ab')2 fragments are "bivalent." "Bivalent" means that the antibody and F(ab')2 fragment have two antigen-binding sites. In contrast, Fab, Fv, ScFv, dAb fragments, and minimal recognition units are monovalent and have only one antigen-binding site. In some embodiments, the ligand moiety enables targeting to tumor cells and can recognize and bind to molecules associated with the tumor state, such as tumor-specific antigens, cellular proteins differentially or overexpressed in tumor cells, or gene products of cancer-associated viruses. An example of a tumor-specific antigen is MUC-1, which is associated with breast cancer (Hareuven et al., 1999). 0, Eur. J. Biochem 189, 475-486), associated with breast and ovarian cancer The products encoded by the mutated BRCA1 and BRCA2 genes (Miki et al., 1994, Science 226, 66-71; Fuireal et al., 1994, Science 226, pp. 120-122; Wooster et al., 1995, Nature 378, pp. 789-7 92), APC, which is associated with colon cancer (Poiakis, 1995, Curr. Opin. Genet. Dev. 5, 66-71), and prostate-specific antigen (P), which is associated with prostate cancer. SA) (Stamey et al., 1987, New England J. Med. 317, 909), carcinoembryonic antigen (CEA) associated with colon cancer (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinase associated with melanoma (Vile et al., 1993, Cancer Res. 53, 3860-3864), and tyrosinase associated with melanoma cells (Vile et al., 1993, Cancer Res. 53, 3860-3864). The receptor for melanocyte-stimulating hormone (MSH), which is highly expressed in breast and pancreatic cancers, and ErbB-2, which has been implicated in breast and pancreatic cancers (Harris et al., 1994, Gene Therapy vol. 1, 170-175), and alpha-fetoprotein, which has been linked to liver cancer (Kanai et al., 1997, Cancer Res. 57, 461-465). In some embodiments, the ligand moiety is a fragment of an antibody that can recognize and bind to the MUC-1 antigen, and thus can target MUC-1-positive tumor cells. In some embodiments, the ligand moiety is an scFv fragment of the SM3 monoclonal antibody that recognizes the tandem repeat region of the MUC-1 antigen (Burshell et al., 1987, Cancer Res. 47, 5476-5482; Girling et al., 1989, Int. J. Cancer 43, 1072-1076; Dokurno et al., 1998, J. Mol. Biol. 284, 713-728). Differential expression in tumor cells Examples of cellular proteins that are overexpressed or over-expressed include the receptor for interleukin 2 (IL-2), which is overexpressed in some lymphoid tumors, and GRP (gastrin-releasing peptide), which is overexpressed in lung cancer cells, pancreatic, prostate, and gastric tumors (Michael et al., 1995). Gene Therapy, Vol. 2, pp. 660-668), TNF (tumor necrosis factor) receptor, epithelial Growth factor receptors, Fas receptor, CD40 receptor, CD30 receptor, CD27 receptor, OX-40, α-v integrin (Brooks et al., 994, Science 264, 569); and receptors of certain angiogenic growth factors (Hanahan, 1997, Science 277, 48). Based on these indicators, it is within the skill of those in the art to recognize such proteins and define suitable ligand moieties that can bind to such proteins. For example, IL-2 is a suitable ligand moiety for binding to the TL-2 receptor. For receptors specific to fibrosis and inflammation, these include TGF beta receptors or adenosine receptors, which have been identified above and are suitable targets for the compositions of the present invention. Cell surface markers for multiple myeloma include, but are not limited to, CD56, CD40, FGFR3, CS1, CD138, IGF1R, VEGFR and CD38, and are suitable targets for the compositions of the present invention. Suitable ligand moieties that bind to these cell surface markers include, but are not limited to, anti-CD56, anti-CD40, PRO-001, Chir-258, HuLuc63, anti-CD138-DM1, anti-IGF1R, and bevacizumab. target

