Enhancing endonuclease based gene editing in primary cells

Abstract

Disclosed herein are nuclease-based systems for genome editing and methods of using the system for genome editing. Also, disclosed are approaches to enhance Cas9-mediated gene editing efficiency in primary human cells with minimal toxicity when using adeno-associated virus vectors (AAV) to express the guide RNAs necessary for CRISPR/Cas9-based genome editing in the presence of helper proteins.

1 . A method for editing a target gene in a cell, the method comprising:
 introducing into a cell a first nucleic acid wherein the first nucleic acid encodes a CRISPR guide RNA complimentary to the target gene;   introducing into the cell a Cas9 protein or a second nucleic acid encoding a Cas9 protein;   introducing into the cell a third nucleic acid encoding a first adenoviral protein; and   introducing into the cell a fourth nucleic acid encoding a second adenoviral protein.   
     
     
         2 . The method of  claim 1 , wherein the first adenoviral protein and the second adenoviral protein are from a serotype 5 adeno-associated virus (AAV). 
     
     
         3 . The method of  claim 1 , wherein the first adenoviral protein comprises a wild type E40RF6 protein, or an AXA mutant E40RF6 protein. 
     
     
         4 . The method of  claim 3 , wherein the first adenoviral protein comprises the AXA mutant E40RF6 protein, and wherein the AXA mutant E40RF6 protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO:23. 
     
     
         5 . The method of  claim 1 , wherein the second adenoviral protein comprises a wild type E1B55K protein, or a mutant EB55K protein. 
     
     
         6 . The method of  claim 5 , wherein the second adenoviral protein comprises the mutant E1B55K protein, and wherein the mutant E1B55K protein is selected from the group consisting of an H373A mutant E1B55K protein, an H354 mutant E1B55K protein, and an R240A mutant E1B55K protein. 
     
     
         7 . The method of  claim 6 , wherein the mutant E1B55K protein is the H373A mutant E1B55K protein and the H373A mutant E1B55K protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO:02. 
     
     
         8 . The method of  claim 6 , wherein the mutant E1B55K protein is the H354 mutant E1B55K protein and the H354 mutant E1B55K protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO:04. 
     
     
         9 . The method of  claim 1 , wherein the CRISPR guide RNA comprises the nucleotide sequence of any one of SEQ ID NO:05, and SEQ ID NOs:15-21. 
     
     
         10 . The method of  claim 1 , wherein a vector comprises the first nucleic acid. 
     
     
         11 . The method of  claim 10 , wherein the vector is an adeno-associated virus (AAV) vector. 
     
     
         12 . The method of  claim 11 , wherein the AAV vector is a self-complementary vector, or a single stranded vector. 
     
     
         13 . The method of  claim 1 , wherein the second nucleic acid, the third nucleic acid and the fourth nucleic acid comprise RNA. 
     
     
         14 . The method of  claim 1 , wherein a vector comprises the third nucleic acid and fourth nucleic acid. 
     
     
         15 . The method of  claim 1 , wherein the Cas9 protein is a  S. pyogenes  Cas9 protein. 
     
     
         16 . The method of  claim 1 , wherein introducing the first nucleic acid, introducing the second nucleic acid, introducing nucleic acid the third nucleic acid, and introducing the fourth nucleic acid, each comprises transiently introducing the nucleic acid into the cell. 
     
     
         17 . The method of  claim 1 , wherein the first nucleic acid, second nucleic acid and third nucleic are introduced into the cell in an order selected from:
 (i) the second nucleic acid is introduced into the cell prior to a vector comprising the first nucleic acid, the second nucleic acid, and the third nucleic acid is introduced into the cell;   (ii) a vector comprising the first nucleic acid, the second nucleic acid, and the third nucleic acid is introduced into the cell prior to introducing the second nucleic acid into the cell; and   (iii) the second nucleic acid and a vector comprising the first nucleic acid, the second nucleic acid, and the third nucleic acid are simultaneously introduced into the cell.   
     
     
         18 . The method of  claim 1 , wherein the cell is a mammalian cell. 
     
     
         19 . The method of  claim 1 , wherein the cell is selected from the group consisting of a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell, and an intestinal cell. 
     
     
         20 . A method of treating, ameliorating, and/or inhibiting a disorder in a subject, the method comprising:
 editing a target gene in cell according to the method of  claim 1  to obtain an edited cell; and   administering the edited cell to the subject.   
     
     
         21 . The method of  claim 20 , wherein the disorder is selected from the group consisting of cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative disease, vascular disease, cystic fibrosis, stroke, hyper IGE syndrome, hemophilia achondroplasia, pseudoachondroplasia, multiple epiphyseal dysplasias, chondrodysplasias, osteogenesis imperfecta, Marfan syndrome, polydactyly, hereditary motor sensory neuropathies I and II (Charcot-Marie-Tooth disease), myotonic dystrophy, and neurofibromatosis.