Compositions and uses thereof for treating cancer with KRAS mutation

Abstract

To provide a composition for treating cancer having KRAS mutation, and to provide use thereof.SOLUTION: The present application provides guide RNAs and genome editing complexes or nanoparticles useful for specifically targeting mutated KRAS. Exemplary genome editing complexes or nanoparticles include a cell-penetrating peptide, and optionally a DNA nuclease (e.g., Cas9) or a polynucleotide encoding a DNA nuclease. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the nucleotide sequence that is substantially complementary to the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to French Patent Application No. FR2004126, filed April 24, 2020, the entire contents of which are incorporated by reference for all purposes.

[0002] Technical field of the application The present application relates to guide RNAs and genome editing complexes or nanoparticles useful for specifically targeting mutated KRAS.

[0003] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 7373720001241SEQLIST.TXT, Recorded: April 23, 2021, Size: 87KB). [Background technology]

[0004] Background of the application KRAS, a member of the RAS subfamily, is the most frequently mutated oncogene in cancers, including highly lethal lung, colon, and pancreatic cancers (Cox et al. 2014 Nat Rev Drug Discov 13, 828). Activating mutations in KRAS play a critical role in the initiation, propagation, and maintenance of cancer, making them important therapeutic targets (Cox et al. 2014). Common cancer-associated mutations occur at codon -12, encoding glycine, in KRAS. Specifically, the single-nucleotide missense substitutions c.35 G>T and c.35 G>A replace the glycine at position 12 with valine (G12V) and aspartic acid (G12D), respectively. The G12V and G12D substitutions are among the most commonly observed mutations in pancreatic (30% and 51%, respectively) and colorectal (27% and 45%, respectively) adenocarcinomas and are associated with poor prognosis (Jones, S. et al. 2008, Science 321, 1801; Wood, LD et al. 2007, Science 318, 1108).

[0005] Today, there is an urgent need for powerful strategies to target KRAS mutations. While several attempts have been made to target RAS-dependent cancers, direct inhibition of the RAS protein has never been successful; most approaches have focused on affecting downstream effectors of mutated RAS. The recent development of KRAS(G12C)-specific inhibitors (Patricelli et al. 2016 Cancer Discov 6: 316) and non-mutation-selective RAS-binding domain inhibitors (Athuluri-Divakar et al. 2016 Cell 165: 643) has demonstrated the feasibility of directly targeting mutated RAS oncogenes. Direct targeting of mutated RAS oncogenes has the potential to disrupt the function of both the aberrant RAS protein and its downstream effector pathways. However, generating these compounds is challenging due to their complex structure, and mutation-specific inhibition has not been achieved using small molecules against KRAS(G12D) or KRAS(G12V), which occur more frequently than KRAS(G12C). KRAS silencing using small interfering RNA (siRNA) to selectively inhibit mutant KRAS mRNA has also been reported; however, given the continuous expression of KRAS mutants, persistent delivery of targeted RNA suppression is required to maintain complete knockdown (Brummelkamp et al. 2002 Cancer Cell 2: 243) (Zorde Khvalevsky et al. 2013 Proc Natl Acad Sci 110: 20723). 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]

[0006] [Non-Patent Document 1] Cox et al. 2014 Nat Rev Drug Discov 13, 828 [Non-patent document 2] Jones, S. et al. 2008, Science 321, 1801 [Non-patent document 3] Wood, LD et al. 2007, Science 318, 1108 [Non-patent document 4] Patricelli et al. 2016 Cancer Discov 6: 316 [Non-patent document 5] Athuluri-Divakar et al.2016. Cell 165: 643 [Non-patent document 6] Brummelkamp et al. 2002 Cancer Cell 2: 243 [Non-Patent Document 7] Zorde Khvalevsky et al. 2013 Proc Natl Acad Sci 110: 20723 Summary of the Invention [Means for solving the problem]

[0007] A brief summary of the application In one aspect, the present application provides a non-naturally occurring polynucleotide comprising a guide RNA for targeting a mutated KRAS, the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the nucleotide sequence substantially complementary to the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the nucleotide sequence is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the nucleotide sequence is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 3, 19, and 34.

[0008] In some embodiments of any one of the above non-naturally occurring polynucleotides, the polynucleotide is chemically modified.

[0009] In some embodiments according to any one of the above non-naturally occurring polynucleotides, the guide RNA has a length of about 200 nucleotides or less.

[0010] In another aspect, the present application provides a genome editing complex comprising: a) a first cell-penetrating peptide; and b) a guide RNA targeting mutated KRAS, wherein the guide RNA comprises any one of the above-mentioned polynucleotides. In some embodiments, the genome editing complex further comprises a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof. In some embodiments, the DNA nuclease comprises a Cas polypeptide. In some embodiments, the Cas polypeptide is Cas9 or Cas12a.

[0011] In some embodiments of any one of the above genome editing complexes, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide.

[0012] In some embodiments of any one of the genome editing complexes described above, the first cell-penetrating peptide further comprises one or more moieties covalently linked to the N-terminus of the first cell-penetrating peptide, wherein the one or more moieties are selected from the group consisting of acetyl, fatty acid, cholesterol, polyethylene glycol, nuclear localization signal, nuclear export signal, antibody, polysaccharide, linker moiety, and targeting moiety. In some embodiments, the first cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240.

[0013] In some embodiments of any one of the above genome editing complexes, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the PEG moiety consists of 2 to 7 ethylene glycol units.

[0014] In some embodiments of any one of the above genome editing complexes, the first cell-penetrating peptide comprises, from the N-terminus, an acetyl group, a targeting moiety, and a linker moiety covalently linked to the N-terminus of the first cell-penetrating peptide.

[0015] In some embodiments of any one of the genome editing complexes described above, the first cell-penetrating peptide further comprises one or more moieties covalently linked to the C-terminus of the first cell-penetrating 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, oside derivative, lipid, phospholipid, fatty acid, cholesterol, polyethylene glycol, nuclear localization signal, nuclear export signal, antibody, polysaccharide, linker moiety, and targeting moiety. In some embodiments, the first cell-penetrating peptide comprises a cysteamide group covalently linked to its C-terminus.

[0016] In some embodiments of any one of the above genome editing complexes, the first cell-penetrating peptide further comprises a carbohydrate moiety. In some embodiments, the carbohydrate moiety is GalNAc.

[0017] In some embodiments of any one of the above genome editing complexes, the first cell-penetrating peptide is a retro-inverso peptide.

[0018] In some embodiments of any one of the above genome editing complexes, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-195. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270.

[0019] In some embodiments of any one of the above genome editing complexes, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1: 1 and about 80: 1. In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 2: 1 and about 50: 1.

[0020] In some embodiments of any one of the above genome editing complexes, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1: 1 and about 80: 1. In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 2: 1 and about 50: 1.

[0021] In some embodiments of any one of the above genome editing complexes, the guide RNA is complexed with a first cell-penetrating peptide.

[0022] In some embodiments of any one of the above genome editing complexes, the nucleotide sequence encoding the DNA nuclease is complexed with a first cell-penetrating peptide.

[0023] In some embodiments of any one of the above genome editing complexes, the genome editing complex further comprises one or more additional guide RNAs comprising different guide sequences.In some embodiments, at least two of the two or more guide RNAs target a single KRAS mutation.In some embodiments, at least two of the two or more guide RNAs target two or more different KRAS mutations.In some embodiments, at least two of the two or more guide RNAs target G12D, G12V, and / or G12C.

[0024] In some embodiments according to any one of the above genome editing complexes, the average diameter of the genome editing complex is between about 10 nm and about 300 nm.

[0025] In another aspect, the present application provides a nanoparticle comprising a core comprising any one of the above-described genome editing complexes. In some embodiments, the core further comprises one or more additional genome editing complexes, such as any one of the above-described genome editing complexes. In some embodiments, the one or more additional genome editing complexes comprise at least one or more guide RNAs targeting different KRAS mutations. In some embodiments, the core is complexed with a second cell-penetrating peptide. In some embodiments, the second cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the second cell-penetrating 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-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-175.

[0026] In some embodiments of any one of the above nanoparticles, the second cell-penetrating peptide in the nanoparticle is covalently linked to the targeting moiety by a linking moiety.

[0027] In some embodiments of any one of the above genome editing complexes, the core is coated with a shell comprising a peripheral cell-penetrating peptide. In some embodiments, the peripheral cell-penetrating 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-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-175.

[0028] In some embodiments of any one of the above genome editing complexes, the peripheral cell-penetrating peptide in the shell is covalently linked to the targeting moiety by a linking moiety.

[0029] In some embodiments of any one of the above genome editing complexes, the average diameter of the nanoparticles is between about 10 nm and about 400 nm.

[0030] In another aspect, the present application provides a pharmaceutical composition comprising any one of the above guide RNAs, any one of the genome editing complexes, or any one of the nanoparticles, and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises two or more nanoparticles, and the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations.

[0031] In another aspect, the present application provides a method for preparing any one of the above genome editing complexes, the method comprising combining a first cell-penetrating peptide with a guide RNA, thereby forming a genome editing complex.

[0032] In another aspect, the present application provides a method for modifying mutated KRAS in a cell, the method comprising contacting the cell with any one of the above-described guide RNAs, any one of the genome editing complexes, or any one of the nanoparticles.

[0033] In another aspect, the present disclosure provides a method for treating cancer in an individual, comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the method further comprises administering a second agent. The present invention provides, for example, the following items. (Item 1) A non-naturally occurring polynucleotide comprising a guide RNA for targeting a mutated KRAS, the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence substantially complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. (Item 2) 2. The non-naturally occurring polynucleotide of item 1, wherein the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). (Item 3) 3. The non-naturally occurring polynucleotide of item 1 or 2, wherein the nucleotide sequence substantially complementary to the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19 to 21, 23, 29, 31, 33, and 34. (Item 4) 4. The non-naturally occurring polynucleotide of item 3, wherein the nucleotide sequence is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 3, 19, and 34. (Item 5) 5. The non-naturally occurring polynucleotide of any one of items 1 to 4, which has been chemically modified. (Item 6) 6. The non-naturally occurring polynucleotide of any one of items 1 to 5, wherein the guide RNA has a length of about 200 nucleotides or less. (Item 7) 7. A genome editing complex comprising: a) a first cell-penetrating peptide; and b) a guide RNA that targets a mutated KRAS, wherein the guide RNA comprises the polynucleotide described in any one of items 1 to 6. (Item 8) The genome editing complex described in Item 7, further comprising a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. (Item 9) 9. The genome editing complex of Item 8, wherein the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof. (Item 10) The genome editing complex of item 9, wherein the DNA nuclease comprises a Cas polypeptide. (Item 11) The genome editing complex of item 9 or item 10, wherein the Cas polypeptide is Cas9. (Item 12) 12. The genome editing complex of any one of items 7 to 11, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. (Item 13) 13. The genome editing complex of any one of items 7 to 12, wherein the first cell-penetrating peptide further comprises one or more moieties covalently linked to the N-terminus of the first cell-penetrating 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, linker moieties, and targeting moieties. (Item 14) Item 14. The genome editing complex of Item 13, wherein the first cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. (Item 15) 15. The genome editing complex of claim 13 or 14, wherein the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. (Item 16) Item 16. The genome editing complex of item 15, wherein the targeting peptide is selected from the group consisting of SEQ ID NOs: 196 to 205 and 235 to 240. (Item 17) 17. The genome editing complex of any one of items 7 to 16, wherein the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. (Item 18) 18. The genome editing complex of any one of items 13 to 17, wherein the first cell-penetrating peptide comprises an acetyl group, a targeting moiety, and a linker moiety covalently linked from the N-terminus to the N-terminus of the first cell-penetrating peptide. (Item 19) 19. The genome editing complex of any one of items 7 to 18, wherein the first cell-penetrating peptide further comprises a carbohydrate moiety. (Item 20) 20. The genome editing complex of item 19, wherein the carbohydrate moiety is GalNAc. (Item 21) 21. A genome editing complex described in any one of items 7 to 20, wherein the first cell-penetrating peptide is a retro-inverso peptide. (Item 22) 22. The genome editing complex of any one of items 7 to 21, wherein the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89 to 107, 111 to 117, and 153 to 175. (Item 23) 23. The genome editing complex of any one of items 1 to 22, wherein the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1. (Item 24) 24. The genome editing complex of any one of items 8 to 23, wherein the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between about 1:1 and about 80:1. (Item 25) 25. The genome editing complex of any one of items 7 to 24, further comprising one or more additional guide RNAs comprising different guide sequences. (Item 26) 26. The genome editing complex of item 25, wherein at least two of the two or more guide RNAs target a single KRAS mutation. (Item 27) 27. The genome editing complex of claim 26, wherein at least two of the two or more guide RNAs target two or more different KRAS mutations. (Item 28) 28. The genome editing complex of item 26 or 27, wherein at least two of the two or more guide RNAs target G12D, G12V, and / or G12C. (Item 29) 29. The genome editing complex of any one of items 1 to 28, wherein the average diameter of the genome editing complex is between about 10 nm and about 300 nm. (Item 30) 30. A nanoparticle comprising a core containing the genome editing complex described in any one of items 1 to 29. (Item 31) A pharmaceutical composition comprising a guide RNA described in any one of items 1 to 6, a genome editing complex described in any one of items 7 to 29, or a nanoparticle described in item 30, and a pharmaceutically acceptable carrier. (Item 32) 32. The pharmaceutical composition of item 31, comprising two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations. (Item 33) 30. A method for preparing a genome editing complex described in any one of items 7 to 29, comprising a step of combining the first cell-penetrating peptide with the guide RNA, thereby forming the genome editing complex. (Item 34) A method for modifying mutated KRAS in a cell, comprising contacting the cell with a guide RNA described in any one of items 1 to 6, a genome editing complex described in any one of items 7 to 29, or a nanoparticle described in item 30. (Item 35) 33. A method for treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of item 31 or 32. (Item 36) 36. The method of claim 35, further comprising administering a second agent. [Brief explanation of the drawings]

[0034] [Figure 1] Figures 1A-1B show the evaluation of gRNAs targeting the KRAS 35G>T mutant G12V KRAS in SW403, SW480, and HT-29 cells. Cancer cells were treated with different ADGN / Cas9 mRNA / gRNA (0.15 μg / 0.2 μg) complexes. Figure 1A shows the indel frequency in the endogenous target sequence in different cell lines, assessed 72 hours after transfection by the T7E1 method. Figure 1B shows cell proliferation analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax system.

[0035] [Figure 2]Figures 2A-2B show the evaluation of gRNAs targeting the KRAS 35G>A mutant G12D KRAS in Panc1, LS513, PK-45H, PK-1, HS-68, and HT-29 cells. Cancer cells were treated with different ADGN / Cas9 mRNA / gRNA (0.15 μg / 0.2 μg) complexes. Figure 2A shows the indel frequency in the endogenous target sequence in different cell lines, assessed 72 hours after transfection by the T7E1 method. Figure 2B shows cell proliferation analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax system.

[0036] [Figure 3] Figures 3A-3B show the evaluation of gRNAs targeting the KRAS 35G>A mutant G12D KRAS in Mia-PACA, H-23, H-358, PANC1, and HT-29 cells. Cancer cells were treated with different ADGN / Cas9 mRNA / gRNA (0.15 μg / 0.2 μg) complexes. Figure 3A shows the indel frequency in the endogenous target sequence in different cell lines, assessed 72 hours after transfection by the T7E1 method. Figure 3B shows cell proliferation analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax system.

[0037] [Figure 4] Figures 4A-4B show the evaluation of gRNAs targeting KRAS 35G>A, KRAS 35G>T, and KRAS 34G>T mutants in different cell lines. SW403, SW480, PANC1, PK-45H, PK-1, MIA-PACA, NIH-H23, H358, HT-29, PC-9, HS-68, and LS513 cells were treated with ADGN / Cas9 mRNA / gRNA (0.4 μg). Figure 4A shows the indel frequency in the endogenous target sequence in the different cell lines, assessed 72 hours after transfection by the T7E1 method. Figure 4B shows cell proliferation analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax system.

[0038] [Figure 5] Figures 5A-5B show the effects of lead gRNAs targeting the G12D and G12V mutants on the KRAS signaling pathway. Figure 5A shows the evaluation of gRNAs targeting the KRAS 35G>A mutant G12D KRAS in PANC-1 cells. Figure 5B shows the evaluation of gRNAs targeting the KRAS 35G>T mutant G12V KRAS in SW403 cells. Western blot analysis (top) and quantification (bottom).

[0039] [Figure 6] Figure 6 shows quantification of Cas9 protein expression by ELISA in different tissues and tumors after in vivo intravenous administration of Cas9 mRNA / gRNA associated with different ADGN-peptides.

[0040] [Figure 7] Figures 7A-7B show quantification of Cas9 protein expression by ELISA in different tissues and tumors after in vivo IV administration of Cas9 mRNA / gRNA associated with ADGN-100 Hy3 (Figure 7A) and ADGN-100-Hy7 (Figure 7B) peptides.

[0041] [Figure 8] Figures 8A-8B show plasma concentrations of Cas9-mRNA in mice treated intravenously with ADGN-Hy-3 / mRNA Cas9 / gRNA and ADGN-Hy7 / mRNA Cas9:gRNA complexes. Mice bearing Panc1 tumors were treated with a single injection of 0.2 mg / kg, 0.5 mg / kg, and 1.0 mg / kg of ADGN / mRNA Cas9 / gRNA nanoparticles. Cas9 mRNA levels in plasma were analyzed by the Quantigen bDNA method. Graphs show mean values ​​(n=4). Calculated amounts in picograms were normalized to the amount of plasma in the lysate and the amount of lysate applied to the plate.

[0042] [Figure 9]Figures 9A-9B show the in vivo efficacy of ADGN / Cas9 / gRNA35A5 in a pancreatic tumor model. A three-week period was allowed to elapse before treatment began to allow PANC1 tumors to develop. Four groups of mice were identified: untreated control mice (G1), ADGN-100Hy3 / mRNA / gRNA35A5 0.5 mg / kg (G3), ADGN-100Hy3 / mRNA / gRNA35A5 1.0 mg / kg (G4), and ADGN-100Hy3 / mRNA / gRNA34T6 1.0 mg / kg (G5). Animals (six animals per group) were injected intravenously (tail vein) on days 0 and 7. Tumor size was assessed weekly by bioluminescence imaging. 9A and 9B show bioluminescence imaging (FIG. 9B) and quantification of total luminescence (FIG. 9A) for the different groups on days 0, 15, and 30.

[0043] [Figure 10] Figure 10 shows the in vivo efficacy of ADGN / Cas9 / gRNA35T3 in a colorectal tumor model. A 10-day period was allowed to elapse before treatment began to allow SW403 tumors to develop. Four groups of mice were identified: untreated control mice (G1), ADGN-100Hy3 / mRNA / gRNA35T3 0.5 mg / kg (G5), ADGN-100Hy3 / mRNA / gRNA35T3 1.0 mg / kg (G7), and ADGN-100Hy3 / mRNA / gRNA34T6 1.0 mg / kg (G6). Animals (6 animals per group) were injected intravenously (tail vein) on days 0 and 7. Tumor size was assessed weekly using calipers.

