Methods and compositions for homologous recombination in human cells

Abstract

The methods and compositions described herein are based, in part, on the discovery of a stem cell state in human cells that resembles the morphology observed in murine-derived stem cells. Induction of such a state in human stem cells permits an increase in the efficiency of homologous recombination. Thus, the methods and compositions described herein relate to cells and methods for increasing the efficiency of homologous recombination in human stem cells.

1 . A method for generating a genetically-modified human stem cell, the method comprising:
 (a) culturing a human stem cell with a GSK3β inhibitor,   (b) modifying the genome of the stem cell in step (a) by introducing a homologous recombination cassette and inducing homologous recombination,   wherein a genetically-modified human embryonic stem-like cell is generated.   
     
     
         2 . The method of  claim 1 , wherein the homologous recombination event of step (b) adds a nucleic acid sequence, deletes a nucleic acid sequence, removes an exon, removes an intron, introduces a point mutation, or corrects a mutation. 
     
     
         3 . The method of  claim 1 , wherein homologous recombination is induced using electroporation techniques. 
     
     
         4 . The method of  claim 1 , wherein the human stem cell is an iPS cell, an embryonic stem cell, a stem cell line, a pluripotent stem cell, a multipotent stem cell, or an adult stem cell. 
     
     
         5 . The method of  claim 1 , further comprising culturing the human stem cell with a MEK inhibitor. 
     
     
         6 . The method of  claim 5 , wherein the MEK inhibitor is PD98059. 
     
     
         7 . A composition comprising:
 (a) a human stem cell comprising at least each of the reprogramming factors: Oct4, Sox2, Nanog, c-MYC and KLf4; and   (b) a homologous recombination cassette.   
     
     
         8 . The composition of  claim 7 , wherein the homologous recombination cassette targets a non-developmentally regulated gene or a non-embryonic gene. 
     
     
         9 . The composition of  claim 7 , wherein the cell in the composition has an enhanced efficiency of homologous recombination compared to a corresponding human stem cell. 
     
     
         10 . The composition of  claim 9 , wherein the efficiency of homologous recombination for the cell is enhanced by at least 50% compared to a corresponding human stem cell not comprising a reprogramming factor. 
     
     
         11 . The composition of  claim 7 , wherein the cell of the composition can survive expansion from a single cell and wherein the corresponding human stem cell not comprising a reprogramming factor does not survive expansion from a single cell. 
     
     
         12 . The composition of  claim 7 , wherein the cell of the composition survives passaging as a single cell by a non-collagenase digest, and wherein the corresponding human stem cell does not survive passaging as a single cell by a non-collagenase digest. 
     
     
         13 . The composition of  claim 7 , wherein the cell of the composition survives passaging as a single cell by trypsin digest, and wherein the corresponding human stem cell does not survive passaging as a single cell by trypsin digest. 
     
     
         14 . The composition of  claim 7 , wherein the cell of the composition has a faster growth rate compared to the growth rate of a corresponding human stem cell not comprising a reprogramming factor. 
     
     
         15 . The composition of  claim 14 , wherein the growth rate is assessed by measuring doubling time. 
     
     
         16 . The composition of  claim 15 , wherein the doubling time is at least 2 hours faster than the growth rate of a corresponding human stem cell not in an admixture with the GSK3β inhibitor. 
     
     
         17 . The composition of  claim 7 , further comprising LIF. 
     
     
         18 . A kit for enhancing the efficiency of homologous recombination in a human stem cell, the kit comprising:
 (a) a GSK3b inhibitor,   (b) a homologous recombination cassette, and   (c) instructions and packaging thereof.   
     
     
         19 . The kit of  claim 18 , wherein the GSK3β inhibitor is selected from the group consisting of: CHIR99021, BIO, Kenpaullone, and SB216763. 
     
     
         20 . The kit of  claim 18 , further comprising a MEK inhibitor. 
     
     
         21 . The kit of  claim 20 , wherein the MEK inhibitor is PD98059.