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1. (WO2015089419) DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS
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WHAT IS CLAIMED IS:

1. A composition comprising a delivery particle formulation comprising a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence

wherem (a), (b) and (c) are arranged in a 5' to 3' orientation,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence.

2. The composition of claim I further comprising a polynucleotide sequence encoding a CRISPR. enzyme, optionally comprising at least one or more nuclear localization sequences, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.

3. The composition of claim 2, wherein the polynucleotide sequence is comprised within a vector system comprising one or more vectors.

4. The composition of claim 2, wherein the CRISPR enzyme is a Cas 9.

5. The composition of claim 1, wherein the guide sequence, tracr mate sequence or tracr sequence is/are RNA and comprised within a vector system comprising one or more vectors

6. The composition of claim 1 , wherein the delivery particle formulation comprises liposomes, nanoparticles, exosomes, microvesicles, or a gene gun.

7. The composition of claim 6, wherein the delivery particles are less than 500 nm in diameter.

8. The composition of claim 6, wherein the deliver}' particles are less than 250 nm in diameter.

9. The composition of claim 6, wherein the delivery particles are less than 100 nm in diameter.

10. The composition of claim 6, wherein the deliver}' particles are about 35 nm to about 60 nm in diameter.

1 1. The composition of claim 6 wherein the delivery particle formulation is a nanoparticle formulation.

12. The composition of claim 1 1, wherein the nanoparticle formulation comprises a lipid-based nanoparticle.

13. The composition of claim 12, wherein the lipid-based nanoparticle comprises an epoxide-modified lipid polymer.

14. The composition of claim 1 comprising two or more of (a), (b), and/or (c).

15. The composition of claim 1, wherein the target sequence is an endothelial target sequence.

16. The composition of claim 1 , wherem the target sequence is a lung or skin target sequence.

17. The composition of claim 1, wherein the target sequence is a cardiac or muscle target sequence.

18. The composition of claim 1, wherein the deliver particle formulation is provided as a pharmaceutical composition,

19. The composition of claim 2, wherein the delivery particle formulation is provided as a pharmaceutical composition.

20. The composition of claim 18 further comprising pharmaceutically acceptable excipients and or carriers for ex vivo and/or in vivo administration.

21. The composition of claim 19 further comprising pharmaceutically acceptable excipients and or carriers for ex vivo and/or in vivo admin stration,

22. A composition comprising a delivery particle formulation comprising:

I. polynucleotides comprising:

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and

(b) at least one or more tracr mate sequences,

II. a polynucleotide sequence encoding a CRISPR enzyme, and

III. a polynucleotide sequence comprising a tracr sequence,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and

wherein the delivery particles are less than 250 nm in diameter.

23. The composition of claim 22, wherein the deliver particle is a formulation comprises a liposome, a nanoparticle, an exosome, a microvesicle, or a gene gun.

24. The composition of claim 22, wherein the target sequence is an endothelial target sequence.

25. A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising administering a composition comprising administering a delivery particle formulation comprising a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cel l,

(b) a tracr mate sequence, and

(c) a tracr sequence

wherein (a), (b) and (e) are arranged in a 5' to 3' orientation,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence.

26. The method of claim 25, further comprising administering a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences, wherein the CRISPR complex comprises the CRISPR enzyme complexed with ( 1 ) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.

27. The method of claim 26, wherein the the CRISPR enzyme is a Cas9.

28. The method of claim 26, wherein the polynucleotide or enzyme coding sequence encoding the CRISPR enzyme is delivered to the cell by delivering niRNA encoding the CRISPR enzyme to the cell.

29. The method of claim 25, wherem the delivery particle formulation comprises a liposome, a nanoparticle, an exosome, a microvesicle, or a gene gun.

30. The method of claim 29, wherein the delivery particles are less than 250 nm in diameter.

31 . The method of claim 29, wherein the deliver ' particles are less than 100 nm in diameter.

32. The method of claim 29, wherein the deliver}' particles are about 35 nm to about 60 nm in diameter.

33. The method of claim 29, wherem the delivery particle formulation is a nanoparticle formulation.

34. The method of claim 33, wherem the nanoparticle formulation comprises a lipid-based nanoparticle.

35. The method of claim 34, wherein the iipid-based nanoparticle comprises an epoxide-modified lipid polymer.

36. The method of claim 26, wherein the method is carried out in vitro, ex vivo, and/or in vivo.

37. The method of claims 26, wherein the organism or subject is a eukaryote.

38. The method of any of claim 26, wherein the organism or subject is a mammal or a non- human mammal.

39. The method of claim 26, wherein the modification comprises functional gene silencing.

40. The method of claim 39, wherem the functional gene silencing comprises a reduction in rnRNA and/or protein levels of a targeted gene.

41. The method of claim 40, wherem the targeted gene is expressed in endothelial cells.

42. The method of claim 40, wherein the targeted gene is expressed in lung or skin.

43. The method of claim 40, wherein the targeted gene is expressed in heart or muscle.

44. The method of claim 40, wherein the functional gene silencing provides a therapeutic benefit.

45. A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising administering a composition comprising a delivery particle formulation comprising:

L polynucleotides comprising:

(a) a guide sequence capabl e of hybridizing to a target sequence in a eukaryotic cell, and ( b) at least one or more tracr mate sequences,

II. a polynucleotide sequence encoding a CRISPR enzyme, and

III. a polynucleotide sequence comprising a tracr sequence,

wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,

wherein the CRISPR complex comprises the CRISPR enzyme complexed. with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and the polynucleotide sequence encoding a CRISPR. enzyme is DNA or R A, and

wherein the delivery particles are less than 250 nm in diameter.

46. The method of claim 45, wherein the delivery particle formulation comprises a liposome, a nanoparticle, an exosomes, a microvesicle, or a gene gun.

47. The method of claim 45, wherein the target sequence is an endothelial target sequence.

48. A method of preparing an sgRNA~and-Cas9 protein containing particle comprising admixing an sgRNA and Cas9 protein mixture with a mixture comprising or consisting essentially of or consisting of surfactant, phospholipid, biodegradable polymer, lipoprotein and alcohol.

49. An sgRNA-and-Cas9 protein containing particle from the method of claim 48.

50. Use of the particle of claim 49 in a method of modifying a genomic locus of interest, or an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest, comprising contacting a cell containing the genomic locus of interest with the particle wherein the sgRNA targets the genomic locus of interest; or a method of modifying a genomic locus of interest, or an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest, comprising contacting a cell containing the genomic locus of interest with the particle wherein the sgRNA targets the genomic locus of interest.