Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.
Custom 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.
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Fenghua
F
Wang
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Hefei
H
Zhang
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Jingxia
J
Gao
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Fengjiao
F
Chen
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Sijie
S
Chen
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Cuizhen
C
Zhang
Institutes of Brain Sciences, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.
Gang
G
Peng
D060326:Chemistry Techniques, Synthetic / Q000379:methods* / Q000592:standards*; D000072669:Gene Editing; D005813:Genes, Synthetic / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000071336:Transcription Activator-Like Effectors / Q000097:blood* / Q000235:genetics*
D009841:Oligonucleotides; D000071336:Transcription Activator-Like Effectors
TALE; gene synthesis; genome engineering; oligonucleotide