TAL effector science

(via J. Boch, thx)

Luo et al, 2015

Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases (TALENs), and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of plant species (Voytas, 2013; Baltes and Voytas, 2015). However, delivery of these nucleases using traditional transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host genome.

Hutin et al, 2014

Transcription Activator-Like (TAL) effectors are type III-delivered transcription factors that enhance virulence of plant pathogenic Xanthomonas species through the activation of host susceptibility (S) genes. TAL effectors recognize their DNA target(s) via a partially degenerate code whereby modular repeats in the TAL effector bind to nucleotide sequences in the host promoter. While this knowledge has greatly facilitated our power to identify new (S) genes, it can also be easily used to screen plant genomes for variations in TAL effectors target sequences and predict for loss-of-function gene candidates in silico. In a proof-of-principle experiment, we screened a germplasm of 169 rice accessions for polymorphism in the promoter of the major bacterial blight susceptibility (S) gene OsSWEET14 which encodes a sugar transporter targeted by numerous strains of X. oryzae pv. oryzae. We identified a single allele with a deletion of 18-bp overlapping with the binding sites targeted by several TAL effectors known to activate the gene. We show that this allele, which we call xa41(t), confers resistance against half of the tested Xoo strains representative of various geographic origins and genetic lineages, highlighting the selective pressure for the pathogen to accommodate with OsSWEET14 polymorphism and reciprocally the apparent limited possibilities for the host to create variability at this particular S gene. Analysis of xa41(t) conservation across the Oryza genus enabled to raise scenarios as to its evolutionary history prior and during domestication. Our findings demonstrate that resistance through TAL effector-dependent loss of S genes expression can be greatly fostered upon knowledge-based molecular screening of large collection of host plants.

Luo et al, 2015

Here, we assembled improved TALENs targeting the DMY gene and generated XYDMY− mutants to investigate gonadal dysgenesis in medaka. DMY-TALENs resulted in indel mutations at the targeted loci (46.8%). DMY-nanos3UTR-TALENs induced mutations were passed through the germline to F1 generation with efficiencies of up to 91.7%. XYDMY− mutants developed into females, laid eggs, and stably passed the YDMY− chromosome to next generation. RNA-seq generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XYDMY− female medaka (TA_F_OV) gonad libraries. Differential expression analysis identified 144 up- and 293 down-regulated genes in TA_F_OV compared with WT_F_OV, 387 up- and 338 down-regulated genes in TA_F_OV compared with WT_M_TE. According to genes annotation and functional prediction, such as Wnt1 and PRCK, it revealed that incomplete ovarian function and reduced fertility of XYDMY− mutant is closely related to the wnt signaling pathway. Our results provided the transcriptional profiles of XYDMY− mutants, revealed the mechanism between sex reversal and DMY in medaka, and suggested that XYDMY− medaka was a novel mutant that is useful for investigating gonadal dysgenesis in phenotypic female patients with the 46, XY karyotype.

(via T. Schreiber, thx)

Luo et al, 2015

Medaka is an ideal model for sex determination and sex reversal, such as XY phenotypically female patients in humans. Here, we assembled improved TALENs targeting the DMY gene and generated XYDMY− mutants to investigate gonadal dysgenesis in medaka. DMY-TALENs resulted in indel mutations at the targeted loci (46.8%). DMY-nanos3UTR-TALENs induced mutations were passed through the germline to F1 generation with efficiencies of up to 91.7%. XYDMY− mutants developed into females, laid eggs, and stably passed the YDMY− chromosome to next generation. RNA-seq generated 157 million raw reads from WT male (WT_M_TE), WT female (WT_F_OV) and XYDMY− female medaka (TA_F_OV) gonad libraries. Differential expression analysis identified 144 up- and 293 down-regulated genes in TA_F_OV compared with WT_F_OV, 387 up- and 338 down-regulated genes in TA_F_OV compared with WT_M_TE. According to genes annotation and functional prediction, such as Wnt1 and PRCK, it revealed that incomplete ovarian function and reduced fertility of XYDMY− mutant is closely related to the wnt signaling pathway. Our results provided the transcriptional profiles of XYDMY− mutants, revealed the mechanism between sex reversal and DMY in medaka, and suggested that XYDMY− medaka was a novel mutant that is useful for investigating gonadal dysgenesis in phenotypic female patients with the 46, XY karyotype

(via T. Lahaye, thx)

Solin et al, 2015

Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer.

Hubbard et al. 2015

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate transcription activator–like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.

WIlkins et al, 2015

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

Bama pigs, which weigh 35–50 kilograms (by contrast, many farm pigs weigh more than 100 kilograms), have previously been used in research.

To make the smaller, gene-edited micropigs, BGI made cloned pigs from cells taken from a Bama fetus. But before they started the cloning process, they used TALENs to disable one of two copies of the growth hormone receptor gene (GHR) in the fetal cells.  Without the receptor, cells do not receive the ‘grow’ signal during development, resulting in stunted pigs.

BGI then created further micropigs by breeding stunted male clones with normal females. Only half of the resulting, naturally conceived offspring were micropigs, but the process is more efficient than repeating the full cloning procedure, and avoids potential health problems associated with cloning. Among the 20 second-generation gene-edited pigs, BGI has observed no adverse health effects

Rocha-Martins et al, 2015

Genome modification technologies are powerful tools for molecular biology and related areas. Advances in animal transgenesis and genome editing technologies during the past three decades allowed systematic interrogation of gene function that can help model how the genome influences cellular physiology. Genetic engineering via homologous recombination (HR) has been the standard method to modify genomic sequences. Nevertheless, nuclease-guided genome editing methods that were developed recently, such as ZFN, TALEN and CRISPR/Cas, opened new perspectives for biomedical research. Here, we present a brief historical perspective of genome modification methods, focusing on transgenic mice models. Moreover, we describe how new techniques were discovered and improved, present the paradigm shifts and discuss their limitations and applications for biomedical research as well as possible future directions.

Fujita & Fujii (2015)

Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional r

(via T. Schreiber, thx)

Breese et al, 2015

One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partne

(via T. Lahaye, thx)

Yu et al, 2015

Xanthomonas TALE transcriptional activators act as virulence or avirulence factors by activating host disease susceptibility or resistance genes. Their specificity is determined by a tandem repeat domain. Some Xanthomonas pathogens contain 10–30 TALEs per strain. Although TALEs play critical roles in pathogenesis, their studies have so far been limited to a few examples, due to their highly repetitive gene structure and extreme similarity among different members, which constrict sequencing and assembling. To facilitate TALE studies, we developed an efficient and rapid pipeline for genome-wide cloning of tal genes as many as possible from a strain. Here, we report the pipeline and its use to identify all 18 tal genes from a newly isolated strain of the rice pathogen Xathomonas oryzae. Target prediction revealed a number of potential rice targets including several notable genes such as genes encoding SWEET, WRKY, Hen1, and BAK1 proteins, which provide candidates for further experimental functional analysis of the TALEs.

(via T. Lahaye, thx)

LaFountaine et al, 2015

In recent years, several new genome editing technologies have been developed. Of these the Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies.