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.

What is claimed is: 
     
         1 . A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising
 delivering a non-naturally occurring or engineered composition comprising:   
       A)—I. a CRISPR-Cas system chimeric RNA (chiRNA) 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, and 
 II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences, 
 wherein (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, and 
 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, 
 or 
 
       (B) 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, and 
 
       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. 
     
     
         2 . The method of  claim 1 , wherein any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence, is/are RNA. 
     
     
         3 . The method of  claim 1 , wherein the polynucleotides encoding the sequence encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence is/are RNA and are delivered via liposomes or nanoparticles. 
     
     
         4 . The method of  claim 1  wherein the polynucleotides are comprised within a vector system comprising one or more vectors. 
     
     
         5 . A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising
 delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding a composition for expression thereof, wherein the composition comprises:   (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising   I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) 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, and   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,   wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,   wherein components I and II are located on the same or different vectors of the system,   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, and   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,   or   (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising   I. a first regulatory element operably linked to   (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 second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and   III. a third regulatory element operably linked to a tracr sequence,   wherein components I, II and III are located on the same or different vectors of the system,   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, and   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.   
     
     
         6 . The method of  claim 5 , wherein one or more of the viral vectors are delivered via liposomes or nanoparticles. 
     
     
         7 . A method of treating or inhibiting a condition caused by a defect in a target sequence in a genomic locus of interest in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the target sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target sequence comprising providing treatment comprising:
 delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system, comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the target sequence is manipulated by the composition when expressed, wherein the composition comprises:   (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising   I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) 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, and   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,   wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,   wherein components I and II are located on the same or different vectors of the system,   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, and   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,   or   (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising   I. a first regulatory element operably linked to   (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 second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and   III. a third regulatory element operably linked to a tracr sequence,   wherein components I, II and III are located on the same or different vectors of the system,   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, and   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.   
     
     
         8 . The method of  claim 1 ,  5  or  7 , wherein the method is carried out in vitro, and/or ex vivo. 
     
     
         9 . The method of  claim 1 ,  5  or  7  including inducing expression. 
     
     
         10 . The method of  claim 1 ,  5  or  7  wherein the organism or subject is a eukaryote. 
     
     
         11 . The method of  claim 10  wherein the organism or subject is a non-human eukaryote. 
     
     
         12 . The method of  claim 1 ,  5  or  7  wherein the organism or subject is a mammal or a non-human mammal. 
     
     
         13 . The method of  claim 5  wherein the viral vector is an AAV or lentiviral vector. 
     
     
         14 . The method of  claim 1 ,  5  or  7  wherein the CRISPR enzyme is a Cas9. 
     
     
         15 . The method of  claim 1 ,  5  or  7  wherein expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase. 
     
     
         16 . A method of delivering a CRISPR enzyme of  claim 1 ,  5  or  7 , comprising delivering to a cell mRNA encoding the CRISPR enzyme. 
     
     
         17 . The method of  claim 1 ,  5  or  7 , wherein the polynucleotide or enzyme coding sequence encoding the CRISPR enzyme is delivered to the cell by delivering mRNA encoding the CRISPR enzyme to the cell. 
     
     
         18 . A method of preparing the AAV or lentivirus vector of  claim 7  comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV or lentivirus into AAV-infected or lentivirus-infected cells, and supplying AAV AAV or lentivirus rep and/or cap and/or helper nucleic acid molecules obligatory for replication and packaging of the AAV or lentivirus. 
     
     
         19 . A method of preparing an AAV or lentivirus vector for use in the method of  claim 7 , comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV or lentivirus into AAV-infected or lentivirus-infected cells, and supplying AAV AAV or lentivirus rep and/or cap and/or helper nucleic acid molecules obligatory for replication and packaging of the AAV or lentivirus. 
     
     
         20 . The method of  claim 18  wherein the cells are mammalian cells. 
     
     
         21 . The method of  claim 19  wherein the cells are insect cells and the helper virus (where present) is baculovirus. 
     
     
         22 . The method of  claim 1 ,  5  or  7  wherein the target sequence is flanked at its 3′ end or followed by 5′-NRG (where N is any Nucleotide), or where the CRISPR enzyme is (or is derived from) a genus belonging to the group consisting of  Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphyloccoccus, Nitratifractor, Mycoplasma  and  Campylobacer.