title: TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.
abstract: Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Tieli
authors.initials: T
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Youjin
authors.initials: Y
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Xuyun
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Jialun
authors.initials: J
authors.lastname: Pang
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Xue
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China.
authors.forename: Mai
authors.initials: M
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China. Electronic address: wulingqian@sklmg.edu.cn.
authors.forename: Lingqian
authors.initials: L
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan, China. Electronic address: liangdesheng@sklmg.edu.cn.
authors.forename: Desheng
authors.initials: D
authors.lastname: Liang
mesh_terms: D002460:Cell Line; D004275:DNA, Ribosomal / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D055879:Gene Knock-In Techniques / Q000379:methods*; D018390:Gene Targeting; D015316:Genetic Therapy; D057809:HEK293 Cells; D006801:Humans; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016335:Zinc Fingers
chemical_list: D004275:DNA, Ribosomal; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I
keywords: Single strand break; TALE Nickase; Targeted gene modification; rDNA