Gene editing

Abstract

The present invention generally relates to systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that may use vector systems related to recombinases and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.

What is claimed is: 
     
         1 . An engineered, non-naturally occurring system for altering expression of a gene product and/or genome editing comprising:
 one or more nucleic acid sequences comprising a) a nucleic acid molecule encoding an enzyme that introduces a double stranded break in a specific targeted sequence in a gene encoding said gene product in said genome of said cell, b) a nucleic acid molecule comprising a nucleic acid sequence of interest to be inserted into said genome at said double stranded break, said nucleic acid molecule comprising one or more free ends; and c) a nucleic acid sequence encoding a UvsX recombinase;   wherein components (a), (b) and (c) are introduced into a eukaryotic cell so that a targeted double stranded break is introduced into the genome of said cell and said nucleic acid molecule encoding a nucleic acid sequence of interest is inserted by homologous recombination at said double stranded break to effect altered expression of at said at least one gene product and/or genome editing of said gene encoding said gene product of interest and wherein said enzyme that introduces a double stranded break in a specific targeted sequence in a gene encoding said gene product and said nucleic acid molecule encoding a nucleic acid sequence of interest to be inserted into said genome at said double stranded break do not naturally occur together.   
     
     
         2 . The system of  claim 1 , wherein the expression of two or more gene products is altered. 
     
     
         3 . The system of  claim 1 , wherein said enzyme that introduces a double stranded break in a specific targeted sequence in a gene encoding said gene product in said genome of said cell is selected from the group consisting of Cas9 nuclease, meganucleases, Zinc finger (ZNF)-nucleases, and transcription activator-like effector (TALE)-nucleases. 
     
     
         4 . The system of  claim 1 , wherein said Cas9 nuclease is part of a CRISPR-Cas system further comprising a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence. 
     
     
         5 . The system of  claim 4 , wherein the CRISPR-Cas system further comprises one or more nuclear localization signal(s) (NLS(s)). 
     
     
         6 . The system of  claim 4 , wherein the CRISPR-Cas system comprises a trans-activating cr (tracr) sequence. 
     
     
         7 . The system of  claim 4 , wherein the guide RNAs comprise a guide sequence fused to a tracr sequence. 
     
     
         8 . The system of  claim 1 , wherein components (a), (b) and (c) are operably associated with the same or different regulatory elements. 
     
     
         9 . The system of  claim 1 , wherein components (a), (b) and (c) are encoded by mRNA molecules. 
     
     
         10 . The system of  claim 1 , wherein components (a), (b) and (c) are located on the same or different expression vectors. 
     
     
         11 . The system of  claim 10 , wherein said expression vectors are one or more viral expression vectors. 
     
     
         12 . The system of  claim 11 , wherein the one or more viral vectors are selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. 
     
     
         13 . The system of  claim 1 , wherein the nucleic acid sequences encoding components (a), (b) and (c) are codon optimized for expression in a eukaryotic cell. 
     
     
         14 . The system of  claim 1 , wherein said eukaryotic cell is a mammalian cell. 
     
     
         15 . The system of  claim 14 , wherein said mammalian cell is a human cell. 
     
     
         16 . The system of  claim 1 , wherein the expression of one or more gene products is increased. 
     
     
         17 . The system of  claim 1 , wherein the expression of one or more gene products is decreased. 
     
     
         18 . The system of  claim 1 , wherein said nucleic acid molecule encoding a nucleic acid sequence of interest to be inserted into said genome at said double stranded break by homologous recombination is at least 250, 500, or 1000 bases in length. 
     
     
         19 . An engineered, non-naturally occurring CRISPR-Cas system comprising:
 one or more vectors comprising:   a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence of a DNA molecule in a eukaryotic cell that contains the DNA molecule, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product,   b) a second regulatory element operable in said cell operably linked to a nucleotide sequence encoding a Cas9 protein, and   c) a third regulatory element operable in said cell operably linked to a nucleotide sequence encoding a UvsX recombinase,   wherein components (a), (b) and (c) are located on same or different vectors of the system, whereby the guide RNA targets and hybridizes with the target sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.   
     
     
         20 . A cell comprising the system of  claim 1 . 
     
     
         21 . A cell comprising the system of  claim 19 .