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Transcription activator–like effector nucleases (TALENs) enable genetic modification at specific sites in a genome. Reyon et al . present a method for high-throughput generation of TALENs, facilitating large-scale genome engineering. Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.

A library of DNA constructs enables high-throughput, ligation-free production of transcription activator–like effector (TALE) genes for genetic engineering. Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly of >600 TALEN genes in a single day. Given its simplicity, throughput and fidelity, LIC assembly will permit the generation of TAL effector gene libraries for large-scale functional genomics studies.

Dear Editor, The traditional approach for the generation of gene- targeted animals is homologous recombination （HR） conducted in embryonic stem （ES） cells followed by chimera technology, such as in mice and rats, or HR in somatic cells combined with nuclear transfer in those animals in which germline-competent ES cells are not available, such as pigs, sheep, goats, and cattle. How- ever, no germline-competent rabbit ES cells are yet available. Furthermore, the efficiency of rabbit cloning is so low that no gene-targeted rabbits have been produced thus far by somatic cell nuclear transfer aooroaches.