[0044] [Figure 11]Figures 11A-11B show the in vivo efficacy of co-treatment with ADGN / Cas9 / gRNA35A5 and Abraxane in a pancreatic tumor mouse model. A 3-week period was allowed to elapse before treatment began to allow PANC1 tumors to develop. Seven groups of mice were identified: untreated control mice (G1), ADGN / mRNA Cas9 / control gRNA (G2), ADGN-100Hy3 / mRNA / gRNA35A5 0.5 mg / kg (G3), ADGN-100Hy3 / mRNA / gRNA35A5 1.0 mg / kg (G4), Abraxane (50 μg) and ADGN-100Hy3 / mRNA / gRNA35A5 0.5 mg / kg (G5), Abraxane (50 μg) and ADGN-100Hy3 / mRNA / gRNA35A5 1.0 mg / kg (G6), and Abraxane (50 μg) alone (G7). For G2–G6, animals (six animals per group) were intravenously (tail vein) injected with ADGN / Cas9:sgRNA on days 0 and 7. For G5-G7, animals were injected intravenously (tail vein) with Abraxane (50 μg) once a week. Tumor size was assessed weekly by bioluminescence imaging. Figures 11A and 11B show bioluminescence imaging and quantification of total luminescence for different groups on days 0, 15, and 30.

[0045] [Figure 12]Figure 12 shows the in vivo efficacy of co-treatment with ADGN / Cas9 / gRNA35T3 and capecitabine in a mouse colorectal tumor model. Seven groups of mice were identified: untreated control mice (G1), ADGN / mRNA Cas9 / control gRNA (G2), ADGN-100Hy3 / mRNA / gRNA35T3 0.5 mg / kg (G3), ADGN-100Hy3 / mRNA / gRNA35T3 1.0 mg / kg (G4), capecitabine (200 μg) (G5), ADGN-100Hy3 / mRNA / gRNA34T3 1.0 mg / kg (G6), and capecitabine (200 μg) and ADGN-100Hy3 / mRNA / gRNA34T3 0.5 mg / kg (G7). Animals (6 animals per group) were pre-inoculated with SW403 tumors and treated by IV tail vein injection with ADGN / Cas9 / gRNA complexes (G2-G4, G6, and G7) on days 0 and 7 and capecitabine (200 μg) once a week (G5 and G7). Tumor size was assessed weekly using calipers.

[0046] [Figure 13] Figures 13A-13B show the expression levels of candidate housekeeping genes in different tissues and tumors after treatment. Animals (6 animals per group) were inoculated with PANC1 tumor cells (Figure 13A) or SW403 tumor cells (Figure 13B), and then injected intravenously (tail vein) with the ADGN / Cas9 / gRNA complex on days 0 and 7.

[0047] [Figure 14] 14A-14B show the levels of gene editing associated with gRNAs targeting KRAS 35G>A and KRAS 35G>T mutants in PANC1 and SW403 tumors. After 50 days of treatment, the indel frequencies in the endogenous target sequences in PANC1 and SW403 tumors were determined by deep sequencing and compared to untreated mice.

[0048] [Figure 15]Figures 15A-15B show the effects of in vivo treatment with ADGN / Cas9 / gRNA targeting KRAS 35G>A and KRAS 35G>T mutants on the KRAS signaling pathway in PANC1 (Figure 15A) and SW403 tumors (Figure 15B).

[0049] [Figure 16] Figures 16A-16B show the effect of in vivo treatment with ADGN / Cas9 / gRNA targeting KRAS 35G>A and KRAS 35G>T mutants on animal body weight in animals inoculated with PANC1 tumor cells (Figure 16A) or SW403 tumor cells (Figure 16B).

[0050] [Figure 17] Figures 17A-17D show the effects of in vivo treatment with ADGN / Cas9 / gRNA targeting the KRAS 35G>A mutant on liver and kidney markers in animals inoculated with PANC1 tumor cells. Four groups of mice were identified: untreated control mice (G1), ADGN-100Hy3 / mRNA / gRNA35A5 0.5 mg / kg (G2), ADGN-100Hy3 / mRNA / gRNA35A5 1.0 mg / kg (G3), and ADGN-100Hy3 / mRNA / gRNA34T6 1.0 mg / kg (G4). Animals (six animals per group) were intravenously (tail vein) injected with the ADGN / Cas9 / gRNA complex on days 0 and 7. Blood samples were collected in heparinized tubes on D7, D15, and D30 and analyzed for plasma concentrations of blood urea nitrogen (BUN) (Figure 17D), creatinine (Figure 17C), aspartate aminotransferase (AST) (Figure 17A), and alanine aminotransferase (ALT) (Figure 17B).

[0051] [Figure 18] Figure 18 shows various target sequences for designing sgRNAs targeting KRAS wild-type or KRAS with G12D, G12V, or G12C mutations.

[0052] [Figure 19] Figures 19A-19B show particle size and aggregation levels of ADGN / mRNA / gRNA complexes measured with a DLS NanoZS (Malvern Ltd). ADGN-Hy3 / mRNA / gRNA particles were prepared in either 5% glucose or DMEM at a molar ratio of 20 / 1 / 1. Data are reported as size distribution by intensity (Figure 19A) and size distribution by volume (Figure 19B).

[0053] [Figure 20] Figure 20 shows the evaluation of ADGN-121 gRNA targeting the KRAS 35G>T mutant in different cell lines. SW403, SW480, PANC1, LS-513, HT-29, H-441, and H-2444 cells were treated with free mRNACas9-gRNA or ADGN-121 (ADGN / mRNACas9-gRNA) complexes (0.1 nM to 10 μM) on day 1. Cell proliferation was analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax (Promega) system.

[0054] [Figure 21] Figure 21 shows the evaluation of ADGN-123 gRNA targeting the KRAS 35G>A mutant in different cell lines. PANC1, PK-45H, PK-1, ASPC-1, MIA-PACA, LS-513, H358, HT-29, and H-441 cells were treated with free mRNACas9-gRNA or ADGN-123 (ADGN / mRNACas9-gRNA) complexes (0.1 nM to 10 μM) on day 1. Cell proliferation was analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax (Promega) system.

[0055] [Figure 22]Figure 22 shows the evaluation of ADGN-122 gRNA targeting the KRAS 34A>T mutant in different cell lines. SW403, PANC1, H358, CALU-1, H-2122, H-441, MIA-PACA, and H-1299 cells were treated with free mRNACas9-gRNA or ADGN-122 (ADGN / mRNACas9-gRNA) complexes (0.1 nM to 10 μM) on day 1. Cell proliferation was analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax (Promega) system.

[0056] [Figure 23] Figure 23 shows the evaluation of AMG-510 and ADGN-122 targeting the KRAS 34A>T mutant in different cell lines. H358, CALU-1, MIA-PACA, and H-2122 cells were treated with ADGN-122 (ADGN / mRNACas9-gRNA) complex and AMG-510 (0.1 nM to 1000 nM) on day 1. Cell proliferation was analyzed over a 5-day period using the CellTiter Glow kit on a GlowMax (Promega).

[0057] [Figure 24] FIG. 24 shows a table listing cancer cell lines that have mutant KRAS genes and / or mutant p53 genes.

[0058] [Figure 25] Figure 25 shows the IC50 and CC50 parameters of ADGN-121 in cancer cell lines.

[0059] [Figure 26] Figure 26 shows the IC50 and CC50 parameters of ADGN-123 in cancer cell lines.

[0060] [Figure 27] Figure 27 shows the IC50 and CC50 parameters of ADGN-122 in cancer cell lines.

[0061] [Figure 28] Figure 28 shows the IC50 parameters of ADGN-122 and AMG-510 in cancer cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0062] Detailed description of the application In one aspect, the present application provides a novel guide RNA that targets specific KRAS mutation sequences (such as KRAS with G12V, G12D, or G12C mutations).As demonstrated in Examples, exemplary guide RNAs can specifically target KRAS genes with specific mutations (for example, G12V, G12D, or G12C) without affecting the KRAS wild-type sequence.

[0063] In another aspect, the present application provides a genome editing complex, comprising a) a first cell-penetrating peptide as described herein, and b) guide RNA.As demonstrated in Examples, administration of the exemplary genome editing complex, comprising the cell-penetrating peptide and guide RNA as described herein, has been successful in treating individuals with tumors that have KRAS mutation, without inducing any serious toxicity, off-target effects, or the appearance of other KRAS mutations.In some cases, administration of the exemplary genome editing complex once or twice has resulted in complete tumor regression.

[0064] Also provided herein are nanoparticles comprising genome editing complexes, guide RNAs, methods for preparing and using the genome editing complexes or nanoparticles, as well as kits and articles of manufacture useful for the methods. I. Definition

[0065] The term "guide RNA" refers to a polynucleotide that cuts, inserts, or joins target DNA in a cell by RNA editing. The guide RNA may be a single-stranded guide RNA (sgRNA). The guide RNA may be a CRISPR RNA (crRNA) specific for a target nucleotide sequence. The guide RNA may further comprise a trans-activating crRNA (tracrRNA) that interacts with Cas9 nuclease. The tracrRNA may comprise a polynucleotide that forms a loop structure. The guide RNA may have a length of 10 to 30 nucleotides. The guide RNA may have a length of, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0066] The guide RNA may comprise RNA, DNA, PNA, or a combination thereof. The guide RNA may be chemically modified.

[0067] The guide RNA may be a component of molecular scissors (programmable nuclease). Molecular scissors refer to all types of nucleases that can recognize and cleave specific sites on the genome. Molecular scissors may be, for example, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, RNA-guided engineered nucleases (RGENs), Cpf1, and Ago homologs (DNA-guided endonucleases). RGENs refer to nucleases that contain guide RNAs specific to target DNA and Gas proteins as components. Polynucleotides may be, for example, components of RGENs.

[0068] In the present application, 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 an approximately 20 bp sequence within the guide RNA that identifies the target site. The term "tracr mate sequence" can be used interchangeably with the term "direct repeat."

[0069] As used herein, the term "wild-type" is a term understood by those skilled in the art and means a typical, naturally occurring form of an organism, strain, gene or characteristic, as distinguished from mutant or variant forms.

[0070] As used herein, the term "variant" should be construed as indicating the quality of having a pattern that deviates from that found in nature.

[0071] The terms "non-naturally occurring," "synthetic," or "engineered" are used interchangeably and indicate the involvement of the hand of man. When referring to a nucleic acid molecule or polypeptide, this term means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is naturally associated and found in nature.

[0072] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers 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 can include modified nucleotides, such as methylated nucleotides and their analogs. The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides, in either single- or double-stranded form, including DNA and RNA. DNA can be in the form of, for example, antisense molecules, plasmid DNA, precondensed DNA, PCR products, vectors (PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can 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, and non-naturally occurring nucleic acids, and have similar binding properties 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 specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitution 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)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked through the phosphate group. "Base" includes purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that introduce new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylase, and alkyl halides. "Oligonucleotide" as used herein generally refers to a short, generally synthetic polynucleotide, generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

[0073] Generally, a "CRISPR system" collectively refers 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 "direct repeats" and partial direct repeats resulting from tracrRNA processing in the context of an endogenous CRISPR system), 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 that contains an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, CRISPR systems are characterized by a molecule (also referred to as a protospacer in the context of endogenous CRISPR systems) that promotes the formation of a CRISPR complex at the site of the target sequence. In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Perfect complementarity is not necessarily required, as long as there is sufficient complementarity to result in hybridization and promote the formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. In some embodiments, the target sequence is present in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence can 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 present application, the exogenous template polynucleotide may be referred to as an editing template.In an aspect of the present application, the recombination is homologous recombination.

[0074] Generally, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands 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 from the target sequence). A tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, about or more than about 26, about or more than about 32, about or more than about 45, about or more than about 45, about or more than about 48, about or more than about 54, about or more than about 63, about or more than about 67, about or more than about 85, or more than about 85 nucleotides of the wild-type tracr sequence) can 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 a guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex. As with the target sequence, full complementarity may not be required, as long as it is sufficient to function. In some embodiments, when optimally aligned, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence.In some embodiments, one or more molecules of the CRISPR system are introduced into host cells so that the formation of CRISPR complexes can occur at one or more target sites.For example, Cas nuclease, the guide sequence linked to the tracr mate sequence, and the tracr sequence can be introduced into host cells, respectively, to allow the formation of CRISPR complexes at the target sequence in host cells that is complementary to the guide sequence.

[0075] "Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick base pairing or other non-conventional methods. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary," as used herein, 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, or more nucleotides, or that two nucleic acids hybridize under stringent conditions.

[0076] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on several factors. Generally, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993). Laboratory Techniques In Biochemistry And Molecular Biology—Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0077] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of nucleotide residues. The hydrogen bonds can occur via Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex may contain two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a larger process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing to a given sequence is called the "complement" of the given sequence.

[0078] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (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 the encoded polypeptide may be 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.

[0079] The terms "subject," "individual," and "patient" are used interchangeably herein and 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. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0080] The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a subject. Beneficial effects include enabling a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder, or pathological condition.

[0081] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results, including but not limited to, therapeutic benefit. Therapeutic benefit means any treatment-related improvement or effect on one or more diseases, conditions, or symptoms under treatment.

[0082] The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent that is sufficient to produce beneficial or desired results.The therapeutically effective amount can vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., and can be easily determined by those skilled in the art.This term also applies to the dose that produces an image for detection by any one of the imaging methods described herein.The specific dose can vary depending on one or more of the specific agent selected, the dosing regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the physical delivery system carried.

[0083] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise specified.

[0084] Reference to "about" a value or parameter herein includes (and describes) embodiments directed to that value or parameter per se. For example, a description referring to "about X" includes a description of "X."

[0085] The compositions and methods of the present application can include, consist of, or consist essentially of the essential elements and limitations of the present application described herein, as well as any additional or optional ingredients, components, or limitations described herein or otherwise useful.

[0086] Unless otherwise noted, technical terms are used according to conventional usage.

[0087] In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the guide RNA is a single guide RNA (sgRNA).

[0088] In some embodiments, provided are polynucleotides (e.g., non-naturally occurring polynucleotides) comprising a guide RNA for targeting mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 1. In some embodiments, provided are polynucleotides (e.g., non-naturally occurring polynucleotides) comprising a guide RNA for targeting mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 3. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 6. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 8. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 15.In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 16. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 19. In some embodiments, provided are polynucleotides (e.g., non-naturally occurring polynucleotides) comprising a guide RNA for targeting mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 20. In some embodiments, provided are polynucleotides (e.g., non-naturally occurring polynucleotides) comprising a guide RNA for targeting mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 21. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO:23.In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 29. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 31. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 33. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 34.

[0089] In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 2. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 4. In some embodiments, provided are polynucleotides (e.g., non-naturally occurring polynucleotides) comprising a guide RNA for targeting mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 5. In some embodiments, provided are polynucleotides (e.g., non-naturally occurring polynucleotides) comprising a guide RNA for targeting mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 7. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO:9.In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 10. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 11. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 12. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 13. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 14.In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 17. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 18. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 22. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 24. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO:25.In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 26. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 27. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 28. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 30. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 32.In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 35. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO: 36. In some embodiments, a polynucleotide (e.g., a non-naturally occurring polynucleotide) is provided that comprises a guide RNA for targeting a mutated KRAS, comprising a specificity-determining CRISPR RNA (crRNA) that comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to the target sequence set forth in SEQ ID NO:37. Guide RNA targeting G12V

[0090] In some embodiments, a guide RNA (e.g., a single guide RNA) for targeting a mutated KRAS comprising a G12V mutation is provided, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1-14. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 6, and 8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8. In some embodiments, the target sequence is set forth in SEQ ID NO: 3.

[0091] In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, and 8. In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to a target sequence of SEQ ID NO: 3.

[0092] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12V comprises a guide sequence complementary to the target sequence adjacent to the PAM sequence AGG at positions 42 to 44. In some embodiments, the guide sequence has a length of about 20 to 24 base pairs, 20 to 22 base pairs, or 20 to 21 base pairs.

[0093] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12V comprises a guide sequence complementary to the target sequence adjacent to the PAM sequence of TAG at positions 41 to 43. In some embodiments, the guide sequence has a length of about 20-24 base pairs, 20-22 base pairs, or 20-21 base pairs.

[0094] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12V comprises a guide sequence complementary to the target sequence adjacent to a PAM sequence of TGG at positions 36 to 38. In some embodiments, the guide sequence has a length of about 20 to 24 base pairs, 20 to 22 base pairs, or 20 to 21 base pairs.

[0095] KRAS G12V mutation has been present in various diseases (for example, solid cancer or liquid cancer such as myelodysplastic syndrome).Exemplary cancers include lung cancer (for example, NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, multiple myeloma, and glioma.In some embodiments, cancer is malignant cancer or advanced cancer.The guide RNA described herein can be used to treat any of the above diseases (for example, by the methods described herein). Guide RNA targeting G12D

[0096] In some embodiments, a guide RNA (e.g., a single guide RNA) for targeting a mutated KRAS comprising a G12D mutation is provided, wherein the guide sequence comprises a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 15-28. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 19.

[0097] In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to the target sequence of SEQ ID NO: 19.

[0098] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12D comprises a guide sequence complementary to the target sequence adjacent to the PAM sequence AGG at positions 42 to 44. In some embodiments, the guide sequence has a length of about 20 to 24 base pairs, 20 to 22 base pairs, or 20 to 21 base pairs.

[0099] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12D comprises a guide sequence complementary to the target sequence adjacent to the PAM sequence of TAG at positions 41 to 43. In some embodiments, the guide sequence has a length of about 20-24 base pairs, 20-22 base pairs, or 20-21 base pairs.

[0100] KRAS G12D mutation has been present in various diseases (for example, solid cancer or liquid cancer such as myelodysplastic syndrome).Exemplary cancers include lung cancer (for example, NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, multiple myeloma, and glioma.In some embodiments, cancer is malignant cancer or advanced cancer.The guide RNA described herein can be used to treat any of the above diseases (for example, by the methods described herein). Guide RNA targeting G12C

[0101] In some embodiments, a guide RNA (e.g., a single guide RNA) for targeting a mutated KRAS comprising a G12C mutation is provided, wherein the guide sequence comprises a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 29-37. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the target sequence is set forth in SEQ ID NO: 34.

[0102] In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to the target sequence of SEQ ID NO: 34.

[0103] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12C comprises a guide sequence complementary to the target sequence adjacent to the PAM sequence AGG at positions 42 to 44. In some embodiments, the guide sequence has a length of about 20 to 24 base pairs, 20 to 22 base pairs, or 20 to 21 base pairs.

[0104] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12C comprises a guide sequence complementary to the target sequence adjacent to the PAM sequence of TAG at positions 41 to 43. In some embodiments, the guide sequence has a length of about 20-24 base pairs, 20-22 base pairs, or 20-21 base pairs.

