Optimization of scarless human stem cell genome editing

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.

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines

“…To increase the stringency of the mismatch calls, the user can nominate a phred score threshold. Optionally, singlenucleotide exchanges introduced by targeted mutagenesis approaches using donor oligonucleotides can also be analyzed (Supplemental Note 1) (Chen et al 2011;Bedell et al 2012;Yang et al 2013).…”

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

“…1e) [48,71]. Generally, the distance of SNV from the DSB should be minimized for efficient SNV incorporation [72,73]. This method is routinely performed in early embryos of many animal species [74][75][76].…”

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

“…Currently, the main use of HDR is not to insert exogenous DNA, but to perform in ovo site-directed mutagenesis. A systematic analysis in cultured cells indicates that a 50-mer oligonucleotide with a 2 nt central mismatch already gives an HDR frequency of 1% (compared with NHEJ) (Yang et al 2013b), whereas a 100-mer oligonucleotide yields higher efficiencies. The PAM should be absent from the exogenous template, to prevent subsequent DNA cleavage and NHEJ after HDR.…”

“…Based on an immune defense mechanism discovered in bacteria (Terns & Terns 2011), the method was adapted and first used in mammalian cells in 2013 (Cong et al 2013, Mali et al 2013b. These initial studies opened a field of very intense investigation and technological development making genome editing very efficient in a number of animal species.…”