Crispr/cas fusion proteins and systems

Abstract

Engineered Cas9 systems are disclosed herein.

What is claimed is: 
     
         1 . A system comprising a fusion protein and an engineered guide RNA, wherein the fusion protein comprises a  Streptococcus pyogenes  Cas9 protein; a marker protein; at least one first linker between the Cas9 protein and the marker protein, wherein the at least one first linker comprises SEQ ID NO:35 or SEQ ID NO:36; and a first heterologous domain that is a nuclear localization signal; wherein the fusion protein is a nuclease and cleaves both strands of a double-stranded sequence, is a nickase and cleaves one strand of a double-stranded sequence, or has no nuclease or nickase activity. 
     
     
         2 . The system of  claim 1 , wherein the engineered guide RNA is a single molecule. 
     
     
         3 . The system of  claim 1 , wherein the engineered guide RNA is two separate molecules. 
     
     
         4 . The system of  claim 1 , wherein the engineered guide RNA sequence is optimized to facilitate base-paring within the engineered guide RNA, minimize base-paring within the engineered guide RNA, increase stability of the engineered guide RNA, facilitate transcription of the engineered guide RNA in a eukaryotic cell, or a combination thereof. 
     
     
         5 . The system of  claim 1 , wherein the marker protein is at the N-terminus of the fusion protein. 
     
     
         6 . The system of  claim 1 , wherein the fusion protein further comprises an optional second linker, wherein the nuclear localization signal, the marker protein, the first linker, the optional second linker (if present), and the Cas9 protein are arranged in the following order (N-terminus to C-terminus):
 marker protein-first linker-nuclear localization signal-Cas9 protein;   marker protein-nuclear localization signal-first linker-Cas9 protein;   nuclear localization signal-marker protein-first linker-Cas9 protein;   marker protein-first linker-nuclear localization signal-first linker-Cas9 protein; or   nuclear localization signal-second linker-marker protein-first linker-Cas9 protein.   
     
     
         7 . The system of  claim 6 , wherein the nuclear localization signal, the marker protein, the first linker, and the Cas9 protein are arranged in the following order (N-terminus to C-terminus):
 marker protein-first linker-nuclear localization signal-Cas9 protein.   
     
     
         8 . The system of  claim 1 , wherein the fusion protein further comprises at least one additional heterologous domain other than the first heterologous domain that is a nuclear localization signal, wherein the at least one additional heterologous domain is a cell-penetrating domain, a chromatin modulating motif, an epigenetic modification domain, a transcriptional regulation domain, an RNA aptamer binding domain, or combination thereof. 
     
     
         9 . The system of  claim 1 , wherein the first heterologous domain that is a nuclear localization signal comprises the sequence PAAKRVKLD (SEQ ID NO:6). 
     
     
         10 . The system of  claim 9 , wherein the fusion protein further comprises two nuclear localization signals comprising the sequence PKKKRKV (SEQ ID NO:1). 
     
     
         11 . The system of  claim 1 , wherein the marker protein has an amino acid sequence comprising SEQ ID NO:19 or 20. 
     
     
         12 . The system of  claim 1 , wherein the marker protein has an amino acid sequence consisting of SEQ ID NO:19 or 20. 
     
     
         13 . The system of  claim 1 , wherein the fusion protein has an amino acid sequence having at least 95% sequence identity with SEQ ID NOS:48, 49, or 50. 
     
     
         14 . The system of  claim 1 , wherein the fusion protein has an amino acid sequence having at least 99% sequence identity with SEQ ID NOS:48, 49, or 50. 
     
     
         15 . The system of  claim 1 , wherein the fusion protein has an amino acid sequence of SEQ ID NOS:48, 49, or 50. 
     
     
         16 . A plurality of nucleic acids encoding the system of  claim 1 , the plurality of nucleic acids comprising at least one nucleic acid encoding the fusion protein, and at least one nucleic acid encoding the engineered guide RNA. 
     
     
         17 . The plurality of nucleic acids of  claim 16 , wherein the at least one nucleic acid encoding the fusion protein is RNA. 
     
     
         18 . The plurality of nucleic acids of  claim 16 , wherein the at least one nucleic acid encoding the fusion protein is DNA. 
     
     
         19 . The plurality of nucleic acids of  claim 16 , wherein the at least one nucleic acid encoding the fusion protein is codon optimized for expression in a eukaryotic cell. 
     
     
         20 . The plurality of nucleic acids of  claim 19 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism. 
     
     
         21 . The plurality of nucleic acids of  claim 16 , wherein the at least one nucleic acid encoding the engineered guide RNA is DNA. 
     
     
         22 . The plurality of nucleic acids of  claim 16 , wherein the at least one nucleic acid encoding the fusion protein is operably linked to a phage promoter sequence for in vitro RNA synthesis or protein expression in a bacterial cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a phage promoter sequence for in vitro RNA synthesis. 
     
     
         23 . The plurality of nucleic acids of  claim 16 , wherein the at least one nucleic acid encoding the fusion protein is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell. 
     
     
         24 . At least one vector comprising the plurality of nucleic acids of  claim 16 . 
     
     
         25 . The at least one vector of  claim 24 , which is a plasmid vector, a viral vector, or a self-replicating viral RNA replicon. 
     
     
         26 . A eukaryotic cell comprising at least one system as defined in  claim 1 . 
     
     
         27 . The eukaryotic cell of  claim 26 , which is a human cell, a non-human mammalian cell, a plant cell, a non-mammalian vertebrate cell, an invertebrate cell, or a single cell eukaryotic organism. 
     
     
         28 . The eukaryotic cell of  claim 26 , which is in vivo, ex vivo, or in vitro. 
     
     
         29 . A method for determining chromosome identity and location within a living eukaryotic cell or chemically fixed eukaryotic cell, the method comprising introducing the system of  claim 1  into the living or chemically fixed eukaryotic cell and detecting a signal from the marker protein. 
     
     
         30 . The method of  claim 29 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a plant cell, a non-mammalian vertebrate cell, an invertebrate cell, or a single cell eukaryotic organism.