Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

BMC Molecular Biology

Philippe Duchateau¹  Fayza Daboussi¹  Aymeric Duclert¹  George H Silva¹  Fabian Bietz¹  Jérome Mikolajczak¹  Mikhail Zaslavskiy¹  Gwendoline Dubois¹  Séverine Thomas¹  Julien Valton¹  Marine Beurdeley¹  Alexandre Juillerat¹ 

[1] CELLECTIS S.A, 8 Rue de la Croix Jarry, Paris 75013, France

关键词: Genome editing;    Protein engineering;    TALEN;    TALE;    Transcription activator-like effectors;   

Others  :  1090392
DOI  :  10.1186/1471-2199-15-13

 received in 2014-04-11, accepted in 2014-07-02,  发布年份 2014

【 摘 要 】

Background

The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases.

Results

In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines.

Conclusion

Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases.

【 授权许可】

   
2014 Juillerat et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

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