• Fenghua Wang, Hefei Zhang, Jingxia Gao, Fengjiao Chen, Sijie Chen, Cuizhen Zhang, and Gang Peng.
• Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
• BioTechniques. 2016 Jan 1; 60 (6): 299-305.

AbstractCustom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

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