De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Desert Agriculture Initiative
Plant Science Program
Permanent link to this recordhttp://hdl.handle.net/10754/561706
MetadataShow full item record
AbstractSite-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.
PubMed Central IDPMC3038751
- Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.
- Authors: Li L, Piatek MJ, Atef A, Piatek A, Wibowo A, Fang X, Sabir JS, Zhu JK, Mahfouz MM
- Issue date: 2012 Mar
- Off-target effects of engineered nucleases.
- Authors: Yee JK
- Issue date: 2016 Sep
- Targeting DNA double-strand breaks with TAL effector nucleases.
- Authors: Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF
- Issue date: 2010 Oct
- Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea.
- Authors: Sun Z, Li N, Huang G, Xu J, Pan Y, Wang Z, Tang Q, Song M, Wang X
- Issue date: 2013 Nov
- A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.
- Authors: Mussolino C, Morbitzer R, Lütge F, Dannemann N, Lahaye T, Cathomen T
- Issue date: 2011 Nov