title: A suicidal zinc finger nuclease expression coupled with a surrogate reporter for efficient genome engineering.
abstract: Genome editing with engineered nucleases, such as zinc-finger nucleases (ZFNs) and TALE nucleases, remains confronted with a high risk of cellular toxicity induced by off-targeting. Here we describe the construction of a suicidal nuclease expression vector in which a pair of ZFNs genes were flanked of its target sites. To further enrich the targeted cells, the suicidal ZFN expression cassette was also inserted within an eGFP reporter, to disrupt the ORF of eGFP gene. ZFN-associated toxicity was reduced by ~40 % with this new system, and the activities of ZFNs were ~4.5 % lower. We conclude that using this new suicidal ZFN expression and surrogate reporter system represents an improvement for genomic editing by reducing toxicity and allowing easy detection of edited cells by eGFP analysis.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, 712100, Shaanxi, China, cfzhang@nwipb.cas.cn.
authors.forename: Cunfang
authors.initials: C
authors.lastname: Zhang
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.forename: Linyong
authors.initials: L
authors.lastname: Hu
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.forename: Chonghua
authors.initials: C
authors.lastname: Ren
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D001483:Base Sequence; D002470:Cell Survival; D003851:Deoxyribonucleases / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases