Project name: Homo sapiens

Description: Precise genome editing in human induced pluripotent cells (hiPSCs) with targeted nucleases promises to advance basic biomedical research and to enable personalized therapies. However, technical barriers towards rapid and simple nuclease synthesis, and the lack of tools for accurate assessment of genome editing continue to impede progress. In addition, current methods still rely on drug selection cassettes to obtain edited cells, leaving undesired foreign elements in the host genome. Therefore, development of efficient and precise approaches for genome editing is needed to deliver the clinical promise of hiPSCs. To address these problems, we have invented a robust and fast pipeline for conducting scarless genome editing in hiPSCs, including four novel and modular components:(1) Software for designing re-coded TALEs (re-TALEs) that minimizes DNA repeats(2) A protocol for rapid one-pot assembly of re-TALE nucleases (re-TALENs).(3) A sensitive and accurate genome editing assessment platform(4) A robust screening protocol for isolation of edited hiPSCsUsing our pipeline, we tested genome editing efficiency of 15 pairs of re-TALENs and corresponding single stranded oligodeoxynucleotides (ssODNs) donors targeting the CCR5 locus. Re-TALENs achieved robust targeting efficiencies of 0.3~1.8% homology directed repair (HDR) in hiPSCs and 3-39% HDR in K562 cells. Elimination of repeats also enabled generation of functional lentiviruses coding for re-TALEs. We discovered that genome editing efficiency is not correlated with DNaseI hypersensitivity but instead inversely correlated with nucleosome occupancy. In addition, we provide novel insights into the still elusive mechanism and functional parameters governing re-TALEN-mediated genome editing with ssODNs. Finally, using our integrated pipeline, we isolated scarlessly genome-edited hiPSCs without selection within 3 weeks.

Data type: targeted loci

Sample scope: Monoisolate

Relevance: Medical

Last updated: 2012-11-26