[0105] In some embodiments, the guide RNA (e.g., single guide RNA) for targeting a mutated KRAS containing G12C comprises a guide sequence complementary to the target sequence adjacent to a PAM sequence of TGG at positions 36 to 38. In some embodiments, the guide sequence has a length of about 20 to 24 base pairs, 20 to 22 base pairs, or 20 to 21 base pairs.

[0106] KRAS G12C mutation has been present in various diseases (for example, solid cancers or liquid cancers such as myelodysplastic syndromes).Exemplary cancers include lung cancer (for example, NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.In some embodiments, the cancer is malignant cancer or advanced cancer.The guide RNA described herein can be used to treat any of the above-mentioned diseases (for example, by the methods described herein).

[0107] In some embodiments, the guide RNA is present in the form of RNA.

[0108] In other embodiments, the guide RNA is present in the form of DNA encoding the RNA (i.e., gDNA). In some embodiments, the DNA is plasmid DNA. In some embodiments, the plasmid DNA further comprises DNA encoding a DNA nuclease (e.g., Cas9).

[0109] In some embodiments, the guide RNA further comprises a DNA nuclease recruitment sequence.

[0110] In some embodiments, the guide RNA is a single guide RNA (sgRNA) that further comprises an auxiliary trans-activating crRNA (tracrRNA).

[0111] In some embodiments, the guide RNA further comprises a tracr mate sequence, a tracr sequence, and / or a tail sequence. Generally, the tracr mate sequence comprises any sequence that has sufficient complementarity to the tracr sequence to promote one or more of the following: (1) deletion of the guide sequence flanked by the tracr mate sequences in cells containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at the target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized with the tracr sequence. Generally, the degree of complementarity refers to optimal alignment of the tracr mate sequence with the tracr sequence along the length of the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm and can further account for secondary structure, such as self-complementarity, within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence along the shorter length of the two when optimally aligned is about 25% or greater than about 25%, about 30% or greater than about 30%, about 40% or greater than about 40%, about 50% or greater than about 50%, about 60% or greater than about 60%, about 70% or greater than about 70%, about 80% or greater than about 80%, about 90% or greater than about 90%, about 95% or greater than about 95%, about 97.5% or greater than about 97.5%, about 99% or greater than about 99%, or more.In some embodiments, the tracr sequence is about or greater than about 5, about or greater than about 6, about or greater than about 7, about or greater than about 8, about or greater than about 9, about or greater than about 10, about or greater than about 11, about or greater than about 12, about or greater than about 13, about or greater than about 14, about or greater than about 15, about or greater than about 16, about or greater than about 17, about or greater than about 18, about or greater than about 19, about or greater than about 20, about or greater than about 25, about or greater than about 30, about or greater than about 40, about or greater than about 50, or more nucleotides in length. In some embodiments, the guide sequence, tracr sequence, and tracr mate sequence are contained within a single RNA (referred to herein as a "single guide RNA" or "sgRNA"), such that hybridization of the tracr sequence and the tracr mate sequence results in a secondary structure, such as a hairpin. A preferred loop-forming sequence for use in a hairpin structure is four nucleotides in length and most preferably has the sequence GAAA. However, longer or shorter loop sequences can be used as alternatives. The sequence preferably includes a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In some embodiments, the sgRNA has at least two or more hairpins. In some embodiments, the sgRNA has two, three, four, or five hairpins. In some embodiments, the sgRNA has up to five hairpins. In some embodiments, the sgRNA further comprises a transcription termination sequence, which is preferably a poly-T sequence, e.g., 6 T nucleotides.

[0112] In some embodiments, the guide RNA is a prime editing guide RNA (pegRNA), which further comprises a primer binding sequence and / or a desired RNA sequence (e.g., at the 3' end of the guide RNA). The pegRNA forms a complex with a prime editor (e.g., a fusion protein comprising a modified Cas9 protein and a reverse transcriptase), thereby enabling prime editing of the target sequence. See, for example, Anzalone & Liu et al., Nature. 2019 Dec;576 (7785):149-157.

[0113] In some embodiments, the guide RNA comprises one or more modifications (e.g., chemical modifications). In some embodiments, the gRNA has one or more modified nucleotides, including nucleobase modifications and / or backbone modifications. Exemplary modifications to the guide RNA include, but are not limited to, phosphorothioate backbone modifications, 2' substitutions in ribose (e.g., 2'-O-methyl and 2'-fluoro substitutions), LNA, and L-RNA. In some embodiments, the guide RNA does not have modifications to the nucleobase or backbone.

[0114] In some embodiments, the guide RNA comprises a portion that facilitates annealing of the guide sequence. In some embodiments, the portion comprises a synthetic nucleotide sequence, the synthetic sequence being about 1 to 200 nucleotides, such as about 5 to about 100 nucleotides, for example, about 8 to about 80 nucleotides, for example, about 10 to about 50 nucleotides, for example, about 12 to about 40 nucleotides.

[0115] In some embodiments, the guide RNA (e.g., a single guide RNA) has a length of about 200 nucleotides or less, such as about 5 to about 100 nucleotides, such as about 8 to about 80 nucleotides, such as about 10 to about 50 nucleotides, such as about 12 to about 40 nucleotides.

[0116] Also provided herein are complexes, nanoparticles, and compositions comprising any of the above guide RNAs, including, but not limited to, the complexes, nanoparticles, and compositions described below.

[0117] Many delivery systems, including but not limited to viruses, liposomes, electroporation, microinjection, and conjugation, can be used to deliver any of the guide RNAs, complexes, nanoparticles, and compositions described herein to achieve introduction of gRNAs into host cells. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Using such methods, nucleic acids encoding the gRNAs of the present invention can be administered to cells in culture or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g., transcripts of the constructs described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes for delivery to the host.

[0118] Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistic bombardment, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, electroporation, nanoparticles, exosomes, microvesicles, or gene guns, naked DNA, and artificial virions.

[0119] The use of RNA or DNA virus-based systems for delivery of nucleic acids has high efficiency in targeting the virus to specific cells and transporting the viral payload to the cell's nucleus. Complex

[0120] The present application provides a genome editing complex comprising: a) a first cell-penetrating peptide; and b) a polynucleotide comprising a guide RNA targeting a mutated KRAS, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease. In some embodiments, the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof. In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.

[0121] In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide; and b) a guide RNA targeting KRAS G12V, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8. In some embodiments, the target sequence is set forth in SEQ ID NO: 3. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (e.g., GalNAc). In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide.In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0122] In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide; and b) a guide RNA targeting KRAS G12D, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 19. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (e.g., GalNAc). In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide.In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0123] In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide; and b) a guide RNA targeting KRAS G12C, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the target sequence is set forth in SEQ ID NO: 34. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (e.g., GalNAc). In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134.In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0124] In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide that is an ADGN-100 peptide; and b) a guide RNA that targets a mutated KRAS, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 153-175. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0125] In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide that is a VEPEP-6 peptide; and b) a guide RNA that targets a mutated KRAS, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, 92-103, 105-107, and 111-114. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0126] In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide that is a VEPEP-9 peptide; and b) a guide RNA targeting a mutated KRAS comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0127] In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide that is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide; b) one or more guide RNAs targeting a mutated KRAS that include a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease) or a polynucleotide encoding a DNA nuclease. In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide that is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide; b) one or more guide RNAs targeting a mutated KRAS that contain a nucleotide sequence that is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease) or a polynucleotide encoding a DNA nuclease. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (e.g., an amino acid sequence selected from the group consisting of 153-175, e.g., an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide.In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, and the fusion protein further comprises a second enzyme that enables base editing or primed editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the one or more guide RNAs comprise at least two guide RNAs that specifically target at least two different KRAS mutations selected from G12V, G12D, and G12C. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide.

[0128] In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide; and b) one or more guide RNAs targeting mutated KRAS that comprise a nucleotide sequence that is 100% complementary to the target sequence set forth in SEQ ID NO:3. In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide; b) one or more guide RNAs targeting mutated KRAS, the guide RNA comprising a nucleotide sequence 100% complementary to the target sequence set forth in SEQ ID NO: 3; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas12a) or a polynucleotide encoding a DNA nuclease. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (e.g., an amino acid sequence selected from the group consisting of 153-175, e.g., an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide.In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide.

[0129] In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide; and b) one or more guide RNAs targeting mutated KRAS that comprise a nucleotide sequence that is 100% complementary to the target sequence set forth in SEQ ID NO: 19. In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide; b) one or more guide RNAs targeting mutated KRAS, the guide RNA comprising a nucleotide sequence 100% complementary to the target sequence set forth in SEQ ID NO: 19; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas12a) or a polynucleotide encoding a DNA nuclease. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (e.g., an amino acid sequence selected from the group consisting of 153-175, e.g., an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide.In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide.

[0130] In some embodiments, a genome editing complex is provided that includes: a) a first cell-penetrating peptide selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide; and b) one or more guide RNAs targeting mutated KRAS that comprise a nucleotide sequence that is 100% complementary to the target sequence set forth in SEQ ID NO: 34. In some embodiments, a genome editing complex is provided, comprising: a) a first cell-penetrating peptide selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide; b) one or more guide RNAs targeting mutated KRAS, the guide RNA comprising a nucleotide sequence 100% complementary to the target sequence set forth in SEQ ID NO: 34; and c) a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., a Cas polypeptide, e.g., Cas9 or Cas12a) or a polynucleotide encoding a DNA nuclease. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide, a VEPEP-6 peptide, or a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175 (e.g., an amino acid sequence selected from the group consisting of 153-175, e.g., an amino acid sequence selected from the group consisting of 154, 155, 157, 158, 162, 167-170, and 172). In some embodiments, the first cell-penetrating peptide comprises a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of 81, 92-103, 105-107, and 111-114. In some embodiments, the first cell-penetrating peptide comprises a VEPEP-9 peptide.In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the molar ratio of the first cell-penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA forms a complex with the first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide. Cell-penetrating peptides

[0131] Cell-penetrating peptides (CPPs) are a promising nonviral strategy. While the definition of a CPP continues to evolve, they are generally described as short peptides of less than 30 amino acids derived from proteins or chimeric sequences. CPPs are typically amphipathic and possess a net positive charge (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206). CPPs are capable of permeabilizing biological membranes to induce the translocation of various biomolecules across the membrane into the cytoplasm and improve their intracellular routing, thereby facilitating their interaction with targets. CPPs can be subdivided into two major classes: those requiring chemical conjugation to the cargo and those involving the formation of stable noncovalent complexes. Both CPP strategies have been reported to be advantageous for the delivery of a large panel of cargoes (plasmid DNA, oligonucleotides, siRNA, PNA, proteins, peptides, liposomes, nanoparticles...) to a wide variety of cell types and in vivo models (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206; Mickan et al. (2014) Curr Pharm Biotechnol 15, 200-209; Shukla et al. (2014) Mol Pharm 11, 3395-3408).

[0132] The concept of protein transduction domains (PTDs) was first proposed based on the observation that some proteins, primarily transcription factors, can shuttle into cells and from one cell to another (for reviews, see Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton); Heitz et al. (2009) Br J Pharmacol 157, 195-206). The first observation was made by Frankel and Pabo in 1988, who showed that the HIV-1 transcriptional transactivator (Tat) protein could enter cells and translocate into the nucleus. In 1991, Prochiantz's group reached the same conclusion using the Drosophila Antennapedia homeodomain and demonstrated that this domain is internalized by neurons. These studies led to the discovery of the first protein transduction domain in 1994: a 16-mer peptide derived from the third helix of the homeodomain of Antennapedia, named penetratin. In 1997, Lebleu's group identified the minimal sequence of Tat required for cellular uptake, and Dowdy's group reported the first proof-of-concept for the application of PTDs in vivo for the delivery of small peptides and large proteins (Gump JM, and Dowdy SF (2007) Trends Mol Med 13, 443-448). Historically, the concept of cell-penetrating peptides (CPPs) was introduced by Langel's group in 1998, when the first chimeric peptide carrier, transportan, was designed, derived from the N-terminal fragment of the neuropeptide galanin linked to the hornet venom peptide mastoparan. Transportan was originally reported to improve the delivery of PNAs (peptide nucleic acids) both in cultured cells and in vivo (Langel U (2007) Handbook of Cell-Penetrating Peptides (CRC Taylor & Francis, Boca Raton)).In 1997, the Heitz and Divita groups proposed a new strategy involving CPPs to form stable but noncovalent complexes of cargoes (Morris et al. (1997) Nucleic Acids Res 25, 2730-2736). This strategy was initially based on short peptide carriers (MPGs) consisting of two domains: a hydrophilic (polar) domain and a hydrophobic (nonpolar) domain. MPGs were designed for the delivery of nucleic acids. Subsequently, the first amphipathic peptide, Pep-1, was proposed for the noncovalent delivery of proteins and peptides (Morris et al. (2001) Nat Biotechnol 19, 1173-1176). Subsequently, Wender's group and Futaki's group demonstrated that a polyarginine sequence (Arg8) is sufficient to drive small and large molecules intracellularly and in vivo (Nakase et al. (2004) Mol Ther 10, 1011-1022; Rothbard et al. (2004) J Am Chem Soc 126, 9506-9507). Since then, many CPPs derived from natural or non-natural sequences have been identified, and the list continues to grow. Peptides have been derived from the VP22 protein of herpes simplex virus, calcitonin, antibacterial or toxin peptides, proteins involved in cell cycle regulation, and polyproline-rich peptides (Heitz et al. (2009) Br J Pharmacol 157, 195-206). More recently, a new noncovalent binding strategy based on secondary amphipathic CPPs has been described. These peptides, such as the CADY and VEPEP-families, can self-assemble into a helical shape with hydrophilic and hydrophobic residues on different sides of the molecule.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, US2010 / 0099626 discloses a CADY peptide, and U.S. Patent No. 7,514,530 discloses an MPG peptide; the disclosures of which are hereby incorporated by reference in their entireties.

[0133] The cell-penetrating peptides in the genome editing complexes or nanoparticles of the present application are capable of forming 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, e.g., φC31), and nucleic acids (e.g., guide RNAs, guide DNAs, and donor nucleic acids). Any cell-penetrating peptide of any genome editing complex or nanoparticle described herein can comprise or consist of any cell-penetrating peptide sequence described in this section.

[0134] In some embodiments, the genome editing complex or nanoparticle described herein comprises a cell-penetrating 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-penetrating peptide is present in the genome editing complex. In some embodiments, the cell-penetrating peptide is present in the genome editing complex present in the core of a nanoparticle. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and associated with a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., Cas9). In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and associated with a gRNA. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and associated with a guide RNA. In some embodiments, the cell-penetrating peptide is present in the core of a nanoparticle and associated with a donor nucleic acid. In some embodiments, the cell penetrating peptide is present in the middle layer of the nanoparticle. In some embodiments, the cell penetrating peptide is present in the surface layer of the nanoparticle. In some embodiments, the cell penetrating peptide is linked to a targeting moiety. In some embodiments, the linkage is by 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, US2010 / 0099626 discloses a CADY peptide, and U.S. Patent No. 7,514,530 discloses an MPG peptide; the disclosures of which are hereby incorporated by reference in their entireties. VEPEP-3 peptide

[0135] 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: 44), wherein X1 is beta-A ("beta-alanine") or S, X2 is K, R, or L (independently of each other), X3 is F or W (independently of each other), X4 is F, W, or Y (independently of each other), 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: 45), 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 13is R or K. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1KWFERWFREWPRKRR (SEQ ID NO:46), X1KWWERWWREWPRKRR (SEQ ID NO:47), X1KWWERWWREWPRKRK (SEQ ID NO:48), X1RWWEKWWTRWPRKRK (SEQ ID NO:49), or X1RWYEKWYTEFPRRRR (SEQ ID NO:50), 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, wherein the cell-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: 51), wherein X is beta-A or S; 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 X 12is R or F. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWRLWWRSWFRLWRR (SEQ ID NO: 53), X1LWWRRWWSRWWPRWRR (SEQ ID NO: 54), X1LWWSRWWRSWFRLWFR (SEQ ID NO: 55), or X1KFWSRFWRSWFRLWRR (SEQ ID NO: 56), 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: 44 and 52-56, where the cell-penetrating peptide is modified by substitution of amino acids at positions 5 and 12 with unnatural amino acids and addition of a hydrocarbon linkage between the two unnatural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence X1RWWX 14 LWWRSWX 14 RLWRR (SEQ ID NO: 57), wherein X is beta-alanine or serine; 14is an unnatural amino acid, and there is a hydrocarbon linkage between the two unnatural amino acids. In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence beta-AKWFERWFREWPRKRR (SEQ ID NO: 58). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence beta-AKWWERWWREWPRKRR (SEQ ID NO: 59). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence ASSLNIA-Ava-KWWERWWREWPRKRR (SEQ ID NO: 60). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence LSSRLDA-Ava-KWWERWWREWPRKRR (SEQ ID NO: 61). In some embodiments, the VEPEP-3 peptide comprises the amino acid sequence Ac-SYTSSTM-ava-KWWERWWREWPRKRR (SEQ ID NO: 62). In some embodiments, the VEPEP-3 peptide is present in a genome editing complex. In some embodiments, the VEPEP-3 peptide is present in a genome editing complex within 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 the nanoparticle and is associated with a guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of the nanoparticle and is associated with a guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of the nanoparticle and is associated with a guide RNA. In some embodiments, the VEPEP-3 peptide is present in the core of the nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-3 peptide is present in the middle layer of the nanoparticle. In some embodiments, the VEPEP-3 peptide is present in the surface layer of the nanoparticle. In some embodiments, the VEPEP-3 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. VEPEP-6 peptide

