Methods and compositions for homologous recombination in human cells
AssigneeThe General Hospital Corporation
InventorsNIELS GEIJSEN, CHRISTA BUECKER
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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.
Claims41
A composition comprising:
- (a) a human stem cell in admixture with a GSK3β inhibitor, and
- (b) a homologous recombination cassette.
A stem cell composition comprising:
- (a) a human stem cell clone isolated from a population of stem cells treated with a GSK313 inhibitor and having the following characteristics:
- (i) survives propagation from a single cell,
- (ii) an efficiency of homologous recombination at least 1-fold higher than the efficiency of homologous recombination in a corresponding human stem cell not treated with the GSK3β inhibitor,
- (iii) a doubling time at least 2 hours faster than the growth rate of a corresponding human stem cell not treated with the GSK3β inhibitor,
- (iv) survives passaging as a single cell using trypsin digest, and
- (b) a homologous recombination cassette.
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.
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.
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.
Dependencies are inferred from the claim text.