TAL effector science

Ousterout et al, 2016

Neuromuscular diseases are a diverse range of conditions that include myopathic and neuropathic disorders related to muscular dysfunction. Inherited neuromuscular diseases are the result of a broad spectrum of genetic mutations, including point mutations, insertions and deletions, chromosomal rearrangements, epigenetic aberrations, and repeat expansions or contractions. Targeted genome editing is a promising method to correct the inherited mutations underlying these disorders. Over the last decade there have been many significant advances in engineering targeted DNA-binding proteins to manipulate specific sequences of complex genomes. These genome editing tools are rapidly becoming viable therapeutics that will allow the targeted addition, exchange, or removal of almost any genetic sequence in the human genome. In this chapter, selected neuromuscular diseases representing inherited myopathies or neuropathies are discussed. The genome editing tools available to create targeted genetic modifications are reviewed. Promising cell- and gene-based therapies are introduced in the context of the treatment of neuromuscular disorders in combination with genome editing therapies. Finally, specific examples of how genome editing may be applied to correct the genetic basis of particular neuromuscular disorders are presented and discussed.

Jasin 2016

Directed modification of the genome is critical for interrogating gene function and can also be applied for gene therapy. Two decades ago a double-strand break (DSB) in the genome was discovered to induce efficient gene modification, either by homologous recombination with introduced DNA, i.e., gene targeting, or imprecise joining of DNA ends leading to mutagenesis. The accelerating development of technologies—meganucleases, ZFNs, TALENs, and CRISPR/Cas9— to introduce DSBs at specific sites in the genome for the purposes of modification is revolutionizing the biological and biomedical sciences. This chapter provides an overview of the research that led to these advances in gene editing and also summarizes DSB repair mechanisms in mammalian cells.

(via T. Lahaye, thx)

Wonni et al, 2016

Background: Bacterial Leaf Blight (BLB) and Bacterial Leaf Streak (BLS) diseases respectively caused by Xanthomonas oryzae pv oryzae (Xoo) and X. oryzae pv oryzicola (Xoc) have become a real threat to rice production in West Africa. Genetic and pathogenicity studies revealed African X. oryzae (Xo) pathovars to be both fairly distant from their Asian counterparts. It is therefore mandatory to evaluate whether available major resistance genes are adapted for an efficient and durable control of BLB in Africa. As to BLS no resistance sources have been identified in rice so far. Results: We screened eleven upland and lowland elite rice varieties cultivated in Burkina Faso for resistance to bacterial blight and bacterial leaf streak. We show that all five upland varieties including three NERICAs are highly resistant to each of the three races of Xoo identified in Africa and generally susceptible to several strains from Asia. We next demonstrated that resistance is occurring independently of the plant developmental stage. Strikingly, phenotyping and in planta growth curves assays showed that the five varieties are also highly resistant to a set of Xoc strains representative of the diversity in West Africa. Finally, we show that resistance of the NERICA varieties to both Xoo and Xoc is likely to be inherited from the O. sativa ssp. japonica rather than the O. glaberrima parental lines. Conclusion: We identified and characterized rice accessions exhibiting new sources of resistance against African Xoo and Xoc strains specifically. These results provide important perspectives for the control of BLB and BLS in West Africa.

Zhao et al, 2016

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

Sugi 2016

Caenorhabditis elegans, a 1 mm long free-living nematode, is a popular model animal that has been widely utilized for genetic investigations of various biological processes. Characteristic features that make C. elegans a powerful model of choice for eukaryotic genetic studies include its rapid life cycle (development from egg to adult in 3.5 days at 20 °C), well-annotated genome, simple morphology (comprising only 959 somatic cells in the hermaphrodite), and transparency (which facilitates non-invasive fluorescence observations). However, early approaches to introducing mutations in the C. elegans genome, such as chemical mutagenesis and imprecise excision of transposons, have required large-scale mutagenesis screens. To avoid this laborious and time-consuming procedure, genome editing technologies have been increasingly used in nematodes including C. briggsae and Pristionchus pacificus, thereby facilitating their genetic analyses. Here, I review the recent progress in genome editing technologies using zinc-finger nucleases (ZFNs), transcriptional activator-like nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in nematodes and offer perspectives on their use in the future.

Voytas 2016

In the coming decades, plant agriculture must produce the food needed to sustain the world’s burgeoning population. This challenge is made more daunting in light of a changing climate, scarcity of inputs such as water and fertilizer, and the need to grow more crops on increasingly marginal land. To achieve food security, we will need to fully exploit plant genetics to develop new crop varieties that produce higher yields of healthier food. Crop improvement through breeding has been the foundation of modern agriculture, resulting in steady increases in productivity of the major commodity crops (Sleper and Poehlman, 2006). Additional genetic variation of value has been created through mutagenesis strategies (Micke et al., 1990). More recently, gene addition through transgenesis has provided valuable traits that impact yield, including herbicide tolerance and resistance to insect pests (Gepts, 2002). Now, with the advent of genome editing, we can directly alter the genetic blueprints of crop species – a powerful approach to create genetic variation needed to produce abundant, healthy food.

