Gene targeting is a powerful genome engineering tool that can be used for a
variety of biotechnological applications. Genomic double-strand DNA breaks
generated by engineered site-specific nucleases can stimulate gene targeting.
Hybrid nucleases are composed of DNA binding module and DNA cleavage
module. Zinc Finger Nucleases were used to generate double-strand DNA
breaks but it suffers from failures and lack of reproducibility. The transcription
activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a
unique type of DNA-binding domain that bind specific DNA targets. The purpose
of this study is to generate novel sequence specific nucleases by fusing a de
novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage
domain. Our data show that the de novo engineered TALE nuclease can bind to
its target sequence and create double-strand DNA breaks in vitro. We also show
that the de novo engineered TALE nuclease is capable of generating double-strand
DNA breaks in its target sequence in vivo, when transiently expressed in
Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based
hybrid nucleases can be tailored to bind a user-selected DNA sequence
and generate site-specific genomic double-strand DNA breaks. TALE-based
hybrid nucleases hold much promise as powerful molecular tools for gene
targeting applications.
| Date of Award | Jun 6 2011 |
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| Original language | English (US) |
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| Awarding Institution | - Biological, Environmental Sciences and Engineering
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| Supervisor | Jian-Kang Zhu (Supervisor) |
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