Abstract
Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated “user friendly” TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains.
Highlights
Generation of genetically modified animal models, as well as creation of mutations and correction of mutants in human cells, have provided many important tools for investigating gene function, genetic disease and drug development [1,2]
The assembly of Transcriptional activator-like effector nucleases (TALENs) by PCR in the original strategy is straightforward, but we frequently observed mutations or deletions that had been introduced into TALENs, probably because tandem repeats of the repeat variable diresidues (RVDs) sequences caused PCR errors
Most reports have utilized plasmid-based TALEN expression systems and tested function by transfection of TALEN pairs into cell lines which were transfected by lipofection [15,17,18,19,29,57,58], electroporation [16,31,59,60] or direct injection of DNA or mRNAs [21,32,59,60,61]
Summary
Generation of genetically modified animal models, as well as creation of mutations and correction of mutants in human cells, have provided many important tools for investigating gene function, genetic disease and drug development [1,2]. The efficiency of genetic modification at the target locus could be increased hundreds to thousands fold by creation of a site-specific double-stand break (DSB) through designing “customized” nucleases. Tremendous progress has been made to improve the specificity and affinity of zinc finger domains that recognize desired DNA sequences, the target site overlap and crosstalk between individual fingers make it complicated to design and select sequence-specific ZFNs [7], which has impeded researcher access. Homing endonucleases, such as LAGLIDADG family members, have been engineered for genome editing. The drawback is that the production of homing endonucleases targeted to specific sequences appeared to be more complicated [8,9,10,11]
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