[0197] In some embodiments, the genome editing complexes or nanoparticles described herein comprise one or more molecules of a genome editing system (e.g., a whole genome editing system) that target one or more genes, including, but not limited to, adenosine receptor A2A, adenosine receptor A2B, adenylyl cyclase, Akt, ALK, ALK / Met, angiopoietin receptor, angiotensin II, APC, AR, ARK5, arrestin, ATF1, ATF-2, B7-1, B7-h1 (pdl-1), β-catenin, Bcl-2, BCL2L12, Bcl6, Bcr-Abl, BRAF, BRCA1, BRCA2, BTK, caspase-2, caspase-9, CCL2, CCN1, Ccnd2, CDK-activating kinase, CEBPA, Chop, c-Jun, c-Myc, CREB, CREB1, CS1, CTGF, CTNNB1, CXCR4, cyclin d1-2, cyclin-dependent kinases (Cdk1-13), DEPTOR, DNMT3B, DPC4, EBOV polymerase L, EGF, EGFR, eIF5A, Elk-1, ER, Erbb4, ERK, ESR1, Ets1, EWSR1, FAK, FGF, FGFR, FOXO1, Frizzled family receptors (FZD-1-10), Fyn, GATA1, GATA3, GLI1, GM-CSF, GP / sGP, GSK, HBV conserved sequence, HDAC, HDGF, HDGFR, Her2, Her3, hexokinase II, HGF, HGFR, HIF-1, HIF-1α, histone methyltransferase EZH2, HIV TAR RNA, HIV Tat, HLA-B7 / beta2-microglobulin, HMGA2, HNF1A, HNF1B, Hsp27, HSP47, human CCR5, Idh1, Idh2, IGF, IGFR, IKK, IKZF1, IL-12, IL-2, INK4, interferon gamma, IRF1, IRF4, JAK, JNK, keratin 16, keratin 17, keratin K6A, keratin K6B, KGFR, KLF6, KRAS, LMO, L MO1, LMP2, LMP7, LOXL2, LPL, LYL1, MADR2, MAPK, Max, Mcl-1, MDA-7, MDM2, MDR-1, MDS1-EVI1, MECL-1, MEF2C, MEK, MEKK, MKK, MLH1, MLST8, MMP-2, MMP-9, MSFR, MSH2, MSH6, MSIN1, mTOR, MUC-1, mutant DDX3X, MYC, NCAP-D2, NCAP-D3, N CAP-G, NCAP-G2, NCAP-H, NCAP-H2, NF1, NF2, NFAT4, NF-κB, Notch1, NPC1, NR4A3, NRAS, Olig2, osteopontin, p53, PA I-1, PARP-1, Patched, PAX3, PAX5, PAX7, PBX1, PDCD4, PDGF, PDGFR, PDK1, PHOX2B, PI3K, PKA, PKC, PKN3, PLK-1, PM L, PR, PRAS40, PRDM16, Prdx1 and Prdx2 (Burkitt's lymphoma), Pre-gen / Pre-C, Pre-S1, Pre-S2 / S, PTC, PTEN, Pyk2, Rad51, RAF, RAPTOR, Rb, RET, RICTOR, RPN13, RRM2, RSV nucleocapsid, RUNX1, S6 kinase, Sap1a, SETBP1, Shc, SLAMF7, Smad, Smad 3, Smad 4, Smad 7, SMC-2, SMC-4, Smoothened, SOX9, SPARC, Spry2, Src, beta-2 adrenergic receptor (ADRB2), beta-globin, STAT5B, STAT, survivin, Syk, Tal, TAL1, TGFR, TGF-α, TGF-β, TGFβ receptor 1, TGFβ receptor 2, TGFβ receptor 3, TGFβ1, thrombospondin, thymidine kinase, TIM-1, TIMP, TNF-α, TP53, transthyretin, TRPV1, ubiquitin ligase, uPAR, VEGF,VEGFR, VEGFR1, VEGFR2, VEGFR3, VHL, VP24, VP30, VP35, VP40, wnt, WT1, WT2, XBP1 (spliced and unspliced), XIAP, and ZBTB16 (including mutant forms thereof (e.g., mutant PTEN, mutant KRAS, mutant p53, etc.)). For example, in some embodiments, the genome editing complex or nanoparticle comprises one or more molecules of an RGEN-based genome editing system (e.g., CRISPR / Cas9 genome editing system), wherein the RGEN-based genome editing system comprises a gRNA that targets one of the genes described herein. In some embodiments, the genome editing complex or nanoparticle comprises one or more molecules of a DGEN-based genome editing system, wherein the DGEN-based genome editing system comprises a gDNA that targets one of the genes described herein. In some embodiments, the genome editing complex or nanoparticle comprises one or more molecules of a ZFN-based genome editing system, wherein the ZFN targets one of the genes described herein. In some embodiments, the genome editing complex or nanoparticle comprises one or more molecules of a TALEN-based genome editing system, wherein the TALEN targets one of the genes described herein. In some embodiments, the genome editing complex or nanoparticle comprises one or more molecules of a homing endonuclease-based genome editing system, wherein the homing endonuclease targets one of the genes described herein. In some embodiments, the genome editing complex or nanoparticle comprises one or more molecules of an integrase-based genome editing system, wherein the integrase targets one of the genes described herein. Nucleic acid-guided endonucleases

