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Nucleic Acids Res. 2013 Oct;41(19):9049-61. doi: 10.1093/nar/gkt555. Epub 2013 Jul 31.

Optimization of scarless human stem cell genome editing.

Author information

1
Department of Genetics, Harvard Medical School, Boston, 02115 MA, USA, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, 02115 MA, USA, Children's Hospital, Boston, 02115 MA, USA, Chemistry and Chemical Biology program, Harvard, 02138 Cambridge, MA, USA and Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, 02138 MA, USA.

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.

PMID:
23907390
PMCID:
PMC3799423
DOI:
10.1093/nar/gkt555
[Indexed for MEDLINE]
Free PMC Article

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