[0136] In some embodiments, the genome editing complex or nanoparticle described herein comprises a VEPEP-6 cell-penetrating peptide. In some embodiments, the VEPEP-6 peptide comprises an amino acid sequence selected from the group consisting of X1LX2RALWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 63), X1LX2LARWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 64), and X1LX2ARLWX9LX3X9X4LWX9LX5X6X7X8 (SEQ ID NO: 65), 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: 66), where 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 (SEQ ID NO: 67), where 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: 68), X1LWRALWRLWRX2LWRLLWX3A (SEQ ID NO: 69), X1LWRALWRLX4RX2LWRLWRX3A (SEQ ID NO: 70), X1LWRALWRLWRX2LWRLWRX3A (SEQ ID NO: 71), X1LWRALWRLX5RALWRLLWX3A (SEQ ID NO: 72), and X1LWRALWRLX4RNLWRLLWX3A (SEQ ID NO: 73), 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: 74), Ac-X1LWRALWRLWRSLWRLLWKA-cysteamide (SEQ ID NO: 75), Ac-X1LWRALWRLLRSLWRLWRKA-cysteamide (SEQ ID NO: 76), Ac-X1LWRALWRLWRSLWRLWRKA-cysteamide (SEQ ID NO: 77), Ac-X1LWRALWRLLRALWRLLWKA-cysteamide (SEQ ID NO: 78), and Ac-X1LWRALWRLLRNLWRLLWKA-cysteamide (SEQ ID NO: 79), where 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: 63-79, 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: 80), Ac-X1LFLARWR S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 81), Ac-X1LFRALWS S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 82), Ac-X1LFLARWS S LLRS S LWRLLWK-cysteamide (SEQ ID NO: 83), Ac-X1LFRALWRLLRS SLWS S LLWK-cysteamide (SEQ ID NO: 84), Ac-X1LFLARWRLLR S SLWS S LLWK-cysteamide (SEQ ID NO: 85), Ac-X1LFRALWRLLS S SLWS S LLWK-cysteamide (SEQ ID NO: 86), Ac-X1LFLARWRLLS S SLWS S LLWK-cysteamide (SEQ ID NO: 87), and Ac-X1LFAR S LWRLLRS SLWRLLWK-cysteamide (SEQ ID NO: 88), where 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 comprises the amino acid sequence beta-ALWRALWRLWRSLWRLLWKA (SEQ ID NO: 89). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 90-117. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence beta-ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 90). In some embodiments, the VEPEP-6 peptide comprises the retro-inverso amino acid sequence AKWLLRWLSRWLRWLARWLR (SEQ ID NO: 91). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-(PEG)7-βALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 92) or Ac-(PEG)2-βALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 93). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 94-103. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence beta-A-Ac-YIGSR-Ava-ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 96). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence beta-A-Ac-YIGSR-Aun-ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 98). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-YIGSR-Ahx-ALWRALWRLWRSLWRLLWK-NH2 (SEQ ID NO: 100) or Ac-YIGSR-Ahx-ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 101). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence beta-Ac-GYVS-Ahx-ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 102) or Ac-YIGSR-βALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 103).In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence stearyl-βA-ALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 104). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 105-107. In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence ALWRA(GalNac)LWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 111). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-SYTSSTM-ava-βALWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 112). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-THRPPNWSPVWPRALWRLWRSLWRLRWKA-NH2 (SEQ ID NO: 113). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-CKTRRVPWRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 114). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-CKTRRVP-ava-WRALWRLWRSLWRLLWKA-NH2 (SEQ ID NO: 115). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-CARPAR-ava-WRALWRLWRSLWRLLWK-NH2 (SEQ ID NO: 116). In some embodiments, the VEPEP-6 peptide comprises the amino acid sequence Ac-THRPPNWSPV-ava-WRALWRLWRSLWRLRWK-NH2 (SEQ ID NO: 117). In some embodiments, the VEPEP-6 peptide is present in a genome editing complex. In some embodiments, the VEPEP-6 peptide is present in a 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 is associated with a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., Cas9). In some embodiments, the VEPEP-6 peptide is present in the core of the nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-6 peptide is present in the core of the nanoparticle and is associated with a guide RNA.In some embodiments, the VEPEP-6 peptide is present in the core of the nanoparticle and is linked to 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 linkage is by covalent bonding. VEPEP-9 peptide

[0137] 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 W or not present, X 12 is either A, R, or absent, and X 13 is W or F, and if X3 does not exist, X2, X 11 and X 12 In some embodiments, the VEPEP-9 peptide has the amino acid sequence X1X2RWWLRWAX6RWX8X9X 10 WX 12 WX 13 X is beta-A or S, X is L or absent, X is S or P, X is F or A, X is S, L or P, and X is S, L or P. 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: 120), X1LRWWLRWASRWASRWAWFR (SEQ ID NO: 121), X1RWWLRWASRWALSWRWWR (SEQ ID NO: 122), X1RWWLRWASRWFLSWRWWR (SEQ ID NO: 123), X1RWWLRWAPRWFPSWRWWR (SEQ ID NO: 124), and X1RWWLRWASRWAPSWRWWR (SEQ ID NO: 125), where 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: 127), X1WWGSWATPRRRWWR (SEQ ID NO: 128), and X1WWRWWAPWARSWWR (SEQ ID NO: 129), where X1 is beta-A or S. In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence beta-ALRWWLRWASRWFSRWAWWR (SEQ ID NO: 130). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence KSYDTY-ava-ALRWLRWASRWFSRWAWR (SEQ ID NO: 131). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence ac-CKRAVRWWLRWASRWFSRWAWWR (SEQ ID NO: 132). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence beta-A-RWWLRWASRWFSRWAWR (SEQ ID NO: 133). In some embodiments, the VEPEP-9 peptide comprises the amino acid sequence KSYDTYAAETRRWASRWFSRWAWWR (SEQ ID NO: 134). 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 DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., Cas9). In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and associated with a gRNA. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and associated with a guide RNA. In some embodiments, the VEPEP-9 peptide is present in the core of a nanoparticle and associated with a donor nucleic acid. In some embodiments, the VEPEP-9 peptide is present in the middle layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is present in the surface layer of a nanoparticle. In some embodiments, the VEPEP-9 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. ADGN-100 peptide

[0138] In some embodiments, a genome editing complex or nanoparticle described herein comprises an ADGN-100 cell-penetrating peptide comprising the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 135), where X1 is any amino acid or absent, and X2-X8 are any amino acids. In some embodiments, an ADGN-100 peptide comprises the amino acid sequence X1KWRSX2X3X4RWRLWRX5X6X7X8SR (SEQ ID NO: 136), 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: 137), KWRSALYRWRLWRVRSWSR (SEQ ID NO: 138), KWRSALYRWRLWRSRSWSR (SEQ ID NO: 139), or KWRSALYRWRLWRSALYSR (SEQ ID NO: 140). In some embodiments, the ADGN-100 peptide comprises two residues separated by three or six residues linked by a hydrocarbon linkage. ... S AGWR S WRLWRVRSWSR (SEQ ID NO: 141), KWR S SAGWRWR S LWRVRSWSR (SEQ ID NO: 142), KWRSAGWR S WRLWRVR S SWSR (SEQ ID NO: 143), KWRS S ALYR S WRLWRSRSWSR (SEQ ID NO: 144), KWR S SALYRWR S LWRSRSWSR (SEQ ID NO: 145), KWRSALYR S WRLWRSR S SWSR (SEQ ID NO: 146), KWRSALYRWR S LWRSS RSWSR (SEQ ID NO: 147), KWRSALYRWRLWRS S RSWS S R (SEQ ID NO: 148), KWR S SALYRWR S LWRSALYSR (SEQ ID NO: 149), KWRS S ALYR S WRLWRSALYSR (SEQ ID NO: 150), KWRSALYRWR S LWRS S ALYSR (SEQ ID NO: 151), or KWRSALYRWRLWRS S ALYS SIn some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 153-171. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of beta-AKWRSAGWRWRLWRVRSWSR-NH2 (SEQ ID NO: 153). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of beta-AKWRSAGWRWRLWRVRSWSR (SEQ ID NO: 154) or beta-AKWRSALYRWRLWRVRSWSR (SEQ ID NO: 155). In some embodiments, the ADGN-100 peptide comprises the retro-inverso amino acid sequence of RSWSRVRWLRWRWGASRWK (SEQ ID NO: 156). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence Ac-(PEG)7-bA-KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 157) or beta-Ac-(PEG)2-βA-KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 158). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence stearyl-βA-KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 159). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 160-169. In some embodiments, the ADGN-100 peptide comprises the amino acid sequence Ac-YIGSR-Ava-KWRSALWRWRLWRVRSWSR-NH2 (Ava is 5-aminopentanoic acid) (SEQ ID NO: 162). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence Ac-YIGSR-Ahx-KWRSALWRWRLWRVRSWSR-NH2 (SEQ ID NO: 167). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence Ac-YIGSR-(PEG)n-βA-KWRSALWRWRLWRVRSWSR-NH2 (n=2, 4, or 7) (SEQ ID NO: 170). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence Ac-KWRSA(GALNAC)LWRWRLWRVRSWSR-NH2 (SEQ ID NO: 172).In some embodiments, the ADGN-100 peptide comprises the amino acid sequence Ac-CARPARWRSAGWRWRLWRVRSWSR-NH2 (SEQ ID NO: 173). In some embodiments, the ADGN-100 peptide comprises a core motif comprising the amino acid sequence of RWRLWRWSR (SEQ ID NO: 168). In some embodiments, the ADGN-100 peptide comprises the amino acid sequence TGNYKALHPDHNGWRSALRWRLWRWSR-NH2 (SEQ ID NO: 174) or Ac-TGNYKALHPDHNG-ava-WRSALRWRLWRWSR-NH2 (SEQ ID NO: 175). 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 within the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present within the core of a nanoparticle. In some embodiments, the ADGN-100 peptide is present within the core of a nanoparticle and is associated with a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., Cas9). In some embodiments, the ADGN-100 peptide is present in the core of the nanoparticle and is linked to a gRNA. In some embodiments, the ADGN-100 peptide is present in the core of the nanoparticle and is linked to a guide RNA. In some embodiments, the ADGN-100 peptide is present in the core of the nanoparticle and is linked to a donor nucleic acid. In some embodiments, the ADGN-100 peptide is present in the middle layer of the 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 by a covalent bond. VEPEP-4 peptide

[0139] In some embodiments, the genome editing complex or nanoparticle described herein comprises a VEPEP-4 cell-penetrating peptide comprising the amino acid sequence XWXRLXXXXXX (SEQ ID NO: 176), wherein X at position 1 is beta-A or S; X at positions 3, 9, and 10 are, independently of each other, W or F; X at position 6 is R if X at position 8 is S, and X at position 6 is S if X at position 8 is R; X at position 7 is L or absent; X at position 11 is R or absent, and X at position 7 is L if X at position 11 is absent. In some embodiments, the VEPEP-4 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 177-180. In some embodiments, the VEPEP-4 peptide is present in a genome editing complex. In some embodiments, the VEPEP-4 peptide is present in a genome editing complex within the core of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the core of the nanoparticle and is associated with a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., Cas9). In some embodiments, the VEPEP-4 peptide is present in the core of the nanoparticle and is associated with a gRNA. In some embodiments, the VEPEP-4 peptide is present in the core of the nanoparticle and is associated with a guide RNA. In some embodiments, the VEPEP-4 peptide is present in the core of the nanoparticle and is associated with a donor nucleic acid. In some embodiments, the VEPEP-4 peptide is present in the middle layer of the nanoparticle. In some embodiments, the VEPEP-4 peptide is present in the surface layer of the nanoparticle. In some embodiments, the VEPEP-4 peptide is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond. VEPEP-5 peptide

[0140] In some embodiments, a genome editing complex or nanoparticle described herein comprises a VEPEP-5 cell-penetrating peptide comprising the amino acid sequence RXWXRLWXRLR (SEQ ID NO: 181), wherein X at position 2 is R or S, and X at positions 4 and 8, independently of each other, are W or F. In some embodiments, the VEPEP-5 peptide comprises the amino acid sequence of any one of SEQ ID NOs: 182-187. In some embodiments, the VEPEP-5 peptide is present in a genome editing complex. In some embodiments, the VEPEP-5 peptide is present in a genome editing complex within the core of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and associated with a DNA nuclease (e.g., a CRISPR-associated endonuclease, e.g., Cas9). In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and associated with a gRNA. In some embodiments, the VEPEP-5 peptide is present in the core of a nanoparticle and associated with a guide RNA. In some embodiments, the VEPEP-5 peptide is present in the core of the nanoparticle and is associated with the donor nucleic acid. In some embodiments, the VEPEP-5 peptide is present in the middle layer of the nanoparticle. In some embodiments, the VEPEP-5 peptide is present in the surface layer of the nanoparticle. In some embodiments, the VEPEP-5 peptide is linked to a targeting moiety. In some embodiments, the linkage is by covalent bonding. Modification of cell-penetrating peptides

[0141] 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 (e.g., covalently 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, a linker moiety, and a targeting moiety. In some embodiments, the one or more moieties comprise an acetyl group covalently linked to the N-terminus of the CPP.

[0142] 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 (e.g., 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, a linker moiety, and a targeting moiety. In some embodiments, the one or more moieties comprise a cysteamide group.

[0143] In some embodiments, a CPP described herein (e.g., a PEP-1, PEP-2, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide) is stapled. "Stapling," as used herein, refers to a chemical linkage between two residues in a peptide. In some embodiments, a CPP is stapled and comprises a chemical linkage between two amino acids of the peptide. 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 joined by a chemical linkage is R and the other is S. In some embodiments, the chemical linkage is a hydrocarbon linkage.

[0144] In some embodiments, the CPP is an L-peptide comprising L-amino acids. In some embodiments, the CPP is a retro-inverso peptide (e.g., a peptide composed of a reversed sequence of D-amino acids that, when extended, assumes the same side chain topology as its parent molecule, but with the amide peptide bonds inverted). In some embodiments, the retro-inverso peptide comprises the sequence of SEQ ID NO: 91 or 156.

[0145] In some embodiments, the CPP comprises an acetyl group, a targeting moiety, and a linker moiety covalently linked from the N-terminus to the N-terminus of the cell penetrating peptide. targeting part

[0146] In some embodiments, one or more moieties comprise a targeting moiety. In some embodiments, the targeting moiety is conjugated to the N-terminus of the CPP. In some embodiments, the targeting moiety is conjugated to the C-terminus of the CPP. In some embodiments, a first targeting moiety is conjugated to the N-terminus of the CPP and a second targeting moiety is conjugated to the C-terminus of the CPP.

[0147] In some embodiments, the targeting moiety comprises a targeting peptide that targets one or more organs. In some embodiments, the one or more organs are selected from the group consisting of muscle, heart, brain, spleen, lymph nodes, liver, lung, and kidney. In some embodiments, the targeting peptide targets the brain. In some embodiments, the targeting peptide targets muscle. In some embodiments, the targeting peptide targets the heart.

[0148] In some embodiments, the targeting moiety comprises at least about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises no more than about 8, 7, 6, 5, or 4 amino acids. In some embodiments, the targeting moiety comprises no more than about 3, 4, or 5 amino acids. In some embodiments, the targeting moiety comprises a sequence selected from the group consisting of GY, YV, VS, SK, GYV, YVS, VSK, GYVS, YVSK, YI, IG, GS, SR, YIG, IGS, GSR, YIGS, and IGSR. In some embodiments, the sequence (e.g., the targeting sequence) is selected from the group consisting of GYVSK, GYVS, YIGS, and YIGSR.

[0149] In some embodiments, the targeting moiety comprises a targeting sequence selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the targeting moiety comprises the targeting sequence SYTSSTM (SEQ ID NO: 196). In some embodiments, the targeting moiety comprises the targeting sequence CKTRRVP (SEQ ID NO: 197). In some embodiments, the targeting moiety comprises the targeting sequence THRPPNWSPV (SEQ ID NO: 198). In some embodiments, the targeting moiety comprises the targeting sequence TGNYKALHPDHNG (SEQ ID NO: 199). In some embodiments, the targeting moiety comprises the targeting sequence CARPAR (SEQ ID NO: 200). In some embodiments, the targeting moiety comprises the targeting sequence ASSLNIA (SEQ ID NO: 203). In some embodiments, the targeting moiety comprises the targeting sequence LSSRLDA (SEQ ID NO: 204). In some embodiments, the targeting moiety comprises the targeting sequence KSYDTY (SEQ ID NO: 205).

[0150] In some embodiments, the targeting moiety is conjugated to the CPP via a linker moiety, such as any one of the linker moieties described herein. Linker part

[0151] In some embodiments, the one or more moieties comprises a linker moiety.

[0152] In some embodiments, the linker moiety comprises a polyglycine linker. In some embodiments, the linker comprises β-alanine. In some embodiments, the linker comprises at least about 2, 3, or 4 glycines, optionally consecutive glycines. In some embodiments, the linker further comprises serine. In some embodiments, the linker comprises a GGGGS or SGGGG sequence. In some embodiments, the linker comprises a glycine-β-alanine motif.

[0153] In some embodiments, one or more moieties comprise a polymer (e.g., PEG, polylysine, PET). In some embodiments, the polymer is conjugated to the N-terminus of the CPP. In some embodiments, the polymer is conjugated to the C-terminus of the CPP. In some embodiments, a first polymer is conjugated to the N-terminus of the CPP and a second polymer is conjugated to the C-terminus of the CPP. In some embodiments, the polymer is PEG. In some embodiments, the PEG is linear PEG. In some embodiments, the PEG is branched PEG. In some embodiments, the molecular weight of the PEG is about 5 kDa or less, 10 kDa or less, 15 kDa or less, 20 kDa or less, 30 kDa or less, or 40 kDa or less. In some embodiments, the molecular weight of the PEG is at least about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa, or 40 kDa. In some embodiments, the molecular weight of PEG is about 5 kDa to about 10 kDa, about 10 kDa to about 15 kDa, about 15 kDa to about 20 kDa, about 20 kDa to about 30 kDa, or about 30 kDa to about 40 kDa. In some embodiments, the molecular weight of PEG is about 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of PEG is selected from the group consisting of 5 kDa, 10 kDa, 20 kDa, or 40 kDa. In some embodiments, the molecular weight of PEG is about 5 kDa. In some embodiments, the molecular weight of PEG is about 10 kDa. In some embodiments, the PEG comprises at least about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG consists of about 10 or less, 9 or less, 8 or less, or 7 or less ethylene glycol units. In some embodiments, the PEG consists of about 1, 2, or 3 ethylene glycol units. In some embodiments, the PEG moiety consists of about 1-8, or about 2-7 ethylene glycol units.

[0154] In some embodiments, the linker moiety is selected from the group consisting of beta-alanine, cysteine, cysteamide bridge, polyglycine (e.g., G2 or G4), Aun (11-amino-undecanoic acid), Ava (5-aminopentanoic acid), and Ahx (aminocaproic acid). In some embodiments, the linker moiety comprises Aun (11-amino-undecanoic acid). In some embodiments, the linker moiety comprises Ava (5-aminopentanoic acid). In some embodiments, the linker moiety comprises Ahx (aminocaproic acid). Carbohydrate portion

[0155] In some embodiments, the cell-penetrating peptide further comprises a carbohydrate moiety. In some embodiments, the carbohydrate moiety is GalNAc. In some embodiments, the cell-penetrating peptide is an ADGN-106 peptide. In some embodiments, the cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, an alanine in the cell-penetrating peptide is modified with a carbohydrate moiety. In some embodiments, the cell-penetrating peptide is set forth in SEQ ID NO: 111 or 172. Cell-penetrating peptide mixture

[0156] In some embodiments, the cell-penetrating peptide in the genome editing complex is a mixture of a) a first peptide comprising a first cell-penetrating peptide (e.g., any of the cell-penetrating peptides described herein) and b) a second peptide comprising a second cell-penetrating peptide (e.g., any of the cell-penetrating peptides described herein), wherein the second peptide comprises a polyethylene glycol (PEG) moiety covalently linked to the second cell-penetrating peptide, and the first peptide does not have a PEG moiety. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is a PTD-based peptide, an amphipathic peptide, a polyarginine-based peptide, an MPG peptide, a CADY peptide, a PEP-1 peptide, a PEP-2 peptide, or a PEP-3 peptide. In some embodiments, the first cell-penetrating peptide and the second cell-penetrating peptide are selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-4 peptide, VEPEP-5 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the molar ratio of cell-penetrating peptide to cargo (e.g., optionally a DNA nuclease or a guide RNA containing nucleotides encoding a DNA nuclease) is between about 1:1 and about 100:1 (e.g., approximately between about 1:1 and about 50:1, or between about 2:1 and about 50:1). In some embodiments, the average diameter of the genome editing complex is between about 20 nm and about 1000 nm (e.g., about 20 to about 500 nm, about 50 to about 400 nm, about 60 to about 300 nm, about 80 to about 200 nm, or about 100 to about 160 nm). In some embodiments, the PEG moiety consists of about 1 to 10 (e.g., about 1 to 8, 2 to 7, 1 to 5, or 6 to 10) ethylene glycol units. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 50 kDa. In some embodiments, the molecular weight of the PEG moiety is about 0.05 kDa to about 0.5 kDa (e.g., about 0.05 to 0.1 kDa, 0.05 to 0.4 kDa, 0.1 to 0.3 kDa, 0.05 to 0.25 kDa, 0.25 to 0.5 kDa).In some embodiments, the PEG moiety is conjugated to the N-terminus or C-terminus of the second cell-penetrating peptide, hi some embodiments, the PEG moiety is conjugated to a site within the second cell-penetrating peptide.

