DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS
The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery particle formulations and/or systems comprising one or more components of a CRISPR-Cas system, which are means for targeting sites for delivery. The delivery particle formulations of the invention are preferably nanoparticle delivery formulations and/or systems. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.
1 . A composition comprising a polymer particle containing: (a) a CRISPR-Cas system RNA polynucleotide sequence; or (b) Cas9; or (c) both a CRISPR-Cas system RNA polynucleotide sequence and Cas9; or (d) one or more vectors that contain nucleic acid molecule(s) encoding (a), (b) or (c), wherein the CRISPR-Cas system RNA polynucleotide sequence and the Cas9 do not naturally occur together.
2 . The composition of claim 1 , further comprising a surfactant, lipid or protein.
3 . The composition of claim 2 , wherein the surfactant comprises a cationic lipid.
4 . The composition of claim 2 , wherein the lipid comprises a phospholipid.
5 . The composition of claim 2 , wherein the protein comprises lipoprotein.
6 . The composition of claim 2 , comprising the surfactant, the lipid and the protein.
7 . The composition of claim 6 , wherein the surfactant comprises a cationic lipid, the lipid comprises a phospholipid and the protein comprises a lipoprotein.
8 . The composition of claim 7 , wherein the lipoprotein comprises a low density protein.
9 . The composition of claim 3 , wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
10 . The composition of claim 1 , wherein the particle or a component thereof is labeled.
11 . The composition of claim 10 , wherein the label comprises a barcode.
12 . The composition of claim 7 , wherein the polymer comprises ethylene-glycol or PEG
13 . The composition of claim 2 , which comprises 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine, polyethylene glycol or cholesterol.
14 . The composition of claim 1 , comprising (a) the CRISPR-Cas system RNA polynucleotide sequence.
15 . The composition of claim 1 , comprising (b) Cas9.
16 . The composition of claim 1 , comprising (c) both the CRISPR-Cas system RNA polynucleotide sequence and Cas9.
17 . The composition of claim 1 , comprising (d) one or more vectors that contain nucleic acid molecule(s) encoding (a), (b) or (c).
18 . The composition of claim 1 , comprising sgRNA pre-complexed with Cas9.
19 . The composition of claim 18 , comprising a surfactant, a lipid and a protein; and wherein the polymer is biodegradable.
20 . The composition of claim 18 , wherein activity of the complexed sgRNA and Cas9 is controllable.
21 . The composition of claim 19 , wherein the surfactant comprises a cationic lipid and the lipid comprises a phospholipid.
22 . The composition of claim 21 , wherein the lipoprotein comprises a low density protein.
23 . The composition of claim 1 , wherein the CRISPR-Cas system RNA polynucleotide sequence comprises a guide sequence capable of hybridizing to a target in a eukaryotic cell, and the CRISPR-Cas system RNA polynucleotide sequence and the Cas9 are capable of altering expression in a eukaryotic cell and/or the CRISPR-Cas system RNA polynucleotide sequence and the Cas9 form a CRISPR-Cas complex that targets a PAM sequence in a eukaryotic cell.
24 . The composition of claim 23 , wherein the particle or a component thereof is labeled.
25 . The composition of claim 24 , wherein the label comprises a barcode.
26 . The composition of claim 23 , which comprises sgRNA pre-complexed with Cas9, wherein activity of the complexed sgRNA and Cas9 is controllable.
27 . The composition of claim 1 , wherein the CRISPR-Cas system RNA polynucleotide sequence and the Cas9 form a CRISPR-Cas complex, and the complex includes an effector domain.
28 . The composition of claim 1 , wherein the particle includes template DNA and/or the particle includes more than one type of RNA polynucleotide sequence.
29 . The composition of claim 1 , wherein the Cas9 is a nickase, or is mutated, or has reduced or abolished nuclease activity.
30 . A method of altering expression in a eukaryotic cell comprising contacting the cell with the composition of claim 23 , wherein a CRISPR-Cas complex is formed in the cell and a sequence in the cell is targeted.