Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains

Abstract

The invention provides for 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 with additional functional domains. 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 non-naturally occurring or engineered composition for altering expression of at least one gene product in a eukaryotic cell comprising:
 a delivery system operably configured to deliver CRISPR-Cas complex components or polynucleotide sequences encoding said components into a eukaryotic cell, wherein said CRISPR-Cas complex is operable in a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM); said composition comprising:   I. one or more CRISPR-Cas complex polynucleotide sequences comprising or encoding for expression in the eukaryotic cell:   (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 CRISPR enzyme or a polynucleotide encoding a CRISPR enzyme for expression in the eukaryotic cell:   wherein the tracr mate sequence hybridizes to the tracr sequence,   the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,   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,   the CRISPR enzyme comprises one or more mutations such that the enzyme has altered nuclease activity compared to the wild-type enzyme, and   the CRISPR enzyme further comprises one or more heterologous functional domain(s), whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition.   
     
     
         2 . The composition of  claim 1 , wherein the delivery system comprises a vector system comprising one or more vectors, and wherein component (I) comprises a first regulatory element operably linked to a polynucleotide sequence which comprises the guide sequence, the tracr mate sequence and the tracr sequence, and wherein component (II) comprises a second regulatory element operably linked to a polynucleotide sequence encoding the CRISPR enzyme. 
     
     
         3 . The composition of  claim 1 , wherein the delivery system comprises a vector system comprising one or more vectors, and wherein component (I) comprises a first regulatory element operably linked to the guide sequence and the tracr mate sequence, and a third regulatory element operably linked to the tracr sequence, and wherein component (II) comprises a second regulatory element operably linked to a polynucleotide sequence encoding the CRISPR enzyme. 
     
     
         4 . The composition of  claim 2  or  3 , wherein components (I) and (II) are located on same or different vectors of the system. 
     
     
         5 . The composition of  claim 4 , wherein components (I) and (II) are located on the same vector. 
     
     
         6 . The composition of  claim 2  or  3 , wherein the one or more vectors are viral vectors. 
     
     
         7 . The composition of  claim 6 , wherein the one or more viral vectors comprise one or more retrovirus, lentivirus, adenovirus, adeno-associated virus or herpes simplex virus vectors. 
     
     
         8 . The composition of  claim 1 , which is a multiplexed composition comprising multiple guide sequences capable of hybridizing to multiple target sequences. 
     
     
         9 . The composition of  claim 1 , wherein the one or more mutations comprise mutation in D10 SpCas9, H840 SpCas9, N854 SpCas9 or N863 SpCas9, or corresponding residues of other CRISPR enzymes. 
     
     
         10 . The composition according to  claim 1  wherein the one or more mutations comprise D10A, H840A, N854A or N863A. 
     
     
         11 . The composition of  claim 1 , wherein the CRISPR enzyme comprises one or more mutations in two or more catalytically active domains. 
     
     
         12 . The composition of  claim 11 , wherein the CRISPR enzyme comprises D10A SpCas9 in a first catalytically active domain and H840 SpCas9 in a second catalytically active domain or corresponding residues of another other CRISPR enzyme. 
     
     
         13 . The composition of  claim 1 , wherein the one or more mutations is in a catalytically active domain of the CRISPR enzyme comprising RuvCI, RuvCII or RuvCIII. 
     
     
         14 . The composition of  claim 1 , wherein the CRISPR enzyme comprises two or more heterologous functional domains. 
     
     
         15 . The composition of  claim 1 , wherein each heterologous functional domain is independently an epitope tag, a reporter, or a domain having transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, nucleic acid or cellular molecule binding activity, activity as a light-responsive cytochrome heterodimer, transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, nuclease activity, transcription-protein recruiting activity, cellular uptake activity or antibody presentation activity. 
     
     
         16 . The composition of any one of  claim 1 , wherein the one or more protein domain(s) has transcription activation activity. 
     
     
         17 . The composition of  claim 16 , wherein the one or more protein domain(s) comprises VP64. 
     
     
         18 . The composition of  claim 1 , wherein a functional domain is a transcriptional repressor domain. 
     
     
         19 . The composition of  claim 18 , wherein the functional domain comprises a KRAB domain or a SID domain. 
     
     
         20 . The composition of  claim 1 , wherein the one or more protein domains comprises one or more nuclear localization signal(s) (NLS(s)). 
     
     
         21 . The composition of  claim 1 , which comprises two or more nuclear localization sequences. 
     
     
         22 . The composition of  claim 1 , wherein the one or more mutations is in a catalytically active domain of the CRISPR enzyme comprising RuvCI, RuvCII or RuvCIII. 
     
     
         23 . The composition of  claim 1 , wherein the CRISPR enzyme comprises two or more heterologous functional domains. 
     
     
         24 . The composition of  claim 1 , wherein a heterologous functional domain comprises a nuclear localization signal (NLS). 
     
     
         25 . The composition of  claim 1 , which comprises one or more NLS(s). 
     
     
         26 . The composition of  claim 25 , wherein the nuclear localization sequence is at or near the amino-terminus of the enzyme or at or near the carboxy-terminus of the enzyme. 
     
     
         27 . The composition of  claim 26 , which comprises two or more NLSs. 
     
     
         28 . The composition of  claim 27 , wherein at least one of the NLSs is at or near the amino-terminus of the enzyme and at least one of the NLSs is at or near the carboxy-terminus of the enzyme. 
     
     
         29 . The composition of  claim 1 , wherein the eukaryotic cell comprises a mammalian cell. 
     
     
         30 . The composition of  claim 29 , wherein the mammalian cell comprises a human cell. 
     
     
         31 . The composition of  claim 1 , wherein the CRISPR enzyme is codon optimized for expression in a eukaryotic cell. 
     
     
         32 . The composition of  claim 1 , wherein the CRISPR enzyme is a type II Cas9 CRISPR enzyme. 
     
     
         33 . The composition of  claim 1 , wherein the Cas9 enzyme is from an organism selected from the group comprising of genus  Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium  or  Corynebacter.    
     
     
         34 . The composition of  claim 1 , wherein the Cas9 is from  Staphylococcus aureus.