Publications

Compact designer TALENs for efficient genome engineering
Marine Beurdeley, Fabian Bietz, Jin Li, Severine Thomas, Thomas Stoddard, Alexandre Juillerat, Feng Zhang, Daniel F. Voytas, Philippe Duchateau & George H. Silva, Nature Communications, doi:10.1038/ncomms2782, Apr 2013

Transcription activator-like effector nucleases are readily targetable ‘molecular scissors’ for genome engineering applications. These artificial nucleases offer high specificity coupled with simplicity in design that results from the ability to serially chain transcription activator-like effector repeat arrays to target individual DNA bases. However, these benefits come at the cost of an appreciably large multimeric protein complex, in which DNA cleavage is governed by the nonspecific FokI nuclease domain. Here we report a significant improvement to the standard transcription activator-like effector nuclease architecture by leveraging the partially specific I-TevI catalytic domain to create a new class of monomeric, DNA-cleaving enzymes. In vivo yeast, plant and mammalian cell assays demonstrate that the half-size, single-polypeptide compact transcription activator-like effector nucleases exhibit overall activity and specificity comparable to currently available designer nucleases. In addition, we harness the catalytic mechanism of I-TevI to generate novel compact transcription activator-like effector nuclease-based nicking enzymes that display a greater than 25-fold increase in relative targeted gene correction efficacy.

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TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti
Azadeh Aryan, Michelle A. E. Anderson, Kevin M. Myles, Zach N. Adelman, PLoS One, e60082, Mar 2013

In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism
for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its
genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been
slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA
lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in
the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20–40% of fertile survivors produced
kmo alleles that failed to complement an existing khw mutation. Most of these individuals produced more than 20% whiteeyed
progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1–7 bp specifically at the
selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding
several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are
highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are
performed in this important disease vector.

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Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides
Wefers, B. et al., PNAS, 110 (10) 3782-3787, Mar 2013

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions.

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Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes
Mashimo, T. et al. , Scientific reports 3 - Nature, doi:10.1038/srep01253, Feb 2013

TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species.

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Knock-out mice created by TALEN-mediated gene targeting
Sung, Y. H. et al. , Nature biotechnology, 31, 23–24, Jan 2013

Phenotypic analysis of gene-specific knockout mice has transformed our understanding of in vivo gene functions. Generation of knockout mice, however, remains a time-consuming and expensive process. Transcription activator-like (TAL) effector nucleases (TALENs) are highly effective in inducing mutations at specific genomic loci, and consequently TALEN-mediated mutagenesis in zygotes is a potential alternative to conventional gene targeting in mice. However, to the best of our knowledge, gene knockout mice have yet to be created using TALENs. Here, we report the generation of mice with a genetic knockout of the progesterone immunomodulatory binding factor 1(Pibf1) or selenoprotein W, muscle 1 (Sepw1) gene using TALENs.

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Overcoming TALE DNA Binding Domain Sensitivity to Cytosine Methylation
Valton, J., et al., The Journal of Biochemical Chemistry, doi: 10.1074/jbc.C112.408864 , Sep 2012

Within the past two years, TAL DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free and universal method to overcome it.

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