title: Simultaneous screening and validation of effective zinc finger nucleases in yeast.
abstract: Zinc finger nucleases (ZFNs) have been successfully used for genome modification in various cell types and species. However, construction of an effective ZFN remained challenging. Previous studies all focused on obtaining specific zinc finger proteins (ZFPs) first via bacterial 2-hybrid approach, and then fusing selected ZFPs to FokI nuclease domain. These assembled ZFNs have high rate of failing to cleave target sites in vivo. In this study, we developed a simultaneous screening and validation system to obtain effective ZFNs directly in yeast AH109. This system is based on Gal4 reporter system carrying a unique intermediate reporter plasmid with two 30-bp Gal4 homology arms and a ZFN target site. DNA double strand breaks introduced on target sequence by ZFNs were repaired by single strand annealing (SSA) mechanism, and the restored Gal4 drove reporter genes expression. Taking the advantage of OPEN (Oligomerized Pool ENgineering) selection, we constructed 3 randomized ZFNs libraries and 9 reporter strains for each target gene. We tested this system by taking goat α s1-casein as target gene following three-step selection. Consequently, 3 efficient pairs of ZFNs were obtained from positive colonies on selective medium. The ZFNs achieved a 15.9% disruption frequency in goat mammary epithelial cells. In conclusion, we created a novel system to obtain effective ZFNs directly with simultaneous screening and validation.
authors.affiliation: College of Animal Science and Technology, Northwest A & F University, Yangling, Shaanxi, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Juan
authors.initials: J
authors.lastname: Lin
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.forename: Chonghua
authors.initials: C
authors.lastname: Ren
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter; D006041:Goats; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D002364:Caseins; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004720:Endonucleases