[0198] In some embodiments, nucleic acid-guided endonuclease is CRISPR-associated nuclease.Generally, CRISPR (clustered regularly interspaced short palindromic repeats) is also known as SPIDR (spacer interspersed direct repeat), and constitutes a family of DNA loci that are usually specific to certain bacterial species.CRISPR loci include another class of interspersed short sequence repeats (SSR) found in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433

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

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

[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 typically differ from other SSRs in the structure of their repeats, which are termed 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, repeats are substantially They are short elements present in clusters regularly spaced by characteristic intervening sequences of constant length (Mojica et al.,

[2000] , supra). The repeat sequences are highly conserved among lineages, but the number of intervening repeats and the sequence of the spacer region usually vary from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401 [2 CRISPR loci may be used in a variety of organisms, including but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaeroba cter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga have been identified in over 40 prokaryotes, including Salmonella, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga (e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al.,

[2005] ).

[0199] Generally, a "CRISPR system" refers collectively to proteins, transcripts, and other molecules involved in the activity of a CRISPR-associated ("Cas") nuclease (e.g., an RNA-guided endonuclease, or "RGEN"), including Cas gene products, Cas gene sequences, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or an active partial tracrRNA), tracr mate sequences (including, in the context of an endogenous CRISPR system, "direct repeats" and partial direct repeats of processed tracrRNA), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), or other sequences, transcripts, and products derived from a CRISPR locus. In some embodiments, one or more molecules of a CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more molecules of a CRISPR system are derived from a particular organism with an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, CRISPR systems feature a molecule (also referred to as a protospacer in endogenous CRISPR systems) that promotes the formation of a CRISPR complex at the site of a target sequence. With respect to the formation of a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, such that hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is present in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be present in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used for recombination into a target locus containing a target sequence is referred to as an "editing template," "editing polynucleotide," "editing sequence," "donor sequence," or "donor nucleic acid."In aspects of the invention, the exogenous template polynucleotide may be referred to as an editing template. In aspects of the invention, the recombination is homologous recombination.

[0200] Typically, with respect to endogenous CRISPR systems, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). A tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence, or more than about these numbers of nucleotides), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence operably linked to the guide sequence. In some embodiments, the tracr sequence is complementary to the tracr mate sequence, and is sufficiently complementary to hybridize and participate in the formation of a CRISPR complex. As with the target sequence, full complementarity may not be necessary, as long as it functions satisfactorily. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more molecules of the CRISPR system are introduced into host cells to allow the formation of a CRISPR complex at one or more target sites. For example, a Cas nuclease, a guide sequence linked to a tracr mate sequence, and a tracr sequence can each be introduced into a host cell to allow the formation of a CRISPR complex at a target sequence in the host cell that is complementary to the guide sequence.