[0157] In some embodiments, the ratio of the first cell-penetrating peptide to the second cell-penetrating peptide is about 20:1 to about 1:1 (eg, about 15:1 to about 2:1, about 10:1 to about 4:1).

[0158] In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is selected from a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, and an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide and / or the second cell-penetrating peptide is selected from a VEPEP-6 peptide and an ADGN-100 peptide.

[0159] In some embodiments, the PEG moiety is a linear PEG. In some embodiments, the PEG moiety is a branched PEG. cargo molecule

[0160] In some embodiments, the cell-penetrating peptides described herein are complexed with one or more cargo molecules. In some embodiments, the cell-penetrating peptide is non-covalently complexed with at least one of the one or more cargo molecules. In some embodiments, the cell-penetrating peptide is non-covalently complexed with each of the one or more cargo molecules. In some embodiments, the cell-penetrating peptide is covalently complexed with at least one of the one or more cargo molecules. In some embodiments, the cell-penetrating peptide is covalently complexed with each of the one or more cargo molecules.

[0161] As described above, the genome editing complex or nanoparticle described herein comprises the above-mentioned guide RNA. In some embodiments, the genome editing complex or nanoparticle comprises one or more genome editing molecules (e.g., DNA nuclease or a polynucleotide encoding a DNA nuclease). guide RNA

[0162] The genome editing complex or nanoparticle described herein comprises a guide RNA that targets a mutated KRAS, such as any of the guide RNAs described in the "Synthetic Guide RNA" section. In some embodiments, the mutated KRAS comprises one or more mutations selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the mutated KRAS comprises one or more mutations selected from the group consisting of G12D, G12C, G12V, G12A, G12S, G12R, G13D and G13C. DNA nuclease

[0163] In some embodiments, the genome editing complex or nanoparticle described herein further comprises a DNA nuclease or a nucleotide encoding the DNA nuclease. In some embodiments, the DNA nuclease is selected from the group consisting of a CRISPR-associated protein (Cas) polypeptide, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a variant thereof, a fragment thereof, and a combination thereof.

[0164] In some embodiments, the genome editing 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 RGEN (e.g., Cas9). In some embodiments, the protein or polypeptide is between about 10 kDa and about 200 kDa (e.g., approximately any of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 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., approximately any of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, and 200 kDa, including any range between these values).

[0165] In some embodiments, the genome editing complex or nanoparticle described herein further comprises a nucleic acid encoding a DNA nuclease. In some embodiments, the nucleic acid is between about 20 nt and about 20 kb (e.g., approximately 0.02 kb, 0.03 kb, 0.04 kb, 0.05 kb, 0.06 kb, 0.07 kb, 0.08 kb, 0.09 kb, 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.5 kb, 2 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 31 kb, 32 kb, 33 kb, 34 kb, 35 kb, 36 kb, 37 kb, 38 kb, 39 kb, 40 kb, 41 kb, 42 kb, 43 kb, 44 kb, 45 kb, 46 kb, 47 kb, 48 kb, 49 kb, 50 kb, 51 kb, 52 kb, 53 kb, 54 kb,

[0013] In some embodiments, the nucleic acid is DNA, such as 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., approximately any of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, and 20 kb, including any range therebetween). In some embodiments, the nucleic acid is RNA, such as mRNA, that encodes a genome editing molecule. In some embodiments, the mRNA is between about 100 nt and about 10 kb (e.g., approximately any of 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, and 10 kb, including any range therebetween).

[0166] In some embodiments, the genome editing complex or nanoparticle comprises multiple nucleic acids, such as 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 a DNA nuclease). 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).

[0167] In some embodiments, the nucleic acid is a single-stranded oligonucleotide. In some embodiments, the nucleic acid is a double-stranded oligonucleotide. The nucleic acids described herein can be any of a variety of lengths, up to, but not necessarily, 200 nucleotides in the case of antisense oligonucleotides, RNAi, siRNA, shRNA, iRNA, antagomir, or up to 1000 kilobases in the case of plasmid DNA.

[0168] In some embodiments, the nucleic acid is a plasmid DNA or DNA fragment (e.g., a DNA fragment up to about 1000 bp in length). Furthermore, the plasmid DNA or DNA fragment may be highly methylated or lowly methylated. In some embodiments, the plasmid DNA or DNA fragment may encode one or more genes and contain regulatory elements necessary for expression of said one or more genes. In some embodiments, the plasmid DNA or DNA fragment may include one or more genes encoding selectable markers, thereby allowing the plasmid DNA or DNA fragment to be maintained in a suitable host cell. CRISPR-associated nucleases

[0169] In some embodiments, DNA nuclease is CRISPR-associated nuclease.Generally, CRISPR (clustered regularly interspaced short palindromic repeats), also known as SPIDR (spacer-interspersed direct repeats), constitutes a family of DNA loci that are usually specific to certain bacterial species.CRISPR loci comprise a distinct class of interspersed short sequence repeats (SSR) that are recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433

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

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

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

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

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

[1995] ). CRISPR loci generally differ from other SSRs by the structure of their repeats, termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

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

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

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

[2000] ). CRISPR loci are found in Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Myc It has been identified in over 40 species of prokaryotes, including, but not limited to, oplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga (see, e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al.,

[2005] ).

[0170] In some embodiments, the DNA nuclease is a Cas protein. 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, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, 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, & Gersbach, CA (2015). Nature chemical biology, 11(3):198-200;Dow et al. (2015). Nature biotechnology, 33(4):390-394;Zetsche et al. (2015). Nature biotechnology, 33(2):139-142;Kleinstiver et al. (2015). Nature. 523:481-485;Bikard et al. (2013). Nucleic acids research, 41(15):7429-7437;Qi et al. (2013). Cell, 152(5):1173-1183). These enzymes are known to those skilled in the art; for example, the amino acid sequence of the S. pyogenes 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.

[0171] In some embodiments, the DNA nuclease comprises an unmodified or modified CRISPR enzyme with DNA cleavage activity, such as Cas9. 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 the cleavage of one or both strands at the location of the target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs the 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 relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of S. pyogenes Cas9 converts Cas9 from a nuclease that cleaves both strands into a nickase (cleaves a single strand). Other examples of mutations that turn Cas9 into a nickase include, but are not limited to, H840A, N854A, and N863A. In some embodiments, Cas9 nickase can be used in combination with a guide sequence, for example, two guide sequences that target the sense and antisense strands of a DNA target, respectively. This combination allows both strands to be nicked and used to induce NHEJ.

[0172] As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to create a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to create a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, less than 10%, less than 5%, less than 1%, less than 0.1%, less than 0.01%, or less than its unmutated form. Other mutations may also be useful; if Cas9 or other CRISPR enzymes are derived from species other than S. pyogenes, corresponding amino acid mutations can be made to achieve similar effects.

[0173] In some embodiments, the Cas protein (e.g., Cas9) is a split Cas protein comprising an N-terminal Cas protein fragment, Cas(N), and a C-terminal Cas protein fragment, Cas(C), wherein Cas(N) is fused to a first dimerization domain, and Cas(C) is fused to a second dimerization domain, and wherein the first dimerization domain and the second dimerization domain facilitate dimerization of Cas(N) and Cas(C) to form a complex having functional Cas nuclease activity. In some embodiments, dimerization of the first dimerization domain and the second dimerization domain is sensitive to a dimerization agent. For example, in some embodiments, the first dimerization domain and the second dimerization domain comprise FK506-binding protein 12 (FKBP) and the FKBP rapamycin-binding (FRB) domain of mammalian target of rapamycin (mTOR), and the dimerization agent is rapamycin.

[0174] In some embodiments, the complexes or nanoparticles described herein comprise a nucleotide sequence encoding a CRISPR enzyme (e.g., Cas9 endonuclease) that is codon-optimized for expression in a particular cell, e.g., a eukaryotic cell, which may be of or derived from a particular organism, such as a mammal, including, but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon (e.g., about one or more, about two or more, about three or more, about four or more, about five or more, about ten or more, about fifteen or more, about twenty or more, about twenty-five or more, about fifty or more, or more) of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell to enhance expression in the host cell of interest, while maintaining the native amino acid sequence. Different species exhibit particular biases toward certain codons for particular amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of tRNAs selected in a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, for optimal gene expression in a given organism, genes can be tailored based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database," and these tables can be adapted in several ways.See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon-optimizing a particular sequence for expression in a particular host cell are also available, for example, Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0175] In some embodiments, the CRISPR enzymes described herein include one or more nuclear localization sequences (NLSs), e.g., about or more than about 1, about or more than about 2, about or more than about 3, about or more than about 4, about or more than about 5, about or more than about 6, about or more than about 7, about or more than about 8, about or more than about 8, about or more than about 9, about or more than about 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, about or more than about 2, about or more than about 3, about or more than about 4, about or more than about 4, about or more than about 5, about or more than about 6, about or more than about 7, about or more than about 8, about or more than about 8, about or more than about 9, about or more than about 10, or more NLSs at or near the amino terminus, or about 1 or about The CRISPR enzyme may comprise more than 1, about 2 or more than 2, about 3 or more than 3, about 4 or more than 4, about 5 or more than 5, about 6 or more than 6, about 7 or more than 7, about 8 or more than 8, about 9 or more than 9, about 10 or more than 10, or more NLSs, or a combination thereof (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus). When more than one NLS is present, each can be selected independently of the other NLSs, and thus a single NLS may be present in more than one copy and / or may be present in combination with one or more other NLSs present in one or more copies. In some embodiments, the CRISPR enzyme comprises up to 6 NLSs.In some embodiments, an NLS is considered to be near the N- or C-terminus if the nearest amino acid to the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids of the N- or C-terminus along the polypeptide chain. Generally, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface, although other types of NLSs are known. Non-limiting examples of NLSs include the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 212); an NLS derived from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 213)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 214) or RQRRNELKRSP (SEQ ID NO: 215); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 216); the IBB domain from importin-alpha, sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 217); the sequences VSRKRPRP (SEQ ID NO: 218) and PPKKARED (SEQ ID NO: 219) of the fibroid T protein; the sequence PQPKKKPL (SEQ ID NO: 220) of human p53; and the mouse c-ab1 IV sequence SALIKKKKKMAP (SEQ ID NO: 221); influenza virus NS1 sequences DRLRR (SEQ ID NO: 222) and PKQKKRK (SEQ ID NO: 223); hepatitis virus delta antigen sequence RKLKKKIKKL (SEQ ID NO: 224); mouse Mx1 protein sequence REKKKFLKRR (SEQ ID NO: 225); human poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 226); and steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 227).

[0176] In some embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., the CRISPR enzyme plus about or more than about 1, about or more than about 2, about or more than about 3, about or more than about 4, about or more than about 4, about or more than about 5, about or more than about 6, about or more than about 7, about or more than about 8, about or more than about 8, about or more than about 9, about or more than about 10, or more domains). The CRISPR enzyme fusion protein can include any additional protein sequences and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, and autofluorescent proteins, including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to genetic sequences encoding proteins or fragments of proteins that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tags, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.Additional domains that can form part of fusion proteins comprising CRISPR enzymes are described in US20110059502, which is incorporated herein by reference. In some embodiments, tagged CRISPR enzymes are used to identify the location of the target sequence.

[0177] In some embodiments, the cargo comprises a base editor. Base editors have been developed that convert Cas endonucleases into programmable nucleotide deaminases, thereby facilitating the introduction of C-to-T mutations (by deamination of C-to-U) or A-to-G mutations (by deamination of A-to-I) without inducing double-stranded breaks. Base editors include the nickase form of SpCas9 (nSpCas9, which stimulates cellular DNA mismatch repair) fused to a nucleobase deaminase enzyme and an inhibitor of base excision repair, such as uracil glycosylase inhibitor (UGI). See Rees et al., Nature Communications Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery volume 8, Article number: 15790 (2017); Komor et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016).

[0178] In some embodiments, the cargo comprises a prime editor. Prime editing is a versatile and precise genome editing method that writes new genetic information directly into specific DNA sites. It uses a fusion protein consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase and a prime editing guide RNA (pegRNA) that can identify the target site and provides new genetic information to replace the target DNA nucleotide. It mediates targeted insertions, deletions, and base-to-base conversions without requiring double-strand breaks (DSBs) or donor DNA templates. See Anzalone et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature volume 576, pages 149-157 (2019).

[0179] In some embodiments, the cargo comprises a fusion protein comprising a non-catalytic nuclease (such as, for example, a non-catalytic Cas9 endonuclease) and a reverse transcriptase (such as, for example, a penta-mutant of M-MLV reverse transcriptase). See, e.g., Anzalone & Liu et al., Nature. 2019 Dec; 576 (7785): 149-157. In some embodiments, the cargo comprises a polynucleotide encoding the fusion protein.

[0180] In some embodiments, the cargo comprises a fusion protein comprising a catalytically inactivated nuclease (e.g., a catalytically inactivated Cas9 endonuclease) and a nucleobase deaminase enzyme. In some embodiments, the nucleobase deaminase enzyme is APOBEC1 cytidine deaminase. In some embodiments, the nucleobase deaminase enzyme is cytidine deaminase CDA1. In some embodiments, the fusion protein further comprises a DNA glycosylase inhibitor. In some embodiments, the DNA glycosylase inhibitor is uracil DNA glycosylase inhibitor (UGI). In some embodiments, the cargo comprises a polynucleotide encoding the fusion protein. ZFPs and ZFNs; TALs, TALEs, and TALENs

[0181] In some embodiments, the cargo molecule comprises a DNA-binding protein (or a nucleic acid encoding a DNA-binding protein / effector protein fusion), such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs), fused to an effector protein, such as an endonuclease. Examples include ZFNs, TALEs, and TALENs. See Lloyd et al., Frontiers in Immunology, 4(221), 1-7 (2013). In some embodiments, the guide RNA described herein can be in the form of DNA (i.e., guide DNA, gDNA) encoding an RNA that guides a ZFP or TAL to a target set. ZFPs and ZFNs

[0182] In some embodiments, the cargo molecule comprises one or more zinc finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

[0183] Among ZFPs are artificial ZFP domains generated by the assembly of individual fingers that target specific DNA sequences, generally 9–18 nucleotides in length.

[0184] ZFPs include those with two, three, four, five, or six fingers, each of which contains an alpha helix containing two invariant histidine residues that coordinate with two cysteines in a beta turn via zinc, with each finger domain being approximately 30 amino acids long. Generally, the sequence specificity of a ZFP can be altered by making amino acid substitutions at four helix positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is not naturally occurring, e.g., engineered to bind to a selected target site. For example, Beerli et al. (2002) Nature Biotechnol. 20:135-141;Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340;Isalan et al. (2001) Nature Biotechnol. 19:656-660;Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637;Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; and 2005 / 0267061, all of which are incorporated herein by reference in their entireties.

[0185] In some embodiments, the cargo molecule comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) derived from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the type IIS restriction endonuclease Fok I. Fok I catalyzes double-stranded cleavage of DNA, typically 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802, 5,436,150, and 5,487,994, as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982.

[0186] In some embodiments, ZFN targets genes present in target cells. In some aspects, ZFN efficiently generates double-strand breaks (DSBs) at predetermined sites within the coding region of genes, for example. Typical targeted regions include exons, regions encoding N-terminal regions, the first exon, the second exon, and promoter or enhancer regions. In some embodiments, transient expression of ZFN promotes highly efficient and permanent destruction of target genes in target cells. In particular, in some embodiments, delivery of ZFN leads to permanent gene destruction with an efficiency of more than 50%.

[0187] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) has collaborated with Sigma-Aldrich (St. Louis, MO, USA) to develop a platform (CompoZr) for zinc finger construction, allowing researchers to completely avoid the construction and validation of zinc fingers, and providing zinc fingers specifically targeted for thousands of proteins. Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some embodiments, commercially available zinc fingers are used or custom-made. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020). TALE and TALEN

[0188] In some embodiments, the cargo molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, e.g., as in a transcription activator-like protein effector (TALE) protein; see, e.g., U.S. Patent Publication No. 20110301073, the entire contents of which are incorporated herein by reference.

[0189] A TALE DNA-binding domain, or TALE, is a polypeptide containing one or more TALE repeat domains / units. The repeat domain is responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is generally 33-35 amino acids long and exhibits at least some sequence homology with other TALE repeat sequences in naturally occurring TALE proteins. Each TALE repeat unit generally contains one or two DNA-binding residues at positions 12 and / or 13 of the repeat, constituting a Repeat Variable Diresidue (RVD). The natural (canonical) codes for DNA recognition of these TALEs have been determined such that the HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NG binds to T; non-canonical (atypical) RVDs are also known. See, for example, US Patent Publication No. 20110301073. In some embodiments, TALEs can be targeted to any gene by designing TAL arrays with specificity for the target DNA sequence. The target sequence generally begins with a thymidine.

[0190] In some embodiments, the cargo molecule comprises a DNA-binding endonuclease, such as a TALE nuclease (TALEN). In some aspects, the TALEN is a fusion protein comprising a DNA-binding domain and a nuclease catalytic domain derived from a TALE for cleaving a nucleic acid target sequence. In some embodiments, the TALE DNA-binding domain is engineered to bind to a target sequence in a target gene.

[0191] In some embodiments, TALENs recognize and cleave target sequences within genes. In some aspects, DNA cleavage results in double-strand breaks. In some aspects, this cleavage stimulates the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an incomplete repair process that often results in changes at the cleavage site of the DNA sequence. In some aspects, the repair mechanism involves rejoining the remaining parts of the two DNA ends by direct religation (Critchlow and Jackson, Trends Biochem Sci. 1998 Oct;23(10):394-8) or so-called microhomology-mediated end joining. In some embodiments, NHEJ repair results in small insertions or deletions, which can be used to disrupt and thereby silence genes. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells that have undergone cleavage-induced mutagenesis events, i.e., mutagenesis events subsequent to NHEJ events, can be identified and / or selected by methods well known in the art.

[0192] In some embodiments, TALE repeats are assembled to specifically target genes. (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405) A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology. 31, 251-258 (2013)). Custom TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).

