CRISPR/Cas9-mediated genome editing in naïve human embryonic stem cells

Eva Z Jacobs, Sharat Warrier, Pieter-Jan Volders, Eva D'haene, Eva Van Lombergen, Lies Vantomme, Margot Van der Jeught, Björn Heindryckx, Björn Menten, Sarah Vergult
Source: Scientific Reports
Publication Date: (2017)
Issue: 7(1): 16650
Research Area:
Stem Cells
Gene Expression
Basic Research
Cells used in publication:
Species: human
Tissue Origin: kidney
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
4D-Nucleofector® X-Unit

2x10^5 naïve hESC were nucleofected with 0.4 µg plasmid in 20 µl P3 Primary Cell Nucleofector Solution with program DN-100. After nucleofection, cells were plated on inactivated MEF and cultured in medium supplemented with Rock inhibitor.
Additionally, 2x10^5 HEK 293T were nucleofected with 0.4 µg plasmid in 20 µl SF Primary Cell Nucleofector Solution with program CM-130


The combination of genome-edited human embryonic stem cells (hESCs) and subsequent neural differentiation is a powerful tool to study neurodevelopmental disorders. Since the naïve state of pluripotency has favourable characteristics for efficient genome-editing, we optimized a workflow for the CRISPR/Cas9 system in these naïve stem cells. Editing efficiencies of respectively 1.3-8.4% and 3.8-19% were generated with the Cas9 nuclease and the D10A Cas9 nickase mutant. Next to this, wildtype and genome-edited naïve hESCs were successfully differentiated to neural progenitor cells. As a proof-of-principle of our workflow, two monoclonal genome-edited naïve hESCs colonies were obtained for TUNA, a long non-coding RNA involved in pluripotency and neural differentiation. In these genome-edited hESCs, an effect was seen on expression of TUNA, although not on neural differentiation potential. In conclusion, we optimized a genome-editing workflow in naïve hESCs that can be used to study candidate genes involved in neural differentiation and/or functioning.