Optimization of scarless human stem cell genome editing
By Luhan Yang, M. Guell, S. Byrne, Joyce L. Yang, A. De Los Angeles, P. Mali, John D Aach, Caroline Kim-Kiselak, Adrian W. Briggs, Xavier Rios and 3 more. 2013
395 citations recorded by the source corpus.
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.
Subfields: Molecular Biology
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Citations in the corpus
- Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, neutral): “Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in Supplementary Tables 8, 9, 11, 12, 15, and 16.”
- Easy quantitative assessment of genome editing by sequence trace decomposition (2014, methodology): “(b) Overview of TIDE algorithm and output, which consists of three main steps: (1) Visualization of aberrant sequence signal in control (black) and treated sample (green), the expected break site (vertical dotted line) and the region used for decomposition (gray bar); (2) Decomposition yielding the ”