[0201] Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cpf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cm Examples of Cas proteins include Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, such as inducible, inactivated, or split Cas proteins (e.g., Dominguez et al. (2015). Nature Reviews Molecular Cell Biology; Polstein, LR, and Gersbach, CA (2016). 2015). Nature Chemical Biology, Vol. 11(No. 3): pp. 198-200; Dow et al. (2015). Nature biotechnology, vol. 33(4), pp. 390-394; Zetsche (2015). Nature biotechnology, 33(2):139-142; Kleinstiver et al. (2015). Nature. 523:481-485; Bikard et al. (201 3 years). Nucleic acids research, Vol. 41 (No. 15) pp. 7429-7437; Qi et al. ( 2013). Cell, Vol. 152 (No. 5): pp. 1173-1183. These enzymes are known to those skilled in the art. For example, the amino acid sequence of the S. pyrogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2, and the amino acid sequence of the Acidaminococcus sp. Cpf1 protein can be found in the SwissProt database under accession number U2UMQ6. In some embodiments, the unmodified CRISPR enzyme, e.g., Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9 and can be Cas9 from S. pyogenes or S. pneumoniae. In some embodiments, the CRISPR enzyme is Cpf1 and can be Cpf1 from Acidaminococcus or Lachnospiraceae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of the target sequence, for example, within the target sequence and / or within the complementary sequence of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands 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 some embodiments, the CRISPR enzyme is mutated so that the mutated CRISPR enzyme lacks the ability to cleave one or bo...

Claims

1. A genome editing complex for modifying a target polynucleotide, comprising a cell membrane-permeable peptide and one or more genome editing system molecules, wherein the cell membrane-permeable peptide is a VEPEP-6 peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 40 and 77, and the one or more genome editing system molecules are a) i) an RNA-guided endonuclease (RGEN) or a nucleic acid encoding RGEN, and a guide RNA (gRNA) comprising a guide sequence complementary to a target sequence within said target polynucleotide, comprising crRNA and tracrRNA, or ii) an RGEN or a nucleic acid encoding RGEN, and a single guide RNA (sgRNA); b) a guide DNA (gDNA) comprising a DNA-directed endonuclease (DGEN) or a nucleic acid encoding a DGEN, and a guide sequence complementary to a target sequence within the target polynucleotide; c) a zinc finger protein (ZFP) that recognizes a target sequence within the target polynucleotide; d) a transcription activator-like effector nuclease (TALEN) that recognizes a target sequence within the target polynucleotide; e) a homing endonuclease that recognizes a target sequence within the target polynucleotide; and f) an integrase that recognizes a recombination site within the target polynucleotide A genome editing complex selected from the group consisting of:

2. A genome editing complex as described in claim 1, wherein the N-terminus of the cell membrane-permeable peptide is covalently bonded to an acetyl group.

3. A genome editing complex described in claim 1 or 2, wherein the C-terminus of the cell membrane-permeable peptide is covalently bonded to a cysteamide group.

4. A genome editing complex described in any one of claims 1 to 3, wherein the genome editing complex comprises: a) RGEN or a nucleic acid encoding RGEN; and b) a guide RNA (gRNA) comprising a guide sequence complementary to a target sequence within the target polynucleotide, which comprises crRNA and tracrRNA.