[0193] In some embodiments, TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vectors can contain selectable markers that allow for identification and / or selection of cells that have received the vector. Donor nucleic acid

[0194] In some embodiments, the genome editing complex or nanoparticle described herein further comprises a donor nucleic acid. In some embodiments, the donor nucleic acid is designed to serve as a template for homologous recombination within or near the target sequence that is nicked or cut by a CRISPR enzyme, for example, as part of a CRISPR complex. The donor nucleic acid can be of any suitable length, such as, for example, about 10 or more than about 10 nucleotides, about 15 or more than about 15 nucleotides, about 20 or more than about 20 nucleotides, about 25 or more than about 25 nucleotides, about 50 or more than about 50 nucleotides, about 75 or more than about 75 nucleotides, about 100 or more than about 100 nucleotides, about 150 or more than about 150 nucleotides, about 200 or more than about 200 nucleotides, about 500 or more than about 500 nucleotides, about 1000 or more than about 1000 nucleotides, or more than that. In some embodiments, the donor nucleic acid comprises a sequence complementary to a portion of a polynucleotide that comprises a target sequence. In some embodiments, when the donor nucleic acid and a polynucleotide comprising a target sequence are optimally aligned, the donor nucleic acid overlaps with one or more nucleotides of the target sequence (e.g., about or more than about 1, about or more than about 5, about or more than about 10, about or more than about 15, about or more than about 20, about or more than about 25, about or more than about 30, about or more than about 35, about or more than about 40, about or more than about 45, about or more than about 50, about or more than about 60, about or more than about 70, about or more than about 80, about or more than about 90, about or more than about 100, or more than about 100 nucleotides).In some embodiments, when the donor nucleic acid and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the donor nucleic acid within the region of complementarity is within about 1 nucleotide, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 5000 nucleotides, 10000 nucleotides, or more nucleotides of the target sequence. RNAi-targeted mutants of KRAS

[0195] In some aspects, the cargo further comprises one or more RNAi (e.g., siRNA) targeting a mutant form of KRAS. In some embodiments, the mutant form of KRAS comprises an abnormality in KRAS, and the abnormality in KRAS comprises a mutation in codon 12, 13, 17, 34, and / or 61 of KRAS. In some embodiments, the abnormality in KRAS comprises a mutation in codon 12 or 61 of KRAS. In some embodiments, the KRAS abnormality is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12S, G12V, G13C, G13S, G13R, G13A, G13D, G13V, G13P, S17G, P34S, Q61E, Q61K, Q61L, Q61R, Q61P, Q61H, K117N, A146P, A146T and A146V. In some embodiments, the KRAS abnormality is selected from the group consisting of G12C, G12S, G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P, S17G, P34S, Q61K, Q61L, Q61R, and Q61H. In some embodiments, the KRAS abnormality is selected from the group consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T, and A146V. In some embodiments, the KRAS abnormality is selected from the group consisting of KRAS G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, and Q61R. In some embodiments, the KRAS abnormality is selected from the group consisting of KRAS G12C, G12D, G12R, G12S, G12V, and G13D. In some embodiments, the KRAS abnormality is selected from G12C, G12D, and Q61K.

[0196] In some embodiments, the one or more RNAi (eg, siRNA) is selected from the group consisting of SEQ ID NOs: 228-234. Modification

[0197] 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 capable of binding to the ligand with high affinity, preferably with high specificity. The cell membrane surface receptor and / or cell surface marker is preferably specific to a particular cell, i.e., it is found more prevalently in one type of cell than in another (e.g., galactosyl residues targeting the asialoglycoprotein receptor on the surface of hepatocytes). The cell membrane surface receptor facilitates cell targeting and internalization of the ligand into the target cell (e.g., the targeting moiety) and the binding molecule (e.g., the complex or nanoparticle of the present application). Numerous ligand moieties / ligand binding partners that can be used in the context of the present application have been widely described in the literature. Such ligand moieties can confer the ability of the complex or nanoparticle of the present application to bind to a given binding partner molecule or 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, cell receptors, viral antigens, antigenic epitopes, and tumor-associated markers. Furthermore, binding partner molecules may consist of or include, for example, one or more sugars, lipids, glycolipids, antibody molecules or fragments thereof, or aptamers. According to the present application, the ligand moiety may be, for example, a lipid, glycolipid, hormone, sugar, polymer (e.g., PEG, polylysine, PET), oligonucleotide, vitamin, antigen, all or part of a lectin, all or part of a polypeptide, such as JTS1 (WO94 / 40958), an antibody or fragment thereof, or a combination thereof. In some embodiments, the ligand moiety used in the present application is a peptide or polypeptide having a minimum length of 7 amino acids. This may be either a native polypeptide or a polypeptide derived from a native polypeptide."Derived from" means containing (a) one or more modifications relative 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 that serve as ligand moieties encompass variants and chimeric polypeptides obtained by fusing sequences of various origins, such as, for example, humanized antibodies that combine the variable region of a murine antibody with the constant region of a human immunoglobulin. Furthermore, such polypeptides can have linear or cyclized structures (e.g., by flanking both ends of the polypeptide ligand with cysteine ​​residues). Furthermore, 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 application further contemplates modifications that render the ligand moiety detectable. For this purpose, modifications having a detectable moiety can be envisaged (i.e., scintigraphic labels, radioactive labels, or fluorescent or dye labels, etc.). Such detectable labels can be attached to the ligand moiety by any conventional technique and can 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, or an antigen specifically expressed on the surface of engineered target cells), and the ligand moiety is an antibody, a fragment thereof, or a minimal recognition unit (e.g., a fragment that still displays antigen specificity), such as those described in detail in immunology manuals (see, e.g., Immunology, third edition 1993, Roitt, Brostoff and Male, ed. Gambli, Mosby). The ligand moiety can also be a monoclonal antibody. Many monoclonal antibodies that bind to many of these antigens are already known, and antibodies to 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 complementary binding, are eliminated. ScFv and dAb antibody fragments can be expressed as fusions with one or more other polypeptides. The minimal recognition unit can be derived from one or more sequences of the complementarity-determining regions (CDRs) of an Fv fragment. Whole antibodies and F(ab')2 fragments are "bivalent." By "bivalent," it is meant that the antibody and F(ab')2 fragment have two antigen-binding sites. In contrast, Fab fragments, Fv fragments, ScFv fragments, dAb fragments, and minimal recognition units are monovalent, having 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 a tumor state, such as tumor-specific antigens, cellular proteins differentially or overexpressed in tumor cells, or gene products of cancer-associated viruses.Examples of tumor-specific antigens include, but are not limited to, MUC-1 for breast cancer (Hareuven et al., 1990, Eur. J. Biochem 189, 475-486), the products encoded by mutated BRCA1 and BRCA2 genes for breast and ovarian cancer (Miki et al., 1994, Science 226, 66-71; Fuireal et al., 1994, Science 226, 120-122; Wooster et al., 1995, Nature 378, 789-792), APC for colon cancer (Poiakis, 1995, Curr. Opin. Genet. Dev. 5, 66-71), and prostate-specific antigen (PSA) for prostate cancer (Stamey et al., 1987, New England J. Med. 317, 1997). 909), carcinoembryonic antigen (CEA) for colon cancer (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinase for melanoma (Vile et al., 1993, Cancer Res. 53, 3860-3864), the receptor for melanocyte-stimulating hormone (MSH), which is highly expressed in melanoma cells, ErbB-2 for breast and pancreatic cancer (Harris et al., 1994, Gene Therapy 1, 170-175), and alpha-fetoprotein for liver cancer (Kanai et al., 1997, Cancer Res. 57, 46 1-465). In some embodiments, the ligand moiety is a fragment of an antibody capable of recognizing and binding to the MUC-1 antigen and thus targeting MUC-1-positive tumor cells.In some embodiments, the ligand moiety is an scFv fragment of the SM3 monoclonal antibody, which 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). Examples of cellular proteins that are differentially or overexpressed in tumor cells include, but are not limited to, receptors for interleukin 2 (IL-2), which are overexpressed in some lymphoid tumors; GRP (gastrin-releasing peptide), which is overexpressed in lung cancer cells, pancreatic, prostate, and gastric tumors (Michael et al., 1995, Gene Therapy 2, 660-668); TNF (tumor necrosis factor) receptors; epidermal growth factor receptors; Fas receptors; CD40 receptors; CD30 receptors; CD27 receptors; OX-40; α-v integrins (Brooks et al., 994, Science 264, 569); and receptors for certain angiogenic growth factors (Hanahan, 1997, Science 277, 48). Based on these indicators, it is within the skill of those skilled in the art to define suitable ligand moieties that can recognize and bind to such proteins. For example, IL-2 is a suitable ligand moiety for binding to the IL-2 receptor. In the case of receptors specific to fibrosis and inflammation, these include TGF-beta receptors or adenosine receptors, which are identified above and are suitable targets for the compositions of the present application.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 application.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.

[0198] 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, Cho p, c-Jun, c-Myc, CREB, CREB1, CS1, CTGF, CTNNB1, CXCR4, cyclin d1-2, cyclin-dependent kinase (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, LMO1, 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-9MSFR, MSH2, MSH6, MSIN1, mTOR, MUC-1, mutant DDX3X, MYC, NCAP-D2, NCAP-D3, NCAP-G, NCAP-G2, NCAP-H, NCAP-H2, NF1, NF2, NFAT4, NF-κB, Notch1, NPC1, NR4A3, NRAS, Olig2, osteopontin, p53, PAI-1, PARP-1, patched, PAX3, PAX5, PAX7, PBX1, PDCD4, PDGF, P DGFR, PDK1, PHOX2B, PI3K, PKA, PKC, PKN3, PLK-1, PML, 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, Sh c, SLAMF7, Smad, Smad3, Smad4, Smad7, 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, TI M-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., a CRISPR / Cas9 genome editing system), wherein the RGEN-based genome editing system comprises a gRNA targeting 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. nanoparticles

[0199] In one aspect, the present application provides a nanoparticle comprising a core comprising any one or more of the above genome editing complexes.

[0200] In some embodiments, nanoparticles are provided that include a core comprising a genome editing complex described herein, wherein the cell-penetrating peptide in the genome editing delivery complex is associated with a cargo. In some embodiments, the association is not by a covalent bond. In some embodiments, the association is by a covalent bond.

[0201] In some embodiments, the nanoparticle further comprises a surface layer (e.g., shell) composed of peripheral cell-penetrating peptides (i.e., CPPs), coating 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 of the CPPs in the core. In some embodiments, 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-4, VEPEP-5, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides. In some embodiments, the peripheral CPP is VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, or ADGN-100 peptide. In some embodiments, the peripheral cell penetrating peptide is selected from the group consisting of PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, at least a portion of the peripheral cell penetrating peptide in the surface layer is linked to a targeting moiety. In some embodiments, the linkage is by covalent bonding. In some embodiments, the covalent linkage is by chemical coupling. In some embodiments, the covalent linkage is by genetic methods. 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 of the CPPs in the core. In some embodiments, intermediate CPPs include, but are not limited to, PTD-based peptides, amphipathic peptides, polyarginine-based peptides, MPG peptides, CADY peptides, VEPEP peptides (such as, for example, VEPEP-3, VEPEP-6, or VEPEP-9 peptides), ADGN-100 peptides, Pep-1 peptides, and Pep-2 peptides.In some embodiments, the intermediate CPP is a VEPEP-3 peptide, a VEPEP-6 peptide, a VEPEP-9 peptide, or an ADGN-100 peptide.

[0202] In some embodiments, the nanoparticle comprises two or more guide RNAs, such as any one of the guide RNAs described herein.In some embodiments, the two or more guide RNAs target two or more different KRAS mutations.In some embodiments, the two or more different KRAS mutations are selected from the group consisting of G12D, G12V, and G12C.In some embodiments, the two or more guide RNAs are contained in the same genome editing complex.In some embodiments, the two or more guide RNAs are contained in different genome editing complexes.

[0203] In some embodiments, the core of the nanoparticle comprises a plurality of genome editing complexes. In some embodiments, the core of the nanoparticle comprises a plurality of 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 core of the nanoparticle further comprises one or more additional guide RNAs, one or more additional cell-penetrating peptides, one or more additional genome editing nucleases, and / or one or more additional donor nucleic acids.

[0204] In some embodiments, the one or more additional genome editing complexes comprise at least one or more guide RNAs targeting different KRAS mutations. In some embodiments, the nanoparticles described herein comprise a) a first genome editing complex comprising a first guide RNA that specifically targets G12D (e.g., any one of the guide RNAs that target G12D described herein), and b) a second genome editing complex comprising a second guide RNA that specifically targets G12V (e.g., any one of the guide RNAs that target G12V described herein). In some embodiments, the nanoparticles described herein comprise a) a first genome editing complex comprising a first guide RNA that specifically targets G12D (e.g., any one of the guide RNAs that target G12D described herein), and b) a second genome editing complex comprising a second guide RNA that specifically targets G12C (e.g., any one of the guide RNAs that target G12C described herein). In some embodiments, the nanoparticles described herein comprise a) a first genome editing complex comprising a first guide RNA that specifically targets G12C (e.g., any one of the guide RNAs that target G12C described herein), and b) a second genome editing complex comprising a second guide RNA that specifically targets G12V (e.g., any one of the guide RNAs that target G12V described herein). In some embodiments, the nanoparticles described herein comprise a) a first genome editing complex comprising a first guide RNA that specifically targets G12D (e.g., any one of the guide RNAs that target G12D described herein), b) a second genome editing complex comprising a second guide RNA that specifically targets G12V (e.g., any one of the guide RNAs that target G12V described herein), and c) a second genome editing complex comprising a second guide RNA that specifically targets G12V (e.g., any one of the guide RNAs that target G12V described herein).

[0205] In some embodiments, the nanoparticle further comprises one or more additional cell-penetrating peptides. In some embodiments, the one or more additional cell-penetrating 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 peptide, VEPEP-6 peptide, or VEPEP-9 peptide), ADGN-100 peptide, Pep-1 peptide, and Pep-2 peptide. In some embodiments, at least a portion of the one or more additional cell-penetrating peptides is linked to a targeting moiety. In some embodiments, the linkage is by a covalent bond.

[0206] In some embodiments according to any of the nanoparticles described herein, the average size (diameter) of the nanoparticles is about 20 nm to about 1000 nm, including, for example, about 50 nm to about 800 nm, about 75 nm to about 600 nm, about 100 nm to about 600 nm, and about 200 nm to about 400 nm. In some embodiments, the average size (diameter) of the nanoparticles is about 1000 nanometers (nm) or less, e.g., about 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 100 nm or less. In some embodiments, the standard or average diameter of the nanoparticles is about 200 nm or less. In some embodiments, the standard or average diameter of the nanoparticles is about 150 nm or less. In some embodiments, the standard or average diameter of the nanoparticles is about 100 nm or less. In some embodiments, the nanoparticles have a standard or average diameter of about 20 nm to about 400 nm. In some embodiments, the nanoparticles have a standard or average diameter of about 30 nm to about 400 nm. In some embodiments, the nanoparticles have a standard or average diameter of about 40 nm to about 300 nm. In some embodiments, the nanoparticles have a standard or average diameter of about 50 nm to about 200 nm. In some embodiments, the nanoparticles have a standard or average diameter of about 60 nm to about 150 nm. In some embodiments, the nanoparticles have a standard or average diameter of about 70 nm to about 100 nm. In some embodiments, the nanoparticles are sterile-filterable.

[0207] In some embodiments, the zeta potential of the nanoparticles is from about -30 mV to about 60 mV (e.g., approximately any of -30 mV, -25 mV, -20 mV, -15 mV, -10 mV, -5 mV, 0 mV, 5 mV, 10 mV, 15 mV, 20 mV, 25 mV, 30 mV, 35 mV, 40 mV, 45 mV, 50 mV, 55 mV, and 60 mV, including any range therebetween). In some embodiments, the zeta potential of the nanoparticles is from about -30 mV to about 30 mV, including, for example, from about -25 mV to about 25 mV, from about -20 mV to about 20 mV, from about -15 mV to about 15 mV, from about -10 mV to about 10 mV, and from about -5 mV to about 10 mV. In some embodiments, the polydispersity index (PI) of the nanoparticles is from about 0.05 to about 0.6 (e.g., approximately 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 all ranges therebetween). In some embodiments, the nanoparticles are substantially non-toxic. composition

[0208] In some embodiments, a composition (e.g., a pharmaceutical composition) is provided comprising a genome editing complex or nanoparticle described herein. In some embodiments, the composition is a pharmaceutical composition comprising a genome editing complex or nanoparticle described herein and a pharmaceutically acceptable diluent, excipient, and / or carrier.

[0209] In some embodiments, the composition comprises a mixture of two or more nanoparticles, wherein the two or more nanoparticles comprise different guide RNAs that target different KRAS mutations.For example, in some embodiments, the composition comprises: a) the first nanoparticles described above comprising a first guide RNA that specifically targets KRAS G12D, and b) the second nanoparticles comprising a second guide RNA that specifically targets KRAS G12V.In some embodiments, the composition comprises: a) the first nanoparticles described above comprising a first guide RNA that specifically targets KRAS G12D, and b) the second nanoparticles comprising a second guide RNA that specifically targets KRAS G12C.In some embodiments, the composition comprises: a) the first nanoparticles described above comprising a first guide RNA that specifically targets KRAS G12C, and b) the second nanoparticles comprising a second guide RNA that specifically targets KRAS G12V. In some embodiments, the composition comprises: a) a first nanoparticle as described above comprising a first guide RNA that specifically targets KRAS G12D; b) a third nanoparticle comprising a second guide RNA that specifically targets KRAS G12V; and c) a second nanoparticle comprising a second guide RNA that specifically targets KRAS G12C.

[0210] In some embodiments, a composition (e.g., a pharmaceutical composition) is provided comprising a cargo delivery complex or nanoparticle described herein. In some embodiments, the composition is a pharmaceutical composition comprising a cargo delivery complex or nanoparticle described herein and a pharmaceutically acceptable diluent, excipient, and / or carrier. In some embodiments, the concentration of the complex or nanoparticle in the composition is from about 1 nM to about 100 mM, including, for example, from about 10 nM to about 50 mM, from about 25 nM to about 25 mM, from about 50 nM to about 10 mM, from about 100 nM to about 1 mM, from about 500 nM to about 750 μM, from about 750 nM to about 500 μM, from about 1 μM to about 250 μM, from about 10 μM to about 200 μM, and from about 50 μM to about 150 μM. In some embodiments, the pharmaceutical composition is lyophilized.

[0211] The term "pharmaceutically acceptable diluents, excipients, and / or carriers," as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans or other vertebrate hosts. Generally, pharmaceutically acceptable diluents, excipients, and / or carriers are those approved by federal, state, or other regulatory agencies or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, including humans and non-human mammals. The terms diluent, excipient, and / or "carrier" refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline solution, and aqueous dextrose and glycerol solutions can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like, including lyophilization aids. If desired, the compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, sustained-release formulations, and the like. Examples of suitable pharmaceutical diluents, excipients, and / or carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should be suitable for the mode of administration. Suitable diluents, excipients, and / or carriers will be apparent to those skilled in the art and will depend primarily on the route of administration.

