title: Efficient targeting of FATS at a common fragile site in mice through TALEN-mediated double-hit genome modification.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Ma
authors.forename: Jianying
authors.initials: J
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Shen
authors.forename: Li
authors.initials: L
authors.lastname: Qiu
authors.forename: Xingxu
authors.initials: X
authors.lastname: Huang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
mesh_terms: D000818:Animals; D004720:Endonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections
chemical_list: D004720:Endonucleases