5. A genome editing complex described in any one of claims 1 to 4, wherein the RGEN is a CRISPR nuclease.

6. The genome editing complex described in claim 5, wherein the CRISPR nuclease is Cas9.

7. The genome editing complex described in claim 5, wherein the CRISPR nuclease is Cpf1.

8. A genome editing complex described in any one of claims 1 to 7, wherein the genome editing complex targets a gene encoding a protein selected from the group consisting of KRAS, HRAS, NRAS, RET, MYC, PCSK9, PD-1, lipoprotein A, ANGPTL3, β-catenin, BRAF, LDLR, ApoB, LDLRAP1, or mutant forms thereof.

9. A genome editing complex described in any one of claims 1 to 8, wherein the gRNA targets a gene encoding a protein selected from the group consisting of KRAS, HRAS, NRAS, RET, MYC, PCSK9, PD-1, lipoprotein A, ANGPTL3, β-catenin, BRAF, LDLR, ApoB, LDLRAP1, or mutant forms thereof.

10. The genome editing complex described in Claim 9, wherein the gRNA targets the gene encoding PCSK9.

11. The genome editing complex described in claim 10, wherein the target sequence is selected from the group consisting of sequence numbers 131 to 136.

12. A genome editing complex as described in claim 9, wherein the gRNA targets a gene encoding a mutant KRAS.

13. A genome editing complex described in any one of claims 1 to 12, wherein the gRNA is a single guide RNA (sgRNA).

14. A genome editing complex described in any one of claims 1 to 13, wherein the molar ratio of RGEN to gRNA is between 1:10 and 10:

1.

15. A genome editing complex described in any one of claims 1 to 14, wherein the molar ratio of the cell membrane-permeable peptide to the RGEN is between 1:1 and 80:

1.

16. A genome editing complex as described in claim 15, wherein the molar ratio of the cell membrane-permeable peptide to the RGEN is between 5:1 and 20:

1.

17. A genome editing complex described in any one of claims 1 to 16, further comprising one or more additional gRNAs comprising different guide sequences.

18. A genome editing complex described in any one of claims 1 to 17, further comprising a donor nucleic acid for introducing a modification into the target polynucleotide, the donor nucleic acid comprising a sequence corresponding to a portion of the target polynucleotide to be modified to include the modification.

19. A genome editing complex described in any one of claims 1 to 18, wherein the average diameter of the genome editing complex is between 10 nm and 300 nm.

20. A nanoparticle comprising a core containing a genome editing complex described in any one of claims 1 to 19.

21. A pharmaceutical composition comprising a genome editing complex described in any one of claims 1 to 19 or a nanoparticle described in claim 20, and a pharmaceutically acceptable carrier.

22. A method for preparing a genome editing complex described in any one of claims 1 to 19, comprising a step of combining the cell membrane-permeable peptide with one or more genome editing system molecules, thereby forming the genome editing complex.

23. A genome editing complex described in any one of claims 1 to 19 for delivering the one or more genome editing system molecules to a cell, characterized in that the genome editing complex is contacted with the cell, and the genome editing complex comprises the one or more genome editing system molecules.

24. A genome editing complex described in any one of claims 1 to 19 for modifying a target polynucleotide in a cell, characterized in that the genome editing complex is contacted with the cell, and the genome editing complex comprises one or more genome editing system molecules that target a sequence within the target polynucleotide.

25. The pharmaceutical composition of claim 21 for use in a method for treating a disease in an individual, wherein the treatment comprises administering to the individual an effective amount of the pharmaceutical composition.

26. A kit for modifying a target polynucleotide, comprising a composition containing a genome editing complex described in any one of claims 1 to 19 and / or a nanoparticle described in claim 20.

27. A nanoparticle as described in claim 20 for delivering the one or more genome editing system molecules to a cell, characterized in that the nanoparticle is brought into contact with the cell, and the nanoparticle comprises the one or more genome editing system molecules.

28. A nanoparticle as described in claim 20 for modifying a target polynucleotide in a cell, characterized in that the nanoparticle is brought into contact with the cell, and the nanoparticle comprises one or more genome editing system molecules that target a sequence within the target polynucleotide.

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