[0212] In some embodiments, a composition comprising a genome editing complex or nanoparticle described herein further comprises a pharmaceutically acceptable diluent, excipient, and / or carrier. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier affects the level of aggregation of the genome editing complex or nanoparticle in the composition and / or the efficiency of intracellular delivery mediated by the genome editing complex or nanoparticle in the composition. In some embodiments, the extent and / or direction of the effect on aggregation and / or delivery efficiency mediated by the pharmaceutically acceptable diluent, excipient, and / or carrier depends on the relative amount of the pharmaceutically acceptable diluent, excipient, and / or carrier in the composition.

[0213] For example, in some embodiments, the presence of one or more concentrations of a pharmaceutically acceptable diluent, excipient, and / or carrier (e.g., salt, sugar, chemical buffer, buffer solution, cell culture medium, or carrier protein) in the composition does not promote and / or contribute to aggregation of the genome editing complex or nanoparticle, or promote and / or contribute to the formation of aggregates of the genome editing complex or nanoparticle that have a size that is at most about 200% larger than the size of the genome editing complex or nanoparticle (e.g., at most, approximately, any of 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including all ranges between any of these values). In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that does not promote and / or contribute to aggregation of the genome editing complex or nanoparticle, or that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle having a size that is at most about 200% (e.g., at most, approximately, any of 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including any range between any of these values) larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle having a size that is at most about 150% larger than the size of the genome editing complex or nanoparticle.In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 100% larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 50% larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 20% larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 15% larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a pharmaceutically acceptable diluent, excipient, and / or carrier at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 10% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a salt, including but not limited to NaCl. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a sugar, including but not limited to sucrose, glucose, and mannitol. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a chemical buffer, including but not limited to HEPES. In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a buffer, including but not limited to PBS.In some embodiments, the pharmaceutically acceptable diluent, excipient, and / or carrier is a cell culture medium, including but not limited to DMEM.Particle size can be determined using any means known in the art for measuring particle size, such as by dynamic light scattering (DLS).For example, in some embodiments, aggregates whose Z-average measured by DLS is 10% larger than the Z-average measured by DLS of genome editing complexes or nanoparticles are 10% larger than the genome editing complexes or nanoparticles.

[0214] In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that does not promote and / or contribute to aggregation of the genome editing complex or nanoparticle, or that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle that have a size that is at most about 100% (e.g., at most, approximately, any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including any range between any of these values) larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle that have a size that is at most about 75% larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 50% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 20% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 15% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a salt (e.g., NaCl) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 10% larger than the size of the genome editing complexes or nanoparticles.In some embodiments, the concentration of salt in the composition is about 100 mM or less (e.g., approximately any of 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, or 1 mM or less, including all ranges between any of these values). In some embodiments, the salt is NaCl.

[0215] In some embodiments, the composition contains a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that does not promote and / or contribute to aggregation of the genome editing complex or nanoparticle, or that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle having a size that is at most about 25% (e.g., at most approximately any of 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including any range between any of these values) larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 75% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 50% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 20% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 15% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a sugar (e.g., sucrose, glucose, or mannitol) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 10% larger than the size of the genome editing complexes or nanoparticles.In some embodiments, the concentration of sugar in the composition is about 20% or less (e.g., approximately any of 18% or less, 16% or less, 14% or less, 12% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, including any ranges between any of these values). In some embodiments, the sugar is sucrose. In some embodiments, the sugar is glucose. In some embodiments, the sugar is mannitol.

[0216] In some embodiments, the composition comprises a chemical buffer (e.g., HEPES or phosphate) at a concentration that does not promote and / or contribute to aggregation of the genome editing complexes or nanoparticles, or that promotes and / or contributes to the formation of aggregates of the genome editing complexes or nanoparticles that have a size that is at most about 10% (e.g., at most, approximately, any of 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including any range between any of these values) larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a chemical buffer (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of the genome editing complexes or nanoparticles that have a size that is at most about 7.5% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a chemical buffer (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of the genome editing complexes or nanoparticles that have a size that is at most about 5% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a chemical buffer (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 3% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a chemical buffer (e.g., HEPES or phosphate) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 1% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a chemical buffer (e.g., HEPES or phosphate) at a concentration that does not promote and / or contribute to the formation of aggregates of genome editing complexes or nanoparticles. In some embodiments, the chemical buffer is HEPES. In some embodiments, HEPES is added to the composition in the form of a buffer containing HEPES.In some embodiments, the pH of the solution comprising HEPES is between about 5 and about 9 (e.g., approximately any of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9, including any ranges between these values). In some embodiments, the composition comprises HEPES at a concentration of about 75 mM or less (e.g., approximately any of 70 mM or less, 65 mM or less, 60 mM or less, 55 mM or less, 50 mM or less, 45 mM or less, 40 mM or less, 35 mM or less, 30 mM or less, 25 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, or less, including any ranges between any of these values). In some embodiments, the chemical buffering agent is phosphate. In some embodiments, phosphate is added to the composition in the form of a phosphate-containing buffer solution. In some embodiments, the composition does not comprise PBS.

[0217] In some embodiments, the composition comprises cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that does not promote and / or contribute to aggregation of the genome editing complex or nanoparticle, or that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle having a size that is at most about 200% (e.g., at most, approximately, any of 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including any range between any of these values) larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 150% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 100% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 50% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 25% larger than the size of the genome editing complexes or nanoparticles.In some embodiments, the composition comprises a cell culture medium (e.g., DMEM or Opti-MEM) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles having a size that is at most about 10% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the cell culture medium is DMEM. In some embodiments, the composition comprises DMEM at a concentration of about 70% or less (e.g., approximately 65% ​​or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or less, including any range between any of these values).

[0218] In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that does not promote and / or contribute to aggregation of the genome editing complex or nanoparticle, or that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle having a size that is at most about 200% (e.g., at most approximately any of 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, including any range between any of these values) larger than the size of the genome editing complex or nanoparticle. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 150% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 100% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 50% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of genome editing complexes or nanoparticles that are at most about 25% larger than the size of the genome editing complexes or nanoparticles. In some embodiments, the composition comprises a carrier protein (e.g., albumin) at a concentration that promotes and / or contributes to the formation of aggregates of the genome editing complex or nanoparticle that have a size that is at most about 10% larger than the size of the genome editing complex or nanoparticle. In some embodiments, the carrier protein is albumin.In some embodiments, the albumin is human serum albumin.

[0219] In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous, intratumoral, intra-arterial, topical, intraocular, ophthalmic, intraportal, intracranial, intracerebral, intraventricular, intrathecal, intravesicular, intradermal, subcutaneous, intramuscular, intranasal, intratracheal, pulmonary, intracavitary, or oral administration, or for nebulization (NB) or intratracheal instillation.

[0220] Exemplary dosing frequencies include, but are not limited to, once every three days or less. Preparation method

[0221] In some embodiments, methods for preparing the genome editing complexes or nanoparticles described herein are provided.

[0222] In some embodiments, a method for preparing a genome editing complex comprising the above-described peptide and a cargo molecule (e.g., a guide RNA) is provided, the method comprising combining the peptide with the cargo molecule, thereby forming a genome editing complex.

[0223] In some embodiments, a method for preparing a genome editing complex comprising a first cell-penetrating peptide and a second cell-penetrating peptide as described above is provided, the method comprising: a) combining the first cell-penetrating peptide and the second cell-penetrating peptide, thereby forming a peptide mixture; and b) combining the peptide mixture with a cargo, thereby forming a genome editing complex.

[0224] In some embodiments, the peptide or peptide mixture and cargo molecule are combined in a molar ratio of about 1:1 to about 100:1, respectively (e.g., between about 1:1 and about 50:1, e.g., between about 2:1 and about 50:1).

[0225] In some embodiments, the method includes mixing a first solution containing a cargo molecule with a second solution containing a peptide or peptide mixture to form a third solution, where the third solution contains or is adjusted to contain: i) about 0-5% sucrose, ii) about 0-5% glucose, iii) about 0-50% DMEM, iv) about 0-80 mM NaCl, or v) about 0-20% PBS; and incubating the third solution to form genome editing complexes. In some embodiments, the first solution contains the cargo in sterile water and / or the second solution contains the peptide or peptide mixture in sterile water. In some embodiments, the third solution, after incubation to form genome editing complexes, is adjusted to contain: i) about 0-5% sucrose, ii) about 0-5% glucose, iii) about 0-50% DMEM, iv) about 0-80 mM NaCl, or v) about 0-20% PBS.

[0226] In some embodiments, the method further comprises a filtration process of filtering the genome editing complex through a membrane of a pore size. In some embodiments, the diameter of the pore is at least about 0.1 μm (e.g., at least about 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, or 1.2 μm, etc.). In some embodiments, the diameter of the pore is about 1.2 μm or less, 1.0 μm or less, 0.8 μm or less, 0.6 μm or less, 0.5 μm or less, 0.45 μm or less, 0.4 μm or less, 0.35 μm or less, 0.3 μm or less, or 0.25 μm or less. In some embodiments, the diameter of the port is about 0.1 μm to about 1.2 μm (eg, about 0.1 to about 0.8 μm, about 0.2 to about 0.5 μm, etc.).

[0227] In some embodiments, for stable compositions comprising the cargo molecule delivery complexes or nanoparticles of the present application, the average diameter of the complexes or nanoparticles does not vary by more than about 10% and the polydispersity index does not vary by more than about 10%.

[0228] Methods for preparing any of the peptides, including the cell-penetrating peptides described herein, are also provided. Method of use (e.g., treatment method)

[0229] In one aspect, the present application provides a method for treating a disease (e.g., cancer) in an individual, the method comprising administering to the individual a genome editing complex or nanoparticle comprising the guide RNA described above. In another aspect, the present application provides a method for modifying mutated KRAS in a cell, the method comprising contacting the cell with a genome editing complex or nanoparticle comprising the guide RNA described above.

[0230] In some embodiments, provided are methods of treating cancer (e.g., pancreatic, colorectal, or lung cancer) in an individual, comprising administering to the individual a genome editing complex or nanoparticle comprising a guide RNA targeting mutated KRAS, wherein the guide RNA comprises a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the guide RNA comprises a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the genome editing complex or nanoparticle is administered intravenously to the individual.

[0231] In some embodiments, a method of modifying mutated KRAS in a cell is provided, comprising contacting the cell with a genome editing complex or nanoparticle comprising a guide RNA that targets mutated KRAS, the guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the guide RNA comprises a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the guide RNA comprises a nucleotide sequence that is 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 15, 16, 19-21, 23, 29, 31, 33, and 34.

[0232] In some embodiments, the guide RNA targets KRAS G12V. In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8. In some embodiments, the target sequence is set forth in SEQ ID NO: 3. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).

[0233] In some embodiments, the guide RNA targets KRAS G12D. In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 19. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).

[0234] In some embodiments, the guide RNA targets KRAS G12C. In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) to a target sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the target sequence is set forth in SEQ ID NO: 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA).

[0235] In some embodiments, the genome editing complex further comprises a first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the first cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the first cell-penetrating peptide further comprises a carbohydrate moiety (e.g., GalNAc). In some embodiments, the first cell-penetrating peptide is an ADGN-100 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 135-175, 259-260, and 267-269. In some embodiments, the first cell-penetrating peptide is a VEPEP-3 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-62. In some embodiments, the first cell-penetrating peptide is a VEPEP-6 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-117, 261-266, and 270. In some embodiments, the first cell-penetrating peptide is a VEPEP-9 peptide. In some embodiments, the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-134. In some embodiments, the molar ratio of the first cell penetrating peptide to the guide RNA is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1). In some embodiments, the molar ratio of the first cell penetrating peptide to the nucleotide sequence encoding the Cas polypeptide is between about 1:1 and about 80:1 (e.g., between about 5:1 and about 20:1, e.g., between about 2:1 and about 50:1).In some embodiments, the molar ratio of the nucleotide sequence encoding the Cas polypeptide to the guide RNA is between about 1:10 and about 50:1 (e.g., between about 1:1 and about 10:1). In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide. In some embodiments, the guide RNA is complexed with a first cell-penetrating peptide. In some embodiments, the genome editing complex further comprises a DNA nuclease (e.g., Cas9) or a nucleotide sequence encoding a DNA nuclease.

[0236] In some embodiments of the methods described herein, the individual is a mammal. In some embodiments, the individual is a human.

[0237] In some embodiments, methods of treating cancer harboring a KRAS G12V mutation are provided, comprising administering a composition comprising a polynucleotide comprising a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8. In some embodiments, the target sequence is set forth in SEQ ID NO: 3. In some embodiments, the guide RNA further comprises a supplemental trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the composition further comprises a DNA nuclease (e.g., Cas9) or nucleotides encoding a DNA nuclease. In some embodiments, the polynucleotide is chemically modified. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0238] In some embodiments, methods of treating cancer with a KRAS G12D mutation are provided, comprising administering a composition comprising a polynucleotide comprising a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 19. In some embodiments, the guide RNA further comprises a supplemental trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the composition further comprises a DNA nuclease (e.g., Cas9) or nucleotides encoding a DNA nuclease. In some embodiments, the polynucleotide is chemically modified. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0239] In some embodiments, methods of treating cancer harboring a KRAS G12C mutation are provided, comprising administering a composition comprising a polynucleotide comprising a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the target sequence is set forth in SEQ ID NO: 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the composition further comprises a DNA nuclease (e.g., Cas9) or nucleotides encoding a DNA nuclease. In some embodiments, the polynucleotide is chemically modified. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0240] In some embodiments, methods of treating cancer with a KRAS G12V mutation are provided, comprising administering a genome editing complex comprising: a) a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14; and b) a cell-penetrating peptide. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8. In some embodiments, the target sequence is set forth in SEQ ID NO: 3. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-195. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0241] In some embodiments, methods are provided for treating cancer with a KRAS G12D mutation, comprising administering a genome editing complex comprising: a) a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28; and b) a cell-penetrating peptide. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 19. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-195. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0242] In some embodiments, a method of treating a cancer harboring a KRAS G12C mutation is provided, comprising administering a genome editing complex comprising: a) a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37; and b) a cell-penetrating peptide. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the target sequence is set forth in SEQ ID NO: 34. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-195. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.In some embodiments, methods of treating cancer harboring a KRAS G12V mutation are provided, comprising administering a genome editing complex comprising: a) a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-14; b) a cell-penetrating peptide; and c) a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, and 8. In some embodiments, the target sequence is set forth in SEQ ID NO: 3. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, and the fusion protein further comprises a second enzyme that enables base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 195. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0243] In some embodiments, methods of treating cancer harboring a KRAS G12D mutation are provided, comprising administering a) a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15-28, b) a cell-penetrating peptide, and c) a genome editing complex comprising a DNA nuclease or a polynucleotide encoding a DNA nuclease. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 16, 19-21, and 23. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO: 19. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, and the fusion protein further comprises a second enzyme that enables base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 195. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0244] In some embodiments, methods of treating cancer harboring a KRAS G12C mutation are provided, comprising administering a) a guide RNA comprising a nucleotide sequence substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29-37, b) a cell-penetrating peptide, and c) a genome editing complex comprising a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34. In some embodiments, the target sequence is set forth in SEQ ID NO: 34. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, wherein the fusion protein further comprises a second enzyme that enables base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide. In some embodiments, the cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide. In some embodiments, the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240. In some embodiments, the cell-penetrating peptide comprises a linker moiety selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-195.In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0245] In some embodiments, methods of treating cancer with a KRAS G12V mutation are provided, comprising administering: a) a guide RNA comprising a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 6-8 (e.g., SEQ ID NOs: 3, 6, and 8); b) a cell-penetrating peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 92-103, 105-107, 112-114, 137-138, 154-155, 157-158, 162, 167, 170, and 172; and c) a genome editing complex comprising a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the target sequence has the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, and the fusion protein further comprises a second enzyme that enables base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0246] In some embodiments, methods of treating cancer with a KRAS G12D mutation are provided, comprising administering: a) a guide RNA comprising a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 15, 16, 19-21, and 23; b) a cell-penetrating peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 92-103, 105-107, 112-114, 137-138, 154-155, 157-158, 162, 167, 170, and 172; and c) a genome editing complex comprising a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the target sequence has the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, and the fusion protein further comprises a second enzyme that enables base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0247] In some embodiments, methods of treating cancer with a KRAS G12C mutation are provided, comprising administering: a) a guide RNA comprising a nucleotide sequence 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, and 34; b) a cell-penetrating peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89, 92-103, 105-107, 112-114, 137-138, 154-155, 157-158, 162, 167, 170, and 172; and c) a genome editing complex comprising a DNA nuclease or a polynucleotide encoding the DNA nuclease. In some embodiments, the target sequence has the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the DNA nuclease is a Cas9 polypeptide. In some embodiments, the DNA nuclease comprises a modified Cas9 (e.g., a catalytically impaired Cas9). In some embodiments, the DNA nuclease is a fusion protein, and the fusion protein further comprises a second enzyme that enables base editing or prime editing. In some embodiments, the second enzyme comprises a reverse transcriptase or a nucleobase deaminase enzyme. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation.

[0248] In some embodiments, a method for treating a cardiac disease or condition is provided, comprising administering a composition comprising a) a cell-penetrating peptide and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 114, 153, 154, 96, 100, and 101. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0249] In some embodiments, a method of treating a disease or condition in the brain (e.g., brain tumor) is provided, comprising administering a composition comprising: a) a cell-penetrating peptide; and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 97, 112, and 113. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0250] In some embodiments, a method of treating a muscle disease or condition is provided, comprising administering a composition comprising: a) a cell-penetrating peptide; and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 95, 98, 114, 100, and 101. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0251] In some embodiments, a method for treating a lung disease or condition (e.g., lung cancer) is provided, comprising administering a composition comprising a) a cell-penetrating peptide and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 89, 90, 92-94, 96, 98, 99, 100, 101, 105-107, 113, 137, 138, 153-155, 157, 158, 162, 164, 167, and 170. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0252] In some embodiments, a method of treating a liver disease or condition (e.g., liver cancer) is provided, comprising administering a composition comprising: a) a cell-penetrating peptide; and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 89, 90, 95, 96, 98, 112, 137, 138, 153-155, 157, 158, 172, 164, 153, 162, 167, 100, and 101. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 89, 90, 95, 98, 112, 137, 138, 153-155, 157, 158, and 172. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0253] In some embodiments, a method of treating a kidney disease or condition (e.g., kidney cancer) is provided, comprising administering a composition comprising a) a cell-penetrating peptide and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 89, 90, 93, 96-98, 137, 100, 101, 138, 154, 155, and 172. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 93, 97, 98, 137, 100, 101, 138, 154, and 155. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0254] In some embodiments, a method for treating a disease or condition in the pancreas (e.g., pancreatic cancer) is provided, comprising administering a composition comprising a) a cell-penetrating peptide and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 98, 99, 137, 138, 153, 154, 155, and 162. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 98, 99, 137, 138, 153, 154, and 155. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0255] In some embodiments, a method for treating a disease or condition in the spleen is provided, comprising administering a composition comprising a) a cell-penetrating peptide and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 93, 153, 154, and 158. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 93 and 158. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises a supplemental trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide.

