Abstract
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.
Highlights
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of developing pre-implantation mouse or human blastocysts and are characterized by their ability for self-renewal and pluripotency[1,2]
The Cas[9] endonuclease is guided by the single guide RNA (sgRNA) to the target locus and will induce a double-stranded break 3 base pairs upstream of the protospacer adjacent motif (PAM) sequence. Either these breaks will be repaired by non-homologous end joining (NHEJ) often leading to the formation of insertions/deletions near the breakpoints as this process is error-prone, or an exogenous DNA sequence can be inserted during homology-directed repair (HDR)[21,22,23]
Efficient genome-editing in naïve human ESCs (hESCs) with CRISPR/Cas[9] system
Summary
Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of developing pre-implantation mouse or human blastocysts and are characterized by their ability for self-renewal and pluripotency[1,2]. Human ESCs (hESCs) are derived from the pre-implantation blastocyst, they differ from naïve mESCs in morphology, culture requirements, molecular profile, differentiation behaviour and clonogenicity[5]. Instead, these cells show more similarities to the post-implantation EpiSCs and conventional derived hESCs are considered as primed pluripotent stem cells[5,7]. A bottleneck with the culture of conventional hESCs is their low proliferation rate[7,26], their inability for single-cell passaging by trypsin digest[7,25] and the low single-cell cloning efficiency[7] These characteristics complicate fast expansion of the culture and clonal isolation and selection after transfection[25]. Another disadvantage of primed hESCs is the heterogeneity in differentiation potential[6,26]
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