Optimized crispr/cas9 systems and methods for gene editing in stem cells

Abstract

The methods and compositions described herein surprisingly increase CRISPR/Cas-mediated gene editing in stem cells by transiently treating the cells with a stem cell viability enhancer prior to and/or after contacting the cells with one or more CRISPR/Cas9 components. Further, this treatment also surprisingly results in increased engraftment of the stem cells into the target tissue of a subject. The present disclosure also provides one or more modified CRISPR/Cas9 components which, when used in combination with the stem cell viability enhancer, further increases the frequency of gene editing in stem cells, increases stem cell viability, and increases stem cell engraftment.

1 . A method of generating a modified cell for transplantation, comprising:
 (a) contacting a cell with a stem cell viability enhancer for a period not long enough to promote expansion of the cell, wherein the cell does not expand, followed by   (b) contacting the cell with a gRNA molecule and a Cas molecule in the absence of the stem cell viability enhancer,   thereby generating a modified cell for transplantation.   
     
     
         2 . The method of  claim 1 , wherein the step of contacting the cell with the gRNA molecule and the Cas molecule is performed using electroporation. 
     
     
         3 . The method of  claim 2 , further comprising cold-shocking the cell before electroporation and/or after electroporation. 
     
     
         4 . The method of  claim 1 , wherein the cell is contacted with the stem cell viability enhancer for a period of about 72 hours. 
     
     
         5 . The method of  claim 1 , wherein the cell is contacted with the stem cell viability enhancer for a period of about 24-48 hours. 
     
     
         6 . The method of  claim 1 , wherein the cell is contacted with the stem cell viability enhancer for a period of fewer than 120 hours. 
     
     
         7 . The method of  claim 1 , further comprising
 (c) contacting the cell with the stem cell viability enhancer for a period of fewer than 72 hours after step (b).   
     
     
         8 . The method of  claim 1 , wherein the stem cell viability enhancer inhibits differentiation, inhibits programmed cell death, inhibits senescence, or inhibits an innate immune response of the cell. 
     
     
         9 . The method of  claim 8 , wherein the stem cell viability enhancer inhibits programmed cell death by inhibiting autophagy or apoptosis. 
     
     
         10 . The method of  claim 1 , further comprising transferring the modified cell to a subject, wherein the cell engrafts into a target tissue of the subject. 
     
     
         11 . The method of any one of  claim 10 , wherein the target tissue is peripheral blood, bone marrow, or spleen. 
     
     
         12 . The method of  claim 1 , wherein the cell is a stem cell. 
     
     
         13 . The method of  claim 1 , wherein the cell is selected from the group consisting of a circulating blood cell, a mobilized blood cell, a bone marrow cell, a myeloid progenitor cell, a lymphoid progenitor cell, a multipotent progenitor cell, a lineage restricted progenitor cell, an endothelial cell, or a mesenchymal stromal cell. 
     
     
         14 . The method of  claim 1 , further comprising culturing the cell in a medium after step (b), wherein the medium comprises one or more of a cytokines, a basic fibroblast growth factor (bFGF), a vascular endothelial growth factor (VEGF), a Notch signaling modulator, a TGF-β signaling modulator, insulin-like growth factor-binding protein 1 (IGFBP1), insulin-like growth factor binding protein 2 (IGFBP2), insulin-like growth factor 1, insulin-like growth factor 2 (IGF2), insulin-like growth factor 3 (IGF3), an angiopoietin (ANG1), an angiopoietin-like protein (ANGPTL4), a SDF1/CXCR4 axis modulator, a Wnt signaling modulator, or combinations thereof. 
     
     
         15 . The method of  claim 14 , wherein the medium comprises one or more cytokines selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), Flt-3 ligand (FL), interleukin-6 (IL-6), and interleukin-11 (IL-11). 
     
     
         16 . The method of  claim 1 , wherein the stem cell viability enhancer is an aryl hydrocarbon receptor (AhR) antagonist or an innate immune response antagonist. 
     
     
         17 . The method of  claim 16 , wherein the AhR antagonist is selected from the group consisting of StemRegenin-1 (SR1), LGC0006, alpha-napthoflavone, and CH-223191. 
     
     
         18 . The method of  claim 16 , wherein the innate immune response antagonist is selected from the group consisting of cyclosporin A, dexamethasone, reservatrol, a MyD88 inhibitory peptide, an RNAi agent targeting Myd88, a B18R recombinant protein, a glucocorticoid, OxPAPC, a TLR antagonist, rapamycin, BX795, and a RLR shRNA. 
     
     
         19 . The method of  claim 1 , wherein the stem cell viability enhancer is selected from the group consisting of MG132, SB431542, UM171, UM729, and 16, 16-dimethyl prostaglandin E2 (dmPGE2). 
     
     
         20 . The method of  claim 1 , wherein the Cas molecule is a Cas9 polypeptide. 
     
     
         21 . The method of  claim 20 , wherein the gRNA molecule and the Cas9 polypeptide are associated in a pre-formed ribonucleotide complex. 
     
     
         22 . A method of making a hematopoietic stem/progenitor cell (HSC) for transplantation, comprising:
 (a) contacting a HSC with a stem cell viability enhancer for a period of fewer than 72 hours, followed by   (b) electroporating the HSC with a gRNA molecule and a Cas polypeptide in the absence of the stem cell viability enhancer, wherein the gRNA molecule and the Cas polypeptide are associated in a pre-formed ribonucleotide complex.   
     
     
         23 . A method of modifying a target nucleic acid in a cell, the method comprising contacting the cell with a stem cell viability enhancer, a gRNA molecule, and a Cas molecule,
 wherein the contacting step comprises:   (a) contacting the cell with the stem cell viability enhancer for a period not long enough to promote expansion of the cell, wherein the cell does not expand, followed by   (b) contacting the cell with the gRNA molecule and the Cas molecule in the absence of the stem cell viability enhancer,   thereby modifying a target nucleic acid in a cell.