Optimization of scarless human stem cell genome editing.

Yang L, Guell M, Byrne S, Yang JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang PY, Daley G, Church G.
Source: Nucleic Acids Res
Publication Date: (2013)
Issue: 41(19): 9049-61
Research Area:
Stem Cells
Basic Research
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector™ X-Unit
Platform: 4D-Nucleofector System Cells: iPSC Cells/sample: 2x10e6 in 20 µl Solution: P3 Program: CB-150 Cas9/gRNA plasmid: 1 µg of Cas9 and 1 µg of gRNA construct reTALEN Plasmid: 1 µg of each Donor construct: 2 µl of 100 µM ssODN or 2 µg of dsDNA donor Summary (by Lonza): The authors developed functional re-coded TALENs (reTALENs) that simplify TALE synthesis and would also allow TALE-based applications with lentiviral vectors. In addition, they established a new genome editing assessment system using next generation sequencing. They compared genome editing efficiencies of the reTALENs with Cas9-gRNA and also optimized ssODN donor design in combination with both methods. For that purpose they transfected human iPSCs with the respective constructs targeting CCR5 and ssODNs for introducing specific mutations using the 4D-Nucleofector™ System. Both nuclease platforms serve as robust tools for genome editing, but Cas9-gRNA achieved 7-8 fold higher non-homologous end joining (NHEJ) efficiencies than reTALENs (3% vs. 0.4%) and moderately superior homology-directed repair (HDR) efficiencies (1.0 vs. 0.6%) when combined with ssODN donors in hiPSCs.
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.