Optimization of scarless human stem cell genome editing

TLDR

Efficient precise genome editing in hiPSCs is essential for advanced research and clinical applications, yet tools such as TALENs and Cas9‑gRNA still require optimization to broaden their use. The study aims to develop re‑coded TALEs (reTALEs) that enable one‑pot synthesis and lentiviral delivery for streamlined genome editing. The authors compared 15 reTALEN/Cas9‑gRNA pairs targeting CCR5 in hiPSCs and optimized ssODN design to introduce specific mutations. Cas9‑gRNA achieved 7–8× higher NHEJ efficiencies (3% vs 0.4%) and slightly higher HDR efficiencies (1.0% vs 0.6%) with ssODN donors, and the optimized design enabled seamless genome correction of hiPSCs within 3 weeks.

Abstract

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.