TAL effector science

(via T. Lahaye, thx!)

Mendenhall et al, 2013

Mammalian gene regulation is dependent on tissue-specific enhancers that can act across large distances to influence transcriptional activity1, 2, 3. Mapping experiments have identified hundreds of thousands of putative enhancers whose functionality is supported by cell type–specific chromatin signatures and striking enrichments for disease-associated sequence variants4, 5, 6, 7, 8, 9, 10, 11. However, these studies did not address the in vivo functions of the putative elements or their chromatin states and did not determine which genes, if any, a given enhancer regulates. Here we present a strategy to investigate endogenous regulatory elements by selectively altering their chromatin state using programmable reagents. Transcription activator–like (TAL) effector repeat domains fused to the LSD1 histone demethylase efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusion proteins frequently causes downregulation of proximal genes, revealing enhancer target genes. Our study demonstrates the potential of epigenome editing tools to characterize an important class of functional genomic elements.

(via T. Schreiber, thx)

Christian et al, 2013

Here, we used TALENs to modify the genome of the model plant, Arabidopsis thaliana. We engineered seven TALENs targeting five Arabidopsis genes, namely ADH1, TT4, MAPKKK1, DSK2B and NATA2. In pooled seedlings expressing the TALENs, we observed somatic mutagenesis frequencies ranging from 2%-15% at the intended targets for all seven TALENs. Somatic mutagenesis frequencies as high as 41%-73% were observed in individual transgenic plant lines expressing the TALENs. Additionally, a TALEN pair targeting a tandemly duplicated gene induced a 4.4 kb deletion in somatic cells. For the most active TALEN pairs, namely those targeting ADH1 and NATA2, we found that TALEN-induced mutations were transmitted to the next generation at frequencies of 1.5-12%. Our work demonstrates that TALENs are useful reagents for achieving targeted mutagenesis in this important plant model.

(via T. Schreiber, thx)

Noël et al., 2013

During the co-evolution process, bacteria have selected a TAL repertoire to adapt to the diversity of natural hosts and the selection of novel crop species by humans. The latter might be the reason why some strains of the rice pathogens Xoo and Xoc have so many TALs (up to 26) (Schornack et al., 2013). Therefore, the choice of the right host plant genotype to find the genuine TAL targets is critical. One will always find a target for a TAL in any plant or even animal genome. The experimental validation of target gene induction or direct TAL-binding to the promoter still does not indicate that the right biological system was studied. If the tal gene studied contributes significantly to the pathogenicity on the selected plant genotype, one has the chance to find important S genes. Yet, in nature, the contribution of many TAL effectors to disease development will be subtle and dependent on the plant genotype. This means that in the future, Xanthomonas and diseased hosts should be sampled together in epidemics to advance in the identification of genuine TAL targets and in our understanding of Xanthomonas virulence strategies. Combining pathosystems isolated from natural epidemics with in silico, genomic and transcriptomic approaches are certainly the way to go in the next decade. These approaches should yield a large number of targets which contribute quantitatively to susceptibility and resistance for marker-assisted breeding in important crop species.

(via T. Lahaye, thx)

Mali et al (2013)

As a genome editing tool TAL effector–FokI dimers are usually used, and for genome regulation TAL effector–VP64 fusions have been shown to be highly effective. We used the latter as ... this format also reveals the specificity profile of individual TAL-effectors. Examining the TAL effector off-targeting data ... reveals that 18-mer TAL effectors can potentially tolerate 1 or 2 mutations in their target sequences, but fail to activate a large majority of three-base mismatch variants in their targets. They are also particularly sensitive to mismatches nearer
the 5ʹ end of their target sequences. Notably, certain mutations in the middle of the target lead to greater TAL effector activity, an aspect that needs further evaluation. We confirmed a subset of the above results through targeted experiments in a nuclease assay ... We also observed that shorter TAL effectors (14-mer and 10-mer) are progressively less tolerant of mismatches but also reduced in activity by an order of magnitude...
To decouple the role of individual repeat-variable di-residues (RVDs), we confirmed that choice of RVDs does contribute to base specificity but TAL effector specificity is also a function of the binding energy of the protein as a whole ... our data imply that engineering shorter TAL effectors or TAL effectors bearing a judicious composition of high- and low-affinity monomers can potentially yield higher specificity in genome engineering applications, and the requirement for FokI dimerization in nuclease applications enables a further dramatic reduction in off-target effects especially when using the shorter TAL effectors.

