Abstract
Transcription activator-like effector nucleases (TALENs) have emerged as a newly developed approach for genome editing. However, its application in targeting specific genomic loci susceptible to DNA damage remains obscure. Here, we report a modified approach for TALENs-based targeting of FATS, a fragile-site gene whose major introns have AT-rich sequence and di-nucleotide repeats. Two pairs of FATS–TALENs were designed to cleave two sites specifically at a coding exon of FATS. After in vitro transcription, the mRNA from FATS–TALEN pairs was microinjected into mouse zygotes. The targeting efficiency of two FATS–TALEN pairs in vivo was more than threefold higher than that of one FATS–TALEN pair. Moreover, large-size DNA deletions were detected, which were heritable and easily detectable by PCR. Our study indicates that the double-hit TALEN approach enhances targeting efficiency in vivo and provides convenience for monitoring germline transmission of mutations by PCR, which will facilitate the functional research on fragile-site genes.
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Acknowledgments
We are grateful to Yawen Zhao for technical assistance. This research was supported by the Grant from National Natural Science Foundation of China (81272283 to Z.L.).
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Ma, K., Wang, J., Shen, B. et al. Efficient targeting of FATS at a common fragile site in mice through TALEN-mediated double-hit genome modification. Biotechnol Lett 36, 471–479 (2014). https://doi.org/10.1007/s10529-013-1387-z
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DOI: https://doi.org/10.1007/s10529-013-1387-z