A Simple and Efficient Method for Assembling TALE Protein Based on Plasmid Library

Zhiqiang Zhang,Duo Li,Tingting Zhang,Zhilong Chen,Zhiying Zhang,Huarong Xu,Ying Xin,Xin Wang,Lixia Ma,Brian P Chadwick

doi:10.1371/journal.pone.0066459

Abstract

DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.

Highlights

Efficient targeted genome editing relies on the use of engineered nucleases, artificial proteins composed of a customizable sequencespecific DNA-binding domain fused to a nuclease that cleaves DNA in a non-sequence-specific manner
Genome alterations have been generated by repair of DNA double-strand breaks (DSBs) through nonhomologous end-joining (NHEJ) or homologous recombination (HR) induced by customized TALE nucleases (TALENs) in plants [6], yeasts [10], zebrafish [11,12,13], Xenopus embryos [14], rat embryos [15] and human somatic [16,17] and pluripotent stem cells [18]
Engineering Transcription activator-like effectors (TALEs) protein to target specific DNA sequence is much easier than using zinc finger protein, assembling TALE repeats is technically challenging

Summary

Introduction

Efficient targeted genome editing relies on the use of engineered nucleases, artificial proteins composed of a customizable sequencespecific DNA-binding domain fused to a nuclease that cleaves DNA in a non-sequence-specific manner. Deciphering the DNA binding mechanism of TALE repeats opens a new avenue to develop TALE-based technology for genome editing [4,5]. TALE repeats can be adjacent in arrays of custom length with capability to target specific DNA sequences. Artificial TALE transcription factors (TALE-TFs) or TALE nucleases (TALENs) have been constructed by fusing customized repeat arrays to transcriptional activation domain or FokI cleavage domain [6,7]. Genome alterations have been generated by repair of DNA double-strand breaks (DSBs) through nonhomologous end-joining (NHEJ) or homologous recombination (HR) induced by customized TALENs in plants [6], yeasts [10], zebrafish [11,12,13], Xenopus embryos [14], rat embryos [15] and human somatic [16,17] and pluripotent stem cells [18]

Methods

Results

Conclusion