An Efficient Method for Generation of Knockout Human EmbryonicStem Cells Using CRISPR/Cas9 System
2017
Human embryonic stem cells (hESCs) represent a promising tool
to study functions of genes during development, to model
diseases, and to even develop therapies when combined with gene
editing techniques such as CRISPR/CRISPR-associated protein-9
nuclease (Cas9) system. However, the process of disruption of
gene expression by generation of null alleles is often
inefficient and tedious. To circumvent these limitations, we
developed a simple and efficient protocol to permanently
downregulate expression of a gene of interest in hESCs using
CRISPR/Cas9. We selected p53 for our proof of concept
experiments. The methodology is based on series of hESC
transfection, which leads to efficient downregulation of p53
expression even in polyclonal population (p53 Low cells), here
proven by a loss of regulation of the expression of p53 target
gene, microRNA miR-34a. We demonstrate that our approach
achieves over 80% efficiency in generating hESC clonal sublines
that do not express p53 protein. Importantly, we document by a
set of functional experiments that such genetically modified
hESCs do retain typical stem cells characteristics. In summary,
we provide a simple and robust protocol to efficiently target
expression of gene of interest in hESCs that can be useful for
laboratories aiming to employ gene editing in their hESC
applications/protocols
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