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.
Highlights
Precise genome editing in human-induced pluripotent cells will enable functional studies of human genetic variation and enhance the potential use of hiPSCs for regenerative medicine
A nuclease-mediated double-stranded DNA break in the genome can be repaired by two main mechanisms [4]: non-homologous end joining (NHEJ), which frequently results in the introduction of non-specific insertions and deletions, or homology-directed repair (HDR), which incorporates a homologous strand as a repair template
We thought that complete elimination of repeats would enable faster and simple synthesis of extended TALE Repeat Variable Diresidue (RVD) arrays and address this important post-synthesis problem
Summary
Precise genome editing in human-induced pluripotent cells (hiPSCs) will enable functional studies of human genetic variation and enhance the potential use of hiPSCs for regenerative medicine. Genome editing via sequence-specific nucleases represents the most efficient way to precisely edit human cell genomes [1,2,3]. When a sequence-specific nuclease is delivered along with a homologous donor DNA construct containing the desired mutations, gene targeting efficiencies are increased by 1000-fold compared with just the donor construct alone [5]. Despite large advances in gene editing tools, many challenges and questions remain regarding the use of customengineered nucleases in hiPSC engineering. Despite their design simplicity, Transcription Activator-Like Effectors Nucleases (TALENs) target particular DNA sequences with tandem copies of Repeat Variable Diresidue (RVD) domains [6]. The modular nature of RVDs simplifies TALEN design, their repetitive sequences complicate methods for synthesizing their DNA constructs [7,8,9,10] and impair their use with lentiviral gene delivery vehicles, most likely by causing sequence instabilities [11]
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