Highly efficient genome editing and cell engineering in stem cells using CRISPR/Cas9
Advances in genome editing has empowered researchers with highly efficient and versatile gene editing tools like Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system thereby making it relatively easier to target user defined endogenous genes in a sequence specific manner. Stem cells have been a preferred platform for various applications including gene function analysis, drug screening disease modeling, and tissue engineering. Therefore novel tools that enable rapid and precise gene manipulation in stem cells are required. Presented here are CRISPR/Cas9 tools and workflows that allow accurate design and rapid synthesis of gRNA along with delivery of Cas9 protein/gRNA RNP complexes into a variety of cells through optimized transfection reagents or electroporation. Discussed here are the results from different CRISPR/Cas9 formats tested in stem cells. Using these formats we have edited mouse embryonic stem cells (ESCs) and human iPSCs with up to 80% to 60% genomic cleavage efficiencies, respectively. The methods described here facilitate efficient disease model generation thereby accelerating research in the field of gene therapy and regenerative medicine.
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