(via T. Lahaye, thx)

Huang et al, 2016

Mutations in disease susceptibility (S) genes, here, referred to as recessive resistance, have promise for providing broad durable resistance in crop species. At the same time, few recessive disease resistance genes have been characterized. Here, we show that the broadly effective resistance gene xa5 for resistance to bacterial blight of rice is dependent on the effector gene complement of the pathogen. Specifically, xa5 effectiveness in preventing disease by strains of Xanthomonas oryzae pv. oryzae is dependent on major transcription activation-like (TAL) effector genes and correlated with reduced expression of the cognate S genes. xa5 is ineffective in preventing disease by strains containing the TAL effector gene pthXo1, which directs robust expression of the S gene OsSWEET11, a member of sucrose transporter gene family. Incompatibility is associated with major TAL effectors that target the known alternate S genes OsSWEET14 and OsSWEET13. Incompatibility is defeated by transfer of pthXo1 to otherwise xa5-incompatible strains or by engineering a synthetic designer TAL effectors (dTALe) to boost SWEET gene expression. In either case, compatible or incompatible, target gene expression and lesion formation is reduced in the presence of xa5. The results indicate that xa5 functions as a quantitative trait locus, dampening effector function and, regardless of compatibility, target gene expression. Resistance is hypothesized to occur when S gene expression, and by inference, sucrose leakage, falls below a threshold level.

Fine et al, 2016

Genome editing is greatly facilitated by using engineered nucleases to specifically cleave a pre-selected DNA sequence. Cellular repair of the nuclease-induced DNA breaks by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) allows genome editing in a wide range of organisms and cell lines. However, if a nuclease cleaves at genomic locations other than the intended target, known as “off-target sites”, it can lead to mutations, chromosomal loss or rearrangements, causing gain/loss of function and cytotoxicity. Although zinc finger nucleases (ZFNs), TAL effector nuclease (TALENs), and CRISPR/Cas9 systems have been used successfully to create specific DNA breaks in cells, they lack perfect specificity and may result in off-target cleavage. Methods have been developed to predict and to quantify the off-target cleavage events, which are very important for optimizing nuclease design and determining if the gene editing approaches are highly specific. These methods have the potential to significantly facilitate the design of engineered nucleases for genome editing applications.

Juillerat et al, 2016

In 2009 plant geneticists described a novel DNA binding domain derived from transcription activator-like effectors (TALEs) of the plant pathogen genus Xanthomonas. The DNA recognition domain was distinguished by a modular structure in which each building block binds to a single DNA nucleotide. The breakthrough was the identification of the key residues within each block that define its DNA binding properties and to show that specific alterations of these residues allow for the assembly of tailored DNA binding domains able to target any given sequence. This discovery set the stage for the generation of various designer proteins by fusing tailored TALE-based DNA binding domains, with either endonucleases, transcriptional modulators or chromatin remodeling domains, with the final purpose to modify the genome, the transcriptome or the epigenome. In the last few years, the exploitation of designer enzymes has expanded impressively with applications spanning from basic research to systems biology and human gene therapy.

Cloney 2016

The companies Cellectis and Life Technologies
(now Thermo-Fisher), along with the
Two Blades Foundation, invested in the
potential of TALENs after licensing the
rights from patent holders at Martin Luther
University,10 the University of Minnesota,
and Iowa State University.11 However,
in comparison with ZFNs, TALEN arrays
to target specific sequences are far easier
and less expensive to design and produce,
putting them within the abilities of most
molecular biologists to assemble and the
budgets of academic and noncommercial
entities who want to make their own custom
proteins.

Aravalli et al, 2016

Introduction: More than 100 liver-related disorders are caused by mutations in a single gene. These include both inherited and acquired genetic disorders. The development of nucleic acid-based therapies for liver diseases has been severely restricted due to many undesirable side effects and methodological limitations.
Areas covered: In recent years, however, efforts have been intensified to address these issues, and to develop liver-targeted approaches using novel gene editing technologies, including ZFN, TALEN, CRISPR/Cas and PITCh. While each of these methods utilizes a distinct mechanism of gene modification at the genomic level, they all are dependent on an efficient delivery system to the target site within the host cell. This review will provide an overview on current and emerging therapeutic strategies for the treatment of liver diseases.
Expert opinion: Clinical trials for liver gene therapy have entered an exciting stage and are already showing promise with the development of novel technologies and delivery options. A deeper understanding of off-target effects produced by gene editing approaches and immune responses generated in host cells by gene carriers is needed to fully realize their potential.