title: Genetic rearrangements of variable di-residue (RVD)-containing repeat arrays in a baculoviral TALEN system.
abstract: Virus-derived gene transfer vectors have been successfully employed to express the transcription activator-like effector nucleases (TALENs) in mammalian cells. Since the DNA-binding domains of TALENs consist of the variable di-residue (RVD)-containing tandem repeat modules and virus genome with repeated sequences is susceptible to genetic recombination, we investigated several factors that might affect TALEN cleavage efficiency of baculoviral vectors. Using a TALEN system designed to target the AAVS1 locus, we observed increased sequence instability of the TALE repeat arrays when a higher multiplicity of infection (MOI) of recombinant viruses was used to produce the baculoviral vectors. We also detected more deleterious mutations in the TALE DNA-binding domains when both left and right TALEN arms were placed into a single expression cassette as compared to the viruses containing one arm only. The DNA sequence changes in the domains included deletion, addition, substitution, and DNA strand exchange between the left and right TALEN arms. Based on these observations, we have developed a protocol using a low MOI to produce baculoviral vectors expressing TALEN left and right arms separately. Cotransduction of the viruses produced by this optimal protocol provided an improved TALEN cleavage efficiency and enabled effective site-specific transgene integration in human cells.
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Cia-Hin
authors.initials: CH
authors.lastname: Lau
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Haibao
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Johan Chin-Kang
authors.initials: JC
authors.lastname: Tay
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Zhendong
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Felix Chang
authors.initials: FC
authors.lastname: Tay
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Can
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Wee-Kiat
authors.initials: WK
authors.lastname: Tan
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Shouhui
authors.initials: S
authors.lastname: Du
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Vic-Ki
authors.initials: VK
authors.lastname: Sia
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Rui-Zhe
authors.initials: RZ
authors.lastname: Phang
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Shin-Yi
authors.initials: SY
authors.lastname: Tang
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Chiyun
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Zhixia
authors.initials: Z
authors.lastname: Chi
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Chieh-Ching
authors.initials: CC
authors.lastname: Liang
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore.
authors.forename: Er
authors.initials: E
authors.lastname: Ning
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore, Singapore ; Institute of Bioengineering and Nanotechnology , Singapore, Singapore.
authors.forename: Shu
authors.initials: S
authors.lastname: Wang