Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

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 delivery systems and tissues or organ which are targeted as sites for delivery. 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 - 31 . (canceled) 
     
     
         32 . A vector containing nucleic acid(s) encoding inducible Cas9 and at least one nuclear localization signal (NLS), whereby when the vector is delivered to a eukaryotic cell, when induced, Cas9 is expressed in vivo in the eukaryotic cell. 
     
     
         33 . The vector of  claim 32  wherein the nucleic acid encoding the Cas9 encodes two NLSs, whereby a first NLS is situated at or within 50 amino acids of the amino terminal of Cas9 and a second NLS is situated at or within 50 amino acids of the carboxy terminal of Cas9. 
     
     
         34 . The vector of  claim 32  wherein the inducible Cas9 is a Cre recombinase dependent Cas9. 
     
     
         35 . The vector of  claim 34  wherein the vector comprises a nucleic acid encoding a stop cassette element, wherein the stop cassette element is loxP-SV40 polyA x3-loxP. 
     
     
         36 . The vector of  claim 35  wherein the nucleic acid encoding the stop element is between a nucleic acid encoding a promoter and the Cas9. 
     
     
         37 . The vector of  claim 32  wherein the Cas9 is a  Streptococcus pyogenes  Cas9. 
     
     
         38 . The vector of  claim 32  further comprising a nucleic acid encoding a peptide cleavage sequence. 
     
     
         39 . The vector of  claim 32  further comprising a nucleic acid encoding a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). 
     
     
         40 . The vector of  claim 32  further comprising a nucleic acid encoding a bovine growth hormone poly-A signal sequence (bGHpolyA). 
     
     
         41 . The vector of  claim 32  further comprising a nucleic acid encoding a peptide cleavage sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a bovine growth hormone poly-A signal sequence (bGHpolyA). 
     
     
         42 . A cell comprising the vector of  claim 32 , wherein the cell gives rise to a Cas9 knock in transgenic non-human animal. 
     
     
         43 . The cell of  claim 42  wherein the cell is a mouse cell, the transgenic non-human animal is a mouse, and the vector includes nucleic acid(s) to target insertion of the nucleic acid(s) encoding inducible Cas9 and at least one nuclear localization signal (NLS) to a Rosa26 locus. 
     
     
         44 . A cell comprising the vector of  claim 35 , wherein the cell gives rise to a Cas9 knock in transgenic non-human animal. 
     
     
         45 . A method of making a transgenic non-human animal expressing Cas9 comprising crossing the animal from the cell of  claim 44  with an animal that ubiquitously expresses Cre. 
     
     
         46 . A transgenic animal expressing Cas9 produced by the method of  claim 45 . 
     
     
         47 . The cell of  claim 42  wherein the transgenic animal is a transgenic mammal. 
     
     
         48 . The cell of  claim 44  wherein the transgenic animal is a transgenic mammal. 
     
     
         49 . The animal of  claim 46  wherein the transgenic animal is a transgenic mammal. 
     
     
         50 . The animal of  claim 47  wherein the transgenic mammal is a transgenic mouse, rat, or rabbit. 
     
     
         51 . The animal of  claim 50  wherein the mammal is a transgenic mouse. 
     
     
         52 . A cell comprising the vector of  claim 32 , wherein the cell gives rise to a Cas9 knock in transgenic plant. 
     
     
         53 . A Cas9 knock in non-human eukaryote. 
     
     
         54 . The Cas9 knock in transgenic non-human eukaryote of  claim 53  wherein the eukaryote is a Cas9 knock in transgenic non-human animal or a Cas9 knock in transgenic plant. 
     
     
         55 . The Cas9 knock in transgenic non-human eukaryote of  claim 54  wherein the eukaryote is an animal. 
     
     
         56 . The Cas9 knock in transgenic non-human eukaryote of  claim 55  wherein the animal is a transgenic non-human mammal. 
     
     
         57 . The Cas9 knock in transgenic non-human eukaryote of  claim 56  wherein the mammal is a mouse, rat or rabbit. 
     
     
         58 . A method for delivering RNA(s) to guide Cas9 of the mammal of  claim 53  comprising delivering to cell(s) of the mammal vector(s) containing and/or expressing in vivo the RNA(s) to guide the Cas9, or nanoparticle(s) that carry(ies) the RNA(s) or the vector(s). 
     
     
         59 . The method of  claim 58  wherein the delivery is by vector or particle. 
     
     
         60 . The method of  claim 59  wherein vector is a viral vector. 
     
     
         61 . The method of  claim 59  wherein vector is AAV, Adenovirus or Lentivirus.