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Yang et al, 2013

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Wang et al, 2013

In the silkworm Bombyx mori, TALEN has been successfully used to knockout an endogenous Bombyx gene, and shown the huge potential in functional genes research and improvement of the economical characteristics of silkworm. Thus, there is an urgent need to develop an applicable system that permits the efficient construction of customized TALEN with high activity that could efficiently induce the hereditable mutagenesis in the silkworm. In this study, we constructed an efficient assembly and evaluation system of the customized TALEN especiallly for silkworm genome editing by combination of a modified Golden Gate ligation strategy, a luciferase (LUC) reporter system in insect cell culture for binding activity and a surveyor nuclease assay system in silkworm embryos for cleavage efficiency. We showed the reliability of this system by assembling a pair of TALENs targeting a silkworm genome locus and assaying their binding and cleavage activities. The assembly strategy was convenient and efficient which allows the rapid construction of customized TALEN in less than 1 week, and the evaluation system was reliable and necessary for screening of the customized TALEN pair with high binding and cleavage activities. The results showed this system is a reliable and efficient tool for the construction of customized TALEN with high activity for gene targeting of silkworm, and will contribute to the wide application of TALEN technology in the functional gene research of silkworm.

(via T. Lahaye, thx)

Chen et al, 2013

The hepatitis B virus (HBV) is a DNA virus that can cause chronic hepatitis B (CHB) in humans. Current therapies for CHB infection are limited in efficacy and do not target the pre-existing viral genomic DNA, which present in the nucleus as a covalently closed circular DNA (cccDNA) form. The transcription activator-like (TAL) effector nucleases (TALENs) are newly developed enzymes that can cleave sequence-specific DNA targets. Here, TALENs targeting the conserved regions of the viral genomic DNA among different HBV genotypes were constructed. The expression of TALENs in Huh7 cells transfected with monomeric linear full-length HBV DNA significantly reduced the viral production of HBeAg, HBsAg, HBcAg and pgRNA and resulted in a decreased cccDNA level and misrepaired cccDNAs without apparent cytotoxic effects. The anti-HBV effect of TALENs was further demonstrated in a hydrodynamic injection-based mouse model. In addition, an enhanced antiviral effect with combinations of TALENs and interferon-α (IFN-α) treatment was observed and expression of TALENs restored HBV suppressed IFN-stimulated response element (ISRE)-directed transcription. Taken together, these data indicate that TALENs can specifically target and successfully inactivate the HBV genome and are potently synergistic with IFN-α, thus providing a potential therapeutic strategy for treating CHB infection.Molecular Therapy (2013); doi:10.1038/mt.2013.212.

(via T. Strauss, thanks!)

Stella et al., 2013

Here, the crystal structure of AvrBs3 (residues 152-895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold.

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Bacman et al, 2013

Mitochondrial diseases are commonly caused by mutated mitochondrial DNA (mtDNA), which in most cases coexists with wild-type mtDNA, resulting in mtDNA heteroplasmy. We have engineered transcription activator-like effector nucleases (TALENs) to localize to mitochondria and cleave different classes of pathogenic mtDNA mutations. Mitochondria-targeted TALEN (mitoTALEN) expression led to permanent reductions in deletion or point-mutant mtDNA in patient-derived cells, raising the possibility that these mitochondrial nucleases can be therapeutic for some mitochondrial diseases.

Transcription activator-like (TAL) effectors from Xanthomonas pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the C-terminal domain of the citrus RNA polymerase II (CTD) and inhibit the activity of CsCYP, a CTD-associated cyclophilin that functions as a negative regulator of cell growth. Here we show that PthA4 specifically interacted with the citrus MAF1 (CsMAF1) protein, an RNA Pol III repressor that controls ribosome biogenesis and cell growth in yeast and humans. CsMAF1 bound the human RNA Pol III and rescued the yeast MAF1 mutant by repressing the tRNAHis transcription. Expression of PthA4 in the MAF1 mutant slightly restored the tRNAHis synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNAi plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and III to coordinately increase transcriptional of host genes involved in ribosome biogenesis and cell proliferation.

(via T. Lahaye, thx)

Xu et al, 2013

Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.