[0256] In some embodiments, a method of treating a disease or condition in a tumor (e.g., a solid tumor) is provided, comprising administering a composition comprising: a) a cell-penetrating peptide; and b) a genome editing complex comprising a guide RNA and / or a DNA nuclease or a nucleotide sequence encoding the DNA nuclease. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 94, 96, 98-101, 105-107, 153, 154, 162, 164, 167, and 170. In some embodiments, the cell-penetrating peptide comprises the amino acid sequence of any of SEQ ID NOs: 94, 96, 98-101, 105-107, 162, 164, 167, and 170. In some embodiments, the genome editing complex is administered intravenously, intramuscularly, subcutaneously, or via nebulization or intratracheal instillation. In some embodiments, the guide RNA targets a KRAS mutation (e.g., a G12D, G12V, or G12C mutation). In some embodiments, the guide RNA comprises a nucleotide sequence that is substantially complementary (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary) or 100% complementary to a target sequence selected from the group consisting of SEQ ID NOs: 1-37, 241-257, and 271. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, 4, 6-8, 15, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 6, 8, 16, 19-21, 23, 29, 31, 33, and 34. In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 3, 19, and 34. In some embodiments, the guide RNA further comprises an auxiliary trans-activating crRNA (tracrRNA). In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA is chemically modified. In some embodiments, the DNA nuclease is a Cas9 or Cas12a polynucleotide. KRAS abnormality

[0257] In some embodiments, the cancer tissue has KRAS abnormality. In some embodiments, the KRAS abnormality comprises a mutation at codon 12. In some embodiments, the KRAS abnormality is selected from the group consisting of G12C, G12D, and G12V. In some embodiments, the KRAS abnormality is G12C, G12D, and / or G12V.

[0258] The genetic abnormality of KRAS can be evaluated based on a sample, for example, a sample derived from an individual and / or a reference sample. In some embodiments, the sample is a tissue sample or nucleic acid extracted from a tissue sample. In some embodiments, the sample is a cell sample (e.g., a CTC sample) or nucleic acid extracted from a cell sample. In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample is a tumor sample or nucleic acid extracted from a tumor sample. In some embodiments, the sample is a biopsy sample or nucleic acid extracted from a biopsy sample. In some embodiments, the sample is a formaldehyde-fixed paraffin-embedded (FFPE) sample or nucleic acid extracted from an FFPE sample. In some embodiments, the sample is a blood sample. In some embodiments, cell-free DNA is isolated from a blood sample. In some embodiments, the biological sample is a plasma sample or nucleic acid extracted from a plasma sample.

[0259] KRAS genetic abnormality can be determined by any method known in the art.For example, see Dickson et al. Int. J. Cancer, 2013, 132(7): 1711-1717; Wagle N. Cancer Discovery, 2014, 4:546-553; and Cancer Genome Atlas Research Network. Nature 2013, 499: 43-49.Exemplary methods include but are not limited to genomic DNA sequencing, bisulfite sequencing or other DNA sequencing-based methods using Sanger sequencing or next-generation sequencing platforms; polymerase chain reaction assay; in situ hybridization assay; and DNA microarray.The epigenetic features (for example, DNA methylation, histone binding or chromatin modification) of one or more genes from a sample isolated from an individual can be compared with the epigenetic features of one or more genes from a control sample. Nucleic acid molecules extracted from the sample can be sequenced or analyzed for the presence of genetic abnormalities compared to a reference sequence, for example, the wild-type sequence of KRAS.

[0260] In some embodiments, the KRAS genetic abnormality is evaluated using cell-free DNA sequencing. In some embodiments, the KRAS genetic abnormality is evaluated using next-generation sequencing. In some embodiments, the KRAS genetic abnormality isolated from a blood sample is evaluated using next-generation sequencing. In some embodiments, the KRAS genetic abnormality is evaluated using exome sequencing. In some embodiments, the KRAS genetic abnormality is evaluated using fluorescent in-situ hybridization analysis. In some embodiments, the KRAS genetic abnormality is evaluated before the initiation of the treatment methods described herein. In some embodiments, the KRAS genetic abnormality is evaluated after the initiation of the treatment methods described herein. In some embodiments, the KRAS genetic abnormality is evaluated before and after the initiation of the treatment methods described herein. An abnormal level of KRAS can refer to an abnormal expression level or an abnormal activity level. Disease (e.g., cancer)

[0261] In some embodiments, the disease is cancer, hi some embodiments, the disease is myelodysplastic syndrome.

[0262] In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a solid tumor.

[0263] In some embodiments, solid tumors include, but are not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, Kaposi's sarcoma, soft tissue sarcoma, uterine sarcoma, synovial sarcoma, and uterine sarcoma. sacronomasynovioma), mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrocyte glioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0264] In some embodiments, the disease is selected from the group consisting of myelodysplastic syndrome, lung cancer (e.g., NSCLC, small cell lung cancer, squamous cell lung cancer), colorectal cancer, acute myeloid leukemia, pancreatic cancer, rectal cancer, esophageal squamous cell carcinoma, gastrointestinal stromal tumor, head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, multiple myeloma, and glioma.

[0265] In some embodiments, the cancer is pancreatic cancer (e.g., pancreatic ductal adenocarcinoma).

[0266] In some embodiments, the cancer is colorectal cancer.

[0267] In some embodiments, the cancer is lung cancer (e.g., NSCLC).

[0268] In some embodiments, the cancer is a malignant and / or advanced cancer. Combination therapy

[0269] Also provided herein is a combination therapy for treating a disease (e.g., cancer) discussed above in an individual, comprising: a) administering to the individual a genome editing complex or nanoparticle described herein; and b) administering to the individual a second agent or therapy. The second agent described herein can be any medication or therapy useful for treating the disease (e.g., standard treatment for the disease). In some embodiments, the second agent comprises a chemotherapeutic agent. In some embodiments, the second agent comprises a taxane. In some embodiments, the second agent comprises a cytotoxic nucleoside analog.

[0270] In some embodiments, methods of treating cancer (e.g., pancreatic cancer) in an individual are provided, comprising: a) administering to the individual a genome editing complex or nanoparticle comprising an effective amount of a guide RNA described herein; and b) administering to the individual an effective amount of a second agent selected from the group consisting of gemcitabine, 5-FU, oxaliplatin, a taxane (e.g., paclitaxel, docetaxel, albumin-bound paclitaxel (e.g., Abraxane)), capecitabine (e.g., Xeloda), cisplatin, irinotecan (e.g., camptosar), an EGFR inhibitor (e.g., erlotinib), a PARP inhibitor (e.g., olaparib), an NTRK inhibitor (e.g., larotrectinib, e.g., entrectinib), and a checkpoint inhibitor (e.g., a PD-1 inhibitor, e.g., pembrolizumab). In some embodiments, the second agent is a taxane (e.g., paclitaxel, docetaxel, albumin-bound paclitaxel (e.g., Abraxane)). In some embodiments, the second agent is Abraxane. In some embodiments, Abraxane is administered about once per week. In some embodiments, Abraxane is administered to a human at a dose of about 5 to 25 mg. In some embodiments, the second agent is capecitabine (e.g., Xeloda). In some embodiments, capecitabine is administered about once per week. In some embodiments, capecitabine is administered to a human at a dose of about 25 to 100 mg.

[0271] In some embodiments, methods of treating cancer (e.g., pancreatic cancer) in an individual are provided, comprising: a) administering to the individual a genome editing complex or nanoparticle comprising an effective amount of a guide RNA described herein; and b) administering to the individual an effective amount of a nanoparticle composition comprising a taxane (e.g., paclitaxel) or an mTOR inhibitor (e.g., rapamycin) and a carrier protein (e.g., albumin, e.g., human serum albumin). In some embodiments, the cancer tissue has a KRAS G12D mutation. In some embodiments, the taxane is paclitaxel. In some embodiments, the other agent is nab-paclitaxel. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the other agent is nab-rapamycin. In some embodiments, the method further comprises administering a chemotherapeutic agent (e.g., gemcitabine). In some embodiments, the individual is human. In some embodiments, the nanoparticles comprising a taxane (e.g., paclitaxel) or an mTOR inhibitor (e.g., rapamycin) have an average diameter of 200 nm or less. In some embodiments, the taxane (e.g., paclitaxel) or mTOR inhibitor (e.g., rapamycin) in the nanoparticles is coated with a carrier protein (e.g., albumin). In some embodiments, the weight ratio of the carrier protein (e.g., albumin) to the taxane (e.g., paclitaxel) or mTOR inhibitor (e.g., rapamycin) in the nanoparticle composition is about 9:1 or less. In some embodiments, the albumin is human albumin. In some embodiments, the dose of the taxane (e.g., paclitaxel) or mTOR inhibitor (e.g., rapamycin) in the nanoparticle composition for each administration in an individual (e.g., human) is about 1 mg / m 2 ~about 150mg / m 2 is.

[0272] In some embodiments, methods of treating cancer (e.g., colorectal cancer) in an individual are provided, comprising: a) administering to the individual a genome editing complex or nanoparticle comprising an effective amount of a guide RNA described herein; and b) administering to the individual an effective amount of a second agent selected from the group consisting of 5-FU, capecitabine, irinotecan, oxaliplatin, a combination of trifluridein and tipiracil, an angiogenesis inhibitor (e.g., a VEGF or VEGFR antagonist, e.g., bevacizumab, e.g., ramucirumab, e.g., aflibercept), and a checkpoint inhibitor (e.g., a PD-1 or CTLA-4 inhibitor, e.g., pembrolizumab, e.g., nivolumab, e.g., ipilimumab).

[0273] In some embodiments, a method of treating cancer (e.g., colorectal cancer) in an individual is provided, comprising: a) administering to the individual a genome editing complex or nanoparticle comprising an effective amount of a guide RNA described herein; and b) administering to the individual an effective amount of a cytotoxic nucleoside analog (e.g., capecitabine or an analog thereof). A series of capecitabine analogs containing "radicals readily hydrolyzable under physiological conditions" has been claimed by Fujiu et al. (U.S. Patent No. 4,966,891) and is incorporated herein by reference. The series described by Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine, implying that these compounds are activated by hydrolysis under normal physiological conditions to provide 5'-deoxy-5-fluorocytidine. In some embodiments, the cancer tissue has a KRAS G12V mutation. In some embodiments, the dose of capecitabine for each administration in an individual (e.g., a human) is about 1 mg / m 2 ~about 150mg / m 2 is.

[0274] In some embodiments, the dose of the taxane (e.g., paclitaxel) or mTOR inhibitor (e.g., rapamycin) in the nanoparticle composition for each administration in an individual (e.g., a human) is about 10 mg / m 2 ~about 50mg / m 2 In some embodiments, the dose of guide RNA for each administration in an individual (e.g., a human) is about 0.001 mg / kg to about 10 mg / kg (e.g., about 0.01 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg). In some embodiments, the dose of guide RNA for each administration in an individual (e.g., a human) is about 0.01 mg / m 2 ~about 400mg / m 2 (e.g., about 0.1 mg / m 2 ~about 100mg / m 2 , about 1mg / m 2 ~about 50mg / m 2 )

[0275] In some embodiments, the genome editing complex or nanoparticle further comprises a polynucleotide encoding a DNA nuclease (e.g., Cas9). In some embodiments, the dose of the polynucleotide encoding the DNA nuclease (e.g., Cas9) for each administration in an individual (e.g., a human) is about 0.001 mg / kg to about 10 mg / kg (e.g., about 0.01 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg). In some embodiments, the dose of the polynucleotide encoding the DNA nuclease (e.g., Cas9) for each administration in an individual (e.g., a human) is about 0.01 mg / kg to about 10 mg / kg (e.g., about 0.01 mg / kg to about 10 mg / kg). 2 ~about 400mg / m 2 (e.g., about 0.1 mg / m 2 ~about 100mg / m 2 , about 1mg / m 2 ~about 50mg / m 2 ) Combination therapy dosing and methods of administration

[0276] In some embodiments, the genome editing complex or nanoparticle composition and / or the second agent / treatment are administered simultaneously. In some embodiments, the genome editing complex or nanoparticle composition and / or the second agent / treatment are administered sequentially. In some embodiments, the genome editing complex or nanoparticle composition and / or the second agent / treatment are administered concurrently.

[0277] The dosing frequency of the genome editing complex or nanoparticle composition and / or the second agent / treatment can be adjusted over the course of treatment based on the judgment of the administering physician. When administered separately, the genome editing complex or nanoparticle composition and / or the second agent / treatment can be administered at different dosing frequencies or intervals. In some embodiments, a sustained continuous release formulation of the genome editing complex or nanoparticle composition and / or the second agent / treatment can be used. Various formulations and devices for achieving sustained release are known in the art. A combination of the administration configurations described herein can also be used.

[0278] In some embodiments, the genome editing complex or nanoparticle composition is administered to the individual at a frequency of about twice per week to about once every two weeks (e.g., about once per week). In some embodiments, the genome editing complex or nanoparticle composition is administered to the individual at least twice.

[0279] The genome editing complex or nanoparticle composition and / or the second agent / treatment may be administered using the same or different administration routes. In some embodiments of the methods described herein, the genome editing complex or the second agent / treatment described herein is administered to an individual by intravenous administration, intratumoral administration, intraarterial administration, topical administration, intraocular administration, eye drop administration, intraportal vein administration, intracranial administration, intracerebral administration, intraventricular administration, intrathecal administration, intravesicular administration, intradermal administration, subcutaneous administration, intramuscular administration, intranasal administration, intratracheal administration, pulmonary administration, intracavitary administration, or oral administration, or by nebulization (NB) or intratracheal instillation.

[0280] In some embodiments, the genome editing complex or nanoparticle composition and / or second agent / treatment described herein are formulated for systemic or local administration. In some embodiments, the genome editing complex or nanoparticle composition and / or second agent / treatment described herein are formulated for intravenous, intratumoral, intraarterial...

Claims

1. A composition for modifying mutated KRAS in a cell, the composition comprising a non-naturally occurring polynucleotide comprising a guide RNA for targeting mutated KRAS, the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) comprising a nucleotide sequence having 100% complementarity to a target sequence, the target sequence comprising the amino acid sequence of SEQ ID NO: 19; The composition is for use in combination with a DNA nuclease or a nucleotide sequence encoding the DNA nuclease, wherein the DNA nuclease comprises a CRISPR-associated protein (Cas) polypeptide.

2. The composition described in claim 1, wherein the guide RNA further comprises an auxiliary transactivating crRNA (tracrRNA).

3. The composition described in claim 1, wherein the guide RNA is chemically modified.

4. A composition for modifying mutated KRAS in a cell, the composition comprising a genome editing complex including: a) a first cell-penetrating peptide; b) a guide RNA for targeting the mutated KRAS, the guide RNA including a specificity-determining CRISPR RNA (crRNA) consisting of a nucleotide sequence having 100% complementarity to a target sequence, the target sequence consisting of the amino acid sequence of SEQ ID NO: 19; The composition is for use in combination with a DNA nuclease or a nucleotide sequence encoding the DNA nuclease, wherein the DNA nuclease comprises a CRISPR-associated protein (Cas) polypeptide.

5. A genome editing complex comprising: a) a first cell-penetrating peptide; b) a guide RNA for targeting a mutated KRAS, the guide RNA comprising a specificity-determining CRISPR RNA (crRNA) consisting of a nucleotide sequence having 100% complementarity to a target sequence; and c) a DNA nuclease or a nucleotide sequence encoding the DNA nuclease, wherein the target sequence consists of the amino acid sequence of SEQ ID NO: 19 and the DNA nuclease comprises a CRISPR-associated protein (Cas) polypeptide.

6. The genome editing complex described in claim 5, wherein the Cas polypeptide is Cas9, a variant thereof, or a fragment thereof.

7. The composition described in claim 4, wherein the first cell-penetrating peptide is selected from the group consisting of CADY, PEP-1 peptide, PEP-2 peptide, PEP-3 peptide, VEPEP-3 peptide, VEPEP-6 peptide, VEPEP-9 peptide, and ADGN-100 peptide.

8. The composition described in claim 4, wherein the first cell-penetrating peptide further comprises one or more moieties covalently linked to the N-terminus of the first cell-penetrating peptide, the one or more moieties being selected from the group consisting of an acetyl group, a fatty acid, cholesterol, polyethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide, a linker moiety, and a targeting moiety.

9. The composition described in claim 8, wherein the first cell-penetrating peptide comprises an acetyl group covalently linked to the N-terminus of the first cell-penetrating peptide.

10. The composition described in claim 4, wherein the first cell-penetrating peptide comprises a targeting moiety comprising a targeting peptide covalently linked to the N-terminus of the first cell-penetrating peptide.

11. The composition described in claim 10, wherein the targeting peptide is selected from the group consisting of SEQ ID NOs: 196-205 and 235-240.

12. The composition of claim 8, wherein the one or more moieties are covalently linked to the N-terminus of the first cell-penetrating peptide by an optional linker moiety, the optional linker moiety being selected from the group consisting of a polyglycine linker moiety, a PEG moiety, Aun, Ava, and Ahx.

13. The composition described in claim 8, wherein the first cell-penetrating peptide comprises an acetyl group, a targeting moiety, and a linker moiety covalently linked from the N-terminus to the N-terminus of the first cell-penetrating peptide.

14. The composition described in claim 4, wherein the first cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-107, 111-117, and 153-175.

15. The method of claim 1, wherein the molar ratio of the first cell-penetrating peptide to the guide RNA is between 1:1 and 80:1; or ii) the molar ratio of the first cell-penetrating peptide to the nucleotide sequence encoding the DNA nuclease is between 1:1 and 80:1; The genome editing complex described in claim 5.

16. The method of claim 1, further comprising one or more additional guide RNAs comprising different guide sequences; i) at least two of the two or more guide RNAs target a single KRAS mutation and at least two of the two or more guide RNAs target G12D, G12V, or G12C; or ii) at least two of the two or more guide RNAs target two or more different KRAS mutations, and at least two of the two or more guide RNAs target G12D and G12V, G12D and G12C, G12V and G12C, or G12D, G12C and G12V; The composition of claim 4.

17. The composition described in claim 4, wherein the average diameter of the genome editing complex is between 10 nm and 300 nm.

18. A nanoparticle comprising a core comprising the composition of claim 4.

19. A pharmaceutical composition comprising the composition of claim 4 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, wherein the composition comprises two or more nanoparticles, the two or more nanoparticles comprising different guide RNAs that target different KRAS mutations.

21. The pharmaceutical composition of claim 19 for treating cancer in an individual.

22. The pharmaceutical composition of claim 21, wherein the composition is administered in combination with a second drug.

23. A method for preparing the composition described in claim 4, comprising the step of combining the first cell-penetrating peptide with the guide RNA, thereby forming the genome editing complex.

24. A composition as described in claim 4 for use in a method for modifying mutated KRAS in a cell, the method comprising a step of contacting the cell with the guide RNA or the genome editing complex.

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