Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation

Abstract

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 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 prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.

What is claimed is: 
     
         1 . A vector system comprising one or more vectors comprising:
 (a) a promoter operably to a nucleotide sequence encoding a Cas9 protein and at least one nuclear localization signal, and   (b) a bidirectional promoter operably linked (i) in a first direction to a nucleotide sequence encoding a first CRISPR-Cas system RNA and (ii) in a second direction to a nucleotide sequence encoding a second CRISPR-Cas system RNA,   wherein (a) and (b) are located on the same vector or different vectors.   
     
     
         2 . The vector system of  claim 1 , wherein the first CRISPR-Cas system RNA is a chimeric RNA comprising, from 5′ to 3′, a first guide sequence capable of hybridizing to a first target sequence in a eukaryotic cell, a first tracr mate sequence and a first tracr sequence; and wherein the second CRISPR-Cas system RNA is a chimeric RNA comprising, from 5′ to 3′, a second guide sequence capable of hybridizing to a second target sequence in a eukaryotic cell, a second tracr mate sequence and a second tracr sequence. 
     
     
         3 . The vector system of  claim 1 , wherein the first and second tracr mate sequences share 100% identity. 
     
     
         4 . The vector system of  claim 1 , wherein the first and second tracr sequences share 100% identity. 
     
     
         5 . The vector system of  claim 1 , wherein the Cas9 protein is a Cas9 ortholog from  Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacteriimi, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteureila, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema , or  Thermotoga.    
     
     
         6 . The vector system of  claim 1 , wherein the Cas9 protein is  Streptococcus pyogenes  Cas9. 
     
     
         7 . The vector system of  claim 1 , wherein the Cas9 protein is  Staphylococcus aureus  Cas9. 
     
     
         8 . The vector system of  claim 1 , wherein the Cas9 protein is a nuclease. 
     
     
         9 . The vector system of  claim 1 , wherein the Cas9 protein comprises mutation(s) in a catalytic domain and is a nickase. 
     
     
         10 . The vector system of  claim 1 , wherein the Cas9 protein comprises mutation(s) in two or more catalytic domains and substantially lacks all DNA cleavage activity. 
     
     
         11 . The vector system of  claim 1 , wherein the Cas9 protein comprises one or more mutations of D10A, E762A, H840A, N854A, N863A, or D986A, and is fused to a heterologous protein domain. 
     
     
         12 . The vector system of  claim 1 , wherein the bidirectional promoter is a bidirectional U6 promoter. 
     
     
         13 . The vector system of  claim 1 , wherein the bidirectional promoter is a bidirectional U6-8/U6-1 promoter. 
     
     
         14 . The vector system of  claim 1 , wherein the one or more vectors are viral vectors. 
     
     
         15 . The vector system of  claim 1 , wherein the one or more vectors are AAV vectors. 
     
     
         16 . The vector system of  claim 1 , wherein the Cas9 protein is a nuclease; wherein the first CRISPR-Cas system RNA is capable of directing cleavage of both strands of a DNA duplex near a first genomic target sequence in a eukaryotic cell, and the second CRISPR-Cas system RNA is capable of directing cleavage of both strands of the DNA duplex near a second genomic target sequence in the eukaryotic cell, thereby introducing microdeletion of a genomic sequence between the first and second target sequences. 
     
     
         17 . The vector system of  claim 1 , wherein the Cas9 protein comprises mutation(s) in a catalytic domain and is a nickase; wherein the first CRISPR-Cas system RNA is capable of directing cleavage of a first strand of a DNA duplex near a first genomic target sequence in a eukaryotic cell, and the second CRISPR-Cas system RNA is capable of directing cleavage of a second strand of the DNA duplex near a second genomic target sequence in the eukaryotic cell, thereby introducing a double-stranded break having an overhang. 
     
     
         18 . The vector system of  claim 17 , wherein the overhang is a 5′ overhang of 26-200 nucleotides. 
     
     
         19 . The vector system of  claim 17 , wherein the overhang is a 5′ overhang of 34-50 nucleotides. 
     
     
         20 . An isolated eukaryotic cell comprising the